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CHEMOT.ERAPY OF BACTERIAL PLASMIDS,i ' 6. PERFORMING ORG. REPORT NUMDER

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CHEMOTHERAPY, BACTERIAL MULTIPLE DRUG RESISTANCE, R-PLASMIDS, RESTORATION

OF BACTERIAL DRUG SENSITIVITY, ANTI-PLASMID DRUGS, FLAVOMYCIN, CLAVULANICACID, INTERCALANTS

21. ABSTRACT (CQn atm rva sei .e ft ne c meay ad Identlt, by block number)Pharmacological approaches to plasmid chemotherapy are (1) Discovery of novel

antibacterial drugs against which plasmids do not carry resistance genes.(2) Molecular modification of existing drugs to render them non-susceptible to K:

z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4,of plasmid DNA replication. (4) Development of drugs which inhibit phenotypic

as expression of plasmid genes, and (5) Development of drugs which are inhibitors o,drug-inactivating enzymes that are plasmid gene products.

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Chemotherapy of Bacterial PlasmidsFred E. HahnDivision of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington.D.C. 20012, USA

Pharmacological approaches to plasmid chemother- 1974 [1] that among 30 million acute hospital admis-apy are: i) discovery of novel antibacterial drugs sions per year, there occurred 300 000 infections withagainst which plasmids do not carry resistance genes, Gram-negative bacteria in which 100 000 fatalities2) molecular modification of existing drugs to render were caused by bacteremia. Finland and Barnes [2]them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative3) development of drugs which are selective inhibitors bacteremia (per 1000 admissions) in a large city hospi-of plasmid DNA replication, 4) development of drugs tal: In 1935, before the introduction of bacterial che- iwhich inhibit phenotypic expression of plasmid genes, motherapy, the incidence was 0.9 and the case fatality Iand 5) development of drugs which are inhibitors was 50%. From 1951, the incidence steadily increasedof drug-inactivating enzymes that are plasmid gene to attain 8.7 in 1969 with a case fatality not differentproducts. from that of the pre-chemotherapy era. By 1972, the

fatality had risen to 57% [14].R-plasmid-harboring bacteria also have caused largeepidemics in populations at large. The first of thesewas a protracted epidemic of bacillary dysentery,

Bacterial plasmids are extrachromosomal, autono- caused by st s of Shigella, in Japan in the latemous genetic entities which can be isolated as circular 1950..s. This I to the discovery that multiple drug jsupercoiled duplex DNA. They are transmitted, espe- resistance can z transferred among Gram-negativecially among Gram-negative bacteria (Table 1), by bacteria upon nixed cultivation. This history of theconjugation without restrictions of bacterial taxo- discovery of ".,-factors" is summarized in Table 1nomy. R-plasmids possess one series of genes which [3]. In 1969 and the following years, a devastatingdetermine the components of the bacterial conjuga- epidemic of shigellosis occurred in Central Americation system. In addition, they carry multiplicities of [4] which involved more than 150 000 cases and, inresistance genes whose products mediate bacterial re- Guatemala (a country of 5.6 million inhabitants) alone,sistance to a corresponding multiplicity of chemother- caused 12 500 fatalities. "An enormous epidemic ofapeutic drugs, antibiotic and synthetic. typhoid fever has been raging in Mexico [5]" forThis assay presents a review of an emerging field. several years which began in 1972 and abated in 1975.A practical chemotherapy of bacteria which harbor It was caused by a chloramphenicol-resistant straindrug-resistance plasmids (R-plasmids) exists currently of Salmonella o'phi, also resistant .o streptomycin,only in the trivial sense of administering antibiotics sulfonamides and tetracycline, and produced in excessor synthetic drugs against which a given plasmid does of 10 000 reported cases [6] with an initial case fatalitynot confer resistance. The subject of this article is of 13.5% [5]. Coincidentally, an epidemic of chloram-the scientific basis of restoring chemotherapeutic drug phenicol-resistant typhoid fever appeared in India insensitivity to bacteria which owe their drug resistance 1972; the causative strain was different from the Mex-to plasmidic determinants. ican strain [7]. More recent problems of plasmid-Bacterial multiresistance to chemotherapeutic drugs, mediated drug resistance are the emergence of strainsdetermined by R-plasmids, causes treatment failures of Haemophilus influenzae, resistant to ampicillin [8]of hospital infections, foremost in patients with a or chloramphenicol [9] and of Neisseria gonorrhoeae,compromised immune system. The Deputy Assistant resistant to penicillins [10].Secretary of Health of the United States reported in R-plasmids have been found in bacteria which were

Naturwissenschaften 66, 555-562 (1979) © by Springer-Verlag 1979 555

80- - !!- ! -J -I

Table I Discovery of R-factors

Discovery Point in time Authors

Drug resistance is transferable by mixed 1959. November Ochiai et alcultivation 1959. November Akiba et al.

