Phenotypic and Genotypic Characterization of Provisional Serotype

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2003, p. 110–117 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.1.110–117.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 1

Phenotypic and Genotypic Characterization of Provisional Serotype Shigella flexneri 1c and Clonal Relationships with 1a and 1b Strains Isolated in Bangladesh Kaisar A. Talukder,1* Zhahirul Islam,1 M. Aminul Islam,1 Dilip K. Dutta,1 Ashrafus Safa,1 M. Ansaruzzaman,1 A. S. G. Faruque,1 Shamima N. Shahed,2 G. B. Nair,1 and David A. Sack1 International Centre for Diarrhoeal Diseases Research, Bangladesh,1 and Department of Biochemistry, University of Dhaka,2 Dhaka-1000, Bangladesh Received 19 July 2002/Returned for modification 14 September 2002/Accepted 6 October 2002

The serotypes of 144 strains of Shigella flexneri serotype 1 (serotypes 1a, 1b, and 1c) isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, between 1997 and 2001 were serologically confirmed by using commercially available antisera and a panel of monoclonal antibodies specific for S. flexneri group and type factor antigen (MASF). Among serotype 1 isolates, the prevalence of provisional serotype S. flexneri 1c increased from 0 to 56% from 1978 to 2001 in Bangladesh. Detailed biochemical studies revealed that none of the strains of serotype 1 produced indole, while all the strains fermented mannose, mannitol, and trehalose. Twenty percent of the serotype 1c and all the serotype 1a strains fermented maltose and 53% of the serotype 1c strains and 60% of the serotype 1a strains fermented arabinose, whereas all serotype 1b strains were negative for fermentation of these sugars. Only 18% of serotype 1b strains were resistant to nalidixic acid, and most of the serotype 1c and 1b strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. All the strains of serotypes 1a and 1b and about 88% of the serotype 1c strains were found to be invasive by the Sereny test, had a 140-MDa plasmid, and had Congo red absorption ability. Plasmid profile analysis showed that 26% of the strains of serotype 1 contained identical patterns. Most of the serotype 1c strains (72%) had the 1.6-MDa plasmid, which was not found in either serotype 1a or 1b strains. A self-transmissible middle-range plasmid (35 to 80 MDa) was found in some strains carrying the multiple-antibiotic-resistance gene. Pulsed-field gel electrophoresis analysis yielded three types (types A, B, and C) with numerous subtypes among the serotype 1c strains, whereas serotypes 1b and 1a yielded only one type for each serotype, and those types were related to the types for serotype 1c strains. Ribotyping analysis yielded three patterns for serotype 1c strains and one pattern each for serotype 1a and 1b strains which were similar to the patterns for the serotype 1c strains. Overall analysis of the results concluded that subserotype 1c is closely related to serotypes 1a and 1b. Furthermore, the high rate of prevalence of serotype 1c necessitates the commercial production of antibody against this subserotype to allow the determination of the actual burden of shigellosis caused by provisional serotype 1c. strains or newer subserotypes are being isolated from different parts of the world, including Bangladesh (45). Among the different provisional serotypes, S. flexneri serotype 1c is a recent addition. According to our previous study, it has been shown that S. flexneri is the predominant species in Bangladesh and serotype 1 is the second most prevalent group after serotype 2 (44). This high rate of prevalence of serotype 1 is largely attributable to provisional serogroup 1c. It was observed that the prevalence of serotype 1c among all isolates of S. flexneri increased from 0% in 1978 to 1984 to 8.2% in 1997 to 2000, whereas that of serotype 1a decreased drastically from 13.1 to 0.4% (44; K. A. Talukder, Q. S. Ahmad, K. Haider, and M. I. Huq, Abstr. Annu. Meet. Bangladesh Soc. Microbiol., p. 2, 1987). This changing trend is continuing in Bangladesh, emphasizing the importance of continuous monitoring of S. flexneri serotype distributions. Carlin et al. (7) first identified serotype 1c in Bangladesh. An extensive study of the lipopolysaccharide structure of serotype 1c strains was done which showed significant differences between other subserotypes (subserotypes 1a and 1b) and serotype 1c (7, 20). Later, El-Gendy et al. (11) reported on the occurrence of similar

