JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1979, p. 433-436 Vol. 10, No.4 0095-1137/79/10-0433/04$02.00/0

Yersinia intermedia: Temperature-Dependent Bacteriocin Production

EDWARD J. BOTTONE,'* KULDIP K. SANDHU,' AND MICHAEL A. PISANO2 Department ofMicrobiology, The Mount Sinai Hospital, New York, New York 10029,1 and Department of Biology, St. John's University, Jamaica, Queens, New York 114392 Received for publication 18 July 1979

Yersinia enterocolitica and related Yersinia species share many temperature- dependent biological attributes. The present report documents temperature-de- pendent production of a bacteriocin-like substance only among Yersinia strains which ferment L-rhamnose, raffinose, and melibiose and which have been tenta- tively designated Y. intermedia. When tested by the "lawn-spotting" technique at 25 and 370C, 7 of 15 Y. intermedia strains produced a bacteriocin only at 250C with a uniform spectrum of activity against 2 strains of Y. enterocolitica, 1 rhamnose-positive, raffinose-negative, melibiose-negative strain tentatively des- ignated Y. frederiksenii, 8 Y. intermedia, and 3 sucrose- and acetylmethylcarbi- nol-negative yersinial isolates tested. Bacteriocin-like activity was not detected among the Y. enterocolitica, Y. frederiksenii, or sucrose-negative yersinial strains tested. The exclusive activity of the Y. intermedia antibacterial substance only against yersiniae and not against other representative tested supports the placement of these microorganisms within the genus Yersinia and further establishes the singularity of Y. intermedia. is a human pathogen and melibiose. Group 4 strains are both sucrose whose biology is markedly influenced by tem- and acetylmethylcarbinol (Voges-Proskauer) perature. Among the in vitro biological attri- negative. butes enhanced by lowered incubation temper- Subsequent to this report, D. J. Brenner (in ature (25°C) are flagella synthesis and acetyl- Contributions to Microbiology and Immunol- methylcarbinol production (14), enterotoxin ogy, vol. 5, in press) has suggested the name Y. synthesis (12, 13), ability to adhere to and pen- frederiksenii for the members of group two, and etrate epithelial cells (9), and resistance to serum Y. intermedia for the group 3 (L-rhamnose-, bactericidal activity (11). In addition to sharing raffinose-, and melibiose-fermenting) strains. most of the above features, rhamnose-, raffi- The sucrose-negative group 4 strains are as yet nose-, and melibiose-fermenting Yersinia pres- unnamed. For convenience, the terminology Y. ent an even broader range of temperature-re- frederiksenii and Y. intermedia has been main- lated phenotypic traits. Among the latter, ex- tained throughout this publication. pressed at 25 but not at 370C, are growth on There exist, therefore, several temperature- various enteric media (7), expanded fermenta- related and genetic features that simultaneously tive capability (4, 7), and increased resistance to enjoin and yet differentiate typical Y. enteroco- ampicillin, carbenicillin, chloramphenicol, and litica from other Yersinia species. This report the aminoglycosides (7). describes the production of a temperature-de- Taxonomically, on the basis of DNA-DNA pendent bacteriocin-like substance produced homology studies, these Y. enterocolitica-like only among Y. intermedia isolates that further strains have been shown by Brenner and col- differentiates these yersiniae from true Y. enter- leagues (5) to be distinct from typical Y. enter- ocolitica. ocolitica. These investigators showed that mi- croorganisms previously classified as Y. enter- MATERLALS AND METHODS ocolitica could be separated into four distinct Bacterial strains. A total of 31 Yersinia isolates groups. Group one is comprised of biochemically were studied (Table 1). Among these were 13 Y. en- and terocolitica (strains 1 through 12), 1 Y. frederiksenii typical strains both indole positive negative. (Ye 867), 15 Y. intermedia (strains 083 through 0:17), Group two is exemplified by isolates that differ and 3 sucrose-negative isolates (MSH, MH, and QG). from those of group one only by their fermen- O antigens of the isolates were determined by H. H. tation of rhamnose. Group 3 strains, in addition Mollaret (Pasteur Institute, Paris) and S. Toma (Min- to rhamnose fermentation, also utilize raffinose istry of Health, Ontario, Canada) by slide agglutina- 433 434 BOTTONE, SANDHU, AND PISANO J. CLIN. MICROBIOL.

