J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from

J. clin. Path. (1959), 12, 568.

NOVOBIOCIN-TETRATHIONATE BROTH: A MEDIUM OF IMPROVED SELECTIVITY FOR THE ISOLATION OF SALMONELLAE FROM FAECES BY LEONARD JEFFRIES From the Department of Clinical Pathology, Uriversity College Hospital, London

(RECEIVED FOR PUBLICATION APRIL 23, 1959)

The selective cultivation of bacteria by the strengths of all solutions were expressed as incorporating chemicals in the media depends on concentrations of pure novobiocin. As the supplying a substance which either stimulates the novobiocin content varied with different batches of growth of the species to be isolated or inhibits the salt a simple calculation was necessary each time a new sample was used. A stock solution containing unwanted species. Tetrathionate is a good novobiocin, 16,000 ug./ml. of water, was stored in selector of Salmonellae in faeces culture, and is the refrigerator. believed to act in both ways. Tetrathionate MacConkey Agar.-Oxoid. reducers, such as Salmonellae, flourish in broth Desoxycholate-citrate Agar. - Southern a of Group containing it, whereas high concentration Laboratory (Lewisham). tetrathionate inhibits many other faecal organisms

Indicator Serum Broth.-This was prepared by the copyright. (Knox, Gell, and Pollock, 1943). Unfortunately, method of Stokes however, members of the Proteus group are (1955). powerful tetrathionate reducers, and when they Investigadon are present selection of Salmonellae is much For efficient selection a concentration of novobiocin impaired, consequently the addition of a further is required which will inhibit most, if not all, strains selective inhibitor effective against Proteus of Proteus without jeopardizing the survival of small organisms is desirable. So far no such satis- numbers of Salmonellae. factory substance has been found: brilliant green to Novobiocin.-The sensitivities to

Sensitivity http://jcp.bmj.com/ and ox bile (Kauffmann) proved unsuccessful novobiocin of 27 strains of Proteus and of 24 strains (Knox et al., 1942, 1943). of Salmonellae were tested by the paper disc method Antibiotics are becoming increasingly used as and by tube titration. Proteus was always more selective agents in culture media, but most of sensitive than Salmonella (Table I). them are approximately equally active or inactive TABLE I against Salmonellae and Proteus. Novobiocin, NOVOBIOCIN SENSITIVITY TESTS however, inhibits Proteus more than other

Tube Titration (Inhi- on September 30, 2021 by guest. Protected Gram-negative bacilli and seemed therefore a bitory Concentration suitable substance for trial. No. of Disc Test of Novobiocin in Strains g./ml. Indicator Organism D e Serum Broth) Materials Tested _____ Sensi-e ta Average Range Tetrathionate Broth.-This was prepared by adding two "oxoid" tablets to 10 ml. of water in a 1 oz. Salmonella species 24 - 24 >2,400 1,600-3,200 to P. morganii 5 5 - 85 25-200 screw-capped bottle and bringing the boil. The P. mirabilis 13 13 - 170 25-400 medium without iodine can be stored at room P. vulgaris. . 9 9 - 160 25-400 temperature for one once iodine is added the Paracolon bacilli 5 - 5 month; E. coli 5 - 5 complete medium is unstable. Ps. pyocyanea 4 - 4 Novobiocin-tetrathionate Broth.-Novobiocin solu- tion containing the appropriate concentration in Effects of Concentrations of Novobiocin in 0.1 ml. was added to 10 ml. of the incomplete medium, Tetrathionate Broth upon Growth of Salmonellae.-A 0.2 ml. of the specified iodine solution being added plate viable count (Miles and Misra, 1938) was made before use. on blood agar from an overnight broth culture of Novobiocin Solution.-The sodium salt of Salmonella paratyphi B. Drops from the highest novobiocin (Upjohn of England Ltd.) was used, and dilutions were seeded into bottles containing J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from

