Yersinia rohdei sp. nov. Isolated from Human and Dog Feces and Surface Water

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1 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Oct. 1987, p lO27-6$2. 1 Copyright 1987, International Union of Microbiological Societies Vol. 37, No. 4 Yersinia rohdei sp. nov. Isolated from Human and Dog Feces and Surface Water STOJANKA ALEKSIC,l ARNOLD G. STEIGERWALT,2 JOCHEN BOCKEMUHL, GERALDINE P. HUNTLEY-CARTER,3 AND DON J. BRENNER2* Meningitis and Special Pathogens Branch and Enteric Bacteriology Section,3 Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 3333, and National Reference Centre for Salmonella, Medizinaluntersuchungsanstalt, Institute of Hygiene, 2 Hamburg 28, Yersinia ruhdei sp. nov. was isolated from feces of dogs and humans and from surface water. The organisms gave the key biochemical reactions typical of Yersinia species, including greater metabolic activity at 25 to 28 C than at 36 C. Y. ruhdei can be distinguished from other yersiniae by its positive reactions in tests for citrate and sucrose and negative reactions in tests for indole, acetoin (Voges-Proskauer), and rhamnose. Two biogroups were found among seven strains investigated, one of which fermented raffinose and melibiose. Deoxyribonucleic acid hybridization tests (hydroxyapatite method) showed average levels of relatedness of 93% at a 6 C incubation temperature and 97% at 75 C. Labeled Y. ruhdei deoxyribonucleic acid showed 3% to 63% relatedness to all Yersinia species except Yersinia ruckeri. The guanine-plus-cytosine contents of five tested strains were 48.7 to 49.4 rho]%. All strains possessed antigens not typable with antisera of the extended typing scheme of Wauters, but most of them had H antigens typical of Yersinia enteruculitica biotype 1. Five tested strains belonged to lysotype X,. Y. ruhdei strains were susceptible to most antimicrobial agents; resistance or intermediate susceptibility was observed for ampicillin, carbenicillin, cephalothin, penicillin, and sulfonamide. Calcium dependency, autoagglutination, and Congo red pigmentation tests, which are indicative of the presence of a virulence plasmid to Y. enteruculitica, were negative in Y. ruhdei. The clinical significance for humans and animals is uncertain and requires further study. The type strain of Y. ruhdei sp. nov. is strain H 2-36/78 (= CDC = ATCC 38). The bacterium now known as Yersinia enterocolitica was first isolated by Schleifstein and Coleman, who called it Bacterium enterocoliticum (26). Subsequent isolates were called Pasteurella pseudotuberculosis rodentium (17) and Pasteurella X (1). Frederiksen systematically studied a large number of strains, concluding that the organism was a member of the Enterobacteriaceae in the genus Yersinia (15). After the study of Frederiksen in 1964 and until 1979, Yersinia enterocolitica was a repository for a large number of strains that differed from typical strains of the species in tests that included sucrose, raffinose, rhamnose, melibiose, and a-methyl-d-glucoside. These strains were termed Yersinia enterocolitica-like or atypical Yersinia enterocolitica. Further studies demonstrated that included among these strains were four species distinct from Y. enterocolitica (5-7, 27). These were Yersinia aldovae (rhamnose positive, sucrose negative), Yersinia frederiksenii (rhamnose positive, sucrose positive), Yersinia intermedia (rhamnose positive, sucrose positive, melibiose positive, raffinose positive), and Yersinia kristensenii (rhamnose negative, sucrose negative). Serotyping studies have indicated that Yersinia H antigens are highly species specific (1). Several strains isolated from dog feces and water in Germany and single human isolates from Germany and the United States possessed antigens that agglutinated with Y. enterocolitica H antisera, but were untypable with Y. enterocolitica antisera. These strains differed from Y. enterocolitica