tion against 34 Y. enterocolitica 0 antisera. With the acterized (2, 4, 7) and one, an 0-group 17, was received exception of the three strains noted below, obtained from S. Winblad. from, respectively, D. Brenner, S. Toma, and S. Win- Three sucrose- and acetylmethylcarbinol-negative blad, the remaining 28 strains were either isolated at clinical isolates were also tested. Two were recovered The Mount Sinai Hospital, New York, N.Y., or for- from eye cultures and one from blood. warded to our laboratory for confirmation from other Demonstration of bacteriocin activity. The investigators. Twelve were biochemically typical (de- term bacteriocin has been applied to the inhibiting fined to be sucrose positive, but rhamnose, raffinose, substance produced by Y. intermedia strains, although and melibiose negative) as determined by conventional we recognize that in this preliminary report we have methods (14) at 25 and 37°C incubation temperatures. not demonstrated any of the physicochemical or ge- The 0 group of these strains is shown in Table 1. One netic criteria included in the definition of a bacteriocin 0-group 3 and one 0:9 isolate were received, respec- (15). tively, from S. Toma and S. Winblad (University of Each of the 31 Yersinia isolates was first screened Lund, Malmo, Sweden). for bacteriocin production by the "lawn-spotting" Sixteen rhamnose-fermenting (25°C) strains were technique (8). In this procedure, 1 ml of an overnight studied. One (Y. frederiksenii, Institut Pasteur Ye Trypticase soy broth (BBL Microbiology Systems) 867), received from D. Brenner of the Center for culture incubated at either 25 or 37°C was added to 50 Disease Control, Atlanta, Ga., designated Ye 867 in ml of 0.4% melted and cooled Trypticase soy agar Table 1, fermented L-rhamnose but did not ferment (TSA). After mixing, 2.5 ml of this suspension was raffinose or melibiose. The remaining 15 isolates called overlaid onto the surface of duplicate TSA plates Y. intermedia all fermented L-rhamnose, raffinose, containing 5% sheep blood (BBL). After solidification, and melibiose. Of these, 14 have been previously char- 0.001 ml of the test organisms obtained from the

TABLE 1. Source, 0 group, and biochemical reactions (25°C) of 31 Yersinia strains studied Acid (25°C) produced from: Strain Source 0 group" V-P (250C)h Rhamnose Raffinose Sucrose 1 S. Toma 3 -C - + 2 Stool 3 - - + + 3 Stool 3 - - + + 4 Stool 5, 27 - - + + 5 Stool 5, 27 - - + + 6 Stool 5, 27 - - + + 7 Stool 8 - - + + 8 Stool 8 - - + + 9 S. Winblad 9 - - + + 10 Stool NAG* - - + + 11 Sputum NAG - - + + 12 Stool NAG - - + + Ye 867 D. Brenner NAG + - + + 083 Urine 17 + + + + 154 Abscess 17 + + + + 176 Throat NAG + + + + 227 Wound 17 + + + + 257 Urine 17 + + + + 287 Eye 17 + + + + 387 Stool NAG + + + + 424 Eye 17 + + + + 467 Stool NAG + + + + 642 Eye 14 + + + + 786 Stool NAG + + + + 803 Urine 17 + + + + 896 Catheter 17 + + + + 898 Stool NAG + + + + 0:17 S. Winblad 17 + + + + MSH Eye 12 MH Eye NAG QG Blood NAG - " NAG, Nonagglutinable in sera to 0-groups 1 through 34. b V-p, Voges-Proskauer test. '-, Negative at 7 days. ' +, Positive within 24 h. VOL. 10, 1979 Y. INTERMEDIA BACTERIOCIN PRODUCTION 435 overnight broth cultures was spotted onto the two lawns. One blood agar plate was incubated at 25°C, and the other was incubated at 370C for 18 h and then observed for zones of inhibition of the indicator strain around the spot inoculum. This procedure was carried out until all strains of Yersinia had been tested against each other. To determine whether the bacteriocin of Y. inter- media was active against other Enterobacteriaceae or other representative gram-negative and gram-positive species, the producer strains were spotted as described above onto lawns of 12 , 5 , 2 , 1 P. rettgeri, 3 Pseu- domonas aeruginosa, 1 Acinetobacter calcoaceticus var. anitratus, and 3 isolates each of Staphylococcus aureus, S. epidermidis, and Streptococcus faecalis. RESULTS Through the lawn-spotting technique, each of the 31 Yersinia strains was tested against itself and the remaining 30 strains at 25 and 370C. On the basis ofthese reactions, two distinct patterns of bacteriocin activity could be discerned: (i) temperature-dependent bacteriocin synthesis among Y. intermedia strains, and (ii) lack of bacteriocin production among Y. enterocolitica, Y. frederiksenii, and the sucrose-negative iso- lates. Of the 15 Y. intermedia isolates tested, 7 (no. 083, 154, 287, 642, 803, 896, and 0:17) produced a bacteriocin only at 250C, each of which had an identical spectrum of activity against the follow- ing Yersinia strains: two Y. enterocolitica (strain 7, 0 group 8; strain 12, nonagglutinable), one Y. frederiksenii (Ye 867), eight Y. interme- dia strains (no. 176, 227, 257, 387, 424, 467, 786, 898), and the three sucrose-negative isolates. FIG. 1. Bacteriocin-like activity of Y. intermedia Distinct zones of inhibition of the indicator after growth at 25°C (a), as contrasted with absence Yersinia strains occurred when the lawn-spot- ofactivity at 37°C (b). Lawn-spotting technique on 5% ting plates were incubated at 25 but not at 370C sheep blood agar. (Fig. 1). Bacteriocin activity of producer strains was always expressed at the lower temperature duced by Y. intermedia. These included an 0- even when the producer strains were grown at group 8 (strain 7) and a nontypable strain (no. 370C for several days and then tested at 250C. 12). Conversely, bacteriocin was still present at 250C Bacteriocin activity was not detected with the and absent at 370C with producer strains main- Y. frederiksenii isolate (Ye 867) or the three tained at 250C before testing. These results were sucrose- and Voges-Proskauer-negative isolates constant, regardless of whether or not the indi- when tested against the other yersiniae at 25 or cator strains themselves were grown at 25 or 370C. These four isolates were, however, suscep- 370C before testing. tible to the bacteriocin produced by Y. inter- None of the 12 Y. enterocolitica isolates media. tested, irrespective of 0-antigen group, produced The antagonistic substance was bactericidal a bacteriocin at 25 or 370C active against either in action because viable cells could not be re- the other Y. enterocolitica strains tested, or covered on subculture of scrapings from the zone against the other 19 Yersinia isolates including of inhibition. Additionally, the inhibition zone Y. frederiksenii, Y. intermedia, or the sucrose- remained clear of indicator colonies during in- negative isolates. When serving as indicator cubation at 250C for 2 weeks. strains, only two of the typical Y. enterocolitica None of the producer strains was susceptible isolates were inhibited by the bacteriocin pro- to its own active principal, nor was the bacteri- 436 BOTTONE, SANDHU, AND PISANO J. CLIN. MICROBIOL. ocin of Y. intermedia active against the repre- be variably expressed among human isolates of sentative Enterobacteriaceae, P. aeruginosa, this group of yersiniae. Acinetobacter sp., or gram-positive species Despite these considerations, it is nevertheless tested. significant that temperature-dependent bacteri- DISCUSSION ocin production was observed only among Y. intermedia isolates. What remains to be defined Among members of the genus Yersinia, bac- is the prevalence of this antagonistic principle teriocin-like activity has been described for Y. among strains freshly isolated from their natural pestis (1) and Y. pseudotuberculosis (6). To our environmental habitat (10) as contrasted to hu- knowledge, bacteriocin production has not been man isolates. Characterization of the active sub- reported for Y. enterocolitica. As noted from stance also remains to be elucidated. the results obtained in this study, it may be that bacteriocin production was tested by previous LITERATURE CITED investigators only among biochemically and se- 1. Ben-Gurion, R., and I. Hertman. 1958. Bacteriocin-like rologically typical strains (which would exclude material produced by Pasteurella pestis. J. Gen. Micro- Y. intermedia) which seem to be nonproducers. biol. 19:289-297. It is also possible that tested strains of Y. 2. Bottone, E. J. 1977. Yersinia enterocolitica: a panoramic enter- view of a charismatic microorganism. CRC Crit. Rev. ocolitica could have lost their bacteriocin activ- Microbiol. 