NOVOBIOCIN-TETRATHIONA TE BROTH 569

TABLE II RECOVERY OF SALMONELLAE FROM ARTIFICIALLY INFECTED FAECES SUSPENSIONS CONTAINING PROTEUS

Minimal Nos. of Salmonellae/ml. of Faeces Suspension Detectable lProteusProteus ~~~~~~~~~~afterCulture in Experi- Salmonella- ment Approximate Tetrathionate Proteus Novobiocn- Proteus Strain No./rml. tetrathsionate Faeces TerathionatentBrothPrsnBot Prsn a Schwarzengrund Morganii (11,229) 5,000 42 x 105 + + + + + 7 + b ,. ,, 266,000 >28 x 103 + + + 28 or less c ,, Vulgaris 5,000 42 or less + + + 42 or less d Typhi-murium Mirabilis (7,406) 5,000 > 140 x 106 +++++ 140 or less + + + + e Paratyphi B 88 x 106 + + + + + 88 x 104 + + f Enteritidis Morganii 5,000 22 x 104 + + + + + 22 x 104 Thompson . . 30 or less + + + + + 30 or less h TAnaunn Mirabilis 5,000 34 x 102 + + + ++ 35 or less

+ Indicates the number of bottles in which Proteus survived. tetrathionate broth and into bottles containing suspension was delivered into each of 5 x 1 oz. different dilutions of novobiocin in tetrathionate screw-capped bottles. Counted numbers of broth. Each concentration of the organism was Salmonellae were then added to give a series of tested in triplicate in each medium. The test was bottles containing faeces, a constant number of repeated with the same strain of Salmonella paratyphi Proteus, and varying numbers of Salmonellae. Each B and also with a strain of Salmonella enteritidis. bottle was shaken thoroughly on a mechanical shaker, As a result of these tests a concentration of and 1 ml. quantities from each bottle were cultured 40 pg./ml. novobiocin in tetrathionate broth appeared in tetrathionate broth with and without novobiocin. to be the most suitable, since there was no inhibition After overnight incubation subcultures were made when growth of about 3 to 30 organisms was on MacConkey agar and two colonies of each type compared in tetrathionate broth and tetrathionate of non-lactose-fermenter were tested for urease copyright. broth plus 40 ,ug./ml. novobiocin. production. Those which proved to be urease negative were tested by slide agglutination, using the Suppression of Proteus by Tetrathionate Containing appropriate Salmonella antiserum. As the properties Novobiocin.-Diluted overnight broth cultures of and colonial appearances of the Salmonella strains four strains of Proteus were seeded into triplicate were well known, final identification by tube bottles of nutrient broth, tetrathionate broth, and agglutination was considered to be superfluous. The novobiocin tetrathionate broth. Novobiocin at a number of each type of non-lactose-fermenting concentration of 40 pg. /ml. completely suppressed colony examined was purposely limited so that the growth of an inoculum of about 70 organisms during chance of isolating Salmonellae should be http://jcp.bmj.com/ incubation overnight. approximately the same as it would be in routine Stability of Novobiocin iD Selective Media.-It examinations of faeces. seemed likely that if novobiocin proved to be a useful Table II shows that Salmonellae representative of selector in tetrathionate broth it might also prove of groups B, C, D, and E can be isolated more success- value in other enrichment media for Salmonellae. Its fully by enrichment in novobiocin-tetrathionate stability was therefore tested in tetrathionate broth, broth than in plain tetrathionate broth when Proteus and is present. Selenite-F medium, in the medium of Rappaport, on September 30, 2021 by guest. Protected Konforti, and Navon (1956). Group A was not investigated because the strain of The drug was stable at a concentration of Salmonella paratyphi A examined proved to be 40 pg. /ml. in all three media for at least 48 hours at inhibited by tetrathionate broth, which confirmed the 370 C. and for at least one month at 40 C. There findings of Banwart and Ayres (1953). was no loss of potency in tetrathionate broth on Comparison of the results of experiments (a) and (b) storage without iodine at room temperature for one shows that the efficiency of novobiocin-tetrathionate month. Novobiocin at a concentration of 40 pg./ml. broth may be unaffected by a 50-fold increase in the tetrathionate broth was used in all subsequent number of Proteus. experiments. In experiments (c), (f), and (g), novobiocin- tetrathionate broth did not detect smaller numbers of Comparison of Tetrathionate and Novobiocin- Salmonellae than plain tetrathionate broth. The only tetrathionate Broths for Recovery of Salmonellae in non-lactose-fermenters recovered from novobiocin- Presence of Proteus from Artificially Infected Faecal tetrathionate broth, however, were Salmonellae, and Suspensions.-An emulsion was prepared in 40 ml. of growths were always heavier than those from water of approximately 5 g. of faeces from a patient tetrathionate broth. who was not receiving antibiotics. A dilution of an Since it was shown in a separate experiment that overnight broth culture of Proteus was added to give small numbers of the Salmonella enteritidis strain approximately 5,000 bacilli/ml., and 5 ml. of this used in experiment (f) survived 40 lAg. /ml. novobiocin J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from