Shigellosis is one of the major diarrheal diseases in Bangladesh and several other countries and is responsible for a significant number of deaths, especially among children (8, 23, 32). Shigellosis is caused by four species of Shigella, namely, Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei. Each serogroup contains multiple serotypes based on the structure of the O-antigen component of the lipopolysaccharide present on the outer membrane of the cell wall (40). In areas where diarrheal disease is endemic, S. flexneri is usually the most prevalent species (13, 15). Until recently, at least 47 serotypes of Shigella were recognized, of which 15 belong to S. flexneri (8). More elaborately, S. flexneri has eight serotypes, of which serotypes 1 to 5 are further classified into 12 subserotypes. Besides these, there are also some provisional and new subserotypes of S. flexneri (31, 44). Nonetheless, this classification scheme for S. flexneri is not comprehensive because atypical * Corresponding author Mailing address: Enteric Bacteriology Laboratory, Laboratory Sciences Division, International Centre for Diarrhoeal Diseases Research, Bangladesh, GPO Box 128, Dhaka-1000, Bangladesh. Phone: 880 2 8811751-60. Fax: 880 2 8812529 or 880 2 8823116. E-mail: [email protected]. 110

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strains in rural Egypt and concluded that the new antigenic determinant (E1037) and the serotype 4-specific antigen of S. flexneri are present in some strains of serotype 1c together with the common antigen. However, no study has yet extensively characterized these newly emerging strains of subserotype 1c. Therefore, as it is a provisional serotype (subserotype 1c), the high rate of isolation in developing countries like Bangladesh indicated that there is a great necessity to study the overall characteristics of strains of this serotype and to determine the clonal diversity among other subserotypes of S. flexneri, particularly subserotypes 1a and 1b, by various phenotypic and genotypic techniques. MATERIALS AND METHODS Bacterial strains. The 144 clinical isolates of S. flexneri serotype 1, of which 6 strains belonged to serotype 1a, 70 belonged to serotype 1b, and 68 belonged to serotype 1c, used in this study were isolated from patients attending the Dhaka treatment center operated by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), in Dhaka, Bangladesh, between January 1997 and December 2001. These strains were isolated and identified in the Clinical Microbiology Laboratory by standard microbiological and biochemical methods (49). The strains were grown in Trypticase soy broth containing 0.3% yeast extract (TSBY) and stored at ⫺70°C after addition of 15% glycerol. S. flexneri strain YSH6000, serotype 2a (37), and an Escherichia coli (ATCC 25922) strain that lacked the 140-MDa invasive plasmid and that was sensitive to all antibiotics were used as positive and negative controls, respectively, in the Sereny test, the test for Congo red binding ability, and the PCR assay for detection of the ipaH gene and the Shigella enterotoxin gene (set). E. coli strains PDK-9, V-517, Sa, RP4, and R1 were used as plasmid molecular weight standards (14). E. coli K-12 (lac⫹ F⫺), resistant to nalidixic acid, was used as the recipient in the conjugation experiments (14). Serotyping. The serotypes of the 144 strains were confirmed by using two serotyping kits: (i) a commercially available kit (Denka Seiken, Tokyo, Japan) with antisera specific for all type- and group-factor antigens and (ii) monoclonal antibody reagents (Reagensia AB, Stockholm, Sweden) specific for all S. flexneri type- and group-factor antigens. The strains were subcultured on MacConkey agar (Difco, Becton Dickinson & Company, Sparks, Md.) plates, and after about 18 h of incubation, serological reactions were performed by the slide agglutination test, as described previously (44). Biochemical characterization. The biochemical reactions of the strains were determined by standard methods (49). Antimicrobial susceptibility. The susceptibilities of the bacteria to the antimicrobial agents tested were determined by the disk diffusion method, as recommended by the National Committee for Clinical Laboratory Standards (29), with commercial antimicrobial discs (Oxoid, Basingstoke, United Kingdom). The antibiotic discs used in this study were ampicillin (10 ␮g), tetracycline (30 ␮g), mecillinam (25 ␮g), nalidixic acid (30 ␮g), sulfamethoxazole-trimethoprim (25 ␮g), and ciprofloxacin (5 ␮g). E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as control strains for susceptibility studies. Keratoconjunctivitis assay (Sereny test). The Sereny test was performed by a procedure described elsewhere (25, 39). Briefly, an overnight culture of bacteria, suspended to a density of approximately 1010 viable cells in 20 ␮l of phosphatebuffered saline, was dropped into the conjunctival sacs of guinea pigs. One eye served as the control. The guinea pigs were observed daily for 72 h, and their inflammatory responses were graded. Determination of Congo red binding ability. TSBY with 1.5% agar and 0.01% Congo red (Sigma Chemical Co. Ltd.) was used to study the pigment binding abilities of the test strains by previously described procedures (35, 38). Isolation of plasmid DNA. Plasmid DNA was prepared by the alkaline lysis method of Kado and Liu (18), with some modifications as indicated previously (45). The molecular weight of the unknown plasmid DNA was assessed by comparing with the mobilities of the plasmids of known molecular weights (14). The plasmids present in previously described strains E. coli PDK-9, R1, RP4, Sa, and V-517 (45) were used as molecular weight standards. Determination of resistance factor. A conjugation experiment between the multidrug-resistant (Ampr Sxtr Ter) donors, S. flexneri serotype 1c (K-265) and serotype 1b (K-4212) strains, and the recipient, E. coli K-12 (Nalr Lac⫹ F⫺), was carried out by a previously described method (27). Transconjugant colonies were selected on MacConkey agar plates containing nalidixic acid (30 ␮g/ml) and