5:211-241. ity by host adaptation at 37°C or by repeated 3. Bottone, E. J. 1978. Atypical Yersinia enterocolitica: subculture, especially at 37°C. Subculturing at clinical and epidemiological parameters. J. Clin. Micro- 37°C has also been suggested to account for the biol. 7:562-567. 4. Bottone, E. J., B. Chester, M. S. Malowany, and J. loss of enterotoxin production in Y. enterocolit- Allerhand. 1974. Unusual Yersinia enterocolitica iso- ica (16) which, analogous to some bacteriocins lates not associated with mesenteric lymphadenitis. (15), is a plasmid-borne function. In the present Appl. Microbiol. 27:858-861. study, however, several of the Y. enterocolitica 5. Brenner, D. J., A. G. Steigerwalt, D. P. Falcao, R. E. Weaver, and G. R. Fanning. 1976. Characterization tested represented freshly obtained clinical iso- of Yersinia enterocolitica and Yersinia pseudotuber- lates that had been subcultured only three to culosis by deoxyribonucleic acid hybridization and by four times before testing. These, however, were biochemical reactions. Int. J. Syst. Bacteriol. 26:180- all human isolates, and bacteriocin activity could 194. 6. Brubaker, R. R., and M. J. Surgalla. 1961. Pesticins. I. have been lost by passage through such a warm- Pesticin-bacterium interrelationships, and environmen- blooded host. tal factors influencing activity. J. Bacteriol. 82:940-949. The lack of evidence ofbacteriocin production 7. Chester, B., and G. Stotzky. 1976. Temperature-de- among typical Y. enterocolitica could also be pendent cultural and biochemical characteristics of rhamnose-positive Yersinia enterocolitica. J. Clin. Mi- explained on the basis of failure to select an crobiol. 3:119-127. appropriate indicator strain. It would seem, how- 8. Fredericq, P. 1957. Colicins. Annu. Rev. Microbiol. 11: ever, that at least one indicator strain should 7-22. have been encountered among the 30 other Yer- 9. Lee, W. H., P. P. McGrath, P. H. Carter, and E. L. Eide. The ability of some Yersinia enterocolitica sinia strains tested, as well as the 20 other strains to invade HeLa cells. Can. J. Microbiol. 23: Enterobacteriaceae. 1714-1722. Temperature-dependent bacteriocin produc- 10. Mollaret, H. H. 1976. Contribution a l'etude epidemiolo- tion only among Y. intermedia strains has tax- gique des infection a Yersinia enterocolitica. III. Bilan provisoire des connaissances. Med. Mal. Infect. 6:442- onomic and evolutionary significance. Because 448. the bacteriocin of Y. intermedia was active only 11. Nilehn, B. 1973. The relationship of incubation temper- against other Yersinia traversing DNA-DNA ature to serum bactericidal effect, pathogenicity and in homology groups 1, 2, 3, and 4, this finding vivo survival of Yersinia enterocolitica, p. 85-92. In S. Winblad (ed.), Contribution to microbiology and im- taxonomically both reinforces the grouping of munology, vol. 2. S. Karger, Basel. these diverse microorganisms within the genus 12. Pai, C. H., and V. Mors. 1978. Production of enterotoxin Yersinia and further establishes the singularity by Yersinia enterocolitica. Infect. Immun. 19:908-911. of Y. intermedia. 13. Pai, C. H., V. Mors, and S. Toma. 1978. Prevalence of enterotoxigenicity in human and nonhuman isolates of The fact that only 7 of the 15 Y. intermedia Yersinia enterocolitica. Infect. Immun. 22:334-338. strains tested elaborated the antagonistic sub- 14. Sonnenwirth, A. C. 1974. Yersinia, p. 222-229. In E. H. stance may not be inconsistent when compared Lennette, E. H. Spaulding, and J. P. Truant (ed.), with the results obtained with Y. enterocolitica. Manual of clinical microbiology, 2nd ed. American So- With the ciety for Microbiology, Washington, D.C. exception of the 0-group 17 isolate of 15. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker. Winblad, the remaining 14 isolates tested were 1976. Bacteriocins of gram-positive . Bacteriol. all recovered from human subjects in which they Rev. 40:722-756. produced mild infections (3). Depending on the 16. Zink, D. L., J. C. Feeley, J. G. Wells, J. C. Vickery, G. duration of such an A. O'Donovan, and C. Vanderzant. 1978. Possible adaptation in these hosts, plasmid-mediated virulence in Yersinia enterocolitica. temperature-dependent bacteriocin activity may Tex. J. Sci. Special Publ. no. 5.