in vitro sensitivity of leptospira to various antimicrobial agents
TRANSCRIPT
IN VITRO SENSITIVITY OF LEPTOSPIRA TO VARIOUS ANTIMICROBIAL AGENTS'
Abstract The in vitro sensitivity of Leptospira icterohaentorrkagiae was checked against
14 antimicrobial agents to find which 'night be added to leptospira culture media to control contamination without seriously depressing growth of lepto- spires. Estimates of the amounts of leptospira growth resulting in these modified media suggested that five agents were worthy of further study. The determina- tion of leptospira population by microscopic counts showed that three of these compounds produced only slight to moderate inhibition. Two of these three anti~nicrobial compounds plus a fungicide were tested in combinations. Quali- tative tests on these media were also carried out employing L. ballz i t , L . catti- cola, L . pyrogenes, and L. sejroe.
Based on results of these tests, a new selective medium for growing leptospires is suggested; i t consists of a standard leptospira liquid medium to which are added 50 mg/l. sulfathiazole, 5 n~g/ l . neornycin sulphate, and 0.5 mg/l. actidione.
I t is well known that contamination of cultures of pathogenic leptospires with other bacteria or fungi often results in overgrowth and destruction of the leptospires. Isolation of the etiologic agent in cases of leptospirosis is thereby rendered difficult. Contamination also tends to affect the accuracy of iilcidellce values obtained in leptospira surveys of wildlife. The advantage of having available a selective medium for growing leptospires is, therefore, obvious. The only linown media suggested for this purpose are those of Sta- vitslty (8), Stuart (9), and Dominguez (4). Stavitslcy used sulfanilamide in a concentration of 4 g/l. of medium. Stuart found sulfaguanidine much Inore effective than either sulfanilainide or sulfadiazine. Doininguez observed tha t suppression of contamination is most effectively accomplished by addition of either dihydrostreptoinycii~ a t concentrations of 100-200 mg/ml or chlor- amphenicol a t 200-500 mg/ml.
The study here reported was carried out to obtain illformatioil on the effects of a variety of antimicrobial agents on leptospires with a view to developing an improved selective medium.
The antimicrobial agents investigated included crystal violet, basic fuchsin, potassium tellurite, sodium azide, desoxycorticosterone, sulfaguanidine, sulfathiazole, furoxone, actidiom,? b a ~ i t r a c i n , ~ neornycin s ~ l p h a t e , ~ ltana- mycin su lp l~a t e ,~ and polyinysin B.4
Method The serotypes used in this study were Leptospira icterohaemorrhagiae, L.
ballum strain M-127, L. canicola strain Hond Utrecht, L. pyrogenes strain
'Manuscript received May 15, 1961. Contribution from the Laboratory of Hygiene, Department of National Health and Wel-
fare, Ottawa, Ontario. ZUpjohn Co. 3Bristol Laboratories. 'Burroughs Wellcome Co.
Can. J. Microbiol. Vol. 7 (1961)
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752 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 7. 1961
Salinem, and L. sejroe strain Mallersdorf. The first four serotypes were ob- tained from Walter Reed Army Medical Centre, \iVasl~ington, D.C. L. sejroe was obtained from the Animal Diseases Research Institute, Canada Depart- ment of Agriculture, Hull, Que. They were maintained in the laboratory by monthly passage in I<orthof's medium plus rabbit serum (1). R stock solution of each of the agents to be tested was prepared and added to Korthof's medium to provide a t least three concentrations for testing. Crystal violet was dis- solved in 47y0 ethyl alcohol to a concentration of 1% and steained for 10 minutes, as recommencled by Salle (7). Basic fuchsin (0.03 g/l.) was dissolved in 10% ethyl alcohol. Desoxycorticosterone (0.025%) was dissolved in 1 : 1 ethanol-chlorofoi-111 and then heated a t 30' C overnight to reinove the sol- vents (6). Other substal~ces tested were clissolved in sterile I<orthofls basic inedium (peptonc salt solution). These other substances wcre: sodium azide (50 mg per 100 ml), potassiuin tellurite (1% solution), actidione (1 g per 100 ml), sulfaguanidine (5 g/l.), sulfathiazole (5 g/l.). Antibiotic solutions were also prepared with this medium as the solvent.
Media with added test substances were dispensed in 5-cc atnounts in 16-min sci-eiv-cap tubes and incubated for 24 hours a t 37' C as a sterility checlr. Three tubes were inoculated with 0.5 in1 of a 5-day-old leptospira growth for each concentration tested in order to determine whether these concen- trations would cause an inhibitory effect on L. icterohaemorrhagiae. Also one tube of standard I<orthof's medium was inoculated as a control. The tubes were incubated a t 29' C and checlted daily for growth of leptospires by placing a loopful of culture on a glass slide and exanlining it by darlt-field microscopy a t 400 X inagnifi cation. Growth was recorded as + (1-10 organ- isins per field), + + (1 1-50 organisms per field), or + + + (more than 50 organisms per field). Each of the antimicrobial agents was tested a t least twice against L. icterohaemorrlzagiae according to the above method. The numbers of pluses recorded for all six tubes used in each test were averaged in order to express the result of each test as a single value for presentatioil in Tables I1 and 111.
Froin this screelling procedure, four coinpouilds (sulfaguanidine, sulfa- thiazole, neomycin sulphate, and actidione) were selected for more critical evaluation using L. icterohaemorrhagiae as the test organism. Leptospira populations were detei-inined by ineans of Chnng's technique (2) einploying calibrated pipettes to apply a ltnown volume of formalized culture over a fixed area of a inicroscope slide. Duplicate preparations were made and, for each, 15 to 20 fields were counted daily for 4 days.
Combiilations of sulfathiazole, neomycin sulphate, and actidione, a t various concentrations, were also tested against L. icterohaemorrhagiae. Results were recorded qualitatively in terins of pluses and, in addition, population counts were inade for two combinations, those with inaximuin and with ininiinum concentratioils of the agents. A transfer was made after 72 hours to standard ICorthof's medium to establish the leptospires' ability to multiply after having been grown in a medium with the antimicrobial agents.
To investigate possible differences between several serotypes in their reactions to these compounds, sulfaguailidine (1 g/l.), sulfathiazole (600 mg/l.), neomycin sulphate (8.5 mg/l.), and a combination of sulfathiazole
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COUSINEAU AND McKIEL: LEPTOSPIRA SENSITIVITY 753
(50 mg/l.), ~ l e o n ~ y c i ~ l sulphate (5 mg/l.), and actidione (0.5 mg/l.) were tested as well against L. bal l z~m, L. canicola, L. pyrogenes, and L. sejroe.
Results Agents Tested Singly
I n the screen test, inarlted inhibition in the growth of L. icterohae)~zorrhagiae was caused by the following nine c o ~ n p o u ~ l d s (the co~lce~ltration listed in bracltets was the lowest of three tested): crystal violet (0.1 mg/l.), basic fuchsin (7.5 mg/l.), sodium azide (10 mg/l.), potassiu~ll tellurite (28.5 mg/l.), desoxycorticostero~ie (1.25 mg/l.), furoxone (25 mg/l.), cl~lora~nphenicol (25 mg/l.), polymyxi~l B (3 units/ml), and 1;anamycin sulphate (20 units/ml). Since these concentrations are lower than those enlployed to inhibit organisms most commonly encountered a s con tami~la~ l t s of leptospira cultures, these compouilds were not considered further in this study.
TABLE I Concentrations a t which certain agents commonly produce an antimicrobial effect
Antimicrobial aaent Concn. Effective against: References
Sulfaguanidine 125-200 mg/l. Enterobacteriaceae 5
Sulfathiazole
Actidione
50 mg/l. Streptococci, staphylococci, salnlonellae, Eschericheae
0.1-1 mg/l. Molds, fungi, 11 Enterobacteriaceae, bacilli, streptococci, staphylococci
Neomycin sulphate 5-10 mg/1. Enterobacteriaceae 10 (except E. coli) bacilli, staphylococci
Bacitracin 5-10 units/ml Bacilli, 10 staphylococci, streptococci
The following five compouilds were found not to inhibit growth of L. icterokaef?zorrhagiae when used a t collce~ltrations equal to or greater than those shown in Table I as being suitable for inhibiting coiltamillallts: neoinycin sulphate, bacitracin, sulfaguailidine, sulfathiazole, and actidione. I n Table I1 it is seen tha t growth of L. icterokaemorrhagiae in standard I<orthof's medium is comparable with growth of this organism in media coiltailliilg sulfaguallidi~le (2 g/l.), sulfathiazole (600 mg/l.), actidione (1.5 mg/l.), neomyciil sulphate (5 mg/l.), or bacitracin (15 units/ml). When higher concentrations of these compounds are used the rate of growth is seen to be reduced. For example, when sulfaguanidine, sulfathiazole, actidione, and ileomycin sulphate were added a t concentrations of 3 g/l., 1 g/l., 2 mg/l., and 5.5 mg/l. respectively, a + ++ growth was attained only after 7 days of incubation. L. icterohaemor- rhagiae populations exceeding ++ were never seen when concentrations
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754 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. i. 1961
TABLE I1 Growth of Leptospira icterohae~norrrlzugiae in modified Korthof's medium
Growth after:
Compound Concn. 3 days 5 days 7 days
Sulfaguanidine
Sulfathiazole
Actidione
Neomycin sulphate 10 mg/l. 8 .5 mg/l. 6.75 mg/l. 5 ing/l.
Bacitracin 25 units 15 units
Control medium
were 4 g/1. for sulfagua~lidi~le and 10 lng/l. for neoinycin sulphate. There was no growth after 3 days when sulfathiazole or bacitracin was added a t a concentration of 2 g/l. or 25 units/cc respectively.
The results of the quantitative study 011 L. icterohaemorrhagiae growth deterinined in standard and lnoclified I<orthof's media are shown in Fig. 1. I t lnay be seen that in media containing neomycin sulphate, only slight inhibition was produced by 5 and 6.75 n~g/l . , moderate inhibition by S.5 mg/l., and inarlied inhibition by 10 ing/l. Sulfaguanidine a t the concentra- tions tested caused inarlted inhibition of L. icterohaemorrhagiae while sulfa- thiazole a t 600 mg/l. and bacitracin a t 10 units/ml produced moderate inhibition.
Media containing neoinycin sulphate (8.5 mg/l.), bacitraci~l (10 units/ml), sulfaguanidine, or sulfathiazole (600 mg/l.) supported growths of L. ballum, L. canicola, L. pyrogenes, and L. sejroe qualitatively similar to those obtained with L. icterohaemorrkaeiae. -
Growth of such contaminants as Psez~domonas, staphylococci, and others may be suppressed by neoinycin sulphate a t 7 mg/l., sulfathiazole a t 50 mg/l., or bacitracin a t 10 units/ml (Table I). Results given above indicate tha t incorporation of one or the other of these substances a t these concen- trations in a selective medium may be expected to produce from slight to moderate inhibition of growth of leptospires.
Agents Tested in Combination Since a variety of microorganisms may be ellcountered as contaminants,
it seems reasoilable to expect that incorporation of more than one antimi- crobial agent should provide a selective medium superior to those which
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COUSINEAU AND McKIEL: LEPTOSPIRA SENSITIVITY 755
POPULATION
I 2 4 HR
48 HR
72 HR
FIG. 1. Leplospire iclerohae~rzorrkegiee growth in modified I<orthofls media.
include only one of these agents. To this end, three of the antimicrobials used above were chose~l for study when employed in combination.
Neonlycill sulphate and bacitracin have similar antibacterial spectra. Of these two, neomj.cin sulphate was selected for further study because ( a ) i t produced a lesser inhibitory effect on leptospiral growth whe11 used a t the concentration reconlnlended in Table I , and (b) solutions of ileo~llyci~l sulphate are stable for 1 year whereas solutions of bacitracin are stable for only 1 weelr. The renlaini~lg two agents included for study in conlbi~latioll were sulfathiazole to inhibit growth of E. coli and a few other organisms, and acti- dione to inhibit growth of fungi.
The results of tests on growth of L. icterohaemorrhagiae in media containing these three agents, each a t two concentrations, are shown in Table 111. Those concentratio~ls to be tested were chosen according to the results obtained above in this study and those published by various authors (3, 10, 11). I t may be seen tha t growth in media incorporating all eight co~llbinations was similar to that in the control medium. During the third and fourth days however, growth was greater in media containing sulfathiazole a t 50 mg/l.