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TABLE 1. Agglutination reactions of S. flexneri serotype 1 strains tested with MASF Reaction with MASF S. flexneri subserotype

Type antigen specific I

1a 1b 1c

II

⫹ ⫺ ⫹ ⫺ ⫺ ⫺

Group antigen specific

IV-2

V

VI

B

⫺ ⫺ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺

⫹ ⫹ ⫹

Y-5

6

7,8

IV-1

⫹ ⫺ ⫺ ⫹ ⫺ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺

Provisional serotype 1c

⫹

ampicillin (50 ␮g/ml). Plasmid analysis and antimicrobial susceptibility testing of the transconjugants were carried out to determine the transfer of plasmids with antibiotic resistance. Determination of transfer frequency and curing of the resistance plasmid were carried out by a method described earlier (27). Detection of Shigella enterotoxin genes (set1 and sen) and ipaH by PCR. Detection of the set1 gene (ShET-1), the sen gene (ShET-2), and the ipaH gene was performed by amplification by PCR with primers set1A, set1B, sen, and ipaH by previously described procedures (47). All these primers were synthesized with an Oligo 1000 DNA synthesizer (Beckman), available in our laboratory at ICDDR,B. PFGE. Intact agarose-embedded chromosomal DNA from clinical isolates of S. flexneri serotype 1 were prepared, and pulsed-field gel electrophoresis (PFGE) was performed with a contour-clamped homogeneous electric field (CHEFDRII) apparatus from Bio-Rad Laboratories (Richmond, Calif.) by procedures described earlier (2, 30, 43, 45), but with different pulse times: 3 to 28 s for 8 h, 5 to 50 s for 8 h, 20 to 80 s for 11 h, and 60 to 120 s for 11 h. Genomic DNA was digested with the NotI restriction enzyme (GIBCO-BRL, Gaithersburg, Md.). The restriction fragments were separated by using the CHEF-DRII system apparatus in 1% pulsed-field-certified agarose in 0.5⫻ TBE (Tris-borate-EDTA) buffer; the gel was stained, destained, and photographed on a gel documentation system by procedures described earlier (45). The DNA size standards used were the bacteriophage lambda ladder (size range, 48.5 to 1,000 kb; Bio-Rad Laboratories) and Saccharomyces cerevisiae chromosomal DNA (size range, 225 to 2,200 kb; Bio-Rad Laboratories). Band patterns were established by use of the criteria described previously (46). Ribotyping. Total cellular DNA was extracted and purified by procedures described previously (45). DNA was digested with the HindIII restriction enzyme overnight at 37°C according to the instructions of the manufacturer (GIBCOBRL) and separated by gel electrophoresis in 0.8% agarose in TBE buffer for 18 h at a constant voltage of 35 V. Southern blotting to a positively charged nylon membrane (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) was performed with a vacuum pump unit (Bio-Rad Laboratories), and the DNA fragments were fixed to the membrane by exposure to UV light for 3 min. A digoxigenin (DIG)-labeled cDNA probe specific for 16S ribosomal DNA (19) was constructed by the procedure described earlier (45). Hybridization of the membrane with the DIG-labeled probe for 18 h at 42°C and development of the membrane with anti-DIG-alkaline phosphatase were performed according to the instructions provided in a DIG DNA labeling and detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The results were documented by taking photographs of the membrane in which the probe had hybridized with the separated DNA fragments.

RESULTS Serological typing and prevalence. All 68 strains of S. flexneri serotype 1c agglutinated with the monoclonal antibody specific for S. flexneri (MASFB), but none of the strains could be serotyped with certainty by using the commercially available antiserum kit (Denka Seiken). All strains reacted strongly with only the provisional serotype S. flexneri serotype 1c-specific monoclonal antibody. The serotypes of strains of serotypes 1a (n ⫽ 6) and 1b (n ⫽ 70) were confirmed by using the commercially available antiserum kit (Denka Seiken) as well as a monoclonal antibody specific for S. flexneri group and type factor antigen (MASF) (Table 1). The prevalence of provisional serotype S. flexneri 1c had increased from 0 to 56% from

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TABLE 2. Incidence of S. flexneri serotype 1 strains isolated from 1978 to 2001 No. (%) of strains isolated in the following yr: Subserotype

1978–1984a (n ⫽ 240)

1985–1987b (n ⫽ 45)

1997–2000c (n ⫽ 108)

2001c (n ⫽ 56)

1a 1b 1c

154 (64) 86 (36) 0 (0)

24 (53) 19 (43) 2 (4)

2 (2) 69 (64) 37 (34)

4 (7) 21 (37) 31 (56)

a Results reported by Talukder et al. (Abstr. Annu. Meet. Bangladesh Soc. Microbiol., p. 2, 1987). b Results reported by Carlin et al. (7). c Results of the present study.

1978 to 2001 in Bangladesh, while at the same time the prevalence of subserotype 1a had decreased from 64 to 7% (Table 2). Biochemical characterization. All strains of S. flexneri serotype 1 (serotypes 1a, 1b, and 1c) examined possessed the biochemical characteristics typical of S. flexneri (10). None of the strains of serotype 1 produced indole, while all of the strains fermented mannose, mannitol, and trehalose. About 22% of serotype 1c strains and all of the strains of serotype 1a fermented maltose. Arabinose was fermented by 53% of the serotype 1c strains and 60% of the serotype 1a strains (Table 3). All strains of serotype 1b were negative for arabinose and maltose fermentation. Serotype 1 strains did not utilize sodium acetate, rhamnose, sorbitol, dulcitol, xylose, raffinose, arginine, lysine, or ornithine. The overall biochemical reactions grouped all serotype 1c strains into four biotypes and all serotype 1b strains into a single biotype (Table 4).

TABLE 3. Biochemical characteristics of S. flexneri serotypes 1a, 1b, and 1c % Positivea Substrate or test

Serotype 1a (n ⫽ 6)

Serotype 1b (n ⫽ 70)

Serotype 1c (n ⫽ 68)

Methyl red Citrate (Simmon’s) Indole Lysine decarboxylase Arginine dehydrolase Ornithine decarboxylase Glucose (acid) Glucose (gas) Sucrose Mannitol Dulcitol Salicin Inositol Adonitol Sorbitol Arabinose Raffinose Rhamnose Trehalose Sodium acetate Maltose Xylose Mannose

100 0 0 0 0 0 100 0 0 100 0 0 0 0 0 0 (60) 0 0 100 0 0 (100) 0 100

100 0 0 0 0 0 100 0 0 45 (55) 0 0 0 0 0 0 0 0 100 0 0 0 100

100 0 0 0 0 0 100 0 0 40 (60) 0 0 0 0 0 0 (53) 0 0 100 0 0 (22) 0 100

a

The results are for times of 24 to 48 h (⬎48 h to 14 days).

TABLE 4. Biochemical patterns of S. flexneri serotype 1 strains Subserotype

Biotypea

No. (%) of strains

1b

B1

70 (100)

1c

B1 B2 B3 B4

27 (40) 26 (38) 8 (11) 7 (11)

a Biotype B1, mannose, mannitol, and trehalose positive; biotype B2, arabinose, mannose, mannitol, and trehalose positive; biotype B3, arabinose, mannose, mannitol, maltose, and trehalose positive; biotype B4, mannose, mannitol, maltose, and trehalose positive.