Transfer of drug resistance is not mediated 1960. January Mitsuhashi et al.b) filtrable agents 1960 Akiba et al.

Drug resistance is transmitted regardless 1960. January Mitsuhashi et al.of the polarity of F agent

Transferable drug resistance property 1960. March Mitsuhashi et al.is lost spontaneously during storage

Transferaole drug resistance property 1960. June Mitsuhashi et al.is eliminated by treatment with acriflavine Fukasawa. Watanabe

Transmission of drug resistance is interrupted 1960 Fukasawa. Watanabeby blender treatment of mixed cultures

The term " R-factor" is introduced for the 1960 M!Isahashitransferable resistance property

The range of transmission includes allspecies of the

Enterobacteriaceae 1960 Haradd et al.Vibrio comma 1961 Baron, FalkowPasteurella pestis 1963 Gmoza. MatneyPseudomonas aeruginosa 1971 Roe et al.. lyobe et al.Aeromonas group 1972 Aoki es al.Bordetella bronchiseptica 1973 Terakado et al.

.solated and lyophilized before antibiotic therapy be- of antibiotics is discontinued [14]. While particularcame prevalent. One strain of Escherichia col, exhibit- ecological or genetic conditions may be responsibleing transferable resistance to streptomycin, tetracy- 'or observations of R-plasmid persistence in the hu-cline and bluensomycin, had been isolated prior to man or animal bacterial flora [13, 15], this does not1937 and lyophilized in 1946 [11]. Streptomycin was detract from the practical importance of the persis-marketed from 1947, and tetracycline and bluensomy- tence phenomenon. The multiresistance problem,cin had not bf. n discovered in 1946. While R-plas- once created by extended and excessive drug pressure,mids, thus, have not arisen de novo in response to may not disappear readily under limited efforts tobacterial exposure to chemotherapeutic drugs, mul- lessen this pressure.tiresistance as a global medical problem has beengenerated by the selective pressure of antibiotic usage, 1either in hospital practice or at large in societies with Pharmacological Approachesmedically uncontrolled availability of antibiotics. Oneof the reservoirs of R-plasmids are farm animals, The practical solution of the multiresistance problemraised on antibiotics-supplemented feed [12]. Between should be the discovery and development of phar-1960 and 1970, the production of antibiotics as feed- macological approaches, i.e., of a chemotherapy ofadditives in the Unitc States increased from 0.5 to bacterial R-plasmids. The feasibility of anti-plasmid3.3 million kg per year L.l]. chemotherapy is being disputed (e.g., [16]). The cur-There has been no lack of suggestions to restrict the rent situation is comparable to that which has pre-medical ase of antibiotics, topically, prophylactically, ceded most major advances in chemotherapy. Evenor without clearcut bacteriological rationale. Like- 10 years after the introduction of salvarsan into prac-wise, the agricultural use of antibiotics in animal hus- tical medicine, the selective chemotherapeutic actionbardry "has come under criticism. Unfortunately, of this drug as well as the basic theory of chemother-there exists a perturbing trend of R-plasmid-harbor- apy were questioned [1], and the advent of antibacterialing bacteria to persist, for example in the intestinal and antiviral-drugs [17] was preceded by the expres-tract of inidividuals no longer receiving antibiotics sions of opinions that such drugs could not exist be-[131. R-plasmid-containing bacteria in antibiotic-fed cause of the prohibitively small size of their metabolicfarm-animals likewise persist after the administration targets. J

