Molecular and Phenotypic Characterization of Potentially New ...

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2001, p. 618–621 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.2.618–621.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 2

Molecular and Phenotypic Characterization of Potentially New Shigella dysenteriae Serotype RONEY S. COIMBRA,1 PASCAL LENORMAND,1 FRANCINE GRIMONT,1 PHILIPPE BOUVET,1 SHIGERU MATSUSHITA,2 AND PATRICK A. D. GRIMONT1* Unite´ des Ente´robacte´ries, INSERM U389, Institut Pasteur, Paris, France,1 and Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan2 Received 7 July 2000/Returned for modification 28 August 2000/Accepted 4 November 2000

From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96–204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96–204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes.

Shigella dysenteriae is the causative agent of severe bacillary dysentery. Until 1958, the time of the last major classification of the genus Shigella (12), S. dysenteriae consisted of 10 serotypes. Subsequently, Ewing et al. (13) described provisional serotypes 3873-50 and 3341-55, which were later proposed for addition to the Shigella scheme as S. dysenteriae serotypes 11 and 12, respectively (5, 29). In 1985, Shmilovitz et al. (31) isolated 31 strains of a new provisional serotype represented by strain I9809-73. This provisional serotype was included in the S. dysenteriae scheme as serotype 13 after further characterization by Wathen-Grady et al. (33). Provisional serotypes E22383 and E23507 were originally reported in 1989 (19). After extensive characterization and isolation from various geographical locations, Ansaruzzaman et al. (1) recommended that they be designated serotypes 14 and 15, respectively. In recent years, three nonagglutinating strains of S. dysenteriae have been reported. Kuijper et al. (E. J. Kuijper, A. van Eeden, B. de Weber, R. van Ketel, and J. Dankert, Letter, Eur. J. Clin. Microbiol. Infect. Dis. 16:553–554, 1997) described a nonserotypeable S. dysenteriae strain isolated from a Dutch patient returning from India. This strain was antigenically related to Escherichia coli O159. In 1997 and 1998, Matsushita et al. (26, 27) reported the isolation of two unrelated nonagglutinating S. dysenteriae groups from stool cultures of imported diarrheal cases in Japan. These two potentially new serotypes are represented by strains 93–119 and 96–204. Serological identification is a crucial step in the diagnosis of Shigella infections. Although the slide agglutination test used to serotype Shigella isolates is easy and rapid, some critical aspects must be considered: (i) its success depends on the

quality of the antisera used (6, 7), although there is no international consensus on the choice of Shigella strains for the production of the antisera (14); (ii) none of the commercially available antisera recognizes S. dysenteriae serotypes 13 to 15 or provisional serotypes; (iii) numerous intra- and interspecific cross-reactions have been reported, requiring multiple absorption steps during antiserum production (1, 15, 19); and (iv) the transition from smooth to rough forms, which do not produce O antigens, makes such isolates untypeable by serotyping. To overcome these troublesome constraints, new molecular methods whose results approach those of serotyping have been developed in recent years. In previous work (26), an MluI ribotyping scheme, correlated overall with serotyping, was described. A database is now available for computer-assisted ribopattern identification (10). Since the chromosomal region called the rfb cluster contains most of the genes coding for the enzymes responsible for O-antigen synthesis, a new method based on restriction of the amplified rfb cluster has been proposed for the genetic identification of O serogroups (8, 9). This method is referred to as rfb-restriction fragment length polymorphism (RFLP). The purpose of the present work was to characterize nonagglutinating Shigella isolates by biochemical tests, MluI ribotyping, and rfb-RFLP. As a result, 22 French isolates tested and Japanese strain 96–204 (27) were found to constitute a new serotype of S. dysenteriae. MATERIALS AND METHODS Bacterial strains. From September 1997 to November 1998, the French National Center for Salmonella and Shigella (FNCSS) (Institut Pasteur, Paris, France) received 22 nonagglutinating Shigella isolates from 22 patients suffering from dysentery. For five patients, a history of recent travel to North Africa (Egypt, Tunisia, and Algeria) was known. Strain 96–204, isolated in Japan from a patient coming from India and Nepal (27), was also included in this study. Biochemical tests. The 22 French isolates were identified as S. dysenteriae by following the routine protocol of the FNCSS (23) (see Table 1).

* Corresponding author. Mailing address: Unite´ des Ente´robacte´ries, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 145688340. Fax: 33 145688837. E-mail: pgrimont @pasteur.fr. 618

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VOL. 39, 2001 TABLE 1. Biochemical reactions of 22 French isolates and strain 96–204 Test