Antibiotic susceptibility test. The percentage of strains of serotypes 1a, 1b, and 1c resistant to a particular antibiotic is described in the Table 5. Eighteen percent of serotype 1b strains were found to be resistant to multiple antibiotics (ampicillin, tetracycline, trimethoprim-sulfamethoxazole, and nalidixic acid). Similarly, 47% of serotype 1b strains and 40% of serotype 1c strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. On the contrary, no multidrug-resistant serotype 1a strains were found. Test for invasiveness. With the exception of eight serotype 1c strains, all strains of serotype 1 harbored the 140-MDa invasive plasmid, had the ability to bind to Congo red, and were positive for the ipaH gene; and guinea pig eyes were positive for keratoconjunctivitis caused by representative strains, attesting to their invasive trait. Eight strains were noninvasive by the Sereny test and did not contain the 140-MDa plasmid, nor did they bind to Congo red. Plasmid profile analysis. Analysis of plasmid DNA revealed that all serotype 1a and 1b strains and about 88% of the serotype 1c strains harbored the 140-MDa plasmid. Heterogeneous plasmid patterns were obtained among the serotype 1b and 1c strains, while only two patterns were observed among the serotype 1a strains (Fig. 1). Interestingly, 26% of the serotype 1 strains (serotypes 1a, 1b, and 1c) had identical patterns (140, 2.8, 2.1, 1.8, and 1.0 MDa). Most of the serotype 1c strains (72%) contained the 1.6-MDa plasmid, which was not found in strains of either serotype 1a or serotype 1b (Fig. 1). Similarly, 78% of the serotype 1b strains contained the 4-MDa plasmid, which was found in only 22% of serotype 1c strains (Fig. 1). Beside these small plasmids, a middle-range plasmid (80 to 35 MDa) was found in 50% of serotype 1a strains, 7% of serotype 1b strains, and 20% of serotype 1c strains. Determination of resistance factor. Strains (one each) of serotypes 1c and 1b with the same antibiotic resistance pattern (Ampr Sxtr Ter) were selected for the conjugation experiment with E. coli K-12 (Lac⫹ F⫺ Nalr). After conjugation, the 50TABLE 5. Antibiotic resistance of S. flexneri serotype 1 strains Serotype

1a (n ⫽ 6) 1b (n ⫽ 70) 1c (n ⫽ 68)

% of strains resistant toa: Te

Amp

Sxt

Nal

Mec

Cip

33 100 97

33 94 97

17 72 43

0 18 0

0 0 0

0 0 0

a Te, tetracycline; Amp, ampicilin; Sxt, trimethoprim-sulfamethoxazole; Nal, nalidixic acid; Mec, mecillinam; Cip, ciprofloxacin.


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FIG. 1. Agarose gel electrophoresis of plasmid DNA showing the representative patterns among isolates of S. flexneri serotype 1c along with those of serotype 1a and 1b strains. Lanes: B and C, S. flexneri serotype 1a (patterns P1a and P2a); D and E, serotype 1b (patterns P2b and P1b); F, serotype 1c (pattern P1c); G, serotype 1c (pattern P8c); H, serotype 1c (pattern P3c); I, serotype 1c (pattern P5c); J, serotype 1c (pattern P4c); K, serotype 1c (pattern P7c); L, serotype 1c (pattern P6c); M, serotype 1c (pattern P2c); N, serotype 1c (pattern P9c); O and A, strains PDK-9 and strains R1 and V-517 (marker). CHR, banding position of chromosomal DNA.

MDa plasmid from serotype 1c strains and the 60-MDa plasmid from serotype 1b strains were transferred independently with the complete spectrum of drug resistance (Ampr Sxtr Ter). The transfer frequency was very high for all transmissible plasmids. All the transconjugants were cured by loss of the plasmids and again became sensitive to all antibiotics (Table 6). Detection of Shigella enterotoxin genes (set1 and sen) and ipaH gene by PCR assays. The Shigella enterotoxin 1 gene (set1) was absent from all strains of serotype 1, while the Shigella enterotoxin 2 gene (sen) and ipaH gene were present in all strains except eight (12%) strains of serotype 1c, which did not harbor the 140-MDa plasmid. PFGE. PFGE analysis of NotI-digested chromosomal DNA of the S. flexneri serotype 1 strains yielded 13 to 18 reproducible DNA fragments ranging in size from approximately 20 to 1,050 kb (Fig. 2). Three major PFGE types designated A, B, and C were obtained among the serotype 1c strains, of which 71% of the strains were of type A, 19% were of type B, and 10% were of type C (Fig. 2). Type A was further subdivided into three subtypes (subtypes A1 to A3). Type B was also subdivided into two subtypes (subtypes B1 and B2). On the other hand, strains of serotypes 1b and 1a yielded only one type each (types D and E, respectively). Type D of subserotype 1b