by their negative reactions for indole production and the Voges-Proskauer test and their utilization of citrate. Most of these strains also fermented * Corresponding author raffinose and melibiose. Further studies showed that these strains represented a single new Yersinia species. In this report we describe this new species, for which the name Yersinia rohdei sp. nov. is proposed. MATERIALS AND METHODS Bacterial strains. The Y. rohdei strains are listed in Table 1. The strains of other Yersinia species that were used in deoxyribonucleic acid (DNA) studies have been previously described (3-7, 13, 27). Strains were maintained at room temperature on nutrient agar (Centers for Disease Control [CDC]) or on tryptone soy agar (Institute of Hygiene). Biochemical tests. Biochemical tests were done on all strains at the Institute of Hygiene and at the CDC. At the CDC biochemical test preparations were incubated at 36 and 25 C, unless otherwise specificed (see Tables 2 and 3), by using the methods of Edwards and Ewing (12) with some previously described modifications (18, 19). Unless otherwise specified, all biochemical tests were done at 28 C at the Institute of Hygiene, using the same methods with the following exceptions: carbohydrate fermentation media contained bromthymol blue as an indicator, and media for tartrate utilization were prepared as described by Kauffmann and Petersen (22). Phage typing. Phage typing of five strains was done at the World Health Organization Collaborating Centre for Yersinia (H. H. Mollaret) at the Institut Pasteur, Paris, France. Antimicrobial agent susceptibility tests. Antimicrobial agent susceptibility was determined on Y. rohdei strains by using the disk method of Bauer et al. (2) at the CDC and the Federal German Standard Methods (11) at the Institute of 327 IP: On: Wed, 6 Dec :44:1

2 328 ALEKSIC ET AL. INT. J. SYST. BACTERIOL. Straina Antigenic formula and lysotypeh TABLE 1. Y. rohdei strains studied Source Location H2-36/78T (= T NT: b, d, e, f, i; lysotype X, Dog feces = ATCC 38T) H274-36/78 ( = ) NT: b, i; lysotype X, Dog feces H276-36/78 ( = ) NT: b, d, e, f, i; lysotype X, Dog feces H277-36/78 ( = ) NT: a, b, d, f, i; lysotype X, Dog feces H111-36/8 (= ) NT: b, d, e, f, k; lysotype X, Surface water H128-36/ ( = 35-85) NT: a, b, i' 37-year-old woman, feces United States H7-36/ ( = 3216-) NT: a, b, i" 3-year-old child, feces ~ ~~~ The designation used at the Institute of Hygiene precedes the designation used at the CDC. ATCC, American Type Culture Collection. NT, Nontypable antigen. Lysotype not determined. Hygiene. The antimicrobial agents and concentrations used are listed in Table 4. Serotyping. The strains were serotyped by slide agglutination as described previously (l), using the growth of 18- to 24-h cultures from nutrient agar or Kligler iron agar for antigens and soft agar as described by Gard for H antigens. Antisera against 6 Yersinia antigens and 38 H antigens were used (1). Virulence factors. Tests to assess virulence included calcium dependency at 37 C on magnesium-oxalate medium (2), autoagglutination at 37 C but not at 22 C in MR-VP medium (Difco) (23), Congo red pigmentation medium at 28 C (25), and pyrazinamidase activity (21). Production of heat-stable enterotoxin was examined by the suckling mouse test (16), and formation of a heat-labile, choleralike toxin was tested by a GM1 enzyme-linked immunosorbent assay on microtiter plates, using Escherichia coli anti-lt (). G+C content of DNA. The guanine-plus-cytosine (G+C) contents of Y. rohdei DNAs were determined spectrophotometrically by thermal denaturation (24), with Y. enterocolitica DNA included as a control. At least four separate determinations were done on DNA from each strain. DNA hybridization. The hydroxyapatite method was used to determine the DNA relatedness of Y. rohdei strains to each other and to representative strains of other Yersinia species. The methods used have been described previously (8). RESULTS AND DISCUSSION Y. rohdei strains were isolated first from feces of healthy dogs and then from surface water in Germany (Table 1). A computer search of the biochemical characteristics of all strains in the Enteric Laboratory Section at the CDC revealed no strains similar to Y. rohdei. Shortly thereafter a strain isolated from the stool of a 37-year-old woman in California was identified as Y. rohdei. The woman was suffering from diarrhea and abdominal cramps. A second human strain was isolated from feces of a healthy 3-year-old child in Germany. Later, six additional strains of Y. rohdei, not included in this study, were identified among strains of the culture collection of the Institute of Hygiene which were biochemically consistent with biogroup 1. These organisms originated from feces of healthy dogs (strains H269-36/78 and H275-36/78), surface water (H694-36/83 and. H75-36/83), and stool specimens from patients with enteritis (H375-36/84 and H96-36/85). It is not known whether Y. rohdei was the cause of diarrhea in the human cases. We hypothesize that the natural habitat of the organisms is water, leading to fecal carriage in dogs and humans and possibly an occasional infection. Y. rohdei strains showed the key biochemical reactions typical of the Y. enterocolitica- Y. aldovae- Y. frederiksenii- Y. intermedia- Y. kristensenii group of yersiniae. These include greater metabolic activity at 25 to 28 C than at 36"C, motility at 25 to 28 C but not at 36"C, positive reactions at 25 to 28 C in tests for methyl red, citrate, urea, and ornithine decarboxylase, negative reactions in tests for HZS, lysine decarboxylase, arginine dihydrolase, phenylalanine deaminase, lipase, and deoxyribonuclease, and production of little, if any, gas from the fermentation of D-glucose and other carbohydrates. The biochemical reactions of Y. rohdei strains at both 25 to 28 and 36 C are shown in Table 2. Five Y. rohdei strains fermented raffinose and melibiose (biogroup l), and two strains (biogroup 2) did not. The differentiation of these biogroups from one another and from other Yersinia species is shown in Table 3. Y. rohdei is separable from Y. enterocolitica by its positive citrate reaction and negative reactions for indole and Voges-Proskauer tests. Positive citrate and sucrose reactions distinguish Y. rohdei from Y. kristensenii. Positive reactions for sucrose and citrate tests and negative reactions for rhamnose, indole, and Voges-Proskauer tests differentiate Y. rohdei from Y. aldovae, Y. frederiksenii, and Y. intermedia. Positive citrate, ornithine decarboxylase, sorbitol, and sucrose tests separate Y. rohdei from unnamed Yersinia group X1 (3). A complete comparison of the biochemical chracteristics of Y. rohdei with those of other Yersinia species can be obtained by comparing the data in Table 2 obtained at 25 to 28 C with previously published data in references 3 through 7, 13, and 27. A comparison of biochemical characteristics obtained at 36 C may be obtained by comparing the data in Table 2 with previously published data in reference 13 and. Y. rohdei strains were susceptible to apalcillin, trimethoprim plus sulfamethoxazole, chloramphenicol, colistin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfadiazine, and tetracycline and were resistant or showed intermediate susceptibility to ampicillin, carbenicillin, cephalothin, penicillin, and sulfonamide (Table 4). There were no significant differences in susceptibility patterns in the two Y. rohdei biogroups. All strains of Y. rohdei possessed antigens that were not typable with 56 antisera of the extended typing scheme of Wauters (28) and 4 provisional group antisera of AleksiC and co-workers (1). Three strains gave weak cross-reactions with :18 of Y. enterocolitica and :38 or :6 or Y. frederiksenii, which were removed by absorption without significant reduction of the homologous titer. All of the strains which we studied and two additional isolates from the IP: On: Wed, 6 Dec :44:1

3 VOL. 37, 1987 YERSINIA ROHDEI SP. NOV. 3 Test Indole production Methyl red Voges-Proskauer Citrate (Simmons) H2S on triple sugar iron agar H2S on peptone iron agar Urea hydrolysis Phenylalanine deaminase L-Lysine-decarboxylase (Moeller) L-Arginine deaminase (Moeller) L-Ornithine decarboxylase (Moeller) Motility Gelatin hydrolysis KCN, growth Malonate utilization D-Glucose, acid D-Glucose, gas Acid from: Adonitol L-Arabinose D- Arabitol Cellobiose Dulcitol Erythritol D-Galactose Glycerol i(rnyo)-inositol Lactose Maltose D-Mannitol D-Mannose Melibiose a-ch3-d-glucoside Raffinose L-Rhamnose Salicin D-Sorbitol Sucrose Trehalose D-Xylose Esculin hydrolysis Mucate, acid Tartrate (Jordan) Acetate utilization Lipase (corn oil) Deoxyribonuclease N3- + NO2- Oxidase o-nitrophenyl-p-d-galactopyranoside Citrate (Christensen) Tyrosine clearing Pigment production TABLE 2. Biochemical reactions of seven Y. rohdei strains and of the type strain Cumulative % of strains positive" 25 to 28 C" 36 C Reaction of type strain ATCC 38 at:" 2 Days 7 Days 1 Day 2 Days 7 Days 25 to 28 C 36 C d a A blank space indicates that the test was not read at this time or, in the case of gelatin hydrolysis, was not done at this incubation temperature. Tests were done at 25 C at the CDC and at 28 C at the Institute of Hygiene. Identical results were obtained at both laboratories for most tests. -, Negative after 2 days; +, positive within 2 days; ( + 1, positive in 3 to 7 days. Small amounts of gas Institute of Hygiene culture collection had H antigens typical of Y. enterocolitica biatype 1. These findings are in contrast to results of previous studies which demonstrated species specificity of H antigens in Yersinia species. Five strains tested belonged to lysotype X, (Table 1). Tests for calcium dependency, autoagglutination, and Congo red pigmentation, which indicate the presence of a virulence plasmid in Y. enterocolitica, were negative in Y. rohdei, as were tests for the detection of known enterotoxins. These results, tqgether with the demonstration of pyrazinamidase activity, suggest that Y. rohdei is an environmental organism rather than a pathogen for humans and warm-blooded animals. G + C contents were determined spectrophotometrically for DNAs from five Y. rohdei strains. The G+C range was 48.7 to 49.4 mol%, There were no significant differences in IP: On: Wed, 6 Dec :44:1

4 ~~~~ 33 ALEKSIC ET AL. INT. J. SYST. BACTERIOL. Test TABLE 3. Biochemical differentiation of Y. rohdei from other Yersinia species" rohdei biogroups Y. entero- coliticci biotypes h Indole - - (+) d Yersinia spnii group Y. pseudo- Y. j?ederik- y. illtermediei spnii senii ''isten- Y. ddowie g!yyp tuberculosis Y. pestis Y. ruckeri x1 A (+) d (-) Voge s-proskauer Citrate (Simmons) (+) + - d + Ornithine decarboxylase d + Cellobiose Melibiose a-ch3-d-glucoside Raffi n o s e L-Rhamnose D-Sorbi to Sucrose d + + a All incubations were done at 25 to 28 C for 3 days (2 days of incubation for Y. ruckeri). -. to 1% positive; (-1, 11 to 25% positive: d, 26 to 74% positive; (+), 75 to 89% positive; +, 9 to 1% positive. Data for Yersinia species other than Y. rohdei are from references 3 through 7, 13, and 27. In addition to results shown, Y. enterocoliticci biotype 5 is negative in tests for trehalose and reduction of nitrate to nitrite: Y. rohdei has the reverse reactions. G+C content between human and animal isolates or between strains from biogroup 1 and biogroup 2 (strains and ). The G+C range for Y. rohdei strains was quite similar to the average of 48.5 k 1.5 mol% reported for 22 strains representing Y. enterocolitica, Y. frederiksenii, Y. intermedia, and Y. kristensenii (19) and the vplue of 48 mol% reported for Y. aldovae (2). DNA hybridization was done with labeled DNAs from four Y. rohdei strains, including the type strain, a biogroup 2 strain, and a human strain (not all strains were tested with any one labeled DNA [Table 51). All strains of Y. rohdei were highly interrelated. The average level of relatedness under optimal DNA reassociation criteria (6 C incubation temperature) was 93%; the average level of relatedness in reactions done at 75"C, where only closely related DNA sequences can reassociate, was 97%, and the percent divergence of nucleotides within related DNA sequences was.5%. These high relatedness values indicate that all Y. rohdei strains, reagardless of source, geographic origin, and biogroup, belong to a single species. Labeled Y. rohdei DNA showed 3% or more relatedness to all Yersinia species other than Y. ruckeri (which most reasearchers feel should not be in the genus Yersinia []). The highest level of relatedness (55%, average of three Y. rohdei strains) was to the type strain of Y. frederiksenii. These data are quite comparable to previously obtained values for relatedness among Y. enterocolitica, Y. aldovae, Y. frederiksenii, Y. intermedia, and Y. kristensenii (45 to 68%), as are the values obtained between species for percent divergence in the present study (7.5 to 13.%) and in previous studies (7. to 13.% [5-7, 13, 2). Since the previously described Yersinia species are 3% or less related to other members of the Enterobacteriaceae (5, 9) and Y. rohdei is as much as 55% related to Yersinia species, it was not necessary to determine the DNA relatedness of Y. rohdei to other Enterobacteriaceae. On the basis of DNA relatedness Y. rohdei is clearly a new species in the genus Yersinia. Description of Yersinia rohdei sp. nov. Yersinia rohdei (roh de. i. M.L. gen. n. rohdei, of Rohde) is named in honor of the late Rolf Rohde, who founded the National Reference Centre for Salmonella in Hamburg, Federal Republic of Germany, and who made many significant contributions to the diagnostic and serological identification of Enterobacteriaceae, especially in the genus Salmonella. Strains of Y. rohdei are gram-negative, oxidase-negative, fermentative, nonpigmented rods with the general characteristics of the family Enterobacteriaceae and of the genus Yersinia (Table 2). They are motile at 25 to 28 C but not at 36 C. All Y. rohdei strains give positive results in tests at 25 to 28 C for methyl red, citrate utilization (Simmons [may be delayed positive] and Christensen), urea hydrolysis (may be delayed positive), L-ornithine decarboxylase (Moeller) (may be delayed positive) tartrate fermenation (Jordan) (may be delayed positive), o-nitrophenyl-p-d-galactopyranoside, reduction of nitrate to nitrite, and acid from fermenation of D-glucose, L-arabinose, cellobiose, D-galactose (may be delayed positive), glycerol (may be delayed positive), D- mannitol, D-mannose, D-sorbitol, sucrose, trehalose, and D-xylose. All Y. rohdei strains give negative results after 48 h of incubation at 25 to 28 C in tests for indole production, the Voges-Proskauer reaction, H2S production (triple sugar iron agar and peptone iron agar), phenylalanine deaminase, L-lysine decarboxylase (Moeller), L-arginine dihydrolase (Moeller), gelatin hydrolysis, malonate utilization, esculin hydrolysis, acid from mucate, acetate utilization, lipase (corn oil), deoxyribonuclease, tyrosine clearing, gas from D-glucose, and acid from the fermentation of adonitol, D-arabitol, dulcitol, erythritol, i(rnyo)-inositol, a-methyl-d- TABLE 4. Susceptibility of seven Y. rohdei strains to antimicrobial agents as determined by agar diffusion Antibiotic(s) Zone diam (mm) % of strains (disk concn) Range Mean SD susceptible Ampicillin (1 pg) Apalcillin (3 pg) Carbenicillin (1 pg) Cephalothin (3 pg) Chloramphenicol (3 pg) Colistin (1 pg) Gentamicin (1 pg) Kanamycin (3 pg) Nalidixic acid (3 pg) Penicillin (1 U) Streptomycin (1 pg) Sulfadiazine (25 pg) Sulfonamide (3 pg) Tetracycline (3 pg) Trimethoprim + sulfamethoxazole ( pg) IP: On: Wed, 6 Dec :44:1

5 VOL. 37, 1987 YERSINIA ROHDEI SP. NOV. 331 Source of unlabeled DNA Y. rohdei 322-ST Y. rohdei Y. rohdei Y. rohdei Y. rohdei Y. rohdei Y. rohdei Y. aldovae Y. enterocolitica Y. frederiksenii IP 6175 Y. frederiksenii Y. frederiksenii IP 7 Y. intermedia IP 48T Y. kristensenii IP 74 Y. pestis 1122 Y. pseudotuberculosis P65 Y. ruckeri Y. group XI strain IP 776 TABLE 5. DNA relatedness of Y. rohdei strains Relatedness to 32P4-labeled DNAs from Y. ruhdei strains Strain V Strain PBR at 6 C %D RBR at 75 C RBR at 6 Cb %D RBR at 75 C d P4-labeled DNAs from strains of Y. rohdei were reacted with unlabeled DNAs from the same strain (homologous reaction), from other Y. rohdei strains, and from strains of all previously described Yersinia species. Each reaction was done at least twice. The average level of reassociation in homologous reactions was 55% before normalization. Control reactions, in which labeled DNA was incubated in the absence of unlabeled DNA, showed an average level of binding to hydroxyapatite of 1.%. The control value was subtracted before normalization. Relative binding ratio (RBR) = [(percentage of DNA bound to hydroxyapatite in heterologous reactions)/(percentage of DNA bound to hydroxyapatite in homologous reactions)] x 1. Percent divergence (%D) was calculated on the assumption that a 1% decrease in the thermal stability of a heterologous DNA duplex compared with that of the homologous duplex was caused by 1% of the bases within the duplex that were unpaired; it was calculated to the nearest.5%. This value was obtained with labeled DNA from strain and unlabeled DNA from strain 322-UT. This value was obtained with labeled DNA from strain and unlabeled DNA from strain P65. Labeled DNA from strain was 92 to 96% related to DNAs from other Y. ruhdei strains (data not shown) glucoside, L-rhamnose, and salicin. Six of our seven strains produce acid from fermentation of lactose and maltose. Strains vary in reactions for growth in KCN and acid production from fermentation of raffinose and melibiose. The strains formed 2 biogroups on the basis of positive (biogroup 1) or negative (biogroup 2) reactions for both raffinose and melibiose. Tests that differentiate Y. rohdei from other Yersinia species are shown in Table 3. Y. rohdei strains were resistant to or showed intermediate susceptibility to ampicillin, carbenicillin, cephalothin, and penicillin and were susceptible to the other antimicrobial agents tested (Table 4). Y. rohdei strains had G+C content of 48.7 to 49.4 mol%. Y. rohdei strains were isolated from the feces of dogs and humans and from surface water. The clinical significance of Y. rohdei as a diarrheal agent in humans and animals is uncertain and requires further study. The type strain of Y. rohdei is strain H2-36/78(= = ATCC 38). Description of the type strain. Y. rohdei 322-MT exhibits the characteristics of the species as given in Tables 2, 4, and 5. The G+C content of its DNA is 49.1 mol%. It was isolated from dog feces in Germany in ACKNOWLEDGMENTS We thank H. H. Mollaret for phage typing of Y. rohdei strains and J. J. Farmer 111 for critically reading the manuscript. LITERATURE CITED 1. AleksiC, S., J. Bockemuhl, and F. Lange. 19. Studies on the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 261: Baper, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol Bercovier, H., D. J. Brenner, J. Ursing, A. G. Steigerwalt, G. R. Fanning, J. M. Alonso, G. P. Carter, and H. H. Mollaret Characterization of Yersinia enterocolitica sensu stricto. Curr. Microbiol. 4: Bercovier, H., H. H. Mollaret, J. M. Alonso, J. Brault, G. R. Fanning, A. G. Steigerwalt, and D. J. Brenner Intra- and interspecies relatedness of Yersinia pestis by DNA hybridization and its relationship to Yersinsia pseudotuberculosis, Curr. Microbiol. 4: Bercovier, H., A. G. Steigerwalt, A. Guiyoule, G. Huntley- Carter, and D. J. Brenner Yersinia aldovae (formerly Yersinia enterocolitica-like group X2): a new species of Enterobacteriaceae isolated from aquatic ecosystems. Int. J. Syst. Bacteriol. 34:16& Bercovier, H., J. Ursing, D. J. Brenner, A. G. Steigerwalt, G. R. Fanning, G. P. Carter, and H. H. Mollaret Yersinia kristensenii: a new species of Enterobacteriaceae composed of sucrose-negative strains (formerly called atypical Yersinia enterocolitica or Yersinia enterocolitica-like). Curr. Microbiol. 4: Brenner, D. J., H. Bercovier, J. Ursing, J. M. Alonso, A. G. Steigerwalt, G. R. Fanning, G. P. Carter, and H. H. 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6 332 ALEKSIC ET AL. INT. J. SYST. BACTERIOL. enterocolitica-like organisms. Curr. Microbiol. 4: Daniels, J. J. H. M., and C. Goudzwaard Enkele stammen van een op Pasteurella pseudotuberculosis gelijkende niet geidentifeceerde species geisoleerd bij knaagdieren. Tijdschr. Diergeneeskd. 88: Deutsches Institut fur Normung Methoden zur Empfindlichkeitspriifung von bakteriellen Krankheitserregern (ausser Mykobakterien) gegen Chemotherapeutika. Agar Diffusionstest. DIN 5894, Teil. Beuth Verlag, Berlin. 12. Ewing, W. H. 19. Edwards and Ewing s identification of Enterobacteriaceae, 4th ed. Elsevier Scientific Publishing Co., New York. 13. Ewing, W. H., A. J. Ross, D. J. Brenner, and G. R. Fanning Yersinia ruckeri sp. nov., the Redrnouth (RM) bacterium. Int. J. Syst. Bacteriol. 28: Farmer, J. J., 111, B. R. Davis, F. W. Hlckman-Brenoer, A. McWhorter, G. G. Huntley-Carter, M. A. Asbury, C. Riddle, H. G. Wathen-Grady, C. Elias, G. R. Fanning, A. G. Steigerwalt, C. M. O Hara, G. K, Morris, P. B. Smith, and D. J. Brenner Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microhiol. 21: Frederiksen, W A study of some Yersinia pseudotuberculosis-like bacteria ( Bacterium enterocoliticum and Pasteurella X ). Scand. Cong. Pathol. Microbiol. : Giannella, R. A Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. : Hassig, A., J. Karrer, and F. Pusteria Uber Pseudotuberkulose beim Menschen. Schweiz, Med. Wochenschr. 79: Hickman, F. W., and J. J. Farmer Salmonella typhi: identification, antibiograms, serology, and bacteriophage typing. Am. J. Med. Technol. 44: Hickman, F. W., J. J. Farmer 111, A. G. Steigerwalt, and D. J. Brenner Unusual groups of Morganella ( Proteus ) morganii isolated from clinical specimens: lysine-positive and ornithine-negative biogroups. J. Clin. Microbiol. 12: Higuchi, K., and J. L. Smith Studies on the nutrition and physiology of Pasteurella pestis. VI. A differential plating medium for the estimation of mutation rate to avirulence. J. Bacteriol. 81: Kandolo, K., and G. Wauters Pyrazinamidase activity in Yersinia enterocolitica and related organisms. J. Clin. Microbid. 21: Kauffmann, F., and A. Petersen The biochemical group and type differentiation of Enterobacteriaceae by organic acids. Acta Pathol. Microbiol. Scand. 38: Laird, W. J., and D. G. Cavanaugh Correlation of autoagglutination and virulence in yersiniae. J. Clin. Microbiol. 11: Marmur, J., and P. Doty Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol Prpic, J. K., R. M. Robins-Browne, and R. B. Davey Differentiation between virulent and avirulent Yersinia enterocolitica isolates by using Congo red agar. J. Clin. Microbiol. 18: Schleifstein, J., and M. B. Coleman An unidentified microorganism resembling B. lignieresi and Pasteurella pseudotuberculosis, pathogenic for man. N.Y. State J. Med. 39: Ursing, J., D. J. Brenner, H. Bercovier, G. R. Fanning, A. G. Steigerwalt, J. Brault, and H. H. Mollaret Yersinia frederiksenii: a new species of Enterobacteriaceae composed of rhamnose-positive strains (formerly called atypical Yersinia enterocolitica or Yersinia enterocolitica-like). Curr. Microbiol. 4: Wauters, G Antigens of Yersinia enterocolitica, p In E. Bottone (ed.), Yersinia enterocolitica. CRC Press, Boca Raton, Fla.. World Health Organization Manual for laboratory investigations of acute enteric infections, p Publication CDDB3.3. World Health Organization, Geneva. IP: On: Wed, 6 Dec :44:1