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Enterobacteriaceae and Vibrionaceae SAM1 RAMIA,' ERWIN NETER,'V2 and DON J

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Enterobacteriaceae and Vibrionaceae SAM1 RAMIA,' ERWIN NETER,'V2 and DON J INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1982, p. 395-398 Vol. 32, No. 4 020-7713/82/040395-04$02.M Copyright 0 1982, International Union of Microbiological Societies Production of Enterobacterial Common Antigen as an Aid to Classification of Newly Identified Species of the Families Enterobacteriaceae and Vibrionaceae SAM1 RAMIA,' ERWIN NETER,'v2 AND DON J. BRENNER3 Division of Clinical Microbiology and Immunology, Erie County Laboratory, Erie County Medical Center, Bflalo, New York 14215'; Departments of Microbiology and Pediatrics, State University of New York at Buffalo, and Laboratory of Bacteriology, Children's Hospital of Buffalo, Buffalo, New York 14222'; and Enteric Bacteriology and Epidemiology Branch, Bacterial Diseases Division, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303333 A study on the production of enterobacterial common antigen by recently described or as-yet-undescribed species belonging to the families Enterobac- teriaceae and Vibrionaceae was carried out by hemagglutination and hemaggluti- nation inhibition tests with rabbit antisera specific for this antigen. All 28 strains known or presumed to belong to Enterobacteriaceae produced this antigen, and none of the seven strains belonging to Vibrionaceae did so. The results obtained with Tatumella ptyseos and Xenorhabdus species are particularly noteworthy, since they represent species which are atypical for Enterobacteriaceae. It is suggested that the determination of the production of enterobacterial common antigen is a significant aid to classification, particularly when the assignation of a new genus or species to Enterobacteriaceae or to another family presents a problem. Enterobacterial common antigen (ECA), EDidemioloav Branch, Centers for Disease Control. which is common to members of the family Tipe strains or deoxyribonucleic acid (DNA) refer- Enterobacteriaceae, was first described by ence strains were used for all species. The sources of Kunin et al. (16,17). As reviewed by Miikela and the strains are given in Table 1. Strains were coded at Atlanta, Ga., and tested blind in Buffalo, N.Y. The Mayer (22), this antigen is produced by numer- codes given to these strains were not in the order ous members of the family Enterobacteriaceae shown in Table 1. References to the recently described but not by species belonging to other families. A species are given in Table 1. Enteric groups refer to few strains belonging to the family Enterobac- potential new species now under study in the Enteric teriaceae do not produce the antigen because of Diseases Reference Laboratories. DNA groups refer a documented or presumed mutation. The only to new species that have not yet been published or species of Enterobacteriuceae which apparently named. does not produce ECA is Erwinia chrysanthemi All strains were seeded onto chocolate agar and As far as species belonging to other families incubated at either 20 or 36"C, depending upon the (20). temperature optima of individual species. Since all are concerned, a possible exception to the rule is strains produced heavy confluent growth on chocolate strains previously identified as Aeromonas shi- agar, suspensions were made from this culture medi- gelloides and presently listed as Plesiomonas um incorporating the growth of three agar plates each shigelloides which do produce ECA (20, 29). In in 6 ml of phosphate hemagglutination buffer (Difco). view of the possible usefulness of ECA produc- Based on serial dilution, the number of organisms in tion in the classification of organisms at the each suspension was approximately 10'' cells per ml. family level, recently described and undescribed The suspensions were heated in boiling water for 1 h species belonging to the families Enterobac- and centrifuged at 23,500 X g for 10 min. The superna- teriaceae and Vibrionaceae were investigated. tants were used as antigens. For control purposes, supernatants of an ECA-positive Escherichia coli In this study, strains of 35 newly described or strain and an ECA-negative Pseudomonas aeruginosa as-yet-unnamed species of Enterobacteriaceae strain were used, and these preparations yielded the and Vibrionaceae were tested for the presence expected results. of ECA. The hemagglutination test was carried out as de- scribed previously (23). Briefly, to the sediment of three-times-washed human erythrocytes (blood group MATERIALS AND METHODS 0; Rh-negative; 2.5% suspension) was added antigen All strains were from the culture collection of the in a dilution of 1:lO. The suspension was incubated at Enteric Diseases Reference Laboratories in the Enter- 37°C and washed three times to remove excess anti- ic Bacteriology Section, Enteric Bacteriology and gen. The antigenically modified erythrocytes (0.2 ml) 3% RAMIA, NETER, AND BRENNER INT. J. SYST.BACTERIOL. TABLE 1. Production of ECA in 35 recently described