was further subdivided into nine subtypes (subtypes D1 to D9) (Fig. 2). Ribotyping. Three different reproducible rRNA gene restriction patterns (ribotypes R1, R2, and R3) were obtained among the S. flexneri serotype 1c strains. The sizes of the bands in all patterns ranged from 15 to 5 kb, and the size distribution was optimum for the discrimination of the strains. The ribotyping patterns of S. flexneri serotypes 1a and 1b were found to be the same as patterns R1 and R2 (Fig. 3) of serotype 1c. However, pattern R3 was unique to S. flexneri serotype 1c. DISCUSSION S. flexneri serotype 1 was the second most prevalent serotype isolated between January 1997 and June 2000 from patients at the ICDDR,B diarrhea treatment center in Bangladesh (44). This high rate of prevalence was largely attributable to the provisional subserotype, namely, S. flexneri serotype 1c, whose prevalence increased from 0% (1978 to 1984) to 4.5% (1997 to 2000) of the total isolates of S. flexneri (44). We have observed in this study that the prevalence of subserotype 1c increased sharply from 0 to 56% from 1978 to 2001, while the prevalence

TABLE 6. Transfer of resistance plasmid to E. coli K-12 by conjugation Strain no. (serotype)

K-265 (1c) K-4212 (1b) a

Parent strain

Transconjugant

Resistance patterna

Plasmid profile (MDa)

Resistance pattern


Amp Sxt Te Amp Sxt Te

140, 50, 2.8, 2.1, 1.8, 1.0 140, 60, 2.8, 4.0, 2.1, 1.8, 1.0

Amp Sxt Te Nal Amp Sxt Te Nal

50 60

See footnote a of Table 5 for definitions of abbreviations.

Frequency of R plasmid transfer

5.0 ⫻ 10⫺4 4.8 ⫻ 10⫺4

Cured strain Resistance pattern


Nal Nal

No plasmid No plasmid

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FIG. 2. PFGE patterns of NotI-digested chromosomal DNA from representative strains of S. flexneri subserotype 1c along with serotype 1a and 1b strains. Lanes: A, S. cerevisiae (marker); B, K-212 serotype 1c (PFGE type A1); C, K-325 serotype 1c (PFGE type A1,); D, K-381 serotype 1c (PFGE type A1); E, K-448 serotype 1c (PFGE type A1); F, K-265 serotype 1c (PFGE type C); G, K-482 serotype 1c (PFGE type B); H, K-639 serotype 1c (PFGE type A1); I, K-734 serotype 1c (PFGE type A2); J, K-840 serotype 1c (PFGE type A2); K, K-1622 serotype 1c (PFGE type A1); L, K-1614 serotype 1c (PFGE type A1); M, K-817 serotype 1b (PFGE type D4); N, K-647 serotype 1a (PFGE type E).

of subserotype 1a decreased from 64 to 7% among the subserotypes of S. flexneri serotype 1 in Bangladesh (Table 2). Analysis of the O-antigenic structure reveals that subserotypes 1a, 1b, and 1c share common immunodominant elements of serotype 1, but in addition, they have some side-chain residues which are different from one another (48). Since commercial antisera have not yet been developed for serotype 1c, we used monoclonal antibody reagents to type these strains. Unlike serotypes 1a and 1b, which have specific group- and type-factor antigens, serotype 1c does not possess any; rather, it reacts only with the antibody specifically developed against it (Table 1). All strains of serotype 1c had the biochemical characteristics typical of S. flexneri serotype 1, but there were some differences when serotype 1c strains were compared with serotype 1a and 1b strains. Twenty percent of serotype 1c strains and all strains of serotype 1a fermented maltose. Arabinose was fermented by 53% of the serotype 1c strains and 60% of the serotype 1a strains. All serotype 1b strains were negative for fermentation of these sugars (Table 3). According to Edwards and Ewing (10), 89% of the strains of serotypes 1a and 1b were positive for raffinose fermentation, but none of the strains in this ex-

J. CLIN. MICROBIOL.

periment were positive for raffinose fermentation (Table 3). Analysis of biochemical reactions indicated the presence of four biotypes among serotype 1c strains and one biotype among serotype 1b strains (Table 4). We did not assign a biotype status to serotype 1a strains, even though four (60%) strains fermented arabinose and two (40%) strains did not ferment arabinose, because of the small number of strains analyzed in the study. Interestingly, 40% of the serotype 1c strains showed a biochemical pattern identical to that of serotype 1b strains (Table 4). The prevalence of antimicrobial resistance among Shigella species has been increasing over the last decade (21, 34). Since antibiotic resistance is a major phenotypic trait, particularly for clinical isolates, there is potential interest in exploring the characteristics of S. flexneri serotype 1. None of the strains of serotype 1c were resistant to nalidixic acid, mecillinam, or ciprofloxacin (Table 5). The lack of resistance to the extendedspectrum antibiotics by serotype 1c strains indicates that these strains have not yet been exposed to the selective pressures of these antibiotics. On the other hand, all serotype 1b strains were resistant to tetracycline, 94% were resistant to ampicillin, 72% were resistant to trimethoprim-sulfamethoxazole, and 18% were resistant to nalidixic acid (Table 5). None of the strains were resistant to mecillinam or ciprofloxacin. Serotype 1b has been prevalent in Bangladesh for a long time (44). This continuous prevalence has facilitated the ability of serotype 1b strains to cope with the selective pressures created in the native environment by the overuse of common antibiotics like nalidixic acid. Although S. flexneri has not yet been found to be resistant to ciprofloxacin, nalidixic acid- and mecillinam-resistant strains of S. flexneri have frequently been isolated in Bangladesh (5, 34). The emergence of resistance to multiple antibiotics among Shigella isolates poses a major public health problem (16) throughout the developing world including Bangladesh. In the present study, of all isolates tested, 42% were resistant to three commonly used antibiotics, ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. In fact, these results show that the multidrug resistance among serotype 1 strains could be conferred by plasmids and derived from other organisms by plasmid transfer. Few published data are available on the association of plasmids of S. flexneri and serotypes. However, previously published reports revealed a heterogeneous plasmid population in strains of S. flexneri, with most plasmids being smaller than 6 MDa (17, 42). A correlation between serotype and plasmid pattern was described in a previous report, in which it was suggested that the plasmid profile might be useful in the identification of epidemic clones of S. flexneri as they are introduced in a population (24). In our previous study (45), we have shown that plasmid profiling had been used as a significant method for the identification and characterization of atypical S. flexneri isolates. A good correlation was observed between plasmid profiles and the results of other typing procedures like biotyping, PFGE, and ribotyping. It was also suggested that some small plasmids present in the strains maintain their existence as a stable gene pool, so these could be used as an extrachromosomal marker for identification of new strains and for differentiation of the existing serotypes as well. In the present study, analysis of plasmid DNA of S. flexneri serotype 1 has shown that all strains contained multiple plasmids rang-

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FIG. 3. Ribotyping patterns of representative strains of S. flexneri subserotypes 1a, 1b, and 1c. Lanes: A, B, and C, S. flexneri serotype 1a (K-647, K-480 and K-4077, respectively; ribotype R1); D, E, and F, S. flexneri serotype 1b (K-817, K-828, and K-4214, respectively; ribotype R2); G, H, J, and I, S. flexneri serotype 1c (K-212, K-381, K-840, and K-796, respectively; ribotype R2); K and L, S. flexneri serotype 1c (K-406 and K-265, respectively; ribotype R3); M and N, S. flexneri serotype 1c (K-572 and K-482, respectively; ribotype R1).