Reactiona

Oxidase ............................................................................................. Catalase ............................................................................................ Glucose (acid).................................................................................. Glucose (gas) ................................................................................... Lactose.............................................................................................. H2S .................................................................................................... Urease............................................................................................... Indole production............................................................................ Mannitol ........................................................................................... Mobility............................................................................................. Lysine decarboxylase....................................................................... Arginine dihydrolase....................................................................... Ornithine decarboxylase ................................................................. Phenylalanine deaminase ............................................................... Beta-galactosidase (O-nitrophenyl-␤-D-galactopyranoside) ....... Christensen’s citrate........................................................................ Simmons citrate ............................................................................... Sodium acetate ................................................................................ Gelatin .............................................................................................. Voges-Proskauer (37°C) ................................................................. Salicin................................................................................................ Adonitol............................................................................................ Cellobiose ......................................................................................... Mannose ........................................................................................... Inositol .............................................................................................. Malonate........................................................................................... Mucate .............................................................................................. Sucrose.............................................................................................. Maltose ............................................................................................. Xylose ............................................................................................... Dulcitol ............................................................................................. Sorbitol ............................................................................................. Arabinose ......................................................................................... Raffinose........................................................................................... Rhamnose......................................................................................... Trehalose .......................................................................................... Glycerol ............................................................................................ NO3 from NO2 ................................................................................

⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ (⫹) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ (⫹) ⫹ ⫺ ⫺ ⫹ (⫹) ⫹

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rfb-RFLP. All the strains included in this study were analyzed for polymorphisms in the rfb chromosomal region by the rfb-RFLP method (8). The restriction patterns obtained were compared to those in the previously published Shigella and E. coli databases (8, 9) using Taxotron. The maximum fragment size variation accepted ranged from 7% for 500 bp to 3.5% for 4,000 bp.

RESULTS Biochemical tests. Table 1 presents the biochemical reactions for the 22 French isolates. The results agree with those for strain 96–204 and are typical of S. dysenteriae. The 12 French isolates tested and strain 96–204 utilized the following substrates, as determined with Biotype-100 strips: D-glucose, D-fructose, D-galactose, D-trehalose, D-mannose, Dribose, L-arabinose, glycerol, L-malate, N-acetyl-D-glucosamine, D-gluconate, DL-lactate, succinate, fumarate, D-glucosamine, and L-aspartate. Computer-assisted data analysis confirmed all of these strains as typical Shigella spp. Serological tests. All French isolates agglutinated solely in the presence of an antiserum raised against strain 96–204. Test for invasiveness. All 23 strains tested for the presence of an invasive plasmid gave positive results. Test for the presence of the Shiga toxin gene (stx). The PCR test for the presence of the stx gene was negative for all strains tested.

a ⫹, all strains positive (1 day); ⫺, all strains negative; (⫹), all strains delayed positive (4 to 7 days).

The extended biochemical reaction patterns of 12 randomly chosen isolates and strain 96–204 were determined with Biotype-100 strips (Bio-Me´rieux, La Balme-les-Grottes, France) as previously described (18). Results from Biotype100 strips were interpreted by using the software Recognizer (Institut Pasteur) or the web-based software RecogNet (written by Roney S. Coimbra) available at http://www.pasteur.fr/recherche/banques/recognet. Both programs compare results for test strains with those for reference strains in a matrix containing the entire family Enterobacteriaceae. Serological tests. The 22 French isolates were tested by slide agglutination assays (14) using appropriate antisera-raised against all recognized Shigella serotypes (purchased from Eurobio, Les Ulis, France, or provided by the FNCSS) and antiserum raised against the potentially new serotype represented by strain 96–204. The production of antiserum against strain 96–204 has been described elsewhere (27). Test for invasiveness. The 22 French isolates and strain 96–204 were tested for the presence of the invasive plasmid by the PCR system proposed by Lampel et al. (22). Test for the presence of the Shiga toxin gene (stx). The French isolates and strain 96–204 were tested for the presence of the stx gene by the PCR system described by Lin et al. (24). Ribotyping. All the strains included in this study were ribotyped after digestion of total DNA with endonuclease MluI by following a previously published protocol (10, 17, 25, 30). Ribopatterns were automatically compared to the Shigella and E. coli MluI ribotyping pattern databases using Taxotron software (Institut Pasteur). A maximum fragment size variation of 5.0% was accepted.

FIG. 1. Ribotype shown by the French isolates and strain 96–204. Lanes M, Citrobacter koseri CIP105177 DNA cleaved by MluI; lanes 1, pattern of strain 96–204 and S. dysenteriae serotypes 3 and 12.

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FIG. 2. Unique rfb-RFLP pattern shown by the French isolates and strain 96–204. Lanes M, mix of Lambda/HindIII (Promega, Madison, Wis.) and AmpliSize Molecular Ruler (Bio-Rad Laboratories, Hercules, Calif.) standards; lanes 1, strain 96–204; lane 2, S. dysenteriae serotype 3; lane 3, S. dysenteriae serotype 12.

Ribotyping. The French isolates and strain 96–204 showed the same ribotype (Fig. 1), which corresponded to ribotype A3a/A12b (10) represented by S. dysenteriae serotype 3 (strain NCDC3990-73) and S. dysenteriae serotype 12 (strain 3–89) in our database. rfb-RFLP. The same restriction pattern was shown by all French isolates and strain 96–204. This unique pattern (Fig. 2) was clearly different from any other pattern in our database for Shigella and E. coli. DISCUSSION Shigella infections remain an important public health problem worldwide (7). Shigellae are nonmotile, nonencapsulated bacilli that conform to the definitions of the family Enterobacteriaceae and the tribe Escherichiae. Several studies on nutritional characterization, numerical taxonomy, and enzyme electrophoresis (11, 16, 21, 28, 32) have justified the historical differentiation between Shigella spp. and E. coli and the recognition of four species within the genus Shigella: S. dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei, also known as groups A, B, C, and D, respectively. In contrast to most E. coli strains, shigellae are typically non-lactose fermenting; anaerogenic; and lysine decarboxylase, acetate, mucate, and Christensen’s citrate negative. Exceptions are S. sonnei, which ferments lactose slowly and can be mucate positive, and S. flexneri serotype 6, S. boydii serotype 13, and S. boydii serotype 14 (Sachs/A12 biotype), which can produce gas from glucose (15). DNA hybridization studies revealed more than 73%

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DNA relatedness between Shigella (with the exception of S. boydii serotype 13) and E. coli K-12. In view of this fact, these organisms have been placed in the same genomic group (2, 3, 4). The close relatedness to E. coli makes serological identification a crucial step in the diagnosis of Shigella infections. Based on their somatic antigen (O-antigen) properties, S. dysenteriae, S. boydii, and S. flexneri are subdivided into 15, 18, and 8 serotypes, respectively, which are given numerical designations. S. flexneri serotypes 1 to 5 are subsequently subdivided into subserotypes. S. sonnei has only one serotype, which is divided into biotypes a, d, e, f, and g (1, 20). New strains are described as provisional Shigella serotypes until a sufficient number of isolates have been collected to determine whether a provisional serotype should be assigned to a Shigella nomenspecies. Validation is done by the World Health Organization Collaborating Center. In this study, 22 French isolates characterized by biochemical, serological, and molecular features proved to belong to the potentially new serotype represented by strain 96–204, previously characterized by Matsushita et al. (27). Molecular analysis of ribosomal DNA suggested a relationship of this serotype to S. dysenteriae serotypes 3 and 12. However, rfbRFLP clearly distinguished this new serotype from any other Shigella or E. coli serotype. In conclusion, our data strengthen the prior proposition of Matsushita et al. (27) for a new Shigella serotype represented by strain 96–204. Moreover, the importance of molecular methods as complementary tools for serotyping was highlighted. ACKNOWLEDGMENTS We thank Nancy A. Strockbine (WHO Collaborating Center, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Ga.) for kindly providing some S. dysenteriae strains and Isabelle Carle for valuable technical help. Roney S. Coimbra was the recipient of a CNPq-Brazil fellowship. REFERENCES 1. Ansaruzzaman, M., A. K. M. G. Kibriya, A. Rahman, P. K. B. Neogi, A. S. G. Faruque, B. Rowe, and M. J. Albert. 1995. Detection of provisional serovars of Shigella dysenteriae and designation as S. dysenteriae serotypes 14 and 15. J. Clin. Microbiol. 33:1423–1425. 2. Brenner, D. J., G. R. Fanning, F. J. Skerman, and S. Falkow. 1972. Polynucleotide sequence divergence among strains of Escherichia coli and closely related organisms. J. Bacteriol. 109:953–965. 3. Brenner, D. J., G. R. Fanning, G. V. Miklos, and A. G. Steigerwalt. 1973. Polynucleotide sequence relatedness among Shigella species. Int. J. Syst. Bacteriol. 23:1–7. 4. Brenner, D. J., A. G. Steigerwalt, G. H. Wathen, R. J. Gross, and B. Rowe. 1982. Confirmation of aerogenic strains of Shigella boydii 13 and further study of Shigella serotypes by DNA relatedness. J. Clin. Microbiol. 16:432–436. 5. Brenner, D. J. 1984. Recommendations on recent proposals for the classification of shigellae. Int. J. Syst. Bacteriol. 34:87–88. 6. Carlin, N. I. A., and A. A. Lindberg. 1983. Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri: clones binding to II, II:3, 4, and 7, 8 epitopes. J. Clin. Microbiol. 18:1183–1189. 7. Carlin, N. I. A., T. Wehler, and A. A. Lindberg. 1986. Shigella flexneri Oantigen epitopes: chemical and immunochemical analyses reveal that epitopes of type III and group 6 antigens are identical. Infect. Immun. 53:103–109. 8. Coimbra, R. S., F. Grimont, and P. A. D. Grimont. 1999. Identification of Shigella serotypes by restriction of amplified O-antigen gene cluster. Res. Microbiol. 150:543–553. 9. Coimbra, R. S., F. Grimont, P. Burguie`re, P. Lenormand, L. Beutin, and P. A. D. Grimont. 2000. Identification of Escherichia coli O-serogroups by restriction of amplified O-antigen gene cluster. Res. Microbiol. 151:639–654. 10. Coimbra, R. S., G. Nicastro, P. A. D. Grimont, and F. Grimont. Identification of Shigella species by rRNA gene restriction patterns. Res. Microbiol., in press.

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