556 Naturwissenschaften 66, 555-562 (1979) © by Springer-Verlag 1979

An examination of the prospects of R-plasmid chemo- clinical antibiotics to their nutrition [23]. The reasonstherapy must be based on a definition of its aims. for the hypersensitivity of R + bacteria to these anti-A prophylactic approach in which an anti-plasmid biotics are not known. It is possible that the attach-drug would be administered, for example. to an entire ment of plasmids to a membrane fraction rendershospital population would be unrealistic. The need plasmid bacteria more sensitive to the actions ofis for curative compounds which can be administered flavomycin or macarbomy.in than bacteria whichto patients. suffering from infections with multiresis- have unoccupicd membranes.tant bacteria, in order to resensitize such pathogens Earlier, Kawakami and Landman had shown [261 thatto conventional chemotherapeutic agents, either pre- conversion of a strain of Salmonella paratyphi to sta- -jceding antibacterial chemotherapy or. ideally. in ble L-forms by high concentrations of penicillin elimi-combination with antibacterial drugs. The problem nated in a segregative manner resistance determinantsof the stabilization of R-plasmids in their host bac- fIom the R-plasmid RI I which had been transferredteria when these are grown in the presence of anti- into these bacteria. The authors speculated that thebiotics for which the plasmids contain resistance de- conversion of the bacteria into spheroplasts elimi-terminants, has been cited [18. 191. Such a selection nated not only the cell wall but also the mesosomeof plasmids or of individual resistance genes [18, 19] fraction of the membrane which they proposed to bewill depend upon the mechanism of action of the a potential attachment site of the plasmid.anti-plasmid compound as well as that of the chemo- A general use of flavomycin as feed-additive mightthcrapeutic drug in combination. Nalidixic acid reduce the size of the R-plasmid carrying bacterialremoved seven out of ten resistance genes from a pool in farm animals and certainly would stop theplasmid, carried by Klebsiella pneunoniae, including selective pressure ofclinical antibiotics on this bacterialthat for nalidixic acid itself [201. That plasmid resis- flora. It is not possible to predict whether or nottance genes may "retreat to the safety of the chromo- such widespread use of flavomycin would ultimatelysome [161" by transposition under the pressure of lead to the emergence of plasmids with resistancecombination chemotherapy has not been demon- genes for flavomycin.strated.I shall consider the following pharmacological ap-proaches to plasmid chemotherapy: Molecular Modification qf Drugs1) Discovery and development of novel antibacterialdrugs in the hope that the global plasmid population Certain enzymes that are plasmid gene products at-will not possess resistance genes against them. tack antibiotics by derivatization such as the acetyla-2) Molecular modification of existing drugs in order tion of the two hydroxyl groups of chloramphenicol

Sto render them insensitive to attack by enzymes [2]or adnlltoactito rphshrlto

which are plasmid gene products. of the hydroxyl or amino groups of aminoglycoside3) Development of drugs which are selective inhibitors antibiotics. While acetylation of chloramphenicolof plasmid DNA replication, proceeds to inactivation of all the antibiotic present,4) Development of drugs which selectively inhibit the the derivatization of the aminoglycosides is restrictedphenotypic expression of plasmid genes, i.e., tran- in its extent, and the role of the deriv.:tives in causingscription and translation. bacterial drug resistance is problematic [28].5) Development of inhibitors of those enzymes which An obvious pharmacological approach to overcomingare plasmid gene products and biochemically mediate resistance to those antibiotics which are derivatizeddrug resistance. by plasmid gene products would be molecular modifi-

cation of the antibiotic molecules to delete those func-tional groups which are derivatized unless such deletion

Novel Antibacterial Drugs also causes loss of antibacterial activity.One such experiment was performed inadvertantly

The phosphoglycolipid antibiotics, moenomycin (fla- through the discovery of gentamicin. This aminogly-vomycin) and macarbomycin inhibit the biosynthesis coside, first marke!-j in 1966, shows remarkable cu-

R of the bacterial cell-wall peptidoglycan [21, 221 by rative properties against bacterial infections of burn- interfering with the transfer of the lipid-bound precur- patients, septicemia, meningitis, and urinary-tract in-

IRL sor to the growing peptidoglycan [231. Macarbomycin fections. Waitz and Weinstein [29] called attentionin limited studies [24] and flavomycin in extensive to the absence of a hydroxyl group in the 3-positionwork [25] have been shown to inhibit preferentially of the methyl-aminosugar moiety of gentamicin andbacteria which carry R-plasmids. Flavomycin is usefrI suggested that this rendered the antibiotic non-suscep-as a feed-additive in farm animals in lieu of ad tible to derivatization by plasmid gene products.

Naturwissenschaften 66. 555-562 (1979) CC by Springer-Vt.... ,9 557Natuwisenscaftn 66 55

a kinetic course of plasmid loss from test culturesNi which resembled the growth curse of the plasmid-

( 1 1 . progeny [40. 411: the original plasmid + population0) became progressively diluted with plasmid- cels.

A few authors [42-44] have suggested that R-plas-.- mid bacteria are more sensitive to growth inhibition

by intercalants than the same organisms not carryingplasmids. An apparent elimination of plasmids from

Fig. I. Structure of chloramphcnicol d[33aties, lackmg ahphatic growing cultures would then. in actuality, be the resulthydroxyl groups [33] of a selective propagation of spontaneous plasmid- seg-

regants by differential growth inhibition. Such consid-However. one year after the publication [29]. R-plas- eratio:'s cannot well explain numerous observationsmid-mediated gentamicin resistance appeared. The (rev. in [19]) of selective elimination of individualbiochemical basis of this resistance is N-acetylation. resistance genes from plasmids. For example, the ka-or O-adenylylation of other hydroxyl groups of the namycin resistance gene is most easily eliminated by ,antibiotic [30]. intercalants. and the frequency of this occurrence isThe two hydroxyl groups of the propanediol moiety a function of the relative stoichiometry of bindingof chloramphenicol were considered indispensible for of these compounds to DNA (Fig. 2) [38]. The selec-antibiotic activity [31. 32]. However, Kono and his tive inhibition of R-plasmid DNA replication mayassociates [33] synthesized the two chloramphenicol be based upon the fact that circular supercoiled DNAderivatives (Fig. 1) which lack both hydroxyl groups. has a higher affinity for intercalants than uncon-They are as active as chloramphenicol itself against strained DNA [451 and also is subject to strongersensitive bacteria and exceed the activity of the drug template inhibition by such compounds [461.against resistant Staphylococcus aureus by factors of Among intercalative anti-plasmid compounds are the60 and 38. clinical drugs, daunomycin, quinacrine, quinine, chlot-The overlapping substrate ranges [28] of the approxi- promazine and chloroquine. However. there existmately 12 known aminoglycoside-derivatizing en- severai arguincits against their practical applicationzymes as well as synthetic-chemical problems would as plasmidostatic drugs. Elimination of plasmids bydiscourage efforts at molecular modifications. but the these drugs in vitro does not attain 100% in overnightwork of Kono et al. [33] suggests that congeners of cultures, and such experiments require small inoculachloramphenicol can be developed against which plas- of the order of 101 bacteria per ml. The preferentialmid-determined resistance would be an unlikely pros- suppression of R-plasmid' bacteria by flavomycinpect and which also might carry a lesser risk of induc- was accomplished in continuous culture over a perioding aplastic anemia. e.g.. by replacing the aromatic of 5 days [25] but extensive efforts in the laboratorynitro group with -SO3 -CH 3 [34]. of this writer failed to maintain in a chemostat low

bacterial titers of the order of 103 organisms per ml.

Inhibitors of Plasmid DNA Replication oo_

Growing cultures of plasmid-containing bacteria inbroth media, containing aminoacridines at concentra- 90 *Qu

tions which were not growth-inhibitory, eliminated Xgenetic [35] and phenotypic [191 indications of the "A" BB .AOfunctions of plasmids as well as biophysical evidence z *. Cof the presence of plasmid DNAs [36, 37]. Hahn and , 8 - ui

his associates postulated. tested and showed that sub- zstances which bind to DNA by intercalation, possess - rthe group property of eliminating plasmids from 20 4060 8o 00growing bacterial cultures [19, 38, 39]. In an unfortu- ENDPOINT [%MG DISPLACED]nate choice of expression, this activity has become Fig. 2. Frequencies of elimination of the kanamycin determinantknown as "curing." Kinetic experiments have shown from the R-plasmid RI in Salmonella typhimurium LL-2 by interca-that plasmids were not literally eliminated from their lants as a function of end points of displacement of methyl green

from its complex with DNA. Eliminating concentrations 10- + :host bacteria, but that "curing" compounds block displacing concentrations 5 x 10- s A. EB ethidium bromide: Quaselectively the replication of plasmid DNA. The non- quinacrine: MD miracil D; Pl propidium iodide: CQ chloroquine;

L- segregation of plasmids into daughter cells produced AO acridine orange; BB berberine; Qui quinine [381

558 Naturwissenschaften 66. 555-562 (1979) 3 by Springer-Verlag 1979

JL, , ++ II IF E-.... VI tt+:

Hence. a computer simulation of progressive R-plas- all four antibiotic resistance determinants from themid loss in a continuous-growth system over R-plasmid RI with frequencies between 56 and 70%prolonged incubation periods could not be tested. [19]. It is not known if this anti-plasmid effect ofOne hundred per cent plasmid elimination from nalidixic acid was a result of an inhibition of plasmidstrains of Sahnonella was accomplished in vivo in DNA replication or of a selective inhibition of plas-mice but required treatment with ethidium bromide mid mRNA transcription [54]: it had been reportedfor one week (471. earlier [55] that nalidixic acid inhibited the synthesisFinally, there cxist unexplained taxonomic differences of plasmid mRNA at concentrations which did notamong both plasmids and host bacteria as concerns affect bacterial mRNA synthesis.susceptibility to plasmid elimination by intercalants The anti-plasmid action of sodium dodecyl sulfate[19. 441 For these various reasons. it remains uncer- (SDS) and other anionic detergents of a chain lengthtair if an effective broad-spectrum plasmidostatic of > 10 ca rbon atoms (rev. in [19]) is probably relatedagent can be developed on the basis of the DNA to the DNA nicking action of DNA gyrase [51]. SDSintercalation principle, promotes double-stranded cleavage of circular DNAA recently discovered bacterial enzyme system, DNA by the gyrase and an attachment of the denatured Agyrase [48]. catalyzes the ATP-dependent introduction enzyme to the DNA by covalent bonds [52].of negative superhelical turns into double-stranded The antibiotic. novobiocin (Fig. 3) inhibits the ATP-circular DNA. This activity is required for the replica- dependent introduction of negative supercoils into cir-tion of plasmids [49. 50]. One subunit of the enzyme cular DNA by blocking the binding of ATP to theis responsible for nicking and closing of circular DNA DNA gyrase j521. An anti-plasmid action of novobio-and is inhibited by nalidix,," acid [51]. The second cin has been investigated by McHugh and Swartzsubunit iitroduces negative supercoiling at the ex- [56. 57]. The antibiotic eliminated 8 out of 14 plasmidspense of ATP hydrolysis and is inhibited by the anti-biotic, novobiocin [52]. CHIEmpirical tests of :be elimination of R-plasmids by C, o o?",nalidixic acid were based upon earlier knowledge that c1., o - O ,the d-ug operationally is an inhibitor of bacterial . -.co

DNA biosynthesis (rev. in [53]). At a concentration oI I!319- M tl:2 -,- O Oil

of 6.25x 106 ,l1which did not inhibit the growth Aof Salmonella lyphilnuritun, nalidixic acid eliminated Fig. 3. Structure of no,obiocin

Table 2. Effect of I- and 7-positicn substitution of i.8-naphthyridines on antibacterial actikity

0

C0O11

Rclatii, activity , Relative activity(R2 =CH3) (R, =CH) it_.in vitro' in r.ico' in vitro' in vivo"

H o o H o ++CH3 o ++ CH3(NAL) . . . . . . . .C2 H5(NAL) 4+++ -++ + CH, . . .

CH(A + ++ . C,H, .. oi-C3H, + jiCH 7 +++ 0CH 2CI=CHa 1++ CHCoH, + 0C4 H9 o o CH2CN .. . + +CH.CF3 . +.+ + + COOH o 0CH 2COOH o o CH2OH (hydroxy-NAL) + +++ + + + +CHCII2OH ++++ ++CH2 C H2 N(C,I s) o 0CH2 OCH 3 4- + ±

Minimal inhibitory concentration against either Salmonella tprphi or Escherichia colt where o = > 100: + = 50':+ +=910:+++=5:and ++++=<51g1ml' Oral activity (ED50)vs. lethal Kiebsiellapneumoniae infection in mice where o=>300: +=300-200:

+ + =200- 100: . . . =100-50, + + + + = <50mglkg/day

Naturwissenschaften 66. 555-562 (1979) iV by Springer-Verlag 1979 559

~i

in a series of bacterial strains, but the concentrations In several strains of Sahnonella. carrying an R-plasmidrequired were growth-inhibitory for the host bacteria, with resistance determinants for tetracycline. chlor-By agarose-gel electrophoresis it was shown that bac- amphenicol, kanamycin and streptomycin. subcul-teria which no longer expressed drug resistance, no turing in the presence of tetracycline, a translationlonger contained plasmid DNA [56]. Authors asked inhibitor, for 5 serial passages resulted in the completethe question if novobiocin-insensitive plasmids pos- loss of all resistance determinants, except that forsess specific DNA gyrase systems. The elimination tetracycline itself [59]. Since this phenomenon wasof plasmids from 50% of clinical isolates of E. coli restricted to Salnonela but was not observed with[571 appeared to be a plasmid-related sensitivity to the same R-plasmid in E. coli. Klehsiella. Protew ornovobiocin: it succeeded in novobiocin-resistant Pseudomonas, it was evidently not due to the inhibi-bacterial mutants. tion of a plasmid-specific translation system.Structure-activity data for nalidixic acid have beentabulated only for bacteria (Table 2) [53]. It would lnhibitors of Drug-inactirating En:,mesbe logical, to develop such information for the anti- that Are PlasDid Gene Productsplasmid activities of nalidixic acid congeners in the t r s G oexpectation to detect compounds with selective effects Overcoming R-plasmid-mediated resistance through

on plasmid-associated DNA gyrases. The structural the use of compounds which inhibit plasmid genereasons of why novobiocin inhibits the DNA gyrase products (enzymes) is currently that area of plasmidreaction in competition with A TP are not apparent chemotherapy which appears closest to practical rea-[52]. The antibiotic is not known to affect any other lization. This is the case for R-plasmids whose geneATP-requiring enzyme at low concentrations. The products are Mactamases which destroy penicillinsstructure of novobiocin (Fig. 3) offers no obvious clue and cephalosporins through hydrolysis, i.e., not byto the nature of the competition with ATP nor to derivatization but by degradation. Among such f-lac-molecular modification with a view to broadening tamase inhibitors are the empirically discoveredthe anti-R-plasmid spectrum of the antibiotic. biological product. clavulanic acid [601 from Strepto-

iyces clavuligerus. and a semisynthetic penicillanicDrugs wrhich Inhibit the Phenot'pic Expression acid sulfone. designated as CP-45.899 [61] (Fig. 4).

Additional P'-lactamase inhibitors of natural originand undisclosed structures have been cited in the liter-

Drugs which inhibit the transcription of mRNA from ature [60. 62. 63]. Clavulanic acid. generally, has onlyDNA and its translation into proteins should inhibitthe phenotypic expression of plasmid genes. But there '0o sSare few biochemical indications that transcription or 1.C-C \X .0IAon ".C-Ci C..MI Cm' C_ " i 1t,_

C-N. -Hl -'(' n

translation from plasmid genes differ from the corre- o tc o 'C' 0sponding bacterial processes. The possibility that nali- 1 H OH

dixic acid may function via inhibition of specific gy- Clavulanic acid CP-45.899rase systems as a selective transcription inhibitor for Fig. 4. Structures of claulanic acid and of CP.45.899 160. 611

plasmids [55] has been cited above. Rifampicm, anantibiotic which inhibits bacterial RNA polymerase. Table 3. P-Lactamase inhibitory activity of sodium claulanateexhibits certain anti-plasmid actions (rev. in [19]). t aHowever. these might be attributed to an inhibition Source of p-lactamase Sub- I:oof the synthesis of a primer RNA which appears to strate' fl*gmllbe required to initiate plasmid DNA replication [191.The antibiotic had no anti-plasmid effects in bacterial Escherichias aruginT410 chromosomalbath ty C 160mutants, resistant to rifampicin [41]. encoded QObservations that the 4s and 5s RNA populations of a Enterohacter cloacae P99 C 10.0strain of E. coli which harbored the R-plasmid RI, Klebsiella aerogenes NCTC 418 P 0.03

contained components which hybridized with the EscherchiacoilT4. R-plasmid encoded P 0.08 _"tcherichia coll JT39. R-plasmid encoded P 0.08 V

plasmid DNA [58] have prompted a speculation that Proteus nirabiis C889 p 003this plasmid may generate and utilize "a modified Pseudornonas aeruginosa Daigleish P 0.1

translation system". No miechanistic evidence of the (preparation contained someoperation of such a system is available which might Sabath-type enzme) Hconceivably provide a point of departure into the Staphylococcus aureus Russell P 0.0R acillus cereus (Whatman Biochemicals Ltd.) P 17.0development of selective inhibitors of the biosynthesisof plasmid gene producis. C=Cephaloridinc: P= Benzylpenicillin

AA

560 Naturwissenschaften 66. 555-562 (1979) _r by Springer-Verlag 1979

. i

weak antibiotic activity of its own. It inhibited a series I SirnnonN. 11I . Stolic>. 1P 1) .1 Air \led A- 22. 123 194of isolated bacterial fi-lactamases to the extents shown 2 l-inland. Ml . lBaine,. %I W Antibiot (hemother 21.,

in Table 3 [641. At low concentrations, it sensitized 9615;! .,\litsuhdshi. S . I~ohe. S.. Inoue. M :bi, _"). 131 (1976,

those bacteria to inhibitions by ampicillin and cepha- -. ~ J.. et al .J Infe.ct Diwjeas 126. 215 (19'2loridine whose fi-lactamases were sensitive to cla. ula- i Anon~znou,: Lanet 1975-1. 1230nic acid. Similar sensitizations were reported for fl- 6 Olartc. J . Galindo. M. Antumicrob Ag~ents Cheother 4

lactamase-producing strains of N. gonorrhoeae [6i. -9 I93Anderson. 1. S Top Infect IDieases 1. 39 (1975)66] and for certain strains of S. aureus, H. iq/7:wn:ae. doraCJ.lhe1.F..Iak.S? teerl,2643

Bacteroides fragifis and several Enterobacteriaccae 19,6)while no sensitization was observed in Pseudlnnonas 9Van Klineercn. J_. %in Emhden. J.. IDe~sn,-Kroon. K Ani-

aeruginosa (661. No factorially de,;igned combination 10 ErobI. L.P.t Cheral ulad /. 28, 19-experiments to test for synergistic growth inhibitions 10 L.P et al - ibi 1. 528 11967,

by graded concentrations of clavulanic acid and of 12- S%%anin. .1. M.: Report of the Joint Comttee on the ucofantibiotics with the isobologram method have been antibiotics in animal husbandr% and '%eteririar% medicine Lon-reported. At the time of this writing, no in vivo experi- don: Her.MajesqWs Stationar% 0111cc 1969ments with combinations of clavulanic acid and anti- 11 Richmond. NLIL.: Top. Inf~ect. D)isease 2. 61 11977). Jonison.

biotis ha bee publshed 1%I.: Scand. J Infect lDiseateb 6. 339 (1974)mo bioicshadbee pulised.14McGo%%an. J.F. Barnes. \1.W. Finland. M1.: J Infect Diz.easestCP-45,899 inhibited P-lactamase enzymes. isolated 132'. 116 (1973)

Sfron' 7 different bacterial species. For enzymes whic .. S.D.. et al. To net ieae .4(97werechromosomal gene products, preincubation uith 16 tDa'ics. J..- ibid. 2. 343 01977)the inhibitor was required. For example. the inhibi- 17- D~ale. IL.11: Ph~..iol. Re%. 3. 359 0921). Bauer. 1) J.: Mod.

EK Trends Med. Virol- 1. 49 (1967)tion of a Pseuddmonas enzyme increased with premncu- I S Lebek. G.:- Z. Hsg Infektionskrankh 149. 255 (1963)bation fromr16.to.80%. The-comfpound was 1000 19. Hahn. E.: Anuiiho. Chemother. 2ai. 196 (i976)times more active against'plismid gene products than 20. Tiraub. W.II. Kleber. L:; Z. Bakt. Parasitenk AN 1. Orig.against chromosomal gene--proiizts [671. Sensitiza- A.2.80(93

dion studies--to peicillins-and cephalosporins by CP- 21. Hluber. G.. Neseniann. G : Biocheni. Biophys. Res Commun.3o. 7 (1968)

MW89 navreyo atei 6-0 rdcdi 2 Suzuki. J.. et al.: J. Ant ibiotics (Tok'.o) 2.5. 94 (1972)vit'ro results- comparable to tlose with clavulanic acid. 23. Huber. G.. in: Antibiotics. V'ol, V a. Berlin- I eidelbL ri-Ncev

citt abve.Facorialy esinedexpeimets ith York: Springer (in press)ctd a nde..a peniillin dsowed thatrthe ts druhs -24. Mitsuhashj. S.. babec. S.. Hashimoto. H.. J. Antibiotics (To-CP,4,899k'o) 23. 319 0197i))

acted synergitcal -1.Ssei n oa ne- 2.Wtnh. T.. et al.: Johns IHopkins Mecd J. Suppl. 2. 98 (1973)ticifs:-of-liwith S. aureus aid systemic infections 26. Ka%%akami. M.. Landman. O.E.: Biochem. Bioph~s. Res. Corn-wit Gram-negative pathogens, were successfully mun. /S. 716 (1965)

treaed ithcom ioins of CP-45,899 and ampicil. 2Ahw .V.Bos. R.F.: J. Bacteriol. 95. 28 (1968)

lin-172' 28 Davies. J.. Kaigan. S.A.: Top. Infect Diseases 2. 207 (1977ii. 29. Wait7. J.A.. Weinstein. M.: Med J. Austral. Suppl June 13.

De-rivtization of penicillanic acid by organic-chem- ~ (90

ical methods should--be c;napble of yielding numerous 30W. Bencvistc. R.. Davies. J.: Ann. Rc%. Biochemn. 42. 471 (1073)c&I~enrsof.CP-45,899_ for -testing as potential anti- 3 1. Hahn. F.E.. et al.: Anuihiot. Chemiotber. 6. 531 (1956)j9 ~l~id rug intho~bateia hos reistnceto 32. Hahn. F.E.. Gund. P.: Top. Infect. Disease!, 1. 245 (1975)

antiiotis iscausd p~id~~code ~.. 33. Kono. M.. ct al.:- J. Antibiotics (Tok'.o) 22. 603,'1969)lactam by 4. Man~an. D.R .. Arimura. G.K.. Yunis. A A.: Mol. Pharmacol

ctdamases. The information in [67-72] suggests that 1/. 520 (1975)this-line of drug development may be promising. 315. Hirota. Y.: Proc. %at. Acad Sci. USA 46. 57 (1960)

36. Ro%,.nd. R.. Naka~a. R.. Nakamura. V.: S. Mol. Biol,1.376 (1966)

.'.Courvalin. P.M.. Carlier. C.. Cliabbert. Y.A.~ Ann. Inst. Pas-__ teur 123. 755 (1972)

38. Hahn. F.E.. Ciak. J.: Anuimicrob. Agent3 Chernother. 9. 77-Increasng- problems of bacterial multiresistance to (1976)

chemtheapeticdrus, ause byR-pasmdsare 39. Allison. R.G.. Hahn, F.E.: ibid. 11. 251 (1977)40. Hohn. B.. Kom,. 0.: J. Meti. Biol. 45. 385 (1969)unlikely to- beontained or to disappear through en- 41' Ri, a. S.. et al.: Antimicrob. Agents Chemother. 3. 456 (1973)

___ti*&liitaion inantbioi-usage. They call for 42. Yoshikav~a. M.. Sevag. M.G.: J. Bacterial. 93. 245 (1967)i riin.p a-c~toicl iprah Athe chemother- 43. Yoshikawa. N: Gefict. Res. 17. 9 (1971)ajio~R-~is i tid -h s ffaoncn sa n- 4.Fow, S.- Infectious Multiple Drug Resistance. London.

___ ma e-ddte -n th _eelpmn of _ -i _actamase Pion 19754.Bauer. m.. Vinograd. J.: Progr. MoL Subcell. Diol. 2. 181inlibiiw 6- -ociic use AMn comrbinatfion chemother- (91

apy arure ntl tenstimsj-p ochS. 46. Richardson. J.P.. Parker. S.R.: J. Mol. Biol. 78. 715 '1973) ___

wau~sm~a6 5-52(99 )b pinger-Vrlg 1979 56.1----

4 Ittan h~iud ) II. (ha h~i *\ nn ~i ~ad'~a (11 Ret-uli. J \.c: .1! \ h~1 i ,h I i~'s.Conf. Antimicrob.IC ~li5(1911 gcmni ( heriumhc . 11 143 IFs

4;S (Mlcni \1 el a 1ro \a! %,a ", 6 2 S -s I C I uua S lh. 1! P'ro,',I ~Ihnt. Congr. Chemother..49 Sakakiblir.i. .1m:a j -1 Ibid -, m. i ft 4i p 21 4~( Iudenhau ter. K L %10j (,Lit (jelit 1.-.2 69 '~ t MC-,a II i -r c Ii J \ntt~hzotics (Tokyo) 26. 51 (1973)

52 rot . \.: %-td 'x.: is % 0 4'iPe i'4 q . (oh% \1 Antimicrob. Agents Chemnothcr. MIStlieII. A . et .11 Ihid 0 S3 9-S).3 o'~ X COOLK. I \1 ll XnIHIboM', X ol 111. p 1-4 11cr- W; Mlekr, j M . 11AKer C.N.. Thornsberry. C.: ibid. 14. 794 (1978)thn-11etdclh.crL-\c-. Yo~rk Springeir 19-5 ,6 Xi R . \Idrc%%,. J.M., Bedford. K.A.: ibid. 13. 389 (1978);4, Smith. C L . Kubo. \1 . himoto. I \ature 27. 4240 19-" 6- 1 t:. K P, \cu. 3i.C.: Abstr. 18th Intcrsci. Conf. Antimicrob.

55 PLII. A' cmn I Xl Bw[t~I~ 99 19 _q3 Agent ( hcmrother.. p. 142 (1978)6 McI ii.hl. 6 L M \at.X \ Xniimob~ Agenit, hemoithicr 61, lci.:u.a. J.A.. et al.: ibid . p. 143

1-2. 12" (19-- 69) A'w tpoakec. N.. Neu, H.C: ibid.. p. 144I- -\kIluch!. 6 1 . S%.grl/. X1 N \bstr I.th Inrrc ti nti- 0 1, Khan. WV.N., et al.: ibid.. p. 145imLroh AXIerit, (hernoilicr . 11 19 19-.,j- 1 lIster. C. et al.: ibid.. p. 146

5N 1logemitie- 6 . Ha~rtmann. 6, . Rt. ( Fop Infect Di~zca'' -2. English. A.R., ct al.: ibid.. p. 147 I- Lcdv. Gi Z H%,, Iiifektion~krankii 1ii~. 41 (191W4

60 1l ro-an. A\ Gi e!a J .\itthNo-ics 4 To k~ w2 9. 66S I I q-0) Received .January 29. 3979 1-

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