ing from 140 to 1.0 MDa. All strains of serotypes 1a and 1b and about 88% of the serotype 1c strains harbored the 140-MDa plasmid. Plasmid profile analysis showed that 26% of the strains of serotype 1 (serotypes 1a, 1b, and 1c) had identical plasmid patterns (140, 2.8, 2.1, 1.8, and 1.0 MDa). All 144 strains harbored plasmids of 2.8, 2.1, 1.8, and 1.0 MDa. These four plasmids were present in both susceptible and resistant strains and appeared to constitute a stable gene pool. Therefore, these plasmids might be considered the serotype-specific plasmids or core plasmids of this serotype. Most of the serotype 1c strains (72%) yielded the 1.6-MDa plasmid, which was not found in serotype 1a and 1b strains (Fig. 1). On the other hand, 78% of the serotype 1b strains contained a 4-MDa plasmid, whereas only 22% of the serotype 1c strains harbored the same plasmid. The present study showed that four core plasmids are unique to strains of S. flexneri serotype 1, perhaps reflecting their dissemination from a single origin. Plasmid analysis also revealed that about 50% of the serotype 1a strains, 20% of the serotype 1c strains, and 7% of the serotype 1b strains contained a middle-range plasmid (35 to 80 MDa). Conjugation and curing experiments demonstrated that these plasmids are self-transmissible and confer resistance to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole (Table 6). These results are essentially similar to those presented in reports of previous studies (45). It is suggested by a number of studies (26, 36, 37) that the genetic loci for the invasiveness of S. flexneri are located on the large 140-MDa plasmid. All strains except eight of the serotype 1c strains harbored the 140-MDa plasmid, had the ipaH gene, had the capacity to absorb Congo red dye, and were able to provoke keratoconjunctivitis in guinea pig eyes, attesting to their invasiveness. According to the previous studies, no strain of S. flexneri lacking the 140-MDa invasive plasmid has yet been isolated directly from patients. This may be the first report of the isolation of noninvasive S. flexneri strains from patients. Although the cardinal feature in the pathogenesis of S. flexneri infection involves the invasion of epithelial cells, it nevertheless has been reported that S. flexneri also produces an enterotoxin (22, 33), mainly of two types, Shigella enterotoxin

1 (ShET-1) and Shigella enterotoxin 2 (ShET-2). In this study, we found that none of the strains were positive for the set1 gene, but all representative strains with the 140-MDa plasmid were positive for the sen gene. However, the strains lacking the 140-MDa plasmid were negative for the sen gene as well. It is now known that the sen gene is located on the 140-MDa invasive plasmid and therefore is present in all strains of S. flexneri that harbor this plasmid (28). By the standard method described by Tenover et al. (46), all strains of serotype 1c were grouped into three different PFGE types (types A, B, and C) with numerous subtypes. Similarly, serotype 1b yielded one type (type D) with several subtypes (subtypes D1 to D9), while serotype 1a yielded only one type (type E) (Fig. 2). Interestingly, the two PFGE types of serotype 1c (types A and B) in the present study were closely related to the PFGE types of serotypes 1b and 1a, respectively. However, the remaining type, i.e., type C, was completely different from the others, and this could be defined as a new clone of S. flexneri serotype 1c prevailing in Bangladesh. On the basis of rRNA gene restriction pattern analysis, three patterns (patterns R1, R2, and R3) were obtained for serotype 1c strains, and one pattern (pattern R1) was identical to that shown by serotype 1a and another pattern (pattern R2) was identical to that shown by serotype 1b (Fig. 3). Comparison of the ribotypes of the S. flexneri serotype 1 strains showed that pattern R1 was common in both serotype 1a and 1c strains and that pattern R2 was common in both serotype 1b and 1c strains. However, ribotype R3 was unique to serotype 1c and was different from patterns R1 and R2, thereby correlating with the results of PFGE. Similar relationships between serotypes and ribotypes have previously been documented for S. flexneri strains isolated in Bangladesh (12, 45). The occurrence of isolates with the same ribotype but different serotypes was explained in previous studies (6, 41, 45). Overall analysis of the results concluded that subserotype 1c is genetically closely related to subserotypes 1a and 1b. Interestingly, serotype 1a was one of the predominant group of S. flexneri isolates in Bangladesh but is now scarcely isolated, whereas provisional subserotype 1c is emerging as the predominant serotype but was not

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found a decade ago. It is known that the O polysaccharides of different S. flexneri serotypes and subserotypes are polymers of the same basic tetrasaccharide unit. Group- and type-specific differences result from the addition of ␣-glycosyl or O-acetyl residues at specific positions in the basic tetrasaccharide, a process mediated by phage conversion (9). There is evidence of phage-mediated antigenic conversion in S. flexneri (1, 3, 4). According to our interpretation, 90% of the serotype 1c strains which belonged to the same PFGE and ribotypes as serotype 1a and 1b strains might have originated from the same ancestral clone. For this cluster of serotype 1c strains, phage-mediated serotype conversion would be a plausible explanation for their evolution. However, for the remaining 10% of the serotype 1c strains which possessed the unique PFGE type (type C) and the unique ribotype (ribotype R3), it is more obvious that