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TABLE 111 n 9
Growth of Leptospira icterohaemorrhagiae in modified ICorthof's medium with combination of antimicrobial agents Z 2 z
Antibiotic 5
Population in transfer concentrations added Population a t times indicated n ~ c d i u ~ n a t times L.,
to medium (mg/l.) 0
(numbers shown in brackets are in millions) indicated c W
Sulfathi- Neornycin Acti- azole sulphate dione Day 1 Day 2 Day 3 Day 4 Day 7 Day 1 Day 2 Day 6
? T
300 8 .5 1 ++(1.7) f f ( 3 . 7 ) f f ( 5 ) ++(4.8) 4 0 ) + + - z 300 8 .5 0.5 f+ f+ +f +$ + + - t!
M
++(1.7) f f ( 4 . 7 ) +++( lO. l ) +++(27.7) +++(42.5) ++ + + ++ +++ ,.. Corltrol medium a s
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COUSINEAU AND McKIEL: LEPTOSPIRA SENSITIVITY 757
regardless of the concelltrations of neomycin sulphate and actidione. In inedia coiltaining sulfatl~iazole a t 300 mg/l. or neo~nycin sulphate a t S.5 mg/l., actively motile leptospires were seen but their ability to multiply was suppressed by these concentrations.
The efficacies of these inedia were evaluated further by subculturing to tubes coiltailling coiltrol mediunl (without ailtinlicrobial agents). The popu- latio~ls were recorded qualitatively for all tubes. In addition, the iluinbers of organisins per milliliter were determined in media in which the antimicro- bials were a t maxinlum and a t miniinum concentrations. On the basis of this test, the most satisfactory concentrations to be used in a selective medium appear to be sulfathiazole a t 50 mg/l., neomycin sulphate a t 5 mg/l., and actidione a t 0.5 mg/l.
Once again, the other serotypes tested (L. ballz~m, L. canicola, L. pyrogenes, and L. sejroe) gave results qualitatively similar to those obtained with L. icterolzaemorrhagiae.
Discussion and Summary The isolatioil of leptospires is difficult and often illlpossible from cases of
leptospirosis or in surveys to deter~nine the incidence of these orgailisrns if the inoculum is contaminated since bacteria may overgrow the leptospires. The purpose of this study was to find suitable antimicrobial agents which might be added to standard Korthof's medium to control growth of conta- minants. To this end, the compounds used were added to ICorthof's inedium in increasing coilcentrations beginning a t a level slightly lower than that commonly employed to inhibit growth of liltely contaminants of leptospires. If the maximum co~lce~ltratioil which did not seriously depress growth of the leptospires was found to be as high as or higher than that used to suppress growth of contaminants, that compou~ld was selected for further study.
In the first step, 14 agents were screened including sulfaguanidine and chloramphenicol as recommeilded by Stuart (9) and Doininguez (4) respec- tively. Qualitative estimates of the amounts of leptospira growth resulting suggested that further examination of sulfaguanidine, sulfathiazole, ileomycin sulphate, bacitracin, and actidione was justified.
In the second step, these five co~npounds which loolted promising were checked by determining the resultant growth by inicroscopic counts as a confirmatory procedure. Following this test three agents were coilsidered worthy of further study (sulfathiazole, neomycin sulphate, and actidione).
The third step consisted of studying the effect on leptospira growth of these three compounds added in combination: sulfathiazole for coiltrol of staphylococci, streptococci, and E. coli; neomyciil sulphate for control of bacilli, Pseudomonas species, and staphylococci; and actidione for coiltrol of yeasts and fungi. The results of tests of the eight combiilatio~ls of the three agents, each a t two concentrations, indicated that the combination most suitable for suppressing growth of contaminants liltely to be ellcountered in cultures of leptospires is sulfathiazole, 50 mg/l.; neoinyciil sulphate, 5 mg/l.; and actidione, 0.5 ing/l. I t is planned to test this coinbinatioil under fieId conditions in the near future.
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758 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 7. 1961
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