species or groups of Enterobacteriaceae and Vibrionaceae Reference Source“ ECA Organism present Cedecea davisae 3278-77 7 CDC + (CIP 80.34, ATCC 33431) Cedecea lapagei 485-76 7 CDC + (CIP 80.35, ATCC 33432) Edwardsiella hoshinae 2169-80 9 P. A. D. Grimont + (CIP 78.56, ATCC 33379) Edwardsiella ictaluri 1976-78 11 J. P. Hawke + (GA-77-52, ATCC 33202) Enterobacter amnigenus 1325-79 15 H. Leclerc + (CUETM 78-118, ATCC 33072) Enterobacter gergoviae 604-77 1 C. Richard + (CIP 76.01, ATCC 33028) Enterobacter intermedium 2992-79 14 H. Leclerc + (Leclerc 77-148) Escherichia blattae 9005-74 2 N. R. H. Burgess + Hafnia DNA group 2 3226-74 26 CDC + Kluyvera ascorbata 648-74 5 CDC + (ATCC 33433) Kluyvera cryocrescens 2065-78 5 CDC + (ATCC 33435) Obesumbacteriumproteus 1496-74 25 F. G. Priest + (Priest 502, NCIB 8771) Proteus DNA pup1808-73 CDC + Providencia DNA group 132-68 CDC + Serratia $caria 1165-79 10 P. A. D. Grimont + (CIP 79.23, ICPB 4050, ATCC 33105) Serratia fonticola 2988-79 6 H. Leclerc + (ATCC 29844) Serratia odorifera 1979-77 8 P. A. D. Grimont + (Grimont 1073, ICPB 3995) Tatumella ptyseos H36 13 CDC + (9591-78, M168, ATCC 33301) Xenorhabdus luminescens 9016-80 28 G. M. Thomas + (Thomas Hb, ATCC 29999) Xenorhabdus nematophilus 1184-80 28 G. M. Thomas + (ATCC 19061) Xenorhabdus DNA group 3 1195-80 28 G. M. Thomas + (Thomas NC-19, ATCC 29304) Xenorhabdus DNA group 4 9014-80 28 G. M. Thomas + (Thomas DN) Enteric group 1 875-72 CDC + Enteric group 10 1350-81 CDC + Enteric group 11 980-72 CDC + Enteric group 19 1042-80 CDC + Enteric group 45 329-73 CDC + Enteric group 46 2896-78 CDC + Aerumonas sobria 9538-76 24 M. Popoff - (Popoff 278, CIP 7433) Vibrio damsela 2588-80 21 M. Love - (ATCC 33539) Vibriofluvialis 9555-78 19 J. V. Lee - (VL 5125, NCTC 11327) “Vibrio hollisae” 75-80 12 CDC - (ATCC 33564)b Vibrio metschnikovii 1316-78 18 NCTC - (NCTC 8443) Vibrio mimicus 1721-77 3 CDC - (ATCC 33653) Vibrio vulnificus 9107-79 4 ATCC - (ATCC 27562) a CDC, Centers for Disease Control, Atlanta, Ga.; ATCC, American Type Culture Collection, Rockville, Md.; NCTC, National Collection of Type Cultures, London, England. Not validly published to date. VOL. 32,1982 ECA AS AN AID TO CLASSIFICATION 397 were added to equal volumes of ECA antiserum in These and previously obtained results (16,17, twofold serial dilutions. The mixtures were incubated 20, 22, 23, 27, 29) establish ECA as a powerful for 30 min in a 37°C water bath, and hemagglutination taxonomic tool with which to separate the En- was read grossly after centrifugation at 1,300 x g for 2 terobacteriaceae &om other families. The only min. The hemagglutination inhibition test was performed exceptions (other than a rare negative strain of as follows. To ECA antiserum in twofold serial dilu- Enterobacteriaceae) are the negative results ob- tions (0.2 ml) were added equal amounts of undiluted tained with all six tested strains of Erwinia antigen. The mixtures were incubated for 30 min in a chrysanthemi (20) and the positive results ob- 37°C water bath. Erythrocytes modified with ECA tained with six Plesiomonas shigelloides strains obtainedfrom E. coli were added, and the hemaggluti- (20,29). It is our opinion that ECA production is nation test was completed as described above. a valuable addition to the description of the ECA antiserum was obtained by immunization of family Enterobacteriaceae and should be re- rabbits with either E. coli 014 or the ethanol-soluble quired in those instances where the assignation ECA preparation from Salmonella typhimurium, as Enterobacteriaceae described previously (27). of a new genus to or to The hemolysis test was done as described above another family creates a problem. except that sheep erythrocytes instead of human erythrocytes were used, and guinea pig complement in ACKNOWLEDGMENTS a dilution of 1:lO was added. Hemolysis was read We are grateful to Mary Alyce Asbury, Geraldine P. Hunt- grossly after incubation for 1 h at 37°C. IeyCarter, Betty R. Davis, Frances W. Hickman, and Alma C. McWhorter for providing the strains and to Frances W. Hickman for coding the strains and decoding the results. RESULTS Thanks are expressed also to Helga von Langendorff for assistance with the antigen tests. Table 1 shows the results of independently This research was supported in part by Public Health executed hemagglutination tests on organisms Service grant AI 00658 from the National Institute of Allergy belonging to 35 bacterial species. Positive and and Infectious Diseases (to E.N.). negative control preparations yielded appropri- ate results. All species known or presumed to be WFUNT REQWTS members of the Enterobacteriaceae were posi- Address reprint requests to: Dr. Erwin Neter, Department tive for the production of ECA, and all species of Microbiology, State University of New York at Buffalo, known or presumed to be members of the Vi- and Children's Hospital, Buffalo, NY 14222. brionaceae (genera Vibrio and Aeromonas) were negative. Hemagglutination occurred with all LITERATURE CITED positive strains with E. coli 014 and ECA antise- 1. Brcnner, D.
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