570 LEONARD JEFFRIES

in tetrathionate broth the poor growth of this broth, no Salmonellae were encountered. Novo- Salmonella was not due to a harmful effect of biocin-tetrathionate broth effectively suppressed novobiocin. Proteus when the specimens contained insufficient Incorporation of Novobiocin in Desoxycholate- numbers to give profuse growths on direct cultures. citrate-agar. - Desoxycholate-citrate-agar (D.C.A.) Discusion suffers from the disadvantage that it readily supports the growth from faeces of most strains of Proteus. It In a discussion of the comparative merits of was therefore considered worth while to investigate Salmonella enrichment media Rappaport et al. the possibility of incorporating novobiocin in D.C.A. (1956) assigned to their new medium a "valency in such concentrations that Proteus would be inhibited coefficient" (Lang, 1952, 1954), showing it to be and the medium would not be rendered more toxic superior to the medium of Bierbrauer: these for Salmonellae and Shigellae. results were based on tests in which only one In order to test the ability of desoxycholate-citrate- strain of each of two organisms was used. agar to support the growth of the test organisms, From and with tetra- 18-hour broth cultures were diluted 1/100, and one the experiments (a) (c) loopful was spread over a segment of a D.C.A. plate, thionate broth (Table II), it will be seen that when which was then incubated for 18 hours. Of 14 two different strains of Proteus were tested with strains of Proteus, nine grew well, four grew poorly, a single strain of Salmonella markedly different and one failed to grow. Good growth was obtained valency coefficients were obtained. with 14 Shigella strains (S. sonnei six, S. boydii two, The term " valency coefficient " is thus shown to S. flexneri six) and five strains of Salmonellae, be misleading, since its value will vary according including one strain of Salmonella typhi. to the strains of organisms tested. The con- Growth on Desoxycholate-citrate-agar Containing clusions of Rappaport et al. concerning the Different Concentrations of Novobiocin.- comparative selectivity of their medium may Desoxycholate-citrate-agar containing novobiocin, therefore be erroneous. 100 rig./ml., was inoculated with dilute broth cultures. Although the value of enrichment media for It was found that this medium, although completely isolating members of the Salmonella group is copyright. inhibitory to all the Proteus strains tested, was also established, there is little agreement upon the markedly inhibitory to the strains of Salmonellae and Shigella sonnei. Desoxycholate-citrate-agar plates relative merits of the many types and their were then prepared containing novobiocin in concen- modifications, which have been described. trations from 50 jig./ml. to 2 jig./ml. of medium Experimental work has shown that no one medium and inoculated with dilute broth cultures of the may be suitable for all members of this large following organisms (the number of strains tested is genus and that the maximum number of shown in S. sonnei S. brackets): boydii (2), S. (2), Salmonella isolations is made when several media http://jcp.bmj.com/ flexneri (6), Salm. thompson (1), Salm. typhi (1), are used simultaneously (Collard and Unwin, P. vulgaris (1), P. mirabilis (2), P. morganii (1). 1958). Plates were incubated overnight. novobiocin The highest concentration of novobiocin in D.C.A. It seems likely that the addition of which did not affect the growth of the Shigellae and would improve the selectivity of all liquid media Salmonellae tested was 16 jig./ml. of medium, and for Salmonellae, but controlled trials in each this did not prevent the growth of three-quarters of the medium would be required, as the combination of Proteus strains. Desoxycholate-citrate-agar contain- novobiocin with different selective agents might ing 32 jig. novobiocin/ml. medium, whilst inhibiting inhibit Salmonellae, as was found in the tests on September 30, 2021 by guest. Protected the growth of half the strains of Proteus, was described above with D.C.A. As tetrathionate markedly inhibitory to one strain of S. flexneri, broth is one of the enrichment media commonly slightly inhibitory to one strain each of S. sonnei and used in this country it was chosen for trial in the S. boydii, and without effect upon growth of Salm. thompson and Salm. typhi, but 50 jig. novobiocin/ml. first instance. medium completely inhibited growth of all strains of A comparison of the proposed medium with Proteus and one strain of Salm. typhi. other enrichment media for the isolation of Thus the lowest concentration of novobiocin tested Salmonellae from faeces of infected patients in D.C.A. which effectively inhibited Proteus was was not possible, owing to the infrequency of also markedly inhibitory to Shigellae and Salm. Salmonella infections in this area. typhi, so that the medium is unsatisfactory for routine use. Summary Proteus Suppression in Routine Faeces Cultures in Of the non-lactose-fermenting Gram-negative Novobiocin-tetrathionate Broth.-During a three- bacilli encountered in faeces, members of the month trial, in which 0.5 ml. of a suspension of each Proteus group are the most sensitive and specimen of faeces examined was inoculated into both Salmonellae are amongst the most resistant to novobiocin-tetrathionate broth and tetrathionate novobiocin. J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from

NOVOBIOCIN-TETRATHIONATE BROTH 571

Tetrathionate broth containing 40 ,tg. Salm. typhi and is therefore not useful for the novobiocin /ml. medium has proved superior to direct culture of faeces. plain tetrathionate broth as an enrichment medium for the isolation of Salmonellae from To Professor Wilson Smith, F.R.S., and Dr. E. J. faeces artificially infected with known Stokes I am indebted for helpful criticism and numbers guidance during the course of this investigation and of Salmonellae and Proteus. in the presentation of the results. I am also grateful Novobiocin at a concentration of 40 ,ug. /ml. to Dr. Joan Taylor and Dr. K. P. Carpenter for medium in tetrathionate broth remained stable for providing Salmonella and Shigella strains, and to Dr. at least 48 hours at 370 C., and for one month at A. R. H. Hicks, of Upjohn of England Ltd., for an room temperature. Results of stability tests in ample supply of novobiocin. other enrichment media are reported. REFERENCES The criteria used for the quantitative com- Banwart, G. J., and Ayres, J. C. (1953). Appl. Microbiol., 1, 296. parison of enrichment media are discussed, and Collard, P., and Unwin, M. (1958). J. clin. Path., 11, 426. Knox, R., Gell, P. G. H., and Pollock, M. R. (1942). J. Path. Bact., the term " valency coefficient," as used by 54, 469. - (1943). J. Hyg. (Camb.), 43, 147. Rappaport et al., is shown to be misleading. Lang, K. (1952). Zbl. Bakt., 1. Abt. Orig., 157, 565. Desoxycholate-citrate-agar containing novo- -1(1954). Ibid., 161, 165. Miles, A. A., and Misra, S. S. (1938). J. Hyg. (Camb.), 38, 732. biocin in a concentration sufficiently high to Rappaport, F., Konforti, N., and Navon, B. (1956). J. clin. Path., 9, 261. inhibit Proteus is also inhibitory to Shigellae and Stokes, E. J. (1955). Clinical Bacteriology, Ist ed. Arnold, London. copyright. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected