seventeenth annual congress of the czechoslovak society for microbiology České budějovice...

Folia Microbiol. 32, 485--530 (1987)

Seventeenth Annual of the Czechoslovak ~eskg Bud~jovice ~qeptember 23--25, 1986

Congress Society for Microbiology

ABSTRACTS OF COMMUNICATIONS

Sections: General Microbiology p. 486 Applied Microbiology p. 493 Environmental Microbiology Medical Microbiology p. 511 Virology p. 524

p. 501

GENERAL MICROBIOLOGY

Construction of New Plasmid Vectors for Streptomycctes

PET~IS~K M., TICH~ P. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Two extrachromosomal replicons -- multicopy plasmids pSLG3 and pSLG33 -- were isolated from Streptomyces lavendulae-grasserius. Southern hybridization showed that the two plasmids do not carry homologous DNA sequences. A detailed genetieal map of the plasmids was set up based on double digestion and the Bal31 oxonuclease. Insertion of a fragment of streptomycete DNA carrying the gcne for 23S RNA methylase (coding for thiostreptone resistance) and for tyrosinase into different places on the pSLG3 plasmid defined a nonessential region tha t was deleted from the plasmid, giving rise to a bioad-host-range vector for Streptomycetes.

ldentij~cation of Promoter Regions in the Streptomycete Plasmid pSLG$

RY~Av,2 P., BESrADA O., STAJNER K., PETf~f~K M., TICH3~ P. Institute of Microbiology, Czecho- slovak Academy of Sciences, Prague

Localization of promoter regions on the pSLG3 DNA molecule (10.5 kb) was studied in vivo by cloning restriction fragments of the pSLG3 DNA in the promoter probe pIJ427, and in vitro by electronoptic visualization of the complex of RNA polymerases isolated from Streptomyces granaticolor and E. coli with promoter regions of the pSLG3 DNA. In vitro transcription showed that the pSLG3 DNA contains a strong promoter coding for the synthesis of a 200 bp RNA molecule.

Genetic Manipulations in Streptomyces cinnamonensis

JANDOV~ Z., TOBEK I., T I C ~ P. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Two extrachromosomal plasmid vectors were detected in the mycelium of Streptomyce, cinnamoncnsis producing monensin. Conditions for the preparation of protoplasts were optimized and the protoplasts were used for genetic transformation with the broad-host-range plasmid pIJ622. Transformants were then selected as colonies producing melanin and resistant to thiostreptone. Genetic information can also be transferred to S. cinnamonensis using the broad-host- range phage ~ C31. Gene bank was prepared from S. cinnamonensis.

Induction of ~-.Glucosidase Activity in Streptomyces granatlcolor E TH7347

JIRE~OVA_ M., DOBROV~ Z., J ~ ' ~ E K J. , lqAp~STEK J. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The enzyme activity was induced by cellobiose (C), arbutin (A), salicin (S), methyl-~-D-glueo- side (~-mG1), carboxymethyl cellulose (CMC) and mierocrystalline cellulose (MC); 5 mM 4-nitro- phenyl-~-D-glucoside was used as a reference substrate. A, C, S and ~-mG1 induced the ~-glucosidase activity localized in the periplasmic and cytoplasmic space of the mycelium. The presence of CMC and MC induced the formation of activity bound to the remnants of the lysozyme- digested mycelium with entrapped substrate. Most of the enzyme was found in culture broth. Analysis on 7.5 % polyacrylamide gel showed an identical electrophoretic mobility of these enzyme activities. An a t tempt was made to differentiate them on the basis of different glycosyl- ation of the protein part.

Uptake of 8H--Cellobiose by Streptomyces granaticolor ETH 7347

JANE~EK J., JIRE~OV.~ M., DOBROV~ Z., ~.~PRSTEK J. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The system for 3H-cellobiose uptake in the mycelium is induced by 10 rn~ cellobiose in culture medium; its Km is 0.5 ~ . I t is inhibi~d by 10 ~ CCCP (carbonylcyanide m-chlorophenylhydra- zone) and 1 m~ 2,4-dinitrophenol which indicates an active transport process. The activity of

1987 GENERAL MICROBIOLOGY 487

the cellobioso uptake sys tem synthesized in the presence of l0 mM cellobiose and 10 mM glucose is a more 20 ~o relative to the activi ty induced by cellobiose alone. 100 mM glucose presene during 3H-collobiose up take has no effect. The t ranspor t sys tem thus appears to be subject to glucose catabolite repression via the so-called inducer exclusion effect.

Morphological Differentiation of Act inomyees sp. 0234 During Submerged Cultivation

KOFRO~OV.4 O., ~ST.AVS]~A J. , BLV3~_AU~OV$, M. Institute of Microbiology, Czechoslovak Acad- emy of Sciences, Prague

Submerged cultures of Actinomyccs (sensu Krassilnikov) sp. 0234, facultat ively psychrophilic, producing antibacterial and insecticide antibiotics, form endospores similar to the spores of bacilli and thermophilic actinomycetes. Scanning electron microscopy was used to s tudy the morphological differentiation leading to spore formation. Ul t ra- th in sections exhibit typical surface coat layers, i.e. cortex, laminated coat and surface coats. Spore formation depends on medium composition, especially on the type of the C source. On solid media cultures of s t rain 0234 exhibit morphological features typical of Streptomyces sp.

Irreversible Eliminat ion of Chloroplasts f r o m Euglena gracilis by Some Inhibi tors of DN,4 Gyrase

~RAJ~OVI~ J. Institute of Molecular and Subcellular Biology, UK, Brati~lava

Several compounds among the so-called inhibitors of DNA gyrase induce a permanent loss of chloroplasts in E. gracilis. Their antiplast id effect and the induction of bleaching depends significantly on experimental conditions, especially the composition of the test med ium (growth versus resting), its pH, temperature (heat shock), light intensity, and exposure length. The inhibitors find common targets in chloroplasts and in eubacteria whereas the nucleoplasmic genetic appara tus remains unaffected; this finding supports the notion of the eubacterial character of chloroplasts.

Effect of Benzothiazole Derivat ives on Euglena gracHis

FOLT~NOV.~ P. Institute of Molecular and Subcellular Biology, UK, Bratislava

We examined the effect of biologically active benzothiazole salts on growth, survival and chlorophyll synthesis in E. gracilis and their ability to induce permanent loss of chloroplasts. The effect depends on the chemical s tructure of the agents. Some of t he m induce, mainly under growth conditions, the formation of white (bleached) mutan t s , this act ivi ty has not yet been described in benzothiazole compounds. The m u t a n t s are not formed in green Euglena cells exposed to the agents under rest ing conditions. After exposure to light the agents markedly affect the chlorophyll biosynthesis in temporarily dark-bleached Euglena cells.

Cloning of a Nicotiana tabacum Nuclear Gone Into EMBL3, a Derivat ive of the ~r Phaga

KHHROV.( V., REICH J., KOV~ALOV~ B., BEZD~K M. Institute of Biophysics, Czechoslovak Academy of Sciences, Brno

Fragments for cloning (mean size 20 kb) were obtained by incomplete digestion of nuclear I )NA with BamHI restrictase. The original vector was eliminated by Spi selection. Selected clones were analyzed to determine the size of the insert. Electrophoreograms were blotted on a nitrocellulose membrane and hybridized with labelled nuclear DNA of s tabacum. Individual inserts were classified according to the character of the hybridization signal. The classification was based on the frequency of their occurrence in the genome. Some of the clones were found to carry unique DNA sequences as well as sequences with different degrees of repetition.

Inhibition of SOS Functions by an Increased Level o f the Rec,4 Protein in Escherichia coli KI2

I~O(rKALOV~[ B., SOw J. , K v ~ a o v l V., REIC~ J. Institute of Biophysics, Czechoslovak Academy of Science~, Brno

We studied the SOS induction of the sulA gone and SOS mutagenesis in E. cell K12 in relation to the level of the RecA protein (P-RecA). Work with recombinant plasmids carrying the recA

485 17th COI~GRESS Vol. 32

gone yielded coils with consti tutively increased levels of P-RoeA. The SOS induction of the *ulA gone was determined with the aid of a fused gone sulA ::lacZ from the a moun t of synthesized ~-galactosidase. The mutagonesis was evaluated from the reversion of the argE3 gone to proto- trophy. The efficiency of SOS induction of the two SOS functions is lowered by overproduction of P-RecA. The results point to the regulatory role of the amoun t of P-RecA in SOS induction.

Damage Caused by cis--DDP and Some Other Pla t inum Derivat ives in Bacter ial Cells

JANOVSKA E., KLEr~VC)~CH~ER V., PILLrC~t J . Institute of Biophysics, Czechoslovak Academy of Sciences, Brno

The effect of four Pt([I) compounds on DNA was assessed from the ex ten t of inactivation of E. cell m u tan t s deficient in gone products part icipat ing in known mechanisms of DNA repair- The cytostatically active compounds, trans-DDP and diene-Pt, exhibited no appreciable differences in the inhibition of growth of the s tudied s trains whereas the cytostat ic compounds, cisc DDP and oxoplat inum, strongly inactivated some strains, especially those defective in the ref and lex systems. Products of these genes are obviously impor tant for pro-replication repair o. D2~'A lesions caused by cis-DDP and oxoplat inum.

Suspension Densi ty Affects Active Transpor t o f Nutr ients in Yeasts

MICHALJANI~OVJ~ D., KO~YK A. Institute of Physiology, Czechoslovak Academy of Sciences, Prague

Like aerobic and anaerobic metabolic processes, active t ranspor t of monosaccharides and amino acids in yeast genera Saccharomycez, Rhodotorula and Lodderomyce8 is negatively affected by suspension density. The m a x i m u m t ranspor t ra te of, e.g., 6-deoxyglucose decreases monotonic- ally with increasing suspension density while the accumulat ion ratio (intracellular concentration divided by medium concentration) is very high a t extremely low suspension concentrat ions (below 0.1 mg dry mass per mL) and then exhibits a m a x i m u m at 6 - -8 mg dry mass per mL. Carbon dioxide is one of the inhibitory factors.

Genetic Analysis o f Mucidin-Resis tant Nuclear Yeast Mutants

,~UBIK J. Food Research Institute, Bratislava

Nuclear m u t a n t s of S. cerevisiae selected by growth on glycerol in the presence of mucidin are characterized by the phenotype of a pleiotrophic resistance to several functionally different inhibitors. The muta t ion , denoted pdr3-1, was mapped by tetrad analysis. I t was found to be localized on the left a rm of chromosome II a t a distance of 5.9 ~= 3.3 cM from the contromete and 11.6 • 3.1 cM from the pet9 marker. The results indicate tha t the region of chromosome II between the centromere and the pet9 muta t ion is the site of the pleiotropic resistance locus PDR3 functional in the control of permeabili ty of the plasma membrane.

Effect of Cultivation Conditions on Sensi t iv i ty o f Yeast to Zymocin

CvI~Xov~ F., VONDR~:JS V. Faculty of Science, UK, Prague

On transfer to a minimal medium a number of auxotrophic m u t a n t s slowly lose their sensit ivity to zymocin whereas prototrophic strains retain their sensitivity. This finding can be used for (a) accumulation of yeast m u t a n t s by exposure to zymocin in an a r rangement similar to the peni- cillin method, (b) purification of an auxotrophic culture from prototrophic revertants .

I m p r o v e m e n t of a Hybr id Strain of S a c c h a r o m y c e s uvarum ZIXANOV~ B., VONDR]KJS V., V~'RNL*ROVz~ J. , BE~DOV~ O. Faculty of Science, UK, Prague, and Brewing and Malting Research Institute, Prague

The hybrid strain S. uvarum P9-LK 12/1 obtained by protoplast fusion from S. vereviaiae T 158C (donor of information for zymocin production) and the brewery s train S. uvarum P9

1987 GEl~ERAL MICROBIOLOGY 489

is capable of eliminating the commonly found contamination by Saccharomyces yeast and is promising for the production of biologically stable beer. As its growth rate was lower than tha t of the P9 strain it was subjected to long-term subculturing under conditions simulating beer production. Rapidly growing subclones retaining desirable brewery properties {deep substrate attenuation, sedimentation, zymocin production) were selected by this treatment.

Biotechnological Propert ies of Improved Hybrid Strains of Baker's Yeast

OBERNAUEROVA M., ~V~iX J., GBELSK.~ Y., DUDIKOV.~ E., I-Iv~IKOVX S., JVRiKOVA K., L ~ G - Vh Z., GRECOV~, G., ZAV~ELOV~- M. Food Research Institute, Bratislava and L I K O -- Research Institute, TrenSin

Hybrid strains of S. cerevisiae were obtained by selection of mitechondrial reeombinants after cell conjugation or protoplast fusion of mitochondrial mutants of selected biochemically out- standing industrial strains. Biochemical analysis formed the basis of selection of clones exhibiting higher rates of maltose fermentation and fermentative activity in dough than conventional wild strains. Out of these, strains which could yield superior industrial yeast in the final fermentation were prepared for industrial use.

Selection of an Industrial Strain of S. cerevis iae with Increased Ergosterol Biosynthesis

l~AsxovA J., ~PA~EX B., B~HALOVA B., NOVOTN~ ~., ZAJ~EX J. Distillery and Canning Research Institute, Prague, and Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

S. cerevisiae strain KBD was subjected to selection pressure by six stage culturing under industrial distillery conditions. The final product (pressed yeast) yielded a selectate D7 differing from strain KBD in its greater mean cell size and in increased ergosterol content. Industrial fermentation with strain D7 yielded a 27 ~o higher theoretical mean ergosterol yield per unit fermentation volume than strain KDB did.

Comparison of Fractionation Protocols fo r S. cerevis iae Designed for a Simultaneous Production of Yeast Extract and Ergosterol

B~HALOV~ B., ~ILLINGER V., MACHEK F., PJ, sxov~ J., ~PA~EX B. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, and Distillery and Canning Research Institute, Prague

We compared extracts obtained from yeast suspensions (a) after disintegration and a 3-h autolysis at 50 ~ (b) after addition of ethyl acetate and a 3-h autolysis a t 50 ~ {c) after a heat shock and a 1-h incubation at 50 ~ The yield and content of a amino nitrogen, total nitrogen and reducing sugars in dry matter were the best with procedure (a). None of the procedures led to the liberation or loss of ergosterol; hence ergosterol remained concentrated in cell sediments after separation of yeast extract by centrifugation.

Metabolic Convereion of 1,2--x4C-dcetate in Yeast During Ethanolic Fermentation of Glucose

~OGROVXSOV~ D., AVGUSTiN J. Faculty o/Chemical Technology, Technical University, Bratislava

During ethanotie fermentation of glucose by S. cerevisiae CCY 28 21, S. cerevisiae CCY 28-18, Candida tropicalis CCY 29-7-33 and Hausenula capsulata CCY 38-11-2 in phosphate buffers (pH 5.9) most of 1,2A4C-acetate remains in the cultivation medium, a small part is incorporated into the cell dry mat ter and only a negligible fraction is liberated as 14CO~. The eell-incorporate<l radioactivity is found mostly in the acid-soluble fraction, some proportion being in the macromolecular fraction. The results bear on the processing of secondary raw materials containing lower fatty acids.

Bacteriocin-Like Agents Produced by B u d v i c i a Strains

~ARDA J. Faculty of Medicine, J.E. Purkyn~ University, Brno

Bacteria of nine tested strains of Budvicia aquatica and five strains of "Pragia fontium" produce bacteriocin-like agents different from colicins. Their inhibitory effect is directed specific-

490 17th CONGRESS Vol. 35

ally against other strains of these two species but not against E. coil or Shigella sonnei. Their zones of inhibition are strikingly narrow ( 1 - 2.5 ram). The agents are produced on both solid a n d liquid media and their production can be monitored by UV-light. They are released into ~he medium. They exhibit complete resistance to trypsin and a marked sensitivity to elevated temperatures. These features point to their corpuscular character.

Bacteriocins of Ruminal Lactobacilli, Streptococci and Staphylococci

K M ~ V., JONECOV.i Z., NEMCOVJ~ R . , LAUKOW( A. Institute of Animal Physiology, 6'loval~ Academy of Sciences, Kogice, and Institute of Experimental Veterinary Medicine, Ko~ice

Ruminal strains of lactobacilli produced bacteriocins agains t the indicator strain L. acidophilus RN2 only in 2 ~o of cases while up to 77 ~o of strains of streptococci of group D were bacterio- cinogenic. Ruminal coagulase-negative staphylococci produced a bacteriocin-like activity not only against indicator strains S. aureus OX 209F and C. renale CCM 5740 but also against S. boris.

Glutamicin CBII, a Bacteriocin-Lihe Substance Isolated from Corynebacterium g lutamicum

PATgK M., HOC~MA~NOVX J., IN:g~W~RA J., K~JDELA V. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, and Research Institute of Crop Production, Prague

Glutamicin CBII is a thermoresistant low-molar-mass substance (s20,~ = 1.1 S, M < 10 kg/mol), probably a glycoprotein, which exerts a bactericidal action on eoryneform b~,oCteria. Gtutamicin CBII produced spontaneously by C. glutamicum CBII was partially purified by ammonium sulfate precipitation and gel chromatography on Sephadex G-50. I t is not toxic~ for plants (lucerne) nnd we tested its application in plant protection against tracheobacterioses caused by eorynebacteria and in determining gram-positive isolates from plants attacked by vascular pathogens.

Attempts at Transferring Genetic Information in the Genus Brevibacterium

~:~YTIft V., KONi(~KOVJ.-P~ADOCHOV.~ ~I., Ko~ihEx J. Institute of ~Iicrobiology, Czechoslov~.~k Academy of Sciences, Prague

The pqssibilities of genetic transfer by transformation, protoplast fusion and fusion of liposomes wi th protoplasts were studied in brevibacteria using chromosomal DNA isolated from Brevi- bacterium sp. M27 his stm r and a plasmid DNA from B. subtilis BD366 trpthr (pUB110). A positive result was obtained by protoplast fusion in mutants Brevibacterium sp. M27 his and Brevibacterium sp. M27 arg. The frequency of transfer in the protoplasts was 100-fold higher than the frequen('y of spontaneous reversions of auxotrophic markers.

Acquisition of Resistant Mutant Strains of Brevibacterium f lavum

KONf~EK J., Ko_~i~Kov.I-RADOCHOVJ~ .AT., RYTiI~ V. Institute of Microbiology, CzecI~os!ew~k Academy of Sciences, Prague

Optimum conditions for mutagenesis by a physical and a chemical mutagen were deterndned in the genetically little known species B. flavum ATCC 14067 using as a model the induction of streptomycin resistant mutations. The mutagens, UV light (up to 6.5 kJ /m 2) and N-methyl-N'- nitro-N-nitrosoguanidine (1 mg/mL), were used up to 1 ~o cell survival. The treatment ind,med mutations resistant to 8 antibiotics. Differences in the efficiency of the mutagens were found with respect to the production of strains resistant to oleandomycin, rifampicin and streptomycin. Genes responsihle for kanamycin and gentamicm resistance were not affected by the mutagenesis.

Comparison of Rapid Tests for Detection of Genotoxicity

LAHITOW[ 1~., SE~A~ M. Institute of Molecular and Subcelluar Biology, UK, Bratislava

The genetic activity of newly synthesized derivatives of 5-nitrofuran, benzothiazolium salts, bis-ammonium salts and carbamate local anesthetics was studied on a bacterial level uslhg

1987 G E N E R A L M I C R O B I O L O G Y 45|

Salmonella typhimurium on the A m e s tes t s y s t e m , Bridges s t ra ins of Escherichia colt, a n d the m e t h o d of induc t ion o f p rophage k in E. colt K12. A m o n g the t e s ted set of more t h a n 200 sub- s tances only n i t r o fu r ans exhib i ted posi t ive resu l t s in all tests . All o the r c o m p o u n d s were ent i re ly w i thou t effect.

Isolation, Cultivation, Identification and Preservation of Aquatic Hyphornycetes

MARVA.~OVA_ L. Czechoslovak Collection of Microorganisms, Purkyn~ University, Brno

Aqua t i c h y p h o m y c e t e s form a n ecological g roup decompos ing a l l och thonous organic m a t t e r in ware: . T h e y are i sola ted f rom f loat ing foam, f om tree leaves shed in to wate r , or f om teleo- m o r p h e u s s tages . I so la tes are cu l tu red on conven t iona l nu t r i en t med i a for fung i a t low t empera - tures (10- -15 ~ Ident i f ica t ion of species is carr ied out wi th spo ru l a t i ng cu l tu res which are ob ta ined (a) f rom s t a t i o n a r y aqueous cu l tures , (b) by ae ra t ing of a q u e o u s cu l tu res , (c) in a f e w . t h rough c h a m b e r t h r o u g h which wate r is passed. Pro longed m a i n t e n a n c e o f the cu l tu res is difficult, a p romis ing t echn ique being p rese rva t ion in liquid n i t rogen.

Fluorescent Microscopical Visualization of Fungi

HEJT~/~EK M. Faculty of Medieirle, UP, Olomouc

Blankopho r B A 267 ~ (Bayer) , a de r iva t ive of stiD~enedisulfcnic acid used as a b r igh tene r for texti les, has a h igh aff in i ty for cellulose and chit in. On add i t ion to a cul ture m e d i u m it b inds to chi t in c o m p o n e n t s of funga l cell walls which t h e n exh ib i t a yellow fluorescence in a blue exc i ta t ion l ight wi th a n orange cut-off f l t e r . No quench ing is observed. The dye can be added to 10 o~) N a O H used for sof ten ing ke ra t in m~ter ia ls in the spec imens . The m e t h o d provides a ~a.st and reliable de tec t ion of funga l o rgan i sms in t i ssue slices, sk in scales, hair , pus , ur ine, s p u t u m , vaginal swabs and cerebrospinal fluid.

Hybridization Test for Identification of Dermatophytes

HEJTM.;~NKOV.~ ~., HEJT~$ANEK M., WEIGL E. Foculty of ~iedicine, UP, Olomouc

We tes ted s t ra ins f rom the complex Tr~.chophyton n~entagrophytes i solated f rom paf fen ts suffering f rom t r i chophy tos i s in Czechoslovakia. Their t e l emorphs were found to ke Arthroderma benham~ae and A. vanbreuseghemii. Most s t r a ins are of m a t i n g type + a n d thei r teleomorF~h is A. benhamiae. We also used the Stockdale xexual s t imu la t ion tes t wi th c o m p e t e n t s t ra in c f A. simii. I ts resu l t s corre la ted posi t ively wi th the resul ts of the hybr id izar tes t .

In Vitro Cultivation of the .Fungus Taphrina deformans

BACtOALOVX K. Instit~tte o/ E~'perimental Biology and Ecology, CBEV, Slo' ak Academy of Sci- e~ces, Bratislava

IsoIates of the f u n g u s were ob ta ined by aseptict, lly t l ans fe r r ing ascospores or ge rmina t ing b las tospores f rom asci onto the cul ture sub:~trate (malt, po t a to -dex t ro se or Sebou raud agar) and cu l tu r ing a t 18- -20 ~ Act ively g rowing myceliunJ fo rmed rouP.~!, convex , g lossy colonies, l ight -p ink or l igh t -b rown in co!cur, composed of tt~in-walled oval yeas t - l ike Ludd ing spores. Ageing cu l tures f ea tu red three types of cells of different shapes and size~. True funga l m y e e l i u m was not observed in vitro. Old cul tures (5 - -6 mon ths ) conta ined mycet ia l cells resembl ing funga l asei d i f ferent ia ted on the surface of the hos t leaf t issue. No spore d i f ferent ig t icn was observed in the cells. This signifies t h a t in vitro the f u n g u s is unable ~o t e r m i n a t e t he life cycle and hos t t i ssue is necessa ry for the fb rmat ion of ascospores .

Use of Scanning Electron Microscopy for Characterization of Cultures of Basidiomycete$

BUCHALO A.S., .~ASEK V., ZAKORDONEC O.A. Institute of Botany, Aeeldemy of Sciences, USSR, Kiev, and Institute of Microbiology, Czeehoslovclk Academy of Sciences, Prclgue

Microscopic u n i f o r m i t y of mycelieol cu l tu res of Basidiomycetes h a m p e r s identificrotion and adequa te morphologica l charac te r iza t ion of individual species in pu re l abora to ry cul tures .


However, a detailed study of clamp connections and anamorphs (clamp type, shape and position, spore type, origin and surface structures) sometimes permits a more accurate morphological characterization and exceptionally also identification. Such a study is conveniently performed by scanning electron microscopy: its advantages were demonstrated on cultures of Lepista nuda, Coprinus comettus, Fistulina hepatica, Pholiota adiposa, Pleurotus sp. die., and others.

APPLIED MICROBIOLOGY

Effect of Chlortetracycline on the Production of Clavins in Claviceps fusiformis

B~EMEK J . , MALINKA Z., HARAZliVL P. Galena, Opava-Komdrov

The in t ens i t y of mycel ia l g rowth and p roduc t i on of e rgot a lkaloids of the c lav in t ype by a submerged cu l tu re of t he h igh -p roduc t ion s t ra in C. fusiformis W1 depends on chlor te t racycl ine (CTC) concen t r a t ion in the m e d i u m . Add i t ion of up ~o 1000 m g / L CTC in the inoclula t ion phase causes a 37 % decrease in mycel ia l g rowth b u t has no effect on mycel ia l g rowth in the p roduc t ion phase . Alkaloid p r o d u c t i o n is lowered by 36 %. T he same c o n c e n t r a t i o n of CTC used in bo th the inocula t ion a n d t he p roduc t ion phase r educed t he i n t ens i t y of myeel ia l g r o w t h by as m u c h as 33 % a n d alkaloid p roduc t ion by 44 %.

Effect of Inoculum on the Production of Clavins by Claviceps fusiformis

MALINKA Z., BI~IgMlgK J . , HARAZlM P., PIL~.T P. Galena, Opaca-Komdrov, and Research Institute of Antibiotics and Biotransformation, Roztolcy near Prague

One- a n d two-s tage inocu lum was used for s u b m e r g e d cu l t iva t ion of C. fusiformis W1. W i t h one-s tage i nocu lum t he to ta l con ten t of a lkaloids d ropped to I 1 -- 58 %. The m e d i u m compos i t ion in the ind iv idua l inocu la t ion s tages and d u r a t i o n of i n o e u h n n cu l t iva t ion affect a lkaloid format ion . W h e n one of the var iab le factors was kep t c o n s t a n t the alkaloid fo rma t ion was lowered to 32 ~o of the m a x i m u m value, e lymoc lav in fo rming 60 -- 96 % of the to ta l alkaloids. A two-s tage i noeu lum ensurod t he physiological s t a te of the cu l tu re necessa ry for h igh alkaloid p roduc t ion and regu la t ion of alkaloid b iosyn thes i s .

Effect of Different Carbon Sources in Inoculation Medium on Fermentative Production of cr

HARAZIM P., ~[ALINKA Z., BI~EMEK J. Galena, Opava-Komdrov

S u b m e r g e d cu l t iva t ion of Claviceps paspali CP 2505 and Y U 6 was pe r fo rmed us ing i n o c u l u m cul tured in med i a wi th sucrose, mann i to l , glucose a n d mal tose . The m e d i u m used in the p roduc t ion s tage con ta ined m a n n i t o l and succinate . W i t h inocula cu l t iva ted on mann i to l , glucose or ma l to se the p roduc t ion s tage exh ib i t ed a un i fo rm ut i l iza t ion of bo th carbon sources, a cons tan t , p H of 5.2 and a l inear alkaloid format ion . The h ighes t p roduc t ion was ach ieved wi th ma l to se - cu l t u r ed inoculum. W i t h sucrose-cu l tu red i nocu l um a preferent ia l u t i l iza t ion o f succ ina te occurs in the p roduc t ion s tage , t he p H rises l inearly a n d the p roduced alkaloids are degraded .

Production of Ergot Alkaloids by Immobil ized Cells of Claviceps fusiformis in a Semi- continuous and Continuous Regime

KfiE~r V., K o z o v ~ J., REHJ~E~: Z. Institute of Microbiology, Czechoslovak. Academy of Sciences, Prague

Vegeta t ive m y c c l i u m of C. fusiformis was immobi l ized in 2 - - 4 % ca lc ium alginate. T h e f e rmen ta t i on was pe r fo rmed in a 3-L l abora to ry fe rmente r . A suspens ion of immobi l ized bio- cy ta ]ys t in a special p roduc t ion m e d i u m was s t i r red by blade impel ler and aera ted . The fer- m e n t a t i o n can be p ro tec ted aga ins t p rokaryo t i c c o n t a m i n a t i o n by ch lo ramphen ico l (100- -150 [xg/mL). F e r m e n t a t i o n yields af ter e s t a b l i s h m e n t of m a x i m u m p roduc t ion rate c an be increased by con t inuous m e d i u m feeding. Devices for cu l t i va t ion of immobi l ized live cells have been designed.

Developmental Profile of Cytochrome P-450 in a Submerged Culture of Claviceps sp. SD--58

I~YLKO V. ~iPAL Z., SAJDL P. , i~EHA~IgK Z. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, and Faculty of Science, Charles University, Prague

Different ia l s p e c t r o p h o t o m e t r y was u sed to de t e rmine c y t o c h r o m e p-450 in the mic rosomM fract ion of a s u b m e r g e d cul ture of Claviceps sp. SD-58 and to define i t s deve lopmen ta l profile


during cultivation. The m a x i m u m content of cytochrome P-450 (0.144 nmol/g protein) was found in the late exponential phase (day 5) characterized by synthesis of elymoclavin and agroclavin. Determination of cytochromc P-450 included elimination of the interference by a pigment {probably a hemoprotein) with a m a x i m u m at 423 nm, min imum around 440 nm and an isosbestic point at 460 n m in the CO-differential spect rum of the reduced micros0mal fraction.

Chemical and Enzymic Preparat ion of Deacetylcephalosporin C

KREMrOV.~_ A., DUDEK M., TOM~IKOV.~ D., WIlL,VAliD L. Drug Research I~stitute, Modra, branch Slovenskd Eup~a

Deacetylcephalosporin C (DCPS-C) is one of the intermediates of cephalosporin C (CPS-C) biosynthesis. DCPS-C can be prepared from fermentat ively obtained CPS-C chemically or enzymically. We compared the two methods of DCPS-C preparat ion on a laboratory scale and optimized the conditions for the preparation of a biocatalyst and the course of the two preparation protocols. The enzymic preparation may conveniently use an immobilized biocatalyst. We elaborated a method for isolation of the product and the result ing DCPS-C was used as a s tandard for the analytical assay of cephalosporins.

Requirements f o r the Biosynthesis of Monensin as a Secondary or Excess ive Metaboli te

MATI~J~Y J., MAR~.~LKOV~ J., KAR~'ETOV~_ J-., NOH~NEK M., NEDBALOV-~_ E., VAN~K Z. Institute of Microbiology, Czech.. Acad. Sci., Prague, Research Institute of Biofactors and Veterinary Drugs Kou~im, National Health Service Centre, Prague 10

A low-production strain of Streptomyces cinnamonensis produced a mixture of monensins ~ond phenazine derivatives; the product ion of one or the other group of substances was favoured by the choice of the C source. Production m u t a n t s synthesizing only monensin A exhibited the highest production during fermentations on glucose. The synthesis was s t imulated also by molasses and some vitamins. However, monensin overproduction was achieved only in m u t a n t s capable of utilizing lipids which represent not only a slowly utilizable carbon source but also (in view of the pror of p-oxidation of fa t ty acids) a subst ra te with a direct metabolic relationship to the synthesis of the oligoketide moncnsin.

Biosynthzsis of Monensins a n t Isolation of Rzgulation Mut:~nts of Streptomyces cinnamonensis

POSPIw S., VAN~K Z. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The oligoether antibiotics monensins A and B belong to the oligoketides. Valine is a specific precursor of butyra te which is a crucial building block of monensin A in a novel metabolic path- way -- isomerization of isobutyrate (formed from valine) to n-butyrate . The knowledge of the role of valine permit ted us to prepare regulation m u t a n t s of S. cinnamonensis producing mostly monensin A -- an impor tan t coceidiostatic. We isolated two new monensins, 3-O-demethylmonensin A and 3-O-demethylmonensin B which probably represent the last step in monensin biosynthesis.

Mutant Strains of Streptomyces lasaliensis

STEINEROV-~ N., LIPAVSK~ H., CUDLIN J. , VANCE Z. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Apart from the oligoether coccidiostatic lasalocid, the collection strain S. lasaliensis N R R L 3382 produces also a quinoxaline antibiotic quinomycin A. Natural selection and selection after mutagenesis (UV-light) yielded mu tan t s producing only one of the two substances. Both m u t a n t strains were compared morphologically with the parent strain S. lasatiensis and with the quinomycin producer S. echinatus. We developed a method of isolation, purification and analytical assay (TLC, NMR and mass-spectroscopic) of the two antibiotics.

198~ A P P L I E D MICROBIOLOGY 495

Rellulation of Fatty Acid Formation in the Alga Porphyridium cruentum by Illumination and Cultivation Temperature

PODOJIL M., ~:~EZANKA T., DOUCHA J. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

P. cruentum strains 126 and 39 were autotrophically cultivated in plate cultivators at illumination levels of 30 and 60 W/m 2 and at 21, 24 and 28 ~ The interspecies differences in the proportion of fa t ty acids wore not significant at comparable i l luminat ion levels and temperatures . However, both strains exhibited increasing proportions of fa t ty acids, particularly 20:4 and 20:5, with decreasing cult ivation temperature and an incerasing content of 16:0 acid with increasing temperature. These results indicate differences in the activi ty of desaturase and ad jus tment of viscosity of cell membranes .

A Characteristic Morphological Feature of High-Production Isolates of the Monensin Producer Streptomyces cinnamonensis

~qIARSALKOV2x J. Rese(~rch Institute of Biofactors and Veterinary Drugs, Kou~im

A microscopic control of the submerged culture of S. cinnamonensis revealed two morphologically different variants: the low-production strain forms branched mycel ium whose appearance does not change throughout the cultivation while a high-production s train obtained by muta - genesis exhibits, around the 40th hour of fermentation, a decomposition of the mycel ium to fragments tha t elongate gradually at one end to form typical "maces" characteristic for the end of fermentation. This change in morphology is stable even after fur ther natura l selection, change in the C-source in the medium, and is independent of the fermentat ion regime (flasks, fermenter).

Selection and Preparation of Streptomyces fradiae Strains for Tylosin Production MAR~-LEK J., ~[AR~LKOVA. J. , ~2~ASTNA. J. Research Institute of Biofactors and Veterinary Drugs, Kou~im, and Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Our modification of the slab method of selection of isolates consists in a cult ivation on micro- plates on agar-supplemented fermentat ion medium with subsequent evaluation of production by the plate-diffusion method. A correlation was found between this method and the conventional evaluat ion technique. The method shortens the selection and permits the number of evaluated isolates to be incerased. Tile selected strain is cult ivated on Petri dishes on a sporulation medium covered with cellophane; the harvested spores are mechanically processed and frozen in the presence of a cryoproteetive agent. The strains are stable during storage and rapidly and syn- chronously germinate after inoculation. The method yields s tandard inoculation material for industrial purposes.

Comparison of Methods for Determining the Growth Rate of Phage-Resistant Strains of Streptomyces aureofaciens PILKOV~. D., VYSKO(~IL P., ETTLER P. Research Institute of Antibiotics and Biotransformations, Roztoky near Prague

The growth of CTC-producing phage-resistant strains of S. aureofaciena was monitored in 20-L fermenters by determining (1) the dry mycelial mass, (2) sediment level by centrifugation, (3) sediment level without eontrifugation, (4) content of dehydrogenases and (5) viscosity. Methods (3) and (4) were the best. Growth was also determined by measuring the diameter of growing colonies on two types of solidified culture media on Potri dishes. I t was linear and agreed well with the results obtained in a fermenter.

Purification and Properties of Anhydrotetracycline Oxygenase from Streptomyces aureofaciens VAN~UROV/~ I., NOVOTN~ J. , FLIEGER M., JANE~EK J. , BENE~ M.J., H O ~ L E K Z., B~HAL V. Institute of Microbiology and Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, Prague

The penul t imate enzyme of tetracycline synthase catalyzing the conversion of anhydrote t ra- cycline to dehydrotetracycline was purified and partially characterized. Purification included


ion-exchange chromatography, HPLC, affinity chromatography and preparative isoeleetro- fucussing. The ligand for affinity chromatography was anhydrotetracyeline, elution of the enzyme was performed by tetracycline. HPLC established the molar mass of the enzyme at 100 kg/mol. Preparative isoelectrofueussing determined the isoelectric point of the enzyme in the range of pH 5.0--5.5.

Purification and Properties of Isocitrate Dehydrogenase f rom S t r e p t o m y c e s a u r e o - fac i ens

NOVOTN/[ J, , VANI~UROV~ I., JANE(~EK J., B~HAL V., HO~ALEK Z. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Isocitrate dehydrogenase (NADP +) (EC 1.1.1.42) was isolated from S. aureofaciens mycelium by a 'purification procedure using efficient preparative ItPLC (separation by particle size). The spectrophotometric method of enzyme assay was optimized. The molar mass of the enzyme (~ 120 kg/mol) determined by HPLC and gel filtration was higher than i~ enzymes from pro- karyotes (80--90 kg/mol). The optimum p i t (Tris-HC1 buffer) was 8.3, optimum temperature about 60 ~ The properties of the enzyme are suitable for copurification with anhydrotctracycline oxygenase.

Mass Spectrometric Determination of Sodium Salts of Monensins ,4 and B in the Premix and the Fermentation Fluid

BERAN M., ZIMA J. , SCHON V. Institute of Microbiology, Czechoslovctk Ac(~demy of Sciences, Prague

Mass spectrometry with electron ionization was used to determine the monensin A/monensin B ra~io and the content of monensin A in fermentation liquid extracts and in the premix. The total content of monensins calculated from the data was compared with that obtained by the spectropho~ometric assay. The method is highly specific, accurate (about 5 relative percent) and makes a simultaneous authentication of the determined monensins possible.

Preparation and Selection of A s p e r g i l l u s n iger Strains for the Biosynthesis of Citric Acid on Molasses

MARTiNKOVA_ L., MUSILIKOVA M., UJcov~i E., SEICHERT L., HANH V.T. Institute of Microbiology, Czechoslovak Academy of Seiencrs, Prague, and Centre of Exp. Biol., Hochi-Minh City, Vietnam

UV-light increased the variability of A. niger S 59 (yield about 50 ~o organic acids from sucrose on molasses) and A. niger K 84 (about 20 ~ yield). Productivity rise by 15 ~ at the most in S 59 mutants and by about 200 ~ in K 84 mutants and the yield in the best isolates of the two strains was thus comparable. Fermented molasses medium contained, in addition to citric acid, large amounts of gluconic acid. We evaluated the method of testing the mutants on plates with Czapek agar and methyl red; the results agreed well with those obtained by cultivation of isolates in flasks on a shaker.

Optimization of Submerged Production of Citric Acid on Beet Molasses

SEICHI~RT L., UJCOVA E., MUSiLKOV~- M., MARTiNKOVJ~ L., MACHEK F, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Top yields in molasses fermentations depend on efficient optimization of the process. On defined media its primary target was the metabolic activity of the strain, as affected mostly by phosphofructokinase manipulation. Equally important in molasses media is the prefermentation adjustment of molasses, especially by buffering and eomplexing agents such as K4Fe(CN)~. Optimization of the process at both levels in 250-L fermenters with 152 L molasses medium containing 15 ~o sugar resulted in a 85 ~o yield per introduced sugar within 5 days.

1987 APPLIED MICROBIOLOGY 497

Formation of In termedia tes and Amino Acids During Fermentat ion of L-LyMne in Brevibacterium f lavum

HugwXx M., BtlSKO M., HI:DgC M., Dvof~-6Kov~- L. Biotika, SlovensIcd Eupga

Capil lary i so tachophores i s was used to m e a s u r e the concen t r a t i ons of some organic ac ids fo rmed as p roduc t s of a s s imi la t ion of glucose or sucrose du r ing L-lysine f e rmen ta t ion . Lac t ic acid was found in the exponen t i a l phase of cu l tu re g rowth , ind ica t ing insuff ic ient ae ra t ion of the f e rmen te r . A m i n o acid fo rma t ion was m e a s u r e d on an a u t o m a t i c a m i n o acid ana lyzer . The com- posi t ion of a m i n o acids p roduced du r i ng L-lysine f e r m e n t a t i o n did n o t depend on the raw materials used. An i m p o r t a n t role was p layed by cu l t iva t ion condi t ions re la ted to o x y g e n t r ans fe r in the f e rmen te r .

Fermentat ive Production of Lysine by Multiple Mutants of Corynebacterium g lutamicum

PLACH'~ J. Research It~stitute of Antibiotics and Biotransformations, Roztoky near Prague

M u t a n t s wi th a h igh p roduc t ion of lysine were isolated f rom C. glutamicum and Brevibacterium flavum: t h e y exh ib i t ed dependence on a m i n o acids, res is tance to lysine ana logues a n d sens i t iv i ty to sod ium f luo ropyruva tc . Repress ion of p y r u v a t e k inase led to inc reased lys ine p roduc t ion . The isolated C. glutamicum m u t a n t s were homose r i ne -dependen t , r e s i s t a n t to S- (2-aminoethyl ) - ~-cysteine (a lysine ana logue) and h a d a h igh sens i t i v i ty to f iuo ropyruva te . E v a l u a t i o n in f lasks a n d in f e r m e n t e r s y ie lded a m u t a n t a c c u m u l a t i n g 57 g/L L-lysine in t he m e d i u m af ter a 4-day cul t iva t ion .

Microbial Transformat ion of Cardioglycosides

FUSKA J . , ~TURDIKOV.~ ~i., PJROKSA B. Faculty of Cherniced Technology, and Institute of Chem- istry, Slovak Academy of Sciences, Bratislava

Out of 50 t e s ted microbia l cu l tures , four (Arthrobacter simplex, A. citreus, Aspergillus niger, Penicillium vermiculatum) efficiently t r a n s f o r m e d l ana tos ide A and C. The bac te r ia t r ans fo rmed lana tos ide A to d ig i tox in and lana tos ide C to d igoxin in a single step. F u n g i t r a n s f o r m e d b o t h subs t r a t e s in two s teps . I t , t he first t he s u b s t r a t e s were deg lyeosy la ted a n d in the second t h e y were deaee ty l a t ed to y ie ld the cor respond ing metabol i t es . The t ime of convers ion in the first s tep was 2 4 - - 3 0 h, the yie ld was 50 ~o-

Proteinases in Some Microorganisms Used in Food Industry

L u K ~ o v ~ J . , PAZOUT V. Faculty of Veterinary Medicine, Brno

Po lyac ry l amide gel e lectropboresis was used to s t u d y the pro teo ly t ic ac t iv i ty of r ep resen ta t ives of Pseudomonas, Flavobacter, Bacillus, Escherichia a n d Micrococeus in a 2 ~o sod ium case ina te solut ion du r ing l , 2 a n d 7-day s torage a t 4 ~ a n d a t room temp~ra tu re . Marked changes in casein f rac t ions were recorded af te r l - day s torage in genera Bacillt:s and Flavobacter. Stra ins of t he genus Pseudomonas degraded casein depend i ng on tempera , lure and s to rage t ime. The p ropor t ion of h i g h - m o l a r - m a s s f rac t ions decreased m a r k e d l y only af ter a 7-day s torage. Similar changes were found in the genus Escherichia. Mictococci exh ib i t ed only a low case in -sp l i t t ing abi l i ty .

Microbial Production of [Irate Oxidase

DgM~ROVg_ K. , KR~LOVA B., S:~t~ M. Institute of Chemic,d Techr~ology, Prague, and Lachema, Brno

Ura t e oxidase (EC 1.7.3.3) pa r t i c ipa tes in ox ida t ive degrada t ion of pur incs . I t is used in clinical b iochemis t ry as a c o m p o n e n t of d iagnos t ic k i t s for u r a t e a s s a y in b o d y fluids. Tes t ing of i t s microbial p roducers showed an increased u r a t e ex idase ac t iv i ty in t he te t racyc l ine p roducer Streptomyces aureofacieus ATCC 1062. The p a r e n t s t r a in yie lded th ree m u t a n t s ; a m o n g these the h ighes t u r a t e oxidase ac t iv i ty was exh ib i t ed by s t ra in B-96 which p roduced no te t racycl ine .


Characterization of Submerged Growth of Moraxella bovis

MANDL M., ~I~o~,- V., HO~AVOVA P., ~ i D B., STA~DARDA S. Institute of Biotect~wlogy, Slov,~k Technical U~iversity, Bratislava, and Veterinary Research Institute, Brno

A batch-culture production of vaccine against infectious keratocon~unctivitis in cattle was performed on medium composed of enzymic casein hydrolysate into which sheep blood was ]eft to dialyze. No significant cell death and autolysis occurred during the exponential phase. Growth limitation by amino acids led to an irreversible lowering of metabolic activity, pH rise caused by ammonia did not act as an inhibitor. Formation of fimbriac (imparting to the culture its immunogenic properties) was documented by electron microscopy and hemaglutination.

Verticil l ium chlamydosporium Mutants with Altered Chitinolytic Activity

CHALUPOVA V., LONHART K., KUNERT J., L~SEK H. Faculty of Medicine, UP, Olomouc

UV-light was used to induce 70 mutants of the ovicidal fungus V. chlamydosporium Goddard with a reduced ability to degrade colloid chitin. The ovicidal and chitinolytic activity of these mutants did not correlate and neither did their ovicidal and proteolytic activity. However, a significant positive correlation was found between the chitinolytic and proteolytic activity and between the growth rate and each of these activities.

Production of Cellulases by Aspergillus terreus

KYSLiKOV~ E., VOLFOVA O. Institute of Microbiology, Czechoslovak Academy of Scietwes, Prague

Simultaneous gradual feeding of cellulose and ammonium ions into the mineral medium during cultivation of the overproducing strain ATG-5 brought about a 180 % increase in the production of FP cellulase, 125 ~o rise in endoglucanase and 156 ~/o rise in aryl-~-glucosidase production as compared with batch conditions. Optimization of composition of culture medium (complex medium) combined with the gradual cellulose and ammonium feeding increased the activity of FP-celhflase by 340 ~o, endoglucanaso by 318 ~o and aryl-~-glucosidase by 560 %. The final concentrations achieved were 2 IU/mL FP-cellulase, 90 IU,ImL endoglucanase and 21 IU/mL aryl-~-glucosidase.

Stability of Escherichia coil Mutants Overproducing Some Catabolic Enzymes

PAVLASOV~ E., STEJSKALOVA E., SIKYTA B. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Mutants of E. cell hyperproductive with respect to ~-galactosidase, D-serine deaminase and tryptophanase, were isolated by the chemostat selection method. The stability of hyperproduetion of individual enzymes varied. During subculturing, ~-galaetosidase mutants lost more than one haft of their high specific activity. D-Serine deaminase mutants retained two-thirds of their activity while tryptophanase mutants preserved their activity intact. In the first two types of mutants the high enzyme production could be restored by transfer into the selection conditions in the chemostat.

Permeabilization of Aspergillus niger Cells

VA~URA A., JAR~ J., VOJTfw V., KU~ERA J. , MAREK M., BASA~OVA G. Inst.Chem.Technol., Prague, Inst.Microbiol., Czech.Acad.Sci., Prague, Food Research Inst., Prague

We studied the permeabilization of intact and immobilized ceils of A. niger. Glucose oxidaso activity was used as an indicator of the plasma membrane permeability. Toluene permeabilization caused an increase of the activity to 183 ~o, dimethyl sulfoxide to 189 ~o and heat shock to 117 %. Cell immobilization and permeabilization included cross-linking of intracellular contents with glutaraldehyde and subsequent treatment with a tenside. Intact , immobilized and merely permeabilized cells were characterized by K'M/02 values which were highest in intact cells per meabilized with toluene.

1987 A P P L I E D M I C R O B I O L O G Y 499

An~hraquinone P igmen t s f r o m a Coloured Mutant of Triehoderma viride

BE'r~NA V., S]~DMERA P. , VOKOU~ ~ J. , PODOJIL M. Slovak Technical University, Bratislava, and Institute of Microbiology, Prague

P i g m e n t s respon~ible for in tens ive colour ing of cu l ture m e d i u m were isolated f rom the b rown conid ia t ion m u t a n t T. viride CCM F-742. The yel low p i g m e n t was 1 ,3 ,6 ,8 - t e t r ahydroxyan th ra - quinone prev ious ly k n o w n only as a syn the t i c c o m p o u n d a n d as a me tabo l i t e in Aspergilias versieolor. The orange p i g m e n t was l - a c e t y l - 2 , 4 , 5 , 7 - t e t r a h y d r o x y a n t h r a q u i n o n e identical w i th t he me tabo l i t e s of sea lilies, Heteromera savingii a n d Lamprometra kluzingeri. I t h a d prev ious ly no t been isolated f rom mic roorgan i sms . The p a r e n t s t r a in T. viride CCM F-534 does no t p roduce e i the r of these p igmen t s .

Growth Parameter s and Propert ies of Methanol Oxidase in Candida boidini i Growing on Methanol

I(M~NTOV.6 M., DVO~KOVg J . , VOLFOV2{ O. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

I n con t i nuous cu l tu res C. boidinii l l B h grows a t d i lu t ion ra tes D 0 .03 - -0 .11 /h on m e t b a n o l wi th 40 ~o yield coefficient a n d crude p ro te in c o n t e n t of 60 - -65 ~o. A t D ~ 0.11/h fo rmate , t he ox ida t ion p roduc t of m e t h a n o l , a c c u m u l a t e s in the m e d i u m a n d inh ib i t s m e t h a n o l oxidase ac t iv- i ty. A d a p t a t i o n of the p a r e n t s t ra in to a m i x t u r e of 1 % m e t h a n o l wi th g radua l ly increas ing concen t r a t ions of H C O O H in a minera l m e d i u m a n d to low p H led to t he se lect ion o f C. boidinii 2, syn thes i z ing m e t h a n o l oxidase wi th a l tered r eac t ion c o n s t a n t s -- 1.54-fold lower Km (methanol) a n d 1.5-fold h igher Ki (HCOO~I). I n con t r a s t to t h e p a r e n t s t r a in , C. boidinii 2 d id no t a c c u m u - la te H C O O H in t he m e d i u m at h igher D.

Biodegradation of Heteropolysaccharide Agrobacteran

~iMov~ E. , KLEK~ER V. Institute of Microbiology, Czechoslovak Academy of Scie'nees, Prague

The s t ruc tu re of a g r o b a c t e r a n (of the succ inog lycan type) syn the s i zed b y Agrobaeterium radiobacter CCM 2704 is no t ye t comple te ly known. The po lysacchar ide is r e s i s t an t to k n o w n ,~-glyca- nases ; however , it was found to be ut i l ized by a m i x e d cul ture i so la ted f rom soil in which t he m u s h r o o m Agaricu* arvensis had grown. I n a minera l m e d i u m con t a in ing 1 o~) a g r o b a c t e r a n as a ca rbon source the cu l tu re h a d a specific g rowth r a t e of 0.02 4- 0.004/h, a specific ra te of sub- s t r a t e u t i l i za t ion of 0.033 4- 0.008 g g - l h - 1 a n d b i omass yield coefficient f rom a g r o b a c t e r a n of 0.62 4- 0.12 g/g. The e n z y m e s y s t e m of th i s m i x e d cul ture is expec ted to m a k e the s t u d y of a g r o b a c t e r a n s t r u c t u r e possible.

Naphthalene Degradation by Pseudomonas aeruginosa As Dependent on Inoculum- Utilized Substrate

]'IRu Y. , SLUGE~ D. , HAL'A~IA D. Slovak Technical University, Bratislava

N a p h t h a l e n e deg rada t ion to salicylic acid by P. aeruginosa B-843 was s tud ied wi th different s u b s t r a t e s suppl ied to t he i nocu l um -- 5 g /L glucose, benzoate , ace ta t e , sa l icyla te , P A B A a n d their combina t ions . K a n a m y c i n add i t ion to t he m e d i a was also tes ted . P r o d u c t i o n of sa l icyla te f rom n a p h t h a l e n e was m a r k e d l y s t i m u l a t e d in t he presence of sa l icyla te , k a n a m y c i n a n d the i r comb ina t i on wi th benzoa te in inocu la t ion m e d i u m . A comple te ly s y n t h e t i c p roduc t ion m e d i u m wi th a h igh buf fe r ing capac i ty s t i m u l a t e d t he sa l icyla te yield up to 5 t imes .

Utilization of Symbios i s of P. shermani i with a Yeast for Increasing the Vitamin Content of M i l k

~ a N ~ J. Food Research Institute, Prague

After su i tab le ac t i va t i on a special f a s t -g rowing s t r a i n of Propionibacterium shermanii syn- thes ized large a m o u n t s o f v i t a m i n B12 a n d fola.cin in mi lk . L a c t o s e - f e r m e n t i n g yeas t s p roduce in to mi lk o ther v i t a m i n s of the B group. Kluyveromyces fragilis was chosen as the m o s t su i tab le

$00 17th C O N G R E S S Vol, 32

a m o n g five yea s t species. I n symbios i s wi th P. shermanii it s t imu la t e s folaein b iosyn thes i s a n d also ensures a h igh p roduc t ion of v i t a m i n B12 in mi lk s tar ter , which is o therwise inhib i ted by lact ic bacter ia . I n add i t i on t he yea s t p roduced t h i amine , n iac in and v i t a m i n B6. Selected da i ry p roduc t s can t h u s be cons ide rab ly enr iched wi th va luab le nu t r i en t s .

Veterinary Hygienic Requirements in the Selection of Strains for Fermentative Dairy Technologies

H o I ~ c J . Faculty of Veterinary Medicine, Brno

Pure da i ry cu l tu res ~nd the ensur ing indus t r ia l sto.rtes are ind ispensable in mi lk process ing a n d p roduc t ion of soured da i ry produc ts . Sour ing cu l tu res suppress the g rowth of hygien ica l ly a n d technological ly h a r m f u l mic roo rgan i sms t h a t cause s i m u l t a n e o u s or pos t -p roduc t i on con tami- na t ions . The r educ t ion caused by y o g h u r t s t r a in s in t he g rowth of E. cell and M. luteus was by 5 - - 6 orders of m a g n i t u d e , in some s t ra ins of S. aureus by 2- - 5 orders of m a g n i t u d e . Some s t r a ins of S. aureus were r e s i s t an t to th is effect. These fac ts should be t aken in to accoun t in the search for new da i ry sou r ing cu l tures .

Production of Bacteriocins by C. perfringens Isolated from Foodstuffs of Animal Origin

CIG-)~NEKOV)~ V. Faculty of Science, Kornensk~ University, Bratislava

Produc t ion of bac te r ioc ins (perfringocins) was s tud ied by four t echn iques ; on increased produc t ion was induced by UV-l igh t . Ou t of 45 t e s ted s t r a ins of C. perfringens 6 exh ib i t ed a s t r ong p roduc t ion of bacter ioeins , wi th a m a x i m u m af te r 3- - 4 h of cul~ivat ion u n d e r s t r ic t ly anaerobic condi t ions a t 37 ~ especial ly af ter induc t ion wi th UV-l ight . Bacter iocin t y p i n g of C. perfringens m a y s u p p l e m e n t s e r o t y p i n g in the search for sources of a l i m e n t a r y toxieo- infeet ions .

Ecological Aspects of Microbial Contamination o f F r e s h l y P r o d u c e d M i l k

URBANOV.;, E., 5IALIKOVA M., VYHNALKOV+~ J. Veterinary Researrh Institute, Brno

I n 1984-- 85 a compa r i son was pe r fo rmed of tile e n v i r o n m e n t a l g r a m - n e g a t i v e microflora wi th i ts con ten t in raw cow's mi lk on 39 f a rms be longing to 23 agr icu l tura l e s t a b l i s h m e n t s . Swabs f rom milk con ta ine r s a n d mechan ica l mi lkers before a n d af ter s an i t a t i on yie lded 132 gram- nega t ive t~acteria, mi lk samples yielded 210 bacter ia . These were isolated by inocula t ion to selective media , d i f ferent ia ted by E N T E R O te s t s a n d the d a t a were compute r -p rocessed . Re- s i s tance to an t imierob ia l a g e n t s was d e t e r m i n e d b o t h qua l i ta t ive ly . The effect o f e n v i r o n m e n t a l mieroflora on microbia l c o n t a m i n a n t s in mi lk was conf i rmed in 58.9 o~) of f a r m s u n d e r invest i - ga t ion .

ENVIRONMENTAL MICROBIOLOGY

Fungobacterization o f Wheat Seeds

tSTAN~K M. Institute of Microbiology, Czechoslova~ Ac(tdemy of Sciences, Prague

Using a pot exper iment we studied the effect of artificial application of bacteria (Pseudomonas putida, P. fluorescens and A g robacterium radiobacter ) and fungi ( Trichoderma harzianum, T. longi- brachiatum and Pythium oligandrum) to winter wheat seeds and the effect of various combinat ions of these microorganisms on the emergence and growth of young plants . The best results were obtained after the applicat ion of combinat ions (T. harz~c~n~m + P. fluorescens), (T. longibran- chiatum + P. putida), and (P. olig(~ndrum + A. radiobacter). The s imul taneous application of fungi and bacteria to the seeds ("faugobacter iza t ion") influenced the emergence and growth of the plants more favorably t han the applicat ion of individual microbial strains.

Wheat Bacterizat ion in Pot and Field E x p e r i m e n t s

MO~AVEC V. Institute of ,~licrobiology, Czechoslovak Academy of Sciences, Prague

I t was found in pot exper iments tha t the efl[~ct of bacterization of winter and spring wheat seeds using a suspension of Pseudomonas putida K 11 cells is influenced by the qual i ty of ni t rogen sources in the soil and concentrat ion of Fe 3+ ra ther than tha t of Mg 2+. I n soils contaminated wi th Rhizoetonia solani the application of Trichoderma harzianum + Pseudomonas fluoresce~ts influenced the growth and state of health more favorably t han the bacter izat ion of the see('s by P. putida cells. The efficiency of the seed bac~erization was different in the soils conductive and suppressive towards Gaeuraannomyces graminis. These factors influenced the bacterizat ion in field experiments .

Micromyce tes of Potatoes Bacter ized by Rhizobacteria

VRAN~ J,, DomX~ K. Institute of i]licrobiology, Czechoslovak Academy of Sciences, Prague, Oseva -- Research and Improvement Potato Institute, Havli~k~tv Bred

The effect of rhizobactcria accelerating the growth of plants on microorganisms, viz. Fusarium spp., colonizing underground pota to par ts and the heal th state of the s tored tubers was investigated ill field and vegetat ion experiments. The occurrence of m!cromycetes ranged from --40 to + 36 ~o, t ha t of fusaria f rom --25 to + 7 1 % , t ha t of bacteria from + 15 to + 45 ~ and tha t of Pseudomonas fluorescens from 148 to 437 %. These changes were connected wi th the qual i ty of the prepara t ion used, soil climatic conditions, the var ie ty used, the heal th s tate of pota toes during plant ing and wi th the occurrence of mycolyt ic bacteria.

Non-pathogenic Rhizosphere Microorganisms and P r o b l e m s of Induced Plant Resistance

NovXx K., tSTAN~K M. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The elicitor activity of selected nonpathogenic microorganisms on roots of pea used as a model p lant was investigated. Suspensions of tested bacteria (Agrobacterium, Rhizobium and Pseudo- monas spp.) and fungi (Trichoderma and Rhizoctonia sop. ) were added directly to the hydroponic solution, and: after an incubat ion period, the concentrat ion of the phytoa lex in pisatin was determined. I t made it possible to determine the role of individual components of biocoenosis of the rhizosphere on the induction of adapt ive root reactions. The protect ive reactions induced by the const i tut ively present nonpathogenic microorganisms can contr ibute to the increased resistance of the roots.

Changes of the Hydroxyprol ine Content in the Cell Wall of Pea Infected wi th Fusarium oxysporum f. sp. pisi

KR,~TKX J. Research Institute for Plant Production, Prague

Changes of the content of hydroxyprol ine bound in the cell wall prote in after infection wi th the pa thogen F. oxysporum f. sp. pisi were invest igated in assorted Czechoslovak peas. I t was

$02 17 th C O N G R E S S Vol. 32

found t h a t the h y d r o x y p r o l i n e con ten t increases s ignif icant ly in in fec ted sens i t ive p lan t s a n d does no t change in in fec ted res i s t an t popula t ion . A possible use of the above changes in t h e hyd roxypro l ine c o n t e n t for the eva lua t ion o f res i s tance of pea var ie t ies to vessel wi l t ing is considered.

Biopredisposi t ion of `4lfalfa to Verticillium albo-atrum after Preceding Inoculation wi th Corynebacterium michiganense pv . insidiosum

K~'DgLA V., ~EZ~-(~ A. Research Institute for Plant Production, Prague, Ce~tral Control and Experimental Agricultural Institute, Brno

Using e ight alfalfa var ie t ies , it was e x a m i n e d w h e t h e r the inocula t ion wi th Corynebacterium michiganense pv. (Cmi) predisposes the p l an t s for s u b s e q u e n t inocula t ion wi th Verticillium albo- atrum (Vaa), pa r t i cu la r ly towards a h igher res i s tance or sensi t iv i ty . Af t e r inocula t ion wi th Cmi t he f ract ion of in fec ted p l an t s r anged wi th in 46 - -71 ~o (~5 ~ 58 ~ af te r inocula t ion wi th Vaa wi th in 27 - -82 % (~ = 37.9 %) and af ter inocu la t ion wi th bo th Cmi a n d Vaa wi th in 53 - -88 % (.~ ~ 72.3 ~o). I n all e igh t var ie t ies t e s ted t he success ive inocula t ion wi th Cmi a n d Vaa resul ted in an increased f rac t ion o f infected p lan t s , c o m p a r e d wi th the inocula t ion wi th only a single pa thogen . The inocu la t ion wi th Cmi predisposed the alfalfa t i s sues for a n easier invas ion a n d coloniz~tion by Vaa.

Germinat ing Power of Oospores of Pythium oligandrum in a Powder Preparation

VFo.~Ef=~ D. Research Institute for Plant Production, Prague

Oospores of the mycopa ra s i t i c f u n g u s Pythium oligandrum ob ta ined af te r a l0 d s t a t i ona ry surface cu l t iva t ion , b e g a n to ge rmina t e a l r eady 30 rain a f te r thei r app l ica t ion to the agar nu t r i en t m e d i u m . D u r i n g 24 h 83 ~o of the oospores ge rmi na t ed ; du r ing eve ry s u b s e q u e n t day , fu r the r genera t ions of t he m y c o p a r a s i t e were p roduced , in which 75 ~o oospores ge rmina ted . Af ter a 48 h d ry ing a t 30 ~ to 5 % mois tu re the p r e p a r a t i o n was powdered a n d 78 ~o oospores ger- m i n a t e d on an aga r n u t r i e n t m e d i u m . Af ter a one-year s torage of the p r e p a r a t i o n in suga r beet seed envelops 67 ~o of oospores ge rmina ted . Af t e r 7 yea r s of s torage 30 ~o of oospores ge rmina t ed (luring a 16-h i ncuba t ion on the n u t r i e n t m e d i u m .

Tyrophagus putrescentiae (Acari, Acaridae) and Microorganisms Related to Soil Apple Replant Prob lem

~ATSt,:.~ V,, SMRZ J-. Institute of 2~Iicrobiology, Czechoslovak Academy of Sciences, Prague, Faculty of Sciences, Charles University, Prague

The causes of the soil apple r ep lan t p rob lem are a p p a r e n t l y complex . I n some cases the n u m b e r o f phy to tox i e m i c r o m y c e t e s in the rh izosphere m a y serve as an ind ica tor of the degree of apple r ep lan t problem. I t was found thp, t some m i c r o m y c e t e s can serve as n u t r i e n t sources for soil m i t e s Tyrophagus putreseentiae (Schrank). The inocu la t ion of apple seedl ing roots wi th t.~ese mi t e s resul ted in a decreased n u m b e r of p h y t o t o x i e mic romyce te s and in an increased n u m b e r of bac ter ia of the genus Azospirillum. The resu l t s indicate t h a t biological m e a n s can t:e used suppress or a t least d imin i sh the effect of the biological soil s ickness.

.4 C o m m o n Occurrence of Fungal and Bacter ial Pathogens on the Carnation

Kov_~6IKOV~ E., ]['~I~DELA V., ALTMANOV~ O. Research Institute for Plant Production, Pragve; Public Gardens, Forests and Horticulture, Prague

I n add i ton to Fnsarium oxysporum f. sp. dianthi, Gram-nega t ive , non- f luorescent , oxidase- posi t ive bac te r ia were isolated f rom lower s t e m in t e rnodes of ca rna t ion (Dianthus caryophyllus L.) p l an t s exh ib i t ing wi l t ing s y m p t o m s . The p a t h o g e n i c i t y of the bac te r ia was verified by m e a n s o f in jec t ion inocula t ion o f c a r n a t i o n slices of W hi t e Sim. origin. After 2 - - 5 weeks necrot ic lesions w i th long i tud ina l r u p t u r e s could b e observed in the v ic in i ty of the inject ion. I t t h u s appear s t h a t in add i t ion to fungi , bac te r i a also belong to the po ten t i a l causes of c a r n a t i o n wiltit~g.

1987 ENVIRONMENTAL MICROBIOLOGY 503

Effect of Substrates f o r the Cultivation of Mush room s on the Growth of Cucumber , Rhizospheric Microorganisms and Fall Caused by Rhizoctonia so lani

NGUYEN HONG HA, TEPLi](OVJ~ J . , DOBm4 M., "~STANJ~I,: 31. Biological Instiiute Hanoi, State Farm Votice, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Subs t r a t e s for the cu l t iva t ion of edible ung i are used as organic fertil izers. C h a m p i g n c n sub- s t ra te and w h e a t s t r aw colonized oys te r m u s h r o o m m y c e l i u m first inh ib i ted and t h e n s t imu la t ed g rowth of the cucumber . B o t h the q u a n t i t y a n d the qua l i ty of microbia l popu la t ions in t he rhizosphere c h a n g e d s imu l t aneous ly . I n soil c o n t a m i n a t e d wi th R. sola~d, a cause of p l an t fall, t he c u c u m b e r emerged less readi ly in the presence of the subs t ra t e s . Thei r p ro tec t ive effect aga in s t the p h y t o p a t h o g e n i c f u n g u s could la ter be d e m o n s t r a t e d . The decreeosed occurrence of the p l a n t disease was assoc ia ted wi th the increased n u m b e r of an tagon i s t i c m i c r o o r g a n i s m s in the rhizosphere .

Effect of a Minimal Treatment of the Soil Containing Various A m ou n t s of Pos tharves t Residues on Biological Activi ty of the Soil and Humus Content

APFELTItALER R. , B0~-iscnovA S., POKORN.( d. Research Institute for Plant Production, Prague

A l imited t r e a t m e n t of m e d i u m gleyie p h a e o z e m soil was no t as:~oeit~ted wi th a s ignif icant reduc t ion of the n u m b e r of bacter ia {starch and T h o r n t o n ag~r), m i c r o m y c c t ; s ( Jenscn agar) a n d azo tobae te r a n d biological ac t iv i ty ( resp i ia tory , a m m o n i z a t i o n a n d cellulolytie tes ts) . Biogenic i ty of the soil depended p r imar i ly on the sowing p r o e e d m e aI~,(t increased in the order: suga r beet , spr ing bar ley , win te r whea t , m e a d o w clover. The c o n t e n t of h u m u s c o m p o u n d s extn~ctab}e wi th diphosphc~te increased af ter the m i n i m a l soil t r e a t m e n t , depend ing on the a m o u n t of pos tharves~ res idues , pa r t i cu la r ly on the p roduc t ion of fulvic acids.

Humus-Decompos ing Microorganisms as an Impor tan t Component of Soil Microbial Coenosis

(~ORDIE.~I(O S. l~stitute of Soil Biology, Czechoslovak Academy of Sciaticas, (?eskd Bud~jcrice

The role of m ic roo rgan i sms in the decompos i t ion a n d r e syn thes i s of h u m i c acids and in t he t r a n s f o r m a t i o n of h n m i e acids in the eu l t iva t i m of ehe~nozem and p e a t soils was inves t iga ted . The release of pigJ~,~ents a n d exopolysaeehar ides in mic roo rgan i sms inereaCed the mola r m.ooss a n d decreased the n u m b e r of func t iona l g roups of h u m i e acids. The resu l t s ob ta ined can be appl ied to the t eehnq logy of b iominera l fertilizers.

The Role of Organic Compounds of Industr ial Compos t s in Biologization of Soil Environment and Fertility of Clayeysand Soils

LSBL F., R~-sTabr K., I~RS'~L J. Research Institute for Pla~t Production, Prague

Five ty~es of indus t r ia l e c m p o s t s con ta in ing 23 82 ~ organic c o m p o u n d s in dr 5, m a s s were applied. The compos t s were, ehar~eterize(l" by t he s~abili ty o f the organic c o m p o u n d s expressed by relat ive respiratim~ NG : B wi th in 3.3 -- 7. l , basa l resp i ra t ion 5.5 -- 16.4 m g CO2 per 100 g dry m a s s per h, the eon t en t of 75- - 205 m g N-NH4 + per 100 d ry m a s s and by va r ious mieroflora. The a m o u n t of mic roo rgan i sms of the s tudied eu! t iva t ion g roups exh ib i t i ng va r ious b iochemica l ac t iv i ty increased s igni f icant ly w h e n app ly ing doses of 40 a 120 t / h m 2 in the sowing sequence: po ta toes , win te r whe~t , spr ing bar ley. A dose of 40 t / h m 2 increased wi th s ta t i s t iea l signifieanee the yie lds of all p l an t s a t the signifieanee level of P < 0.1 a n d P < 0.5 aeeord ing to Rod.

Effect of Repeated Application of Industr ial Compos t s on the Changes of Microflora and the Biochemical Act iv i ty of Clayey-Sandy Soil

~TIKOV~- A., I:~.EICHLOV.~ E. , RCSTAM ]~2., KR~!IL J. Research Institute for Plant Production, Prague

Five t ypes of indus t r i a l compos t s were appl ied a t 4(i and 120 t / h m 2 to c l ayey - sandy soil of tile expe r imen t a l s t a t i on H u m p o l e c in 1982 and 1985. After~ tile first app l ica t ion a s ignif icant differ-

$04 17th CONGRESS Vol. 32

entiation of the microbial and biochemical act ivi ty was detected in the first product. During a three-year t rea tment composts with a higher stabil i ty of organic compounds were responsible for the above changes to a more significant extent . A repeated dose of the same composts increased the microbial and biochemical activities more t han after the first application. The high dose of 120 t/hm2 influenced much less the changes of the microbial and biochemical activities, particularly as compared with the dose of 40 t /hm 2.

Application of Industr ial Compos t wi th a High Content of Potential ly Toxic Elements

KRJ, L J. , LSBL F., FIALA J . , ~TIKOVA A., PtT~LOV.~ V. Research Institute for Plant Production, Prague

Industr ial compost from waste and sewage sludges containing 3 m g Z n , 1 mg Cu, 0.2 mg Ni, 0.5 rag Pb and 0.05 rng Cd per kg dry mass was repeatedly applied in the sowing procedure in six different soil-climatic conditions. A dose of 40 t / hm 2 of the compost increased the level of accept- able Zn, Cu, B and Mg by 86 %. Changes of bacterial microfloia signal a s tar t ing inhibition. During vegetation bacteria mult iply significantly, as shown by cultivation on starch agar and MPA, of micromycetes on Jensen ' s agar and later of Azotobacter. Changes of respiration indicate a delayed onset of the conversion of organic compounds associated with the development of the microflora.

Effect of Cd, Cu and Pb Perchlorates on the A m o u n t of Microorganisms, Ammoniza t ion and Nitrification in the Soil

VACt~K O., PA~KOV~ M., KUNC F. Faculty of Sciences, Charles University, Prague, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Samples of sandy-loam brown soil were supplemented with metals at concentrations up to 2000 ppm, the highest available doses being 160 ppm Cd, 70 ppm Cu and l0 ppm Pb. Doses up to 100 ppm increased the number of microorganisms on agar media. High doses reduced the number of individuals and species diversity in the microbial community . Micromycetes, penicillia and pigmenting species in particular, were most resistant , act inomycetes were most sensitive. Ammonizat ion was more resis tant to perchlorates than nitrification; the toxic effect decreased in the order of Cu, Cd and Pb in both cases.

Effect of Herbicides on Nitrification in the Soil of Apple- tree Orchards

ULRICH R. Institute of Soil Biology, Czechoslovak Academy of Sciences, ~eskg Bud~jovice

When studying soils of apple-tree orchards it was found ~hat nitrite nitrogen accumulates. The soil of a 10-year-old apple-tree orchard treated with the herbicide Zeazin 50 contains the highest quant i ty of nitrite nitrogen. The incubation method showed tha t oxidation of nitrite ni trogen to nitrate nitrogen did not take place in this soil.

Effect of Clay Minerals on Soil Dehydrogenase Act iv i ty

KUBISTA K., NOV~KOV/~ J. Agricultural University, Prague

The soil dehydrogenase act ivi ty is an impor tant indicator of the extent of dehydrogenation reactions of the soil microbial population. The addition of caolinite and bentonite influences the mineralization of starch, cellulose, sewage plant sludges, pig slurry and other organic materials added to sandy-loam chernozom soil under model conditions. The extent of dehydrogenat ion reactions expressed by the dehydrogenaso soil act ivi ty was found to be significantly decreased by bentonite during the initial period of decomposition of the materials added.

Effect of Clay Minerals on the Act iv i ty of Soil E n z y m e s

NOVkKOVk J. Agricultural University, Prague

The effect of bentonite, kaolinite, nitrogen, starch and cellulose on the activity of catalase ~-amylase and cellulase in the soil was investigated. Both clay minerals and nitrogen decreased

1987 ENVIROI~MEI~TAL MICROBIOLOGY $08

catalaso activity. This effect was even more pronounced in the presence of s tarch and cellulose. The addition of s tarch alone increased the eatalase activity, cellulose was without effect. The addition of s tarch increased markedly the 0t-amylase activity. Clays and nitrogen did not induce significant changes. Collulase act ivi ty was substant ia l ly decreased by bentonite and increased in the presence of kaolinite; it was unchanged after the addition of cellulose and nitrogen.

Activity of Isolated Soil Cellulase Complex

MA~KOVA. H.l~ VAN(~URA V. Institute of Soil Biology, Czechoslovak Academy of Scie~ces, ~eskd Bud~jovice, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The soil cellulase complex was used as a model for s tudy of the effect of soil environment on ~he activity of enzymes. A protein complex exhibi t ing the activi ty of all cellulase complex components was isolated fl'om soil preincubated with carboxymethyl cellulose using a buffer containing high salt concentrations, EDTA and 2-mercaptoethanol. Comparison of the catalytic properties of the isolated complex with those of non-isolated soft cellulolytic enzymes showed t ha t in the soil environment the activi ty of cellulases decreases, due probably to adsorption on soil particles, and, on the other hand, their resistance against unfavorable effects increases.

Activities of Chitinase and fl-lj3-Glucanase in Soil

JAND~A A., HANZLiKOV.( A., ~OTOLOVA- I. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Air-dried soil did not exhibit a measurable ~-1,3-glucanase activity. Simple wett ing of the soil with water results in the enzyme production, similarly to the addition of glucose or casein hydro- lyzate. The increased act ivi ty was related to the biological activity of the treated soil. Addition of ~-l,3-glucan to the soil resulted in a fur ther act ivi ty increase. The increase was not observed in the presence of glucose. The air-dried soil exhibits a basal chitinase activity. The activity of this enzyme in the soil increases only in the presence of chitin. This increase is not negatively influenced by glucose. As compared with ~-l ,3-glucanase the increase of chitinase activi ty is slow.

Activity of ~-l,J-Glucanases in the RhizoslJhere of Germinating Barley

~OTOLOVi I., JANDER~. A. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The surface of barley roots produces .3-1,3-glucanase. The contribution of the seed microflora is negligible. The enzyme remains bound to ~he surface root s tructures and its act ivi ty depends on the barley variety al~d period of germination. The enzymic act ivi ty of the root surface is 103 times higher than tha t in the rhizosphere soil. The enzymic act ivi ty in the rhizosphere soil is on the average by 50-- 100 ~o higher than in free soil, depending on the barley variety and the soft used. After the addition of glucan to the soil with germinat ing barley the enzyme act ivi ty in the rhizo3phere soil increases significantly. The act ivi ty remains unchanged on barley roots.

Activity of Chitinase in the Rhizosphere of Germinating Barley

HANZLiKOV~- A., JA~'DERA A. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Microorganisms and plant roots contribute to the activity of chitinase in the rhizosphere. The activity of this enzyme is determined on the basis of concentration changes on N-acetyl- glucosamine, a product of the enzymic chitin decomposition. The highest enzyme activi ty was detected in the variety Krystal , the lowest one in the variety Erfa. Chitinase remains bound to the surface root structures. The contribution of the seed microflora to the enzyme activi ty on plant roots amounts to 30--40 %. Chitinase activi ty in tile rhizosphere soil does not significantly differ from tha t in free soil.

$06 17th C01%GI%ESS Vol. 32

The Effect of Inoculation with Vesicular-Arbuscular Mycorrhizal Fungi on the Growth of Strawberries

I-IR~EI, OV~ H.~ VE~SADOVX H. , P~IKRYL Z., VAI~U~A V., VAcItovA J . , ViT A. Institute of Micro- biology, Czechoslovak Academy of Sciences, Prague, Research Institute of Plant and Fruit Breeding, Holovousy

D u r i n g t r ans fe r f rom t he aga r m e d i u m to a s t e a m e d s u b s t r a t e (soil, perl i te , sand) m e r i s t e m e x p l a n t a t c s of s t rawber r ies (Fragaria ananasa Duch . ) were inocula ted wi th the ves icu la r -a rbuscu la r mycor rh iza l fung i Gigaspora margarita, Glomus maerocarpum a n d Glomua catedonium. Myeorrh iza l symbios i s occm~red du r i ng a f i ve -mon th g rowth o f s t rawberr ies in t he greenhouse . Mycorrhiz is developed m o s t s igni f icant ly in roots inocu la ted wi th G. caledonium. C. margarita a n d G. caledonium increased s ta t i s t i ca l ly s igni f icant ly the p l a n t b iomass wi th respec t to the control, wh ich was man i f e s t ed by a h igher con t en t of 1 ~, N a n d Mn.

Mutual Relationships among Vesicular--Arbuscular Fungi, Rhizob ium japonicum and Soya

V~.~ISAI)OVi H. , HR.gELOVg. H . , :P~IKRYL Z., VAN(~URA V. Institute of Microbiology, Czechoslovak ~4cad~my el Sciences, Prague

Soya (Glycine max (L.) Merr. cv. Duna j ka ) was inocu la t ed wi th the ves i cu l a r - a rbuseu la r mycorrhizal (VAM) fung i Glomus caledonium, Glomu8 fasciculatum, Gigaspora margarita a n d by n i t rogen- f ix ing b a c t e r i u m Rhizobium japonicum. T he p l an t s were cu l t iva ted hydropon ica l ly in a p h y t o t r o n in a m i x t u r e of perl i te a n d sand . A modif ied K n o p n u t r i e n t so lu t ion (pH 6.6) con- t a i ned 147 tzM P a n d 630 I• N. All s t ra ins of VAM fungi a n d R. japonicum could colonize soya roo t s a n d increase the u p t a k e of nu t r i en t s .

Natural Occurrence of Vesicular--.4rbuscular Mycorrhizal Fungi in Grape Vine

P~IKRYL Z., VAI~UI~A V., FERRER R.L . Institute of Microbiowgy, Czechoslovak Academy of Sciences, Prag~te, Botanical Institute, Cuban Academy of Sciences, Havana

The n a t u r a l occurrence of ves icu la r -a rbuscu la r mycor rh i za l fung i (VAM) was inves t iga t ed a t th ree localities in the region Lednice and Valt ice in Sou th Moravia . Roo t s of all 14 samples of va r ious g rape vine var ie t ies were myeorrh iza l , 8 of t h e m s t rong ly mycor rh iza l . A to ta l of 15 t y p e s o f spores o f VAM fungi be longing to th ree gene ra were isolated f rom t h e roo t - a t t ached soil a n d identif ied. The genus Glomus inc luding l0 species was m o s t f requent , t he genus Scleroeystis inc luded 3 species a n d t he genus Acaulospora 2 species G. fasciculatum, G. mosseae a n d G. macro- carpum were p re sen t in more t h a n 70 ~o samples .

Algae - - an Important Component of Soil Biocoenosis

LUKE~OV~ A. Institute of Soil Biology, Czechoslovak Academy of Sciences, ~esk~ Bud~jovice

C o m m u n i t i e s of soil a lgae were followed in the following success ive s tages : field, follow field, m e a d o w and forest c o m m u n i t i e s of different age. T he ind iv idua l s tages differed bo th in the to ta l n u m b e r of isolated species -- m i n i m u m 19 (cl imax), m a x i m u m 43 (seven-year-o ld meadow) a n d occurrence of t he m a i n t axonomi ca l g roups of a lgee p re sen t in the soil: Chlorophyta (.29--60 ~ Bacillariophyta (10- -33 ~o), Xanthophyta (10--21 ~ Cyatwphyta ( 5 - -24 ~ Field a n d m e a d o w c o m m u n i t i e s inc lude u charac te r i s t ic h igh p ropor t ion of species of the g roups Bacillario- phyta a n d par t i cu la r ly Cyanophyta, whose r ep re sen ta t ives are pract ica l ly ab sen t in forest com- mun i t i e s .

Determinat ion of the N u m b e r of Bacteria in the Soil by Epifluorescence Microscopy Using Membrane Filters

KRI~T~'FgK V., ~II~EK K . , CRUNDA B., PUN~OCH.~]~ P. Institute of Soil Biology, Czechoslovak Academy of Sciences, Institute of Landscape Ecology, Czechoslovak Academy of Sciences, ~esk~ Bud~jovice, Agricultural University, Brno

The procedure for the d e t e r m i n a t i o n of the n u m b e r of bac te r ia in the soil combines epifluo- rescenee microscopy , s t a in ing wi th acridine orange (AO) and collection of m ic roo rgan i sms on

1987 ENVIRONMENTAL MICROBIOLOGY 507

m e m b r a n e filters S y n p o r 8 (VCHZ Syn thes i a , Czechoslovakia) and includes: (1) d i s in tegra t ion of soil par t ic les , (2) s t a in ing (2 m L of soil suspens ion , 4 m L of 0.01 ~o AO a n d 2 m L of 5 m ~ NaHCOa), (3) f i l t ra t ion t h r o u g h b lack-s ta ined filters. The n u m b e r s of bac te r ia d e t e r m i n e d by the above m e t h o d were h ighe r and less var iable t h a n those de t e rmined in a Bi i rker chamber .

Modification of the Chloroform Fumigation Method of the Determinat ion of Soil Microorganisms B i o m a s s for Operational Purposes

RI~EK L. Agricultural University, Prague

The m e t h o d descr ibed by J e n k i n s o n (1976) was modif ied for the pu rposes of Agr icu l tu ra l regional A C H P labora tor ies and 17KZI)Z labora tor ies , according to the i r personne l possibi l i t ies a n d e q u i p m e n t . The ca rbon of soil m i c roo rgan i sms was followed bo th u n d e r def ined condi t ions (28 ~ 77 ~o WI-IC in homogen ized fine soil 2 ram) a n d unde r undef ined condi t ions (19- -30 ~ 31 - -85 ~o WI-IC, in m i n i m a l l y d a m a g e d m o n o l i t h s o f the arable soil profile 0 - - 15 cm). :No signi- f icant differences could be detected. Values of Cbiomas s de te rmined d u r i n g the per iods of Apri l 20 -- May 20 a n d S ep t embe r 20 -- October 20 are close to the average du r ing the whole vege ta t ion period.

Anaerobic Production of Ethylene by Soil Isolates of the Genus Clostr id ium

Pa~ou~ J. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

The soil isolate Clostridium sp. p roduced up to 600 p p m of e thy lene du r ing a 48-h cu l t iva t ion in a m i n i m a l m e d i u m con ta in ing 1 ~o glucose. The isolate was a m i x e d cul tm'e of C. butyricum with 15 ~o add i t iona l C. follax. The s t i a i n s were sepa ra ted by r epea t ed s u b c u l t u r e s unde r anaerobic condi t ions u s i ng m a n n i t o l a n d s t a r ch med i a for C. butyricum find C. fallax, respect ively. I n purified cu l tures , spo ru la t ion was induced by a slow increase of o x y g e n concen t ra t ion . The s t ra ins were t h e n t r ans fe r r ed to a m e d i u m sui tab le for the minor Clostridium in ruder to induce i ts ge rmina t ion . The cu l tu res were t h e n f luxed wi th pure o x y g e n to kill the mino r s t ra in . I t was found t h a t e thy lene was only p roduced by C. butyrieum.

Nonsymbiotic (Associative) Fixation of Molecular Nitrogen in Soils under Various Grass and Winter Wheat Stands

~I)]EK M. Institute of Soil Biology, Czechoslovak Academy of Sciences, Oeskd Bud~jovice

Nit rogen f x a t i o n was ca lcu la ted f rom the de t e rmined va lues of n i t r c g e n a s e a c e t y l e n e - - e t h y - lene reduc ing ac t iv i ty . Ni t rogenase ac t i v i t y c h a n g e d p ronounced ly du r ing the year , t he deter- m i n e d va lues showed t h a t n i t rogen f ixat ion is low. I n grasses , f ixat ion was e s t i m a t e d to be 0 .50- -3 .82 kg N / h m ~ in the soil layer of 0 - - 3 0 0 r am a n d du r ing a period of 210 days , in win te r w hea t it was 0 .065--0 .123 kg :N/hm 2 in the same soil layer and du r ing l ]4 (lays of the sp r ing a n d s u m m e r period. An effect of soil mo i s t u r e on n i t rogenase ac t iv i ty was descr ibed in meadow- grass a n d d i s t r ibu t ion of n i t rogenase ac t iv i ty in the s t a n d and soil was de te rmined .

Utilization of Alcohols and Saccharides in Azospir i l lum l ipoferum

LASiK J . , CHOTJANOVICH A.V. Institute o.f iYlicrobiology, Czechovlovcrk Ac~derny of Sciet~ces, Prague, Institute of Agricultural Microbiology, VASCHNIL, Lc~i~grl:d

We inves t iga ted w h e t h e r eJ cul ture of the associa t ive :N2 fixing mic roo rgan i sm Azospirillum lipoferum 799 uti l izes bacter ia l o1" p l an t po lysaeebar ides or some alcohols and sacchar ides occur- r ing in p l an t root e x u d a t e s ov p roduced by mic roorgan i sms . It. was found t h a t the mic roo rgan i sm used uti l izes only a smal l n u m b e r of sacclmrides , it does no t grow on ol igosacchar ides and m o s t po lysacchar ides tes ted a n d t h a t it ass imi la tes x y l a n only to a low ex ten t . I t is a s s u m e d t ha t these complex p l a n t or microbia l c o m p o u n d s can be ut i l ized by Azosl)irillum only a f te r their t rans~ fo rma t ion by o ther soil mic roorgan i sms .

508 17th C O N G R E S S Vol. 32

Relationships between Bacteria and Zooplankton in a Valley Reservoir

STRA~KR.~-~OVX V. Institute of Landscape Ecology, Czechoslovak Academy of Sciences, ~eskd Bud~jovice

Rela t ionsh ips be tween the s u p p l y of organic c o m p o u n d s a n d b iomass of p l a n k t o n o rgan i sms in reservoirs were inves t iga ted . Signif icant direct r e la t ionsh ips be tween be tc ro t rophic p l a n k t o n b iomass (bacteria p lus zooplankton) a n d supp ly were d e m o n s t r a t e d in m e a n va lues (energe t ic uni ts) du r ing s e m i a n n u a l w a r m (Apr i l - -Sep tember ) a n d cold (Oc tobe r - -March ) periods. Wise a re la t ive decrease of zoop l ank t on b iomass wi th respect to p h y t o p l a n k t e n the b iomass of p l a n k t o n bac te r ia increases. The signif icance of zoop lank ton e x c r e m e n t s se rv ing as s u b s t r a t e for bac te r i a a n d con ta in ing , according to t he qua l i t a t ive compos i t ion of phy to - and zoop lank ton , va r ious a m o u n t s o f und iges t ed food, is t h u s sugges ted .

Significance and Demonstration of Iodine Bacteria in Water

~VORCOV_~, L. District Hygienic Station Sokol~v

Iod ine bac te r ia conve r t iodides or ioda tes to iodine in n a s c e n t s t a te which is h igh ly toxic for o rgan i sms , e.g. fishes in aquar i a . Iod ine in n a s c e n t s t a t e is p roduced in reservoirs c o n t a i n i n g iodine water , especially by the ac t i v i t y of Pseudomonas iodooxidans i solated f rom these wa te r s on a n u t r i e n t m e d i m n of the fol lowing compos i t ion (g/L): K 2 t t P O 4 0.5, (NHa)2SOa 0.5, MgSO4 0.5, NaNO3 0.5, K I 5, s o d i u m c i t ra te 3, s t a rch 1,5, sucrose 1.5, aga r 10. Colonies of iodine-producing bac te r ia are s t a ined b lue b y t he s t a rch p r e sen t in t he m e d i u m . Ox ida t ion of iodides a n d p r o d u c t i o n of free iodine were d e m o n s t r a t e d ana ly t ica l ly .

Comparison of the Methods Used to Detect Pseudomonas aeruginosa in an Aqueous Environment

:BER~'i, TOV~ V., POKOR~'~ J. Institute of Hygiene and Epidemiology, Prague

Two l iquid n u t r i e n t med i a -- a m e d i u m wi th a spa rag ine a n d n u t r i e n t b r o t h w i th ma l ach i t e green - - a n d two solid med i a -- on aga r m e d i u m wi th a c e t a m i d e a n d ccn t r imide m e d i u m Pseudo- sel were compared . Milk agar , aga r according to K i n g - K l i n g a n d ace t amide aga r wi th pheno l red were used for verif icat ion. Model e x p e r i m e n t s were verified by pract ieM tes ts . N u t r i e n t b r o t h w i th ma l ach i t e green, a n d a s p a r a g i n m e d i u m were m o s t useful ; t he m e d i u m accord ing to K i n g . K l i n g a t 42 ~ was used for ver i f icat ion. Detec t ion in l iquid m e d i a was less efficient.

Comparison of Cha~ges of Microbial Activity in Water Determined Autoradiographically and as Maximum Rate of Glucose Incorporation

,~I~z~: K . , FUKS_~_ J. Ia.~t#~te of Landscape Ecology, Czechoslovak Academy of Sciences, Ceskd Bud~jovice, Construction Geology, Prague

I n the p o n d Velk~ Phlenec we de t e rmi ned the to ta l m l m b e r of bac te r ia a n d au to rad iog raph ic - a l ly , the n u m b e r of bac te r i a incorpora t ing a m i x t u r e of 3H-amino acids and 3H- thymid ine . The m a x i m u m ra te of ]dC-glucos~ incorpora t ion was d e t e r m i n e d in th ree series, i nc lud ing a 24.h cycle. W h e r e a s the n u m b e r s of bac te r ia were conserve: l d u r i n g 24 h, the a c t i v i t y p a r a m e t e r s osci l la ted, depend ing on the cycle of p roduc t ion of soluble organic c o m o o u n d s by the phy to - p l a n k t o n . Dai ly ac t iv i ty changes de t e rmi ned by. bo th m e t h o d s were comparab le . The resu l t s o f au to rad iograph ic m~bhods ind ica t ing changes of in : l iv idual a::tivitie~ osci l la ted more signi- f i can t ly t h a n the ra te of glucose incorpora t ion .

Occurrence of Resistance to Antibiotics in Enterobacteria in Surface and Drinking Waters

KOCMOUD Z. Regional Hygienic Station, ~eskd Budgjovice

The occurrence of s t r a ins of the fami ly Enterobacteriaceae r e s i s t an t to an t ib io t ics a n d chemo- t h e r a p e u t i c a l s (14 compounds ) was followed in samples of sur face a n d d r ink ing waters . Res i s t ance t o 3 an t ib io t i cs was de tec ted in more t h a n 45 ~ s t r a in s of E. cell and to 5 an t ib io t ics in 40 ~

1987 E N V I R O N M E N T A L MICROBIOLOGY 509

s t r a ins of Citrobacter. I n the genus Euterobacter res i s tance to penicil l in an t ib io t ics , eolistin a n d te t racycl ine was c o m m o n . A high pe rcen tage of r e s i s t an t s t ra ins was de tec ted in Providencia rettgeri and Morganella morganii. Res i s t ance to g e n t a m i c i n and oxolinic acid was n o t de tec ted in t he s t ra in collection tes ted .

Survival of I m m o b i l i z e d Microorganisms in Aqueous Environment

To.'~A~ow~ov.~ D. Institute of Experimental Biology and Ecology CBEV, Slova~ ~ Academy of Sciences, Bratislava

Trichosporon eutaneum cells immobi l ized in a lg ina te preserve ful ly the i r physiological s t a t e ( luring surv iva l in a q u e o u s e n v i r o n m e n t . The n u m b e r o f cells able to m u l t i p l y s l ight ly decreases b u t the decrease of the n u m b e r of colonies of free cells is more signif icant . E n d o g e n o u s and sub- s t r a t e resp i ra t ion of immobi l i zed cells h igh ly exceeds t h a t of free cells. E n d o g e n o u s resp i ra t ion is 2 - - 3 t imes h igher , s u b s t r a t e resp i ra t ion is even more p ronounced .

Determinat ion of Nitrification in Surface Waters

Vm3A J. Institute of Landscape Ecology, Czechoslowlk Academy of Sciences, ~eskg Bud~jovice

I n 1985 the n u m b e r s of ni t r i f icat ion bac te r ia were de t e rmined in the wa te r of the Mal~e r iver above Kapl ice and below t he s e w a g e - t r e a t m e n t p l an t (STP; a m m o n i a source) in Kaplice. The modif ied M P N m e t h o d in a l iquid minera l m e d i u m was used. The n u m b e r s var ied wi th in t he range of 1 0 2 - 108/L all t he yea r round , i r respect ive of flow rate , seasonal t e m p e r a t u r e course and o ther pa rame te r s . The n u m b e r s below the S T P were s ignif icant ly higher , which can be due to t h e p e r m a n e n t a m m o n i a source or release of ni t r i f icat ion bac te r ia f rom the STP. The resu l t s indicate t h a t ni t r i f icat ion m a y poten t ia l l~ occur in t he r iver s t r eam.

Time Variability of Microbial Aerosol in the Outer Environment

P u ~ o c H A f ~ P. , BR~HOVA H. Research Institute of Water Economics, Prague, Institute of Land. scape Ecology, Czechoslovak Academy of Sciences, ~esk4 Bud~jovice

Concen t ra t ions of mic roo rgan i sms in the a t m o s p h e r e a t the ma rg in of hous ing qua r t e r s in, P r ague were de te rmined . Samples were t a k e n us ing the f i l t rat ion t echn ique wi th a one-s tage impac tor . I n 1984 1985, t he concen t ra t ions of mic roo rgan i sms in the a t m o s p h e r e (colonies of bac te r ia and mic romyce t e s , CFU) reached 10--104/mS; du r ing 2-h in te rva l s t h e y va r ied wi th in a n order of m a g n i t u d e , du r i ng 24 h wi th in two orders of m a g n i t u d e a n d du r ing one m o n t h wi th in three orders of m a g n i t u d e . The d a t a are d iscussed wi th respect to m o n i t o r i n g the concen t ra t ions of mic roo rgan i sms in the a t mosphe re .

Biodegradat ion of Vulcanization Agents in an Aqueous Environment

KARELOV~ E. Institute of Experimental Biology and Ecology, CBEV, Slovak Academy of Sciences Bratislava

Degrada t i on of 2 -mercoptobenz th iazo le (2-MBT) was followed us ing a n a d a p t e d cul ture o f desul fur ica t ion bac te r ia in a Dav i s minera l m e d i u m wi th glucose ( 0 . 1 % ) a n d w i t h o u t it u n d e r anaerobic condi t ions . T he add i t ion of 0.1 ~o glucose to the a d a p t e d cu l tu re decreases the ra te of 2-MBT deg rada t ion a t concen t r a t ions of 5 and l0 ~zg/mL. In the suspens ion of bac te r ia w i thou t glucose 3.7 [zg and 4.4 ~zg o f 2-MBT decomposed in s amples con ta in ing 5 a n d 10 ~ g / m L of th i s c o m p o u n d , respect ively . I n s amples wi th a n d w i t h o u t glucose 2-MBT decomposed more rap id ly a t h igher concen t r a t ions wh ich is also conf i rmed by h igher va lues of the " m e a n " r a t e cons tan t s .

The Antifungal Effect o f Benzthiazoles and their Possible Utilization f o r Material Protection

ZE~_~NOV~. A., RIDA METWALI M., DROBNICOV~ I. , GVOZDJA:KOV.~ A. Faculty of Natural Sciences, Comenius University, Bratislava

D a t a on the an t i funga l effect of benzth iazoles are briefly reviewed a n d resu l t s ob ta ined w h e n s t u d y i n g the effect of a b o u t 100 benzthiazole de r iva t ives (EDs0, ED100, MIC) on the mul t ip l i ca t ion

$ | 0 17th CONGRESS Vol. 32

and g rowth of yeas ts and f i l amentous microscopic fung i are p resen ted . The m a j o r i t y of c o m p o u n d s exh ib i t ed a lower or comparab le effect wi th respec t to 2 -mercap tobenz th iazo le u sed for compar ison. Some compounds , e.g. 3-p iper id inemethy lene-6 .X-2-benz th ioazo l ine , were more efficient. Ut i l iza t ion of these c o m p o u n d s as indus t r i a l fungicides is r e c o m m e n d e d .

The Antibacterial Effect of Benzthiazole Derivat ives and their Possible Utilization fo r Material Protection

DROB:NICOVg_ I. , ZEMANOV./_ M., SID6OVg_ E. , HOLBOV~- E. , HALGA~ J. Department of Virology and Microbiology, Faculty of Natural Sciences, Comenius University. Bratislava, Chemical Institute, Comenius University, Bratislava, Department of Organic Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava

More t h a n 100 der iva t ives were inves t iga ted wi th r e spec t to the inh ib i t ion of g rowth of r epresen ta t ives of G + and G- bac te r ia charac te r ized b y g rowth curves , MIC, EDs0, ED100 a n d inh ib i t ion of to ta l p ro te in syn thes i s (Lowry) a n d nucleic acid syn thes i s (Kalckar) . Allyl or ha logen der iva t ives conta in ing sul fur b o u n d to ca rbon in the heterocycl ic r ing of benz th iazole were found to be m o s t effective aga ins t G + bacter ia . Growth, t he level of to ta l p ro te ins and nucleic acids were inh ib i ted by a concen t ra t ion of 200 Ixmol/L (the ED100 of mercap tobenz th i azo l e used for compar i son was 80 txmol/L). A possible use of the above c o m p o u n d s for ma te r i a l p ro tec t ion is considered.

Degradation Effect of Soil Microorganisms on Asphal t Insulation Str ips

~TIFT~:R M., WASSERll.~UEa I{., BERANOV-i M. ElectrotechnicaL Testing Institute, Prague

Carrier plates of a spha l t insu la t ion s t r ips were t e s ted in biologically act ive soil u s ing complex resp i ra t ion tests . Glass m a t and polyes te r inser ts were res i s t an t , inser ts f rom syn the t i c text i le fibers were par t ia l ly res i s tan t , ca rdboards , n o n - w o v e n tex t i les were nonres i s t an t . Te s t s of complex insu la t ion s tr ips in which the a spha l t coa t ing was art if icially d a m a g e d f rom one side showed t h a t the effect of readi ly a t t a c k e d inser ts can also be man i f e s t ed in complex str ips. T h u s , a spha l t does no t secure thei r protectic, n aga ins t mic roo rgan i sms b u t is par t ia l ly a t t a c k e d itself.

Biocorrosion of Plastic Materials Based on Polyolefins and Silicone Rubber by the Enzymic Sys t em Localized on Bacterial Cell Mem bran es

WASS~RBAUER R., BERA•OVi M., ~ T I F T ~ M. Electrotechnical Testir~g L~stitute, Prague

The e n z y m e h y d r o x y l a t i o n s y s t e m consis t ing of cy toeh rome P-450 N A D P H - e y t o c h r o m e reduc tase and alcohol dehydrogenase is localized on the cell m e m b r a n e s of Pseudomonas fluorescens, P. putida, Bacillus pumilus and B. brevis. The s y s t e m is induced in the presence of poly- e thy lene , paraff in, ba rb i tu ra t e s , acry la te and silicone rubber . Low- a n d h igh -dens i t y po lye thy lene , po lypropy lene and silicone r u b b e r are oxidized to h y d r o x y l a t e d der iva t ives t h a t are fu r ther conve r t ed to keto der iva t ives a n d to es ters a f te r an add i t iona l g radua l t r an s fo rma t ion . The first changes in I R spec t ra P E Bra len NA 7- -25 a t 1720 cm -1 were a l ready observed af te r a 2-h exposure .

Biocorrosion of Polyethylene Foils by the Enzymic S y s t e m of Bacteria and Eukaryotic Liver Homogenates and Their Microsomal Fractions

BERANOV~ ~I., WASSI~RBAUER R. , ~TIFTER ~[. Electrotechnical Testing Institute, Prague

Bacter ia l cell h o m o g e n a t c s a n d liver h o m o g e n a t e s o f ra t s , mice and ca ts exh ib i t P E degrada t ion ac t iv i ty . B iodegrada t ion of p las t ic ma te r i a l s by p r o k a r y o t e s a n d euka ryo t e s was t h e n modelled. The degrada t ion process is s t a r t ed by a sequence of enzymic reac t ions g iv ing rise to h y d r o x y l a t e d der iva t ives t h a t are f u r t he r conve r t ed to in te rna l ke t ones (1720 era- l ) . The m a x i m u m produc t ion o f h y d r o x y l a n d ca rbony l g roups was de tec ted in t he mic rosoma l l iver f rac t ion ob ta ined by prec ip i ta t ion wi th Ca 2+ ions. T he ac t iv i ty of the mi e ro soma l f rac t ion was b locked by CO and s t i m u l a t e d by the add i t ion o f NADPI-I . The abso rp t ion b a n d of the in te rna l ke tone was grea ter b y 0.1 n m / m m in the v a r i a n t wi th 100 Ixmol N A D P H .

M E D I C A L M I C R O B I O L O G Y

Surface Lectins in K l e b s i e l l a p n e u m o n i a e

~TEFAIgOV& I. Institute of Experimental Medicine, Czechoslovak Academy of Sciences, Prague

The cha rac t e r of adhes i n surface s t ruc tu re (fimbriae) a n d p ro te ins of t he outer m e m b r a n e of K. pneumoniae was s tud ied . U s i n g agg l u t i na t i on o f e r y t h r o c y t e s a n d Saceharomyces cerevisiae cells, the biological a c t i v i t y of bacter ia l surface was de te rmined . The h a e m a g g l u t i n i n tes t a n d pa r t i cu la r ly t he inhibi t ion o f h a e m a g g l u t i n a t i o n by polysacchar ides , conf i rmed the pa r t i c ipa t ion of leetins in the in te rac t ion of bacter ia l surface wi th t he receptor s t ruc tu res . Ou t of a n u m b e r of m o n o s a c c h a r i d e s and ol igosacchar ides tes ted , D-mannose possessed a s igni f icant inh ib i to ry effect. The abi l i ty to interfere w i t h h a e m a g g l u t i n a t i o n sugges t s possible pa r t i c ipa t ion of th i s saccharido or i ts ana logue in the recep tor s t ruc tu re .

Control of Surface Structures in Enterotoxigenic E. col i Z 2 - - a Superproducer of BiJimbrial and Bicapsular Vaccine

SOKOL A. School of Veterinary Medicine, KoSice

Using a phase -c lon ing t echn ique , a Z 2 v a r i a n t was isolated f rom the en te ro tox igen ie E. coli s t ra in 6012, 0101, K99, I t ? , which p roduced in the s tabi l ized l iquid m i n i m u m m e d i u m approxi- m a t e l y a 10-t imes h igher a m o u n t of f imbriae t h a n isolates of o ther 0 types . I n addi t ion , i t p roduced even f imbriae of t ype 1 a n d two t ypes of capsules . E lec t ron-microscopic ana lys i s of n e g a t i v e l y s t a ined p repa ra t i ons revea led t h a t in the 20-h s u b c u l t u r e of the 12-h i nocu lum, 65- - 75 % micro- o rgan i sms possessed two t ypes of m e n t i o n e d f imbriae a n d two types of capsu les t h a t a le i m p o r t a n t for a cheap a n d fas t p roduc t i on of an effective vaccine aga ins t neona t a l E. coli d ia r rhea in calves.

Use of Antifimbrial Serum of Enterotoxigenic E. co i l (ETEC) Strain 443

t ' uzovX H. , JANmovA V., HLAU(~OV~ J . , KERESTIg~OV~ A., KMEqOVX M., IT)UROVI~OVA J. School of iFIedieine, P.J. Safdrik University, Kodiee

Using the ant i f imbr ia l E T E C se rum (s t ra in 443), we e x a m i n e d 57 p l a smid - t e s t ed E. coli s t ra ins a n d 10 o ther s t r a ins of i n t e s t ina l rods: Salmonella, Citrobacter and Klebsiella. The E. coli s t ra ins e x a m i n e d be longed to en te ro tox igenic , nonen te ro tox igen ie a n d en te ro invas ive s t ra ins . I n the agg lu t in in reac t ion (working t i t re 1 : 100), 18 s t r a ins yie lded posi t ive resu l t s , i.e. 31 ~ m o s t cases being en te ro tox igen ie s t ra ins . The s e r u m yie lded no posi t ive reac t ion us ing o ther t ypes of in tes t ina l rods. We p r e s u m e t h a t we are no t dea l ing here wi th a s e rum aga ins t F1 f imbriae; f u r t he r s tud ies are requ i red to es tab l i sh w h e t h e r the s e r u m possesses an ac t iv i ty aga ins t F2 a n d F3 fimbriae.

Characteristics of Adherence of Group B Streptococci to Human Vaginal and Pharyngeal Cells

P ~ c M., JELINKOV~ J . , Bicov.4 R. , GRABOVSKAYA K., BULGAKOVA T., TOTOLIAN A. Institute of Hygiene and Epidemiology, Prague, Institute of Experimental Medicine, Academy of Medical Scienc('s, USSR, Leningrad

Stra ins of g roup B s t reptococci (serotypes Ia, Ib, I I and III) of the vag ina l a n d p h a r y n g e a l origin showed comparab l e adherence to w,g ina l a n d p h a r y n g e a l cells. Ne i the r t i s sue t r op i sm nor type dependence of adherence could be es tabi l ished. I n addi t ion , adhe rence was no t d e p e n d e n t on the v iabi l i ty of bac te r ia a n d adhes in s t ruc tu re ; on t he o ther h a n d , it was the rmosens i t i ve . I n c u b a t i o n of s t reptococci in the presence of pro teo ly t ic e n z y m e s s igni f icant ly reduced adherence . Similar effect y ie lded p re ineuba t ion of hos t cells wi th l ipoteiehoie acid or i n c u b a t i o n o f bac ter ia wi th an t ibodies aga ins t t h i s par t i cu la r acid. U l t r a s t r u c t u r a l s tud ies revea led t h a t adherence is m e d i a t e d by the bacter ia l capsule .


Antigenic Capsular Components of New Serotypes IV and V of Group B Streptococci

JELi~-~:OV.~ J., MOTLOV~ J., R~c M., WAGNE~ M. Postgraduate Medical and Pharmaceutical Institute, Prague, Institute of Hygiene and Epidemiology, Prague, I~stitute of Microbiology and Experimental Therapy, Jena

Out of 1708 strains of clinical origin, 165 strains were selected that could not be typed by antisera of conventional types Ia, Ib, II and III . In 95 strains (out of 165), antigens of newly proposed types IV (52 • ) and V (35 • ) and two candidates (8 • ) could be found. :In the selected strains, belonging to new types (proposed in our laboratory), sialic acid, an integral component of polysaccharide type antigerm was found, using both quantitative (according to Aminoff) and screening (specific lectin from Cepaea hortensis) assay. In prototype strains IV, V and in candidates, capsular structures could be demonstrated electron-microscopically after incubating the cells with type-specific antibodies.

Molecular Structures of M Protein - - Basic Virulence Factor of Group A Streptococci

HAVLi~EK J., .~IoR.~VIgK L., Ki~HNEMUND O. Institute of Hygiene attd Epidemiology, Prague tCesearch L~stitute of Organic Chemistry and Biochemistry, Czechoslowlk Academy of Sciences, Prague, Zentralinstitut fi~r Mikrobiologie und Experimentelle Therapie, Jena

Pepsin splitting of the N-terminal portion of M protein at the hypersensitive site permits its isolation and purification. We designed optimum conditions of splitting for the M1 type and the corresponding pcptide was isolated using affinity chromatography. Sequence studies of 35 N- terminal amino acid residues revealed significant homology with some other types. Thus one can detect a uniform pattern in the build-up of M proteins with a controlled variability.

Structure and Biological Activity of CoxleUa burnetii Lipopolysaccharide

SCHRA.~I~: ~. Institute of Virology, Slova~ Academy of Sciences, Bratislaca

Lipopolysaccharide (LPS) possesses a key role among surface macromolecules of the cell wall of the rickettsia C. burnetii -- the causative agent of Q fever. I t is a virulence factor, carrier of endotoxin moieties; from the immunological point of view, it is the most important antigen of this intraeellular bacterium. Its individual components, i.e. lipid A, the polysaccharide component, immunodominant molecules, possess an atypical composition and structure which can explain the low endotoxin activity of LPS (lethal toxicity in mice) and high resistance of C. burnetii against the defense immune mechanisms of the host.

Some Propert ies of Free and Bound P. aeruginosa Endotoxin BARTKO~-~ G., HO~TAC~ A., MA~A.N V. Research Institute of Preventive :~iedicine, Bratislavr

We compared the immunogenic properties of P. aeruginosa endotoxin, spontaneously released into the environment (LPS-F1) with endotoxin extracted from cells by the phenol procedure (LPS). Both types were tested using a tandem double-spaced immunoelectrophoresis. In addition, we ~ested the effects of organophosphate insecticide and choleragen on its biological activity. Free endotoxin was shown to be a better immunogen in comparison with the phenol extract. The reaction of both LPS types in experimental animals was more significant in combination with enterotoxin and methione.

Effect of Endotoxins on Cytochrome P-450 Content in the Liver

O~tcA,w J., MARA M., SCH~A~EK ~., M~SEL-MIXOLAJCZY~ F. Laboratory of Special Medical Microbiology and Immunology, School of Medicine, Charles University, Prague, Institute of Virology, Slovalc Academy of Sciences, Bratislava, Institute of Biostructure, Academy of Medical Sciences, Warsaw

Using cytochrome P-450 inhibition in mouse liver, the effectiveness of endotoxins of Saline. neUa abortue egui, CoxieUa burnetii, Bactereides ovatus as well as of the Ei factor, prepared by the diethyl e ther - -water procedure from Listeria monocytogenes, was tested. All fractions of C. burnetii

1987 MEDICAL M I C R O B I O L O G Y $13

endotoxin and listcria factor Ei possessed similar act ivi ty as bacterial polysaccharides and pepti- doglycans after adminis t ra t ion of 0.5-- 1.0 mg per mouse. B. ovatus endotoxin was more effective by one order and Sahnonclla LPS was effective even at a dose of 0.1 ~xg per mouse. The pheno- menon of " s a t a r a t i on" of Kupffer cells by a high dose of a h igh-molar-mass substance is valid for all components with the exception of classical LPS.

Interaction of Ant imicrob ia l Components wi th the Bacterial and Model Membrane

LEITMANOV2{ A., DEViNSKY F., ~ERE~EI~I F., MLYNAR~K D. Faculty of Pharmacology, Come~,ius University, Bratislava, I~stitute of Chemistry, Comenius University, Bratislava

The effect of l -methyldodecyl t r imethy a m m o n i u m bromide and ]-mett~yldodecyldimethyl- amine oxide on the cytoplasmic membrane of E. cell and on the propert ies of a model membrane p epared f rom total lipids isolated from E. cell, was tested. T rea tm nt of nat ive cells with both compounds resulted in the release of a UV-absorbing material and K + ions. The effect of the organic a m m o n i u m salt was more sig-nificant. The d ff~rence in the intensi ty of the effect of be th substances was based on their different affinity fo~ lipids which was confirmed by changes in the model membrane , measured by the ESIZ method. The results contr ibute to the unders tanding of the mode of action of a compound of this type on the bacterial cell.

Typing of Legionella pneumophila Strains

BAZOVSK~_ S. School of Medicine, Comenius University, Bratislava

Serological typ ing of Legio~ella strains by microagglut inin reaction wi th rabbi t antisera does no t permit to differentiate strains of serological groups 3 and 6 as well as strains 5 and 8 of L. pneumophil~. Therefore we employed immune sera of other animal species (guinea pig, mouse, hamster) . Mouse serum tu rned out to be most suitable as it yielded in group 3 and 6 positive reactions with the homologous antigen only; in s trains of serological groups 5 and 8, mouse serum produced a significantly higher reaction wi th homologous antigen.

Isolation and Proper t ies of Legionella Strains f r o m Drinking and Industr ial Water

DRA~AR V. District Hygi ne Centre, Vy~kov

Samples were obtained f rom hea~th care centers, industrial plants , hous ing quarters , saunas and swimming pools; we examined hot -water pipelines, swabs from showers and cooling towers. Legionella could be detected in almost all samples. I n cooling towers, L. pneumophila sg. 1 was predominant ; in addition, serological groups 3, 4, 5, 6, 7, 8, 10 and L. gormanii were isolated. I n hot -water tanks, sg. 6 was most frequent , sg. 3 and 5 were found less frequently. In air- conditioning systems, a s t ra in belonging among the ten new spec es of Legionella, was !solated. I n 58 strains seven atypical reactions were found using the diagno-~tic scheme according to Vickers and Y u (1984).

Classification of Legionella f r o m Drinking and Utili ty Water in the ~ S R

POTU~NiKOV~ B., SCHINDLER J . , DRA~AR V. Institute of Medical Microbiology and Immunology, Charles University, Prague

Fifty Legionella strains isolated f rom industr ial cooling towers and ho t -wa te r tanks, together wi th 15 labora tory collection strains (LegioneUa pneumophila serovar 1, 2, 4, 5, 6, 7, 8, L. gormanii, L. bozemanii, L. feeleii, L. dumoffii, L. micdadei, L. jordania, lb. oakridgensis and L. wadsworthii) were submi t ted to cluster analysis using the UPGMA method on the basis of a spec t rum of free amino acids, obtained by electrophoretic separat ion on a polyacrylamide gel. The strains isolated f rom the cooling towers and strains from water heaters consti tute, even according to the amino acids spect rum, independent clusters.

$ | 4 17th CONGI~ESS Vol. 32

Legionella Infection in Laboratory Animals

~I~5EKOV~, M. School of Medicine, Comenius University, Bratislava

We tes ted the suscep t ib i l i ty of gu inea pigs, ra t s , h a m s t e r s a n d mice to Legionella pneumophila ( l s t serological group) infec t ion af te r in t raper i tonea l a d m i n i s t r a t i o n of 108/mL mic roorgan i sms . On t he basis of the clinical p a t t e r n , pa thologica l a n a t o m i c a l f indings a n d microscopic deter- m i n a t i o n of ge rms in o rgan p r in t p repa ra t ions , s t a i ned accord ing to Giemsa a n d e x a m i n e d by immunof luo re seence , the m o s t s ignif icant f indings occur red in ra ts , to a lesser degree in mice. No pos i t ive f indings were ob t a i ned in h a m s t e r o rgans even wi th posi t ive cu l tu re f indings.

Contribution to Legionellosis Serology

PULCHA~TOVA E., SvoJ~E R. District Nationat Health Cantre, Liberec

U s i n g mic roagg lu t ina t ion wi th colour Legionella pneumophila (serotypo l) an t igen , an t ib iodies were detem~ined in 390 h e a l t h y ind iv idua l s and in 115 p a t i e n t s suffering f lora b ronchopnoumon ia . T i t r e s of 1 : 16 and h ighe r were found in 3.3 e/o of h e a l t h y and in 6 % of d iseased indiv iduals . T h e m e a n t i t ro in h e a l t h y ind iv idua l s was 1 : 23, in p a t i e n t s 1 : 61.

Haemolytic Activity of Listeria

5IEN~KOVX E. Institute of Medical Microbiology and Immunology, School of Medicine, Charles University, Prague

I n 85 listeria s t ra ins of all serovars , the effect of ex t race l lu la r p roduc t s of Staphylococcus aureus (phosphol ipase C), Streptec)ceus agalactiae, Rhodococcus equi, Corynebacterium ovis a n d Coryne. bacte~'ium haemolyticum on the haomoly t i c ac t iv i ty was tes ted . No haemoly t ic ac t iv i ty was found wi th L. innocua, L. murrayi a n d L. wetsheimeri. I n 18 Z. monocytogenes s t r a ins (serovar 4a, 4b), isolated f rom sheep, m o s t in tens ive haemolys i s was found in the haemoly t i c zone of C. ovis a n d C. haernolytieum (the CAMP p h e n o m e n o n ) . In 44 .L. monocytogenes s t r ams , isolated f rom h u m a n diseases , haemolys i s was p o t e n t i a t e d by chol inoxidase of R. equi in 14 s t ra ins in 27 s t r a ins even b y p roduc t s of C. ovis and C. haernolyticum. Ten s t r a i n s of L. iv~twvii cons t i t u t e a g roup , charac te r ized by the CAMP p h e n o m e n o n wi th S. agalactiae a n d by haemolys i s inh ib i t ion wi th sphin- gomye l inase D of C. ovis a n d C. haemolyticum. T h u s we p r e smne t h a t L. ivanovii produces phospho l ipase C.

Composition of Fatty Acids of Listeria of Various S e r o v a r s

Jur , . iK J . , MNX~iKOVX E. Institute of Medical Microbiology and Immu~tology, School of Medicine, Chartes University, Prague

U s i n g the capi l lary gas e h r o m a t o g l a p h y of m e t h y l es ters , tile compos i t ion of f~ t r y acids was t es ted . The group inc luded 20 s t r a ins of L. monocytogenes of all known sc rovars a n d 3 s t r a ins of the re la ted spe:~ies Erysipelothrix rhusiopathiae. No differences could be found a m o n g L. monocytogenes serovars , or be tween th i s species and s t r a ins L. innocuet and L. ivanovii. The L. murayi s t r a ins differ s gnif icant ly in the qua l i t a t ive d i s t r ibu t ion of f a t t y aeid~. The s t r a ins E. rhusiopathiae differed s ignif icant ly no t only f rom listeria, b u t also be tween c:~eh other , whzch indicates a n o n h o m o g e n e i t y of th i s species.

Findings of Lactobacillus catenaforme in Clinical Material

ClZ~IEL_~ D., Km~TOC~ViL Z.. URBX~-OV~( P. Regional Hygiene Ce~tre, Oo'trava, District Institute of National Health, Bohumin

Two s t r a ins of G +, anaerobic non - spo ru l a t i ng rods were isolated from two pationos suffer ing f rom severe b r o n c h o p n e u m o n i a . T he successful isolat ion o f these s t ra ins was based on direct microscopic e x a m i n a t i o n of s p u t u m which revealed a l r eady before cu l t iva t ion h igh n u m b e r s of G § rods. The s t ra ins were ident i f ied as L. catenaforme. The course of the disease was ve ry severe a n d a typica l . Penicil l in and aminoglycos ide ant ib io t ics were ineffectivo~. On tile basis of s ens i t i v i ty

1987 MEDICAL MICROBIOLOGY 515

t e s t s of L. catenaforme s t ra ins , e l i ndamye in was used for t h e r a p y wi th a fas t a n d long- las t ing beneficial clinical effect.

Development of Resistance to Trimethoprim in Clinically Significant Strains of Enterobacteria

SCH()N E., MANDLIKOVg Z., PLACttJ~ J . , JIIDLI(~KOVz( A., MIKOLs B. Department of Epide- miology, School of Medicine, Charles University, Prague, Centre of Antibiotics, Region Hygiene Centre, Prague, Centre of A atibioties, Faculty Hospital and Polielinies, Prague

Sys temic foflow-up of res is tance to an t ib io t ics revealed a g radua l increase of res is tance to co t r imoxazol in cl inically s ignif icant s t r a ins of en te robac te r i a , pa r t i cu la r ly in s t ra ins isolated f rom pa t i en t s suffer ing f rom u r i n a r y t r ac t infect ions, Res i s t ance to t r i m e t h o p r i m u s u a l l y occurs in m u l t i r e s i s t a n t s t ra ins ; u s i n g c o m m o n con j uga t i on procedures , i ts t r ans fe rab le cha rac t e r c an be de te rmined . C o m m o n t r ans fe r of d e t e r m i n a n t s amp slf of str-s~f ind ica tes t he presence of t r ansposons Tn7 or even o the r t r ansposons . I n t he cont ro l g roup of s t r a in s isolated f rom stool, t he occurrence of res i s tance to t r i m o t h o p r i m appea r s to be except ional .

Serotypes of Streptococcus agalactiae (Serological Group B)

MOTLOV~ J . Postgraduate Medical and Pharmaceutical Institute, Prague

Using the classical s c h e m e of po lysacchar ide t y p e s Ia , Ib , I I , I I I , m o r e t h a n 10 ~ s t r a ins r ema in un typab l e . A n u m b e r of t h e m con ta in p ro te in an t i gens R , X , c ( = Ibe). U s i n g c o m p a r a t i v e ant igenic s tudies , we selected new se ro types IV, V, a n d de t e rmined the i r t ime a n d geographic d is t r ibut ion . U s i n g th i s sy s t em, we increased t he ident i f ica t ion va l id i ty of the scheme. The cand ida te s " N T 6 " alid "7271" are u n d e r s tudy .

Serological Diagnostics of Human Chlamydia Infections

K^ZAR J . , KOVA(~OV~ E. , KITTLNR J . , SCHRAMEK ~. Institute of'Virology, Slovak Academy of Sciences, Bratislava

Sera of pa t i en t s suffer ing f rom con junc t iv i t i s or i n f l a m m a t i o n o f t he u rogen i t a l t r ac t m u c o u s m e m b r a n e s were e x a m i n e d us ing the compl emen t - f i xa t i on reac t ion (CFR), pass ive h a e m o | y s i s (PH) and e n z y m e - i m m u n o s o r b e n t a s s ay (ELISA). T he sens i t iv i ty a n d specif ici ty of these t e s t s were e v a l u a t e d b y c o m p a r i n g d a t a wi th the resu l t s of serological e x a m i n a t i o n in the micro- immunof iuorescence tes t . The m o s t sens i t ive (92 ~o), b u t a~ t he s ame t ime leant specific (73 ~ p roved to be t he E L I S A tes t . On the o ther h a n d , C F R was h igh ly specific (94 ~o) bu t of vt.ry low sens i t iv i ty (51 o~)). P H was shown to be more sens i t ive t h a n CFI~ (84 ~ ) a n d more spe('ifie t h a n E L I S A ( 9 1 % ) . The resu l t s of the E L I S A tes t , ob ta ined wi th an an t igen p repared m our l abora tory a n d wi th a k i t f rom Fin land , were similar .

Detection of Chlamydia in Clinical Material Obtained from Women and Newborns

(~ISL~-XOVs L., PROKOl" R . , HOLLER J . , PROKOP(~J~KOVJ~ H. , Posl ' iglL R . Chair of Hygie~e aml Epidemiology, School of Medicine, P.J. ~afdrik University, Kogice, Department of Gynaecology at~d Obstetrios, Municipal Institute of N~,tional Health -- Factory Institute of National Health, Ko.$iee

I n order to ob ta in a s u r v e y concern ing the poss ible occurrence of eh l amyd ia , we e x a m i n e d 330 w o m e n in the yea r s 1982--1985. Microscopic a n d immunof luorescence-pos i t ive f indings w~-xe ob ta ined in 20 % women . Us i ng isolat ion of c h l a m y d i a in t h e yo lk sac of chick embryo , one isolat ion was successful , two were suspicious . I n 47 newborns , suffer ing f rom conjunc t iv i t i s , microscopic a n d imnmnof iuo rescence f indings were posi t ive in l l i n f an t s (23 ~o).

Mycoplasma of the Urogenital Tract

BU~'EK R. , LINHARTOV.~- M., P]~ECECHT~L F. School of Medicine, J.E. Purkyn~ University, Brno

Smears f rom the pos ter ior vag ina l vau l t were ob ta ined f rom r a n d o m l y chosen w o m e n in a field gynaecological a m b u l a n c e . Ureaplasma urealyticum was isolated in 60 ~o a n d Myeoplasma homitds


in 27 ~o women. Compar i son of a control group wi th p a t i e n t s wi th posi t ive gynaecologica l f indings revea led no s ignif icant corre la t ion be tween m y c o p l a s m a isola t ion a n d clinical diagnosis . Th0 f indings of M. hominis were more f r equen t in w o m e n over 40 years of age.

Staphylococci in Urine: Species Determination Using the Conventional Method and the S T A P H Y T E S T

VOTAVA M., Sr=ALKA B. Faculty Hospital and Policlinic, Brno, School of Veterinary Medicine, Brno

226 s taphylococca l s t r a in s were isolated f rom r a n d o m l y ob ta ined samples o f h u m a n uciuc. U s i n g t he conven t iona l m e t h o d , inc lud ing up to 18 tes ts , S. haemolyticus was found 81 • S. epidermidis 59 • S. aureus 23 • S. warneri 20 • S. hominis 17 • S. simulans 14 • S. saprophyticus 4 a n d o the r 5 species 8 • U s i n g the L a c h e m a S T A P H Y T E S T (8 tests) , 198 s t r a ins could be classified (up to the level o f species 192); corre la t ion wi th the conven t iona l m e t h o d was found in 163 s t r a ins (82 ~ ). T he real iabi l i ty of t he S T A } ? H Y T E S T was the h ighes t in m o s t f r equen t ly occur r ing s t ra ins (S. haemolyticus 86 %, S. epidermidis 95 ~ S. aureus 100 ~ ).

Bacteriocin Typing of Staphylococci SK.~L~A B. School of Veterinary Medicine, Br ,o

The poss ib i l i ty of u s i ng exosubs t ances , of t he s t aphy loc in charac ter , p roduced b y act ive s t r a in s o f s taphylococci for charac te r iz ing Staphylococcus spp. , was examined . The g roup of p roduce r s compr ised 10 ac t ive s t ra ins , isolated m o s t l y in our l abora tory . The m a j o r i t y of coagu- ]ase-posi t ive, and a h igh n u m b e r of coagulasc-nega t ive s taphylococci could be classified wi th in th is specie~ on the basis of bas ic sens i t iv i ty ; the add i t ive sens i t iv i ty ind ica ted the indiv idual charac te r i s t i c of the s t ra in .

Differentiation of Listeriae Isolated in S l o v a k i a ~ccording to Newly Proposed Classification ELISCIt~ROV/[ K. , ~UPKOV~ E, Research Institute of Preventive Medicine, Bratislava

I n a g r e e m e n t wi th t he proposa l o f new genospecies o f Listeria (L.), we reeva lua t ed t he species c lass i f icat ion of 690 Listeria s t ra ins , isolated in S lovakia f rom var ious sources .Us ing the p h e n o t y p e charac ter i s t ics , i.e. an t igen ic s t ruc tu re , haemolys i s , se lec ted b iochemica l proper t ies a n d vir thence, 352 s t r a in s were identif ied as L. monoeytogenes se~su stricto, 40 s t ra ins as L. ivanovii, 250 s t ra ins as L. innoeua, 37 s t r a ins as L. seeligeri and 3 s t ra ins as L. welsheimeri. No o the r nonhaemoly t i c s t r a ins could no t be classified. The resu l t s d o c u m e n t the c i rcula t ion o f s t ra ins of all k n o w n Listeria species in our terr i tory.

Pathogenicity of Lis ter ia m o n o c y t o g e n e s L-Forms Under Experimental Conditions KALVOI)OV_~ D., JIR~-Sl~K A., Dtrw M. Department of Microbiology, School of Mediein4:, Charles University, Prague, Department of Pathological Anatomy I , School of Medicine, Charles University, Prague

W h i t e mice and chick e m b r y o s were infected wi th s table L - fo rms of Listeria, i solated in vitro in the presence of penicill in. I n f ec t i on wi th t he L - fo rms was n o t le tha l in mice; hcwever , in t ra- cerebral infect ion elicited p u r u l e n t men ingoonccpha l i t i s t h a t could be p roved histologically. The L - fo rms were le tha l to chick e m b r y o s p rov ided t he chor ioal lantoic m e m b r a n e (CAM) a n d a m n i o t i c sac were infected. I n t h e CAM, i n f l a m m a t o r y changes a n d necrosis could be observed. N e i t h e r mul t ip l i ca t ion o f L . fo rms , n o r revers ion to bac ter ia l f o rms could be de tec ted in t he t i ssues o f e x p e r i m e n t a l an imals .

Classification of Klebs i e l l a p n e u m o n i a e Isolated from the Environment and Man by Electrophoresis of Proteins

SCHII~DLER J., POTU~NfKOV.~ B. Institute of Medical Microbiology and Immunology, School of Medicine, Charles University, Prague

T h e a i m o f t he s t u d y was to es tab l i sh w h e t h e r K . pneumoniae s t ra ins , isolated f rom h u m a n disease and s t ra ins p resen t in t he e n v i r o n m e n t o f va r ious i n d e p e n d e n t localities, would be differ-

1987 MEDICAL MICROBIOLOGY $17

ent . 106 s t ra ins , i sola ted f rom the ur ine a n d r e sp i r a t o ry t r ac t of pa t i en t s f r om Prague , O s t r a v a W a r s a w , Copenhagen , a n d f rom the surface wa t e r in B o h e m i a and F in l and , were classified according to the s p e c t r u m of free prote ins . The s t r a in collect ion spl i ts well into s epa ra t ed clusters , t he s t r a ins isolated f rom wa te r differ s ignif icant ly f rom s t r a in s of clinical origin.

Determination of Acid Products of Glucose Fermentation in P r o p i o n i b a c t e r i u m acnes by Capillary Isotachophoresis

I~.~DI~NXL J . , HOLEC V. District Institute of Nationa~ Health, Karvind, Department of Clinical Biochemistry, Havi~ov, District Hygiene Centre, Karvind, DeFars, ent ef Bacteriology, H(tvi;~ov

Capil lary i so tachophores i s was modif ied for the d e t e r m i n a t i o n of acid p r o d u c t s of glucose f e rmen ta t i on in anaerobic bacter ia . An un ive r sa l p rocedure of ident i f ica t ion of acid f e r m e n t a t i o n p roduc t s was e l abora t ed a n d t e s t ed in P. aenes. Al ready wi th in 24 h, the cu l tu re con ta ined suf- ficient a m o u n t s of organic ac ids requi red for ident i f ica t ion by capi l lary I T P . The I T P measure - n m n t s were pe r fo rmed u s i ng a n i so tachophore t ic ana lyze r Z K I 001 by t he jo ined c o l u m n s tech- n ique .

Lysogeny of P r o t e u s m i r a b i l i s and P. m o r g a n i i

MAJT~NOVX L., BORECKX J . , KAROL~EK J. Institute of Preventive Medicine, Bratislava

The f r equency of ocemreneo of lysogenie s t r a ins in P. mirabilis and P. morganii was found to be a p p r o x i m a t e l y 40 %. I n lysogenic s t ra ins of P. mirabilis, repl ica t ion of phage D N A and lib- e ra t ion of m a t u r e phages into the cul ture m e d i u m took place in the presence of sens i t ive s t ra ins . I so la ted phages rep l ica ted up to the critieM di lu t ion 10 - 3 - 10 -4 and t h e y were employed for t y p i n g of P. mirabilis. Simul taneous ly , the ~,elationship be tween lysogony a n d t o x i n o g e n y was followed. I n lysogenic P. morganii s t ra ins , cor re la t ion be tween the presence of t e m p e r a t e phage and haemgly t i c ac t iv i ty was observed.

Toxinogenic Potential of P s e u d o m o n a s a e r u g i n o s a

]~,TAJT.~.N V., HO~TACK~ A., KAROL~EK J. D~stitute of Preventive Medicine, Bratislava

Culture f i l t rates of P. aeruginosa exh ib i t ed toxic ac t iv i ty in a sk in t e s t in rabb i t s , foo t -pad swelling tvst in mice a n d in t he ligo, ted rabbi t in tes t ina l loop. At leas t four f rac t ions were ob ta ined f rom concen t ra t ed cu l tu re f i l t rates following thei l par t ia l pur i f icat ion on a Sephadex G-100 column. The f rac t ions were of p ro te in na tu re , t he mola r m a s s be ing a r o u n d 23 kg /mol ; besides the en tero toxic ac t iv i ty , t h e y possessed s ignif icant hae mo ly t i e ac t iv i ty in va r ious t ypes of e ry th- rocytes . Haemoly t i c a c t i v i t y was exh ib i t ed even by t he f rac t ion possess ing a mola r m a s s of abou t 58 kg/mol . The hagmvly t i c ac t iv i ty is t he rmos t ab l e , two o ther f rac t ions arc biologically in- effective.

Thermostable and Thermolabile Enterotoxins of E. co i l Isolated f r o m Childrzn

HO,~TACKX A., B,~RTOVA L. , 5IAzTX)r V. Research Institute of Preventive ~lledicine, Br(~tislava

The oresence of the rmolab i l e (LT) a n d the rmos t~b le (ST) en te ro tox ins of E. cell, isolated f rom 50 chi ldren wi th a clinical d iagnosis of d ia r rhea l disease and f rom 50 h e a l t h y chi ldren, was examined . Double diffusion in agarose and a tes t accord ing to Elke were used for de tec t ion of LT, de t e rmina t i on of ST was pe r fo rmed in suck l ing mice. I t was found t h a t no s t r a in with the except ion of se ro type 06 could be classified as en t e ropa thogen i e or en te ro invas ive .

Participation of Anaerobic Nonsporulating Bacteria in the Etiology and Pathogenesis of Maxillar Sinusitis

H U D ~ A., ODL]~R I. , BAKYTOVX A., ~IAJTANOV/~ L. Research Institute of Preventive Medicine, Bratislavet

I n clinical ma te r i a l ob t a ined f rom pa t i en t s wi th s inus i t i s maxi l la r i s a cu t a , the occurrence of these s t ra ins was rare (5 eases ou t of 30 ~ 17 %); on the o ther h a n d , in m a t eriM f rom pa t i en t s


wi th sinusitis maxillaris chronica, the occurrence was f requent (67 cases out of 89 = 75 ~o). A pure culture was found in 1 acute case out of 30 (3 ~o), and in 7 chronic cases (8 ~ This finding confirmed tha t anaerobic nonsporula t ing bacteria, as well as a necrotizing agent, are responsible for the severi ty and chronicity of the in f lammatory process. The most effective antibiotics for the t rea tment of these conditions proved to be the wide-spectrum antibiotics clinda- mycin, lineomyein, chloramphenieol , rifampicin, tetracycline and vancomycin.

Electron-Microscopic Examination of the Gastrointestinal Tract after Experimental Infection of Calves with S. typhimurium MIKULA L., PILIt"~I_.~C E., TI3~KOVI(~OVX N., LE VAN TAO School of Veterinary Medicine, Kogice

Pel'orM infection of three groups of calves with S. typhimurium strain 4/5 (106 -- 1st group, 5 • 10 H -- 2nd group) and s t ra in 1344 (5 • 1011 -- 3rd group) elicited a clinical sMmonellosis. Damage to resorption cells, small intestine and colon mucous membrane was studied. I n the 1st group, part ial changes in entorocytes were observed and the challenge strain penet ra ted into the internal layer of lamina propr ia mucosae and was phagoeytized. I n the 2nd and 3rd group, the apical par t of enterocytes was heavily damaged in all pa r t s of the small intestine and even the penetra t ion ability of the challenge strain into the lamina propria nmcosae was significantly higher.

Biotyping of Salmonella typhimurium Ci~;EK A. School of Veterinary Medicine, Brno

Ore" exper iments were based on the studies of Dugid et al. and of Old who tested a new bio- typ ing ~cheme for S. typhimurium. Using fermenta t ion of D-xylose, L-rhamnose, myo-inositol and fimbria determinat ion ( type-l) tests, we divided the s trains of animal origin into seven main bioeypes. Bovine strains belonged mainly to biotypes 1, 7 and 25. Most strains isolated f rom sea gulls lacked type-1 fimbriae and did not ferment myo-inositol and L-rhamnose ( F I R N strains), the s trains of pigeon origin belonged to biotype 27 hi.

Detection of Listeria monocytogenes after Experimental Infection of Vaccinated Sheep S.~OLA J. , ~ I R C J. , GUBRA~ ~ D. High Veterinary School, Brno, Bioveta, Nitre

A highly virulent L. monoeytogenes strain was found in the blood samples of 8 vaccinated and 2 control sheep within 24 h af ter in t ravenous infection. Using various isolation media and procedures, the highest posi t ivi ty was found on nonselective mult ipl icat ion in broth at 37 ~ On the other hand, the lowest isolation posit ivi ty was found on direct cultivation on blood agar and selective multiplication in the med ium according to Fenlon. The challenge strain could be fur ther isoh~ted f rom rectal smears of all sheep within 8 h and f rom nasal secretions of 6 sheep within 28 h of infection.

Conjugative Alpha-Amylase Act ivi ty in Rumen Strains of Lactobacillus plantarum K~IET V., ~'EMCOVX R., JAVORSK5 ~ P. Institute cf Physiology of Farming Animals, Slovak Acad- emy of Scie~ces, Kogice, IJ~stitute of Experimental Veterinary ~Iedicine, KoMce

Amylolytic L. pla~tarum strains f rom the rumen fluid of calves were found to be resistant to tetracycline and s t reptomycin. I n conjugation exper iments , the alpha-amylase act ivi ty was t ransferred together with tetracycline resistance to the recipient strains Lactobacillus lactis RN1 and Streptococcus lactis RS16 at the frequency 10 6_ 10 7 The presence of plasmids in L. planta,~'um strains was detected by agarose electrophoresis.

Growth and Metabolism of the Rumen Bacterium Lachnospira multiparus D 151 in Pectin

MA~O~FK M. Institute of Physiology and Genetics of Farming A,imals, Czechoslovak Academy of Sciences, Prague

L. multiparus is p robab ly the mos t significant pectin-util izing bacter ium in the rumen of ruminan t s . We studied its g rowth and metabol ism in a static culture using a simple medium con-

1 987 MEDI CAL M I C R O B I O L O G Y 5 | 9

raining pectin as a source of energy. The mean doubling -- time was 94 min. The main metaboli te were found to be formate (1.08 mole per mole utilized hexose), acetate (0.64), ethanol (0.51) and lactate (0.12). The growth yields comprised 52 g of dry substance and 25 g protein per mole of utilized hexose. The exper iments were performed in labora tory fermenters LF2 at p H 6.5.

Isolation and Identification of a Morphologically Interest ing Rumen Bacter ium

HODROV~ B. Institute of Physiology and Genetics of Farming Animals, Czechoslowtk Ac~Memy of Sciences, Prague

An anaerobic gram-negat ive rod of cylindrical shape wi th a round end ~as isolated f rom sheep rumen content. I t differed significantly by its size (6--30 ~tm • 1.5--3 ~xm) and by sagit tal seg- menta t ion f rom the commonly occurring rumen bacteria of cattle. High fermenta t ion activity with simple saecharides and pectin, a somewhat lower act ivi ty with other polysaceharides was found in isolated bacteria. On the basis of these data we presume tha t we are dealing here with a bacter ium of the genus Oscillospora which has not been described in a pure culture st) far.

Isolation of an Anaerobic Cellulolytic Fungus f r o m Sheep Rumen

NOVOZ,4.~ISK~ K. h~stit~te of Physiology and Genetics of Farmi~g Animals, Czeehoslowtlc Academy of Sciences, Pc*ague

An anaerobic fungus, morphologically and metabolically closely related to the species Neo- callimastix free, tails (family Neoeallimasticaceac, order Spicellomycetales, class Chytridiomycetes) was isolated from the sheep rumen content. The isolate possessed a high fermenta t ion activity for monosaccharides and polysaecharides (cellulose, starch, pectin). The final products of fermenta t ion wore formate , lactate, acetate and ethanol. The activity of cellulolytic enzymes of the isolate was comparable wi th the activity of the rumen bacterial populat ion, t towever , the activity of cellobiohydrolase, a key enzyme in spli t t ing crystalline cellulose, was significantly higher. Those properties document the part icipation of this organism in the digestion of fibrous ma t t e r in the rumen.

Computer -Ass i s ted Evaluation of Radial Immunodiffusion

S':KOROVX B., VLKOV.4 V. District Hygiene Centre, Liberec

A programme for the processing of results of single radial immunodiffusion, based on the prin- ciple of a calibration curve, is described. A linear dependence of the area of the precipitation ring on concentrat ion is assumed. The programine plots at least three exper imental points of this dependence using the method of least squares. In fo rma t ion concerning deviations of individuM s tandards from the calibration eur~e is provided for the users. I f needed, the computer excludes automatical ly too ext reme values and thus the o p t i m u m course of the line is obtained; this is followed b 3, reading the values and pr int ing the results. The p rogramme is designed for the microcomputer A P P L E !1 using the BASIC compute r language.

Antibacterial Cytotoxici ty Tests in Mononuclear Leuhocytes

PROKE,~OVX L., DINH HUU Dud'S, JO~N C. Institute of Medical Microbiology a~d Immu~ology, School of Medicine, Charles University, Prague

The method of Tagliabu et (d., originally designed for test ing ant isahnonella eytotoxiei ty of h u m a n peripheral blood mononuelear leukoeytes, was modified for test ing antibacterial activity against staphylococci. In cont ras t to the original method, 10 • fewer cells are required. The results are expressed as the ant ihacter ial index ~ AI (% of decrease in n u m b e r of living bacteria). In hea l thy individuals, the ~ AI value is 60--85 in the absence of antibodies; tile presence of antibodies increases the antibacterial activity. Inac t iva ted autologous serum was used as a source of antibodies.


Immunological Characteristics of Cervix Uteri

TREBICHAVSK~" I., N:ZKLi~EK O. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, District Hospital, Ndchod

Twen ty bioptic samples, obtained from the interface region between the squamous and cylindrical epi thel ium at various stages of menstrual cycle or in menopause, were examined. The most impor tan t immunological markers were followed using immunofluorescence in cryostat sections. All samples contained IgA, IgG and C3 complement components; in addition, IgA plasma cells contained even a J-chain. HLA-DR positive cells were found in the submucosa, IgM- positive cells were found in two samples only. Only a few T3 cells were subepithelial; IgD, IgE and dendritic cells were not found.

Changes in the Jejunum of Young Rats after Repeated Gliadin ,4dministration

~T]~PANKOVA R., TLASKALOVA~-HOGENOV.~ I-I., TREBICHAVSK~ I. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

A 0.5 % gliadin solution was administered by the intragastric route to young rats immediately after delivery and on the 17th day of life. Intest inal villi wore wide with abundan t lymphocytic infiltration, the lamina propria contained a high number of IgA-producing ceils. These cells wore not present in the controls. After a long-term immunizat ion with gliadin, administered several t imes a week, an increased number of IgG-producing ceils was found in the lamina propria in two-month-old rats. The serum IgA levels and the Ig intestinal content were significantly higher in immunized animals than in controls. These changes resemble huma n coeliae disease caused by gliadin.

Determination of IgG and Ig~l Antibodies Against E. coil Using the ELISA Test

51o~Novi, H., (~Jxovg_ J. , BAR~OVA M., SALX~ J. Institute of Medical Microbiology, Faculty of Paediatrics, Charles University, Prague

We compared the suitability of two antigens for the ELISA test: LPS prepared according to Wostphal and crude LPS ant igen (haemagglutinat ion antigen) which is much simpler to prepare. Both ant igen types were derived from 10 antigenic E. cell types which occur most frequently in urinary tract infecti+ns (UTI): 01, 02, 03, 04, 06, 07, 012, 022, 023,075. The sandwich detection method was employed. IgM and IgG antibodies against both ant igen types were determined in sera of 299 patients suffering from UTI. Comparison of results obtained with the two ant igens revealed comparable values. In addition, the results of the ELISA test were compared with an indirect haemagglut inin test with polyvalent E. cell antigen; positive correlation was found in the IgM class only.

Cellular and Extracellular Products of Pseudomonas aeruginosa and their Significance in Immunomodulation

KUBi~KOVA~ D., PILLICg J. , JANDOVA_ D., TOUFAROVA D. Institute of Biophysics, Czechoslovak Academy of Sciences, Brtto

Selection of suitable bacterial and phage P. aeruginosa strains permitted the preparat ion of phage lyzates (PL) with immunomodula t ing properties. Selected bacterial strains produce pyocins, can be typed serologically and produce a number of exoproducts. The blastic transformation of mouse spleen cells increased 25 times after a 2-d cult ivation in the presence of PL. This effect is comparable with the mitogenic act ivi ty of LPS. Stimulation of cells of a thymie mice by PL suggests primarily s t imulat ion of non-T cells. The immunopotent ia t ion effect of PL on the phagocytic act ivi ty of mouse neutrophils and spleen cell suspensions was determined by chemiluminescence measurement .

1987 MEDICAL MICROBIOLOGY $2|

Induction of the Immune Response in Mice by ~4ttenuated FranciseHa tularensis Strain

K o v ~ o w [ H., STULi,r J . , M),CELA A. Department of Biochemistry, School of Medicine, Charles University, Hradec KrSlovd, J.E. Purkyn6 Military Postgraduate Medical Institute, Hradec Krdlov$

Immunization with a living tularemia vaccine induces the following gradual changes: increase level of superoxide dismutase in the peritoneal lavage cells, incieased production ef active oxide metabolites, changes in the parameters of phagoeytosis, increased interferon level, cytotoxie activity of macrophages, activity of natural killer cells, production of MIF and MAFc, a com- paratively low level of agglutinating antibodies, a significant hypersensitivity reaction. In addition, change in the procein synthesis by immunocompetent cells took place (SDS-FAGE).

Immunotherapy of Recurrent Candida colpitis

SVOBODOV.~ M., PoRtmSi~r ~., MANDL V., MAK~OVA D. Department of Microbiology, Faculty Hospital and Policlinic, Brno, Centre for Prevention of Female Cancer, Municipal Institute of National Health, Brno, Department of Allergology, Municipal Institute of National Health, Brno

The contemporary t rez tment of Candida infections in gynaecology and obstetrics is unsatis- factory. Active immunization (hyposensitization) using an autovaccine prepaeed from pathogenic candidas seems to be a promising approach. For production of autovaecines, we employed Ca~dida strains, isolated from patients, cultivated on solid media covered with a cellophane foil. The vaccinotherapy was successful in approximately 70 % cases with both subjective and clinical improvement. However in 30 % of patients, candida persisted in vaginal smears, without clinical symptoms.

Analysis of the Immunoregulatory Sys tem of the Host During lntracellular Bacterial Infection

MAC~.LA A., Kov.~f~OVg H., S~ULiK J. J.E. Purkyn~ Military Postgraduate Medical Institute, Hradec Krdlovd, Department of Biochemistry, School of Medicine, Charles University, Hradec Krd- lovd

Immunization with at tenuated Francisella tularensis strain results in changes in the production of immunoregulatory molecules by immunocompetent cells. Transfer ef INT-reductase activity to normal macrophages by supernatants of stimulated lymphocyte cultures serves as an example of regulation of macrophage metabolic activity. Production of oxygen radicals may amplify the induction process at the lymphocyte level. Lymphocyte products of the MAFc type further regul- ate the offoctor functional activity of macrophages. Even the mediators of inflammation, prosta- glandins, play an important regulatory role.

Occurrence of Intestinal Bacteria in the Luns of Precolostral Gnotobiotic l~'glets

TALAFANTOV.~ M., MA]~DEL L., TREBICHAVSK~ I., T~-~V.~I6EK J. Institute of Microbiology, Czechoslovak Academy of Sciences, Nov~ Hrddek

After peroral colonization of germ-free piglets with certain types of intestinal bacteria, their translocation into mesenteric lymph nodes takes place. In addition, the bacteria can be found even in lungs within 24 h after colonization of the gastrointestinal tract. Streptococcus feacalis, for example, was isolated (104/g tissue) as late as two weeks after colonization. After peroral administratien of certain pathogenic E. colt strains, lung lesions could be observed, the haemo. culture being negative at the same time. Stimulation of immunity in the lungs produced an increased expression o ~ class i i ~ransplan~a~lon antigens in alveolar maeropl~ages.

Induction of the Cytotoxic Factor in Mice Sensit ized with Coxiella burneti i Preparations by Bacterial Endotoxin

BAZLIKOV~- M., SCttI~A~EK ~. Institute of Virology, Slovak Academy of Sciences, Bratislava

Mice that had been sensitized to the effect of bacterial endotoxin by injection of killed particles of C. burnetii, phase I, developed hepatosplenemegaly, and an intravenous injection of bacterial


endotoxin produced an ondotoxie shock. During the shock, a " thermostablo factor" is released into the mouse serum; the factor possesses similar properties as the tumorneerotizing factor, i.e. it is cytotoxic for L coils. The liberation of the " thermostable factor" depends on the C. burnetii preparation used, on its concentration and route of immunizat ion of mice.

Comparison of Antibody ,4gainst Double--Stranded Deoxyribonucleic Acid Titration Using Two Methods

VASILUKOVX S., H~NA I. Postgradual Medical and Pharmaceutical Institute, Prague

Antibodies against ds-DNA ~r assayed in sera of 147 pat ients suffering from systemic lupus e ry thematosus using two methods: (1) the locally commercially available quant i ta t ive neoplast tes t with Trypanosoma danilewslcyi, (2) the colorimetric method according to Farr, modified by Cebeeauer, in which over 10 ~/o DNA binding is considered to be positive. Agreement in the results of both tests was found in 79 ~/o of sera. Using pure phage ds-DNA in control experiments , we showed tha t the higher posit ivity rate found by Farr ' s method, is probably due to the presence of ss-DNA in the substrate which results in nonidentical serum activities.

Modulation of NK-Cell Act ivi ty by Interferon (IFN) and Bloekin 8 Factor in Sera of Patients with Sys temic Lupus Erythematosus ( SLE)

LACKOVI~ V., BORECar~ L., ROVENSK~ J. Institute of Virology, Slovak Academy of Sciences, Bratislava, Research Institute of Rheumatic Diseases, Piektany

On the basis of IFN and an t i - IFN activity, 42 pat ients , suffering from SLE, were divided into 3 groups: (1) IFN positive, (2) an t i - IFN positive, (3) IFN negative (lacking both activities). The act ivi ty of the disease was characterized in individual groups and the presence of autologous NK-cells blocking factor (SBF) was examined in the sera of patients. Whereas in IFN posit ive pat ients (8) all autologous sera blocked the NK-cells activity, in the an t i . IFN group only 2 out of 6 sera were positive and in the IFN negative group, SBF was found in 17 out of 32 pat ients . T rea tmen t of NK-eells with IFN of SBF produced a partial decrease in N K activi ty.

Development of Attenuated Vaccines for lmmunoprophylaxis of Salmonellosis in Calves and Fowl

~I~AK F., VALO~KOV~ P., GRAN/[TOV/( M., ROSYPAL S., ROSYPALOV~ A. School of NaturalSciences, J.E. Purkyng University, Brno

A number of Salmonella typhimurium mutan t s , auxotrophic for growth factors, was obtained. Fur thermore , m u t a n t s were selected tha t did not revert spontaneously at the frequency of 10 -s to 10 -11 and possessed low virulence for mice. Two selected m u t a n t strains were shown to be irnmunogenic and harmless in immunizat ion experiments. Peroral immunizat ion of experimental animals with two doses of 10 s microbes st imulated a cellular immune response, i.e. posit ivity of leukocyte migration inhibition (LMI) and leukocyte adherence inhibition (LAI) tests with group B, C and D salmonella antigens. No significant increase of specific O and I t agglutinins in s e r a

could be found. Vaccination of mice did not prevent invasion of the virulent wild challenge salmonella strain, it did, however, protect against lethality.

lmmunogenici ty of Adsorbed Tetanus Anatoxin for Immunizat ion of Horses

TIR(~O S., LYS/[K J. I M U N A , ~Sri~sk~ Michalany, BIOVETA, Ivanovice na Hang

The immunogenici ty of t e tanus anatoxin in an an t i t e tanus vaccine (anti toxin content 30, 60, 90 and 120 binding uni ts per mL) was tested using the challenge method, determinat ion of an t ibody response in immunized guinea pigs and t i t ra t ion of immune uni ts on the basis of an n ternat ional standard. The IU t i trat ion method permits quant i ta t ive evaluat ion of immuno- genicity of adsorbed anatoxin. Vaccines, containing 60-- 120 binding uni ts per mL, adminis tered twice (I0 m L a dose) at a 14-d interval, elicited a significant and long-lasting immuni ty in the immunized horses.

1987 M E D I C A L M I C R O B I O L O G Y 523

Effect of Colostral I m m u n i t y on the Ant ibody Response in Piglets after Vaccination with Inactivated Vaccine Against Aujezsky Disease (AD)

VALi(~EK L. , JUR.~K E. , RODJ~K L., ~MID B. Research Institute of Veterinary Medicine, Br~w

A follow-up s t u d y of pigle ts of sows, i m m u n i z e d wi th inac t iva t ed vacc ine aga ins t AD showed t h e following: 1. Af te r vacc ina t ion of pigle ts wi th two doses a t the age of 8 a n d l l weeks, 73 ~0 a n i m a l s (out o f 45) did no t p roduce v i rus -neu t ra l i z ing (VN) ant ibodies ; u s ing t he E L I S A detec t ion m e t h o d , 22 ~o a n i m a l s lacked ant ibodies . Af te r the th i rd vacc ina t ion (17 weeks of age), l l ~o of p igs developed no an t ibod ies a t the age of 22 weeks; u s ing the E L I S A m e t h o d , however , an t ibod ies could be found in all an imals . 2. Af te r tr iple vacc ina t ion of p ig le ts a t the age of 8, 13 a n d 19 weeks, VN an t ibod ies were p re sen t in all an ima l s on the 23rd week. 3. E v e n af ter vacc ina t ion a t t he age o f 12, 17 and 23 weeks, VN an t ibod ies were de tec ted in all a n i m a l s a t the age of 27 weeks.

VIROLOGY

Electron-MicroscoIn'c Analysis of Cloned EBV DN.4, Employed for Transfection of Human Lymphocytes

REISCKm J., HIRSCH I., BAaTSCH D. Faculty of Medicine, Charles University, Plzeg, Institute oj Sera and Vaccines, Prague

Human primary lymphocytes were transfeeted with EBV DNA (M-ABA) fragments cloned in cosmid pHC 79 or plasmid pPACYC 184. Three fragments (cosmid cMB-14 and cM301-99, plasmid pM966-20) were able to stimulate transient synthesis of cell DNA and blast transformation. The three mentioned fragments were visualized using various electron-microscopic techniques. Using partial denaturation and restriction maps, we succeeded in determining the orientation within the EBV genome. The fragments bear at least one binding site for nuclear antigen of EBV (EBNA-1), possessing a regulatory function.

Antibodies Induced by the Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV) Infection in Leukaemia

FI~Af~KOV~ V., SEDL$,~OV,I E., H~SKOV~ V. Faculty of Medicine, C~arlea University, Prague

The dynamics of antibody response was followed in 21 patients with newly detected acute leukaemia or chronic myeloid leukaemia in acute relapse. First samples were obtained prior to initiation of cytostatic t reatment , further samples at 1--3 month intervals. In comparison with healthy (seropositive) individuals, specific IgA and high IgG levels against EB-VCA and antibodies to the R component of EBV-associated EA, were found significantly more frequently. Anti-EBNA antibodies were mostly absent in patients, or only traces could be found. Specific IgM against EBV was found in three patients; in one patient, both IgM EBV and IgM CMV were detected.

Distribution of Mouse Cytomegalovirus (MCMV) and Mouse Herpes Virus (MHV) After Experimental Infection by Natural Route

SVOBODOVA J., BLAgKOVX6 D., RAJ6A.NI J., HUCKOVg- D. Department of Virology and Micro- biology, Faculty of Natural Sciences, Comenius University, Bratislava, Institute of Virology, Slovak Academy of Sciences, Bratislava

After peroral experimental infection of white mice with MCMV and MHV, different results were obtained in terms of virus localization in individual organs and in the site and duration of long-term persistence. In mice infected with MCMV tha t had survived generalized infection, the virus was found in the lungs and kidney, and permanent virus replication was found in salivary glands (123 d). The persistence of virus in iVIHV-infected mice, surviving acute infection, possessed the character of dynamic latency, localized in Gasser's ganglion and in the lungs (169 d). The presence of virus could be proved in Gasser's ganglion and in the brain after MCMV intbction. No virus could be found after MHV infection in salivary glands.

Models of Latent Herpes Virus Infection

I~_JCANI J. , BLA~KOVI6 D., SVOBODOVA_ J., LEg~O J. Institute of Virology, Slovak Academy of Sciences, Bratislava, Department of Virology, Microbiology and Immunology, Faculty of Natural Sciences, Comenius University, Bratislava

Herpes simplex virus (HSV-1) persists in regional rabbit sensory ganglia mostly in the non- productive form. On the other hand, tile persistence of mouse herpes virus (MHV) in the lungs and spleen is mainly of the dynamic type. MHV spreads primarily by the blood route whereas tISV-1 penetrates mainly along the nerves. Cultivation of fragments of the mouse trigeminus ganglion and kidney revealed that the probability of reisolation of the virus, as compared with non-cul t ivated fragments, increased two-fold. Determination whether the static or the dynamic phase took place requires further analysis.

V I R O L O G Y $25

Defective Synthesis of Polypeptides of the Herpes Virus Simplex, Type I (HSV-1), HSZP Strain

MAT~riS J . , RAj(~AlqI J . , SAB6 A. Institute o/ Virology, Slovak Academy of Scie~wes, Bratislava

H S Z P s t ra in po lypep t ides , syn thes i zed b y cells of the s table line of r abb i t lungs (RL) , inh ib i t t he m e t a b o l i s m of cell p ro t e in a f te r r e m o v a l of a cyc loheximide block. Such inh ib i t ion does n o t occur in p r imocu l tu re s o f r abb i t k idney (RK) cells. I n t he R K a n d R L cells, in fec ted wi th t he ~ISZP s t ra in in t he presence of phosphoace t i c acid -- c o n t r a r y to infect ion of these cells wi th the reference K O S s t ra in -- no differences in the syn t he s i s o f v i rus po lypep t ides could be observed; obviously , D N A repl ica t ion of the H S Z P s t r a in did no t t ake place. I n cells, infec ted wi th the H S Z P s t ra in a t 41 ~ no inh ib i t ion of ve ry la te pep t ides could be observed.

Comparative ,4nalysis of Human (HSV 1, HSV 2), Mouse (MHV) and Owl (OHV) Herpes Viruses with Mouse Cytomegalovirus (MCMV)

STAN(~EKOVX M., MATIS J . , "~RAu(~IlVA J . , BLA~KOVI(~ D. , STAN~EK D. Chair o/ Virology and Microbiology, Faculty of Sciences, Comenius University, Bratislava, Institute of Virology, Slovak Academy of Sciences, Bratislava, tCesearch Institute of Preventive Medicine, Bratislava

The polypept ide compos i t i on of five purif ied M H V vi ruses was ana lyzed us ing S D S - P A G E a n d fu r the r compa re d wi th H S V 1. Elec t rophorot ic profiles of v i ruses showed 24 zones of s t ruc tu ra l po lypep t ides (molar m a s s 25 - -275 kg/mol) . M H V zones were s imilar to H S V 1 zones even t h o u g h t h e y were no t ident ical . Compar i son of M H V po lypep t ides wi th H S V 1, H S V 2, O H V a n d MCMV revea led differences in the local izat ion of po lypep t ide zones in he rpes v i ruses of different origin a n d in MCMV. A u t o r a d i o g r a p h y S D S - P A G E of l aC-amino acid hydro lyza te - l abe l l ed O H V a n d MCMV also showed changes in O H V a n d MCMV polypept ides ; th i s f ind ing was conf i rmed even b y dens i tomet r i e profiles o f H S V l , OI~IV a n d MCMV

Experimental Chemotherapy of Herpetic Keratitis by Nucleoside Analos and their Influence on Latency Induction

RAJ~/~zI J . , R E ~ F S C ~ - ~ G ~ J. l~stitute of Virology, Slovak Academy of Sciences, Bratislava, Institut fi~r Virologie, Humboldt- Universitdt Berlin

The vi rus levels were d e t e r m i n e d in the con j unc t i va l sac du r ing t r e a t m e n t of herpe t ic kera t i t i s ( s t ra in KOS, v i rus HSV- 1, 106 P F U , 50 ~zL in to r igh t cornea) . As compared w i th u n t r e a t e d rabbi t s , t h e t i t res in a n i m a l s t r ea t ed wi th Acyclovi r (ACV) r eached 0 .02- -0 .25 ~o of cont ro l va lues ; u s ing B r V U d R , 0.01 - -0 .1 ~o, V a r a u U decreased t he t i t re to 0 .06 - -2 .8 ~o a n d B r V a r a U to 0.01 -- 1.76 ~o. I n rabb i t s , t r ea ted wi th ACV a n d B r V U d R , no r educ t i on of l a t en t infect ion occurred p rov ided t he t r e a t m e n t s t a r t ed 2 h p.i . I n o ther t r ea t ed an i ma l s , l a t e n t infect ion of t he n. V gang l ion was f o u n d in 1 -- 6 ~o o f t e s t ed f r a g m e n t s (43 ~o of pos i t ive f r a g m e n t s were found in u n t r e a t e d an imals ) .

Influenza Virus-Associated Ribonuclease: Terminal Sequence of 28S RNA Split Products

ROS~NBERGOVA M., SL~VIK I. Institute of Virology, Slovak Academy o/Sciences, Bratislava

In f luenza v i rus -assoc ia ted r ibonuclease was solubil ized a n d par t ia l ly purif ied. The purif ied e n z y m e spl i ts r ibosomal R N A in to h i g h - m o l a r - m a s s po lynuc leo t ides possess ing ident ical specific- i ty as t he v i r ion -bound e n z y m e . The t e rmi na l sequences of spl i t p roduc t s were de te rmined . A t t he 5" end of t he spl i t p roduc t s , a 5 ' -AAG sequence was found . A t t he 3' end , no specific sequence wa s de te rmined , t h e t e rmi na l nuc leo t ide be ing a pyr imid ine . The p r e s u m e d specif ici ty of ribo- nuc lease sp l i t t ing is 5"-Py ~ AAG-3".

Antisenic Changes of Influenza Virus Haemagglutinin in a Mild Acid Environment

KOSTOLA~SX~ F. , :RUSS G., STYK B. Institute of Virology, Slovak Acaden~y of Sciences, Bratislava

Fol lowing p e n e t r a t i o n o f t h e inf luenza v i rus in to the cell, t h e acidic e n v i r o n m e n t of the endosome causes changes in t he s t r uc t u r e of h a e m a g g l u t i n i n (HA), fus ion of the v i rus envelope wi th


the endosome membrane and liberation of virus RNA into the cytosol. We have shown tha t even antigenic changes in the HAt and HA2 portions of the HA molecule occur in the acid envi ronment which is manifested by enhanced binding of some monoclona[ antibodies due to increased availability of the corresponding epitopes. We presume tha t the observed antigenic changes occur in correi~tion with the availability of the N-terminal (fusogenic) HA2 portion of the HA molecule.

Topological C/zaracterization of the Influenza Virus Haemagglutinin Using Monoclonal Antibodies to Haemagglutinin Glycopeptides HAt and HA2

VArCE~KOV~. R., KOSTOLANSK~ F., R ~ s s G. Institute of Virology, Slovak Academy of Sciences, Bratislava

A common procedure was used for topological characterization of haemagglutinin: competi- t ive-binding test with purified and radialabelled monoclonal antibodies (MA). Some MA, however, lost their activity during purification and/or radioactive label. The antigenic sites, corresponding to these antibodies, can be defined using our competit ive-inhibition binding test. The modified test is based on competit ion of two MA (one of t hem being in solid phase) for radiolabelled antigen.

Determination of Specific IgM and IgG Against Epidemic Parotitis (PAR) and Parainfluenza (PI-7"3) Viruses Using a Modified ELISA Method

HoLvBov$, J. , FRA~KOV.~ V., GRUBHOFFER L., KA~OV~ V., SixTovg E., ROZPRIMOV~- L. Institute of Medical Microbiology and Immunology, Faculty of Medicine, Charles University, Prague, Institute of. Parasitology, Czechoslovak Academy of Sciences, ~esk~ Bud~jovice, Regional Hygiene Centre, Usti n. Labem, Clinic of Infectious Diseases, Charles University, Prague, Institute of Sera and Vaccines, Prague

The following reagents wore used in the experiments: 1. Capturing monoclonal and hotoro- clonal antibodies to h u m a n I2M an~i labelled purified whole virion antigen (ag) PAR; 2. un- labelled purified ag, nucleoprotoin ag PAR and glycoprotein ag PI-T3. The findings correspond to the results of examinat ion of paired patient sera using indirect IF of HIT. Higher sensit ivity of detection of specific IgM was found with. glycoprotein ag but , for detection of IgG, the whole virion ag PI-T3 yielded better results. Application of the labelled whole virion ag in the "capture" modification, or the nueleoprotein ag, excluded the heterotypic cross response to the virus PAI~ and PI-T3 infection.

Dependence of the Biological Properties of Parotitis Virus Strains on the Degree of Attenuation

HANOVX J., GRUBI~OFFER L., OTAVOV~ M. Institute of Sera and Vaccines, Prague, Institute of Parasitology, Czechoslovak Academy of Sciences, Ceskd Bud5jovice

We compared the properties of 10 strains representing three specific groups (pathogenic, laboratory and vaccine strains). All strains were purified using a linear sucrose gradient; infection titre, haemagglut inin titre, absorbance at 280 nm, sialidase act ivi ty and haemolytic act ivi ty were examined in the gradient profile. Selected gradient fractions were analyzed by the SDS-PAGE method. The kinetics of N-acetylneuraminidase was studied in detail in individual strains. I t seems tha t the value of the Michaelis--Menten constant best reflects certain interstrain differences.

Detection Method of Synergism of the Antiviral Effect of Two-Substance Combination

RADA B. Institute of Virology, Slovak Academy of ~ciences, Bratislava

The modified plaque-inhibition method was employed for the determinat ion of effectiveness of a combination of two antiviral substances. The first substance (A) diffuses through agar from the centre of the cell culture, the second (B) becomes incorporated into the agar layer a t sub- inhibitory concentrations. The inhibitory effect of the A ~ B combination is demonst ra ted by an enlargement of the inhibition zone in cmnparison with the control zone, formed by substance A above. The ratio the diameter of the inhibition zone formed by A ~ B combination to the dia-

1987 VIROLOGY $27

meter of the control zone (substance A) serves as the degree of interaction index (DI). Quant i ta t ive evaluation of the degree of potentiation by means of isobolograms has shown tha t indexes DI 1.5 induce the synergic effect of the given combination.

Double-Labelling Method for Ultrastructural Localization of Virus Proteins Within Infected Cells

(~XAMPOR F., Bo~N W. Institute of Virology, Slovab Academy of Sciences, Bratislava, Heinrich- Pette.lnstitut fis Expcrimentelle Virologic und Immunologic, Hamburg

HeLa cells, chronically infected with morbilli virus, were fixed in 1 % paraformaldehyde and 0.025 % glutaraldohyde, t reated with rabbit antibodies against the morbilli virus and swine antibodies against pcroxidase-conjugated rabbit Ig. The samples were stained with DAB, fixed in 0 . 1 % OsO4, dehydrated in acetone and mounted in LR Gold medium. Ul t ra th in sections were treated with ant i -P anti .M and ant i -HA nmrbilli virus protein monoclonal antibodies and with protein A labelled with colloid Au. Osmification of tissue preserves the ul t ras t ructure and does not decrease the antigenieity of proteins. In addition, it permits localization of both virus proteins and virus particles a t the op t imum ultrastructural level.

Properties of the Bhandja Virus

HUBALEK Z. Research Institute of Vertebrates, Czechoslovak Academy of Sciences, Brno

The Bhandja arborvirus (Bunyaviridae) possesses almost spherical virions, 90--120 nm; the virus is susceptible to diethyl ether, sodium deoxyeholate; it is relatively thermostablo (survives at 37 ~ for 2 weeks, a t 56 ~ over 3 h and at 65 ~ 5 min), acidolabile (survives up to 1 h at pH 4), resis tant to t rypsin and heparin; chloramino B and formaldehyde (contrary to phenol) t rea tment results in fast inactivation. The virus produces lethal infection in suckling mice regardless of route of administrat ion. In adult mice and guinea pigs, the lethal effect can be obtained after intracerebral and intranasal adminis t ra t ion only. The virus is nonpathogenic in birds, hedgehog and rabbit; as regards sheep, symptoms of CNS infection occur provided high doses are administered by the intracerebral route.

Serological Examination of Free-Living Birds for Arbor~iruses

Ju~Icov~ Z. Research Institute of Vertebrates, Czechoslovak Academy of Sciences, Brno

Using the haemagglut inat ion inhibition test, we examined 557 free-living birds, belonging to 25 species out of 9 families in Southern Moravia. The birds had been captured in the period of a u t u m n migration (Augus t - - September) in 1984, and during June - - August 1985. Blood serum was examined for the presence of antibodies against 6 arborvirus antigens, genus Alphavirus (Sindibis -- SIN), Flavivirus (tick-borne encephalitis -- TBE, West Nile -- WN) and family Bunyaviridae (~ahyfla -- TAH, ~,alovo -- CVO and Bhandia -- BHA). Antibodies against all tested viruses were detected a t various frequencics (in %): SIN 2.5, TBE 5.2, WN 10.1, TAH 14.4, CVO 10.2 and BHA 1.8.

Cytopathic Activity of Human Rotaviruses in Cultures of MA 104 Cells

KORYCI:f B., ~TR:NACT~- I. Institute of Medical Microbiology and Immunology, Faculty of Medicine, Charles University, Prague

Samples of 12 stools, which were positive for rotavirus and its antigens in EM, CIEP, ELISA and PAGE tests, were employed for infection of slide cell cultures, line MA 104. The superna tan t of stool suspensions induced formation of specific ant igens during roller cult ivation in the presence of trypsin. The ant igens could be demonstra ted by immunofluorescenee in 11 out of 12 samples (one sample discarded due to contamination) s tar t ing 2 d after infection. Morphological changes in the 1st passage were visible in 4/11 samples within 1 d, after 2 d in 6/11; in the 2nd passage, the changes were present in 10/11 samples within 10 d of cultivation, in the 3rd pasage, the changes were present in all samples.


Comparison of the Immunoenzyrne Test ELISA and Virus Neutralization Test in the Diagnostics of the BVD/MD Virus

C~LUPOV.~ L. , P ~ I ~ L I . , ~Ti~.pA2UEK J. Research Institute of Veterinary Medicine, Brno

A n i m m u n o e n z y m e E L I S A m e t h o d for de tec t ion of specific a n t i - B V D / M D an t ibod ies was deve loped . The decisive fac tor p roved to be t he p r e p a r a t i o n of an t igen which is b o u n d to t he b o t t o m of po lys ty rene p la tes . The an t igen u sed is soluble a n d was p repa red by nonionic de te rgen t s . The sens i t iv i ty a n d specif ici ty of t he E L I S A te s t was e x a m i n e d in field s e rum samples t h a t were s imu l t aneous ly e x a m i n e d by v i rus neu t r a l i za t ion tes t . E L I S A appea r s to be more sens i t ive t h a n the N T t e s t a n d iw h i g h l y specific.

Detection and Identification of the IBR/IPV Virus by ELIS/I Test

Pw I . , SVOBODA I. , ZRAL:~ Z., CHLV:'OVg L., HA~IxOV/~ E. Research Institute of Veterinary Medicine, Brno, Institute o9 r Sera and Vaccines, Prague

A n E L I S A te s t for d i rect p roo f of bov ine herpes v i rus in b o d y secret ions of expe r imen t a l l y in fec ted a m m a l s was designed. T he sens i t i v i ty a n d s p e c i f c i t y of t he d lagnos tm m e t h o d was t e s ted u s i n g t he g a m m a - f r a c t i o n of r a b b i t h y p e r i m m u n e a n t i s e r u m a n d monospecif ic a n t i s e r u m of co los t rum-f ree calves, i m m u n i z e d in utero a t the ago o f 8 m o n t h s . The usefu lness of t he E L I S A te s t was d o c u m e n t e d b y following t he g rowt h curves o f ex t race l lu la r a n d in t race l lu la r v i rus a t d i f ferent levels of g rowt h a d a p t a t i o n in the M D B K cell line, a n d in a mode l of art if icial ly in fec ted pel le ts of bovine s e m e n a n d f resh bov ine e jacu la te .

Preparation of the Subunit Antigen of Infectious Bovine Rhinotracheitis Virus

CELER V., NES~ALOV~ E. School o] Veterinary Medicine, Brno

The I B R virus , pa s saged on a M D B K cell line, was c o n c e n t r a t e d by u l t r acen t r i fuga t ion . The sur face l ipoprote in coat was spl i t a n d fu r the r isolated by fine solubi l isat ion w i th T r i t o n X-100. Th i s s u b u n i t an t igen con ta ined 200 [zg pro te in per m L . The i m m u n o g e n i e proper t ies of t h e a n t i g e n were t e s t ed in 24 calves. The h i g h e s t t i t res (1024) of v i rus -neu t r a l i z ing an t ibod ies were ob ta ined in c o m b i n a t i o n wi th t he a d j u v a n t A1-Span-Oil. Ne i t he r clinical s y m p t o m s of t h e disease, nor a sh i f t in a n t i b o d y t i t res occurred a f te r chal lenge wi th t h e :[BR virus.

Isolation and Identification of Trout Infectious Pancreas Necrosis Virus

POSPi~IL Z., T O ~ N E K J . , VALI~W.X L. , ZE~DVLXOV~ D. Research Institute of Veterinary Medicine, Brno

U s i n g fish R T G - 2 cell cu l tu res , t he infect ious panc rea s necros is v i rus was isola ted f rom diseased l a rvae o f t he t r ou t Saline gairdneri irideus for t he first t ime in CSSR. The v i rus was ident i f ied b y e lec t ron microscopy, a cross v i rus -neu t ra l i z ing t e s t a n d immunof luorescenee . All i sola tes ob t a ined here were ident ical or closely re la ted to t he reference s t r a in Sp which is widely d i s t r i bu t ed in Europe . The isolat ion p rocedure can be used even in t h e d iagnos is of o ther fish v i rus diseases.

Effect of lonizing~Radiation on Conjugated Resistance Plasmids

K m ~ o v 4 M., Puzov.K H . , REXA 1~. School of Medicine, P .J . ~afdrik University, Ko$ice, Faculty of Science, P.J . ~afSrik University, Ko~ice

The effect o f ionizing r ad i a t i on on t he t rans fe r f r equency of res is tance p l a smids in E. cell was t e s t ed us ing a s imple c o n j u g a t i o n tes t . T he a b s o r b e d dose ra te of g a m m a - r a d i a t i o n (e0Co) was 1.0, 1.6 a n d 2.1 G y / m i n , respect ively . The to t a l ab so rbed dose for each s t r a in was 1 kGy. The t r ans fe r f r equency ra t e o f R p l a s m i d s increased 10-- 100 t imes a f te r i r rad ia t ion in c o m p a r i s o n wi th t he controls .

1987 ' VIROLOGY $29

Exploitation of TnlO Shifts f r o m the F'Plasm it as a Source of Insert ion Mutants of Phage L (Salmonella typhimnrium

~PANOV~ A., KAXLOVS~" P. Institute of Biophysics, Czechoslovak Academy of Sciences, Brnc

Phage L was employed to obtain transposon TnlO from the F ~ plasmid during growth of this phage on strain TT629 with TnlO (and with resistance to the tetracycline gone) at an undefined site of the F" plasmid. Insertion mutants with insertion of intact transposon TnlO were found among phages, transferring the resistance to tetracycline. Restrictase analysis of insertion mutants permitted to map the exact localization and orientation of nonlethal insertion in the EcoRI B and EcoRI E region of the DNA phage L, in which no genes were found when using conditionally lethal mutations and also in the EcoRI C region at the site of localization of flagellar genes.

Function of the T-DN.4 P la smid p Ti37 in the Plant Genome

NE~.S~K J., DUSB~BXOV-~ L. Institute of Experimental Botany, Czechoslovak Academy of Sciences, ~eskd Budgjovice

Tumor transformation was done in tomato line XXIV. Approximately 10 ~ of tumors possessed the ability to form stalks tha t rooted easily in axenic culture. Thus the tomato transformants differrod from most other plant species. According to nopalin synthetase (nos) formation, the T-DNA -- as a component of the plant DNA genome -- is stable during both mitotic and meiotic division. Transformants of the nos + genome are not resistant to further infection with octopin strain B6-806 and nopalin strain T37. Some of these are pollen-sterile and manifest further distur- bances of the fertilization process.

Radiation Damage of Bacter icphag

HRADESN~( Z. Institute of Biophysics, Czechoslovak Academy of Sciences, Brno

Damage, induced by ionizing and nonioinzing radiation was analyzed on the lambda-bacteriophage model. The effect of radiation was followed in terms of phage inactivation, deterioration of phage functions and repair of the damage. Following ionizing radiation, adsorption of the phage to the host cell is damaged as well as injection and replication of phage D~A. Nonionizing radiation induces damage of replication only. The radiation damage induced by nonionizing radiation can be repaired both enzymically and genetically. After ionizing radiation, the repair is restricted a n d successful repair can be obtained by genetic recombination only.

,4ctinophage ~SLT f r o m Streptomyces lavendulae--graserius

BE~ADA O., TICH~ P., J~zovA L., L~DViX J. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague

Actinophage ~)SL7 was isolated from the S. levandulae-graserins strain after induction with mitomycin. Phage particles were multiplied on S. levandulae and isolated in a CsC1 gradient. Using electron microscopy of negatively stained preparations (2 ~o UA), the ultrastructure of phage particles was defined: polyhedral bacteriophage with long non-contractile tail (group B) terminated by a basal plate. The envelope of r DNA is composed of four main proteins (detected by SDS-PAA electrophoresis). The r genome was analyzed by restriction enzymes and electron microscopy. The head of r contains linear DNA with probable terminal re- dundances.

Host Spec t rum of Phage ~ S L 7

TOB~X I., MOSKALE~IXO J.N., J~zovA L., P E ~ i 6 E x M., BENADA O., TICH~ P. Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Institute of Microbiology, Academy of Sciences, Moscow

Lytic actinophage r was multiplied in the mycelium of Streptomyces lavendulae ~7 strain and further isolated. The infection capacity of the phage was tested in 17 S. lavendulae strains;

530 17th CONGRESS Vol. $2

7 strains yielded a lytic response of different effectiveness (restriction 10 - a - 10-5). Simulta- neously, the presence of plasmid DNA was analyzed in all 17 strains. The presence of plasmid DNA pSLG3 in S. lavendulae-grasserius results in lysogenization by phage (I)SL7.

Use of O s m i u m Tetroxide f o r Detection of Conformation Changes in Bacter iophage PM2 Suprahelical DNA

VOJTI~KOV~ M., I~.AV~UKOVA n. , VOGEL K., PALE~E]K E. Institute of Biophysics, Czechoslovak Academy of Sciences, Brno, Zentralinstitut fi~r Molekularbiologie, Berlin

Changes in the structure of suprahelical DNA PM2 after osmium tetroxide t r ea tment were studied by gel electrophoresis, by nuclease t rea tment and electron microscopy. As a result of such t r ea tment the suprahelical DNA-PM2 molecules were relaxed and "denatura t ion bubbles", visible in electron microscope, were formed. The localization of "denatura t ion bubbles" was mapped against the unique spli t t ing site HpaII and PstI. The existence of three sites of pr imary binding was determined, the main being localized in the 0.76 region of map units , i.e. into one region of early denaturation.

Genetic Bas is of Staphylophage Polyvalence and i t s Use in S t a p h y l o c o c c u s a u r e u s Diagnost ics

ROSYPAL S., ROSYPALOVA A., DO~KA~ J., PAKROV.~ E. Faculty of Science, J.E. Purkyn~ University, Brno

I t was found tha t 70 % S. aureus strains (collection strains and those isolated from patients) were sensitive to polyvalent phage 812. In mu tan t s within the host range of this phage, the percentage of susceptible strains increased to 89 %. In correlation with diagnostic markers proposed by Hhjek (1983), the sensi t ivi ty can be employed for differentiation of S. aureu~ strains from other coagulase-positive species of the genus Staphylococcus. The genetic basis of resistance to polyvalent phage appears to be restriction in the host cell. This conclusion follows from studios on r - m u t a n t s and effect of enzyme extracts , prepared from resis tant strains on phage 812 DNA, followed by electrophoresis in agarose gel,

Differences in Cultivation P a r a m e t e r s of Mycop lasma Virus MV-L2-L 1 72 and Virus M V-L 2

LI~KA B. Institute of Biophysics, Czechoslovak Academy of Sciences, Brno

Viruses of the family plasmoviridae, represented by the MV-L2 group, possess a globular shape, double-stranded DNA and are coated by a lipoprotein membrane. The virus, originally designated MV-Lg-L 172, which was classified into the MV-L2 group as strain L 172, exhibits, however, aberrations in the mult ipl icat ion kinetics, in the degree of carrier s tate and in the con- nection of the multiplication cycle with the growth curve of the host culture. Together with the fact t ha t L 172 possesses a single-stranded DNA (Dybvig etal. 1985), it suggests t ha t this virus is the only representative of a new group or subgroup in the systemat ics of viruses so far.

seventeenth annual congress of the czechoslovak society for microbiology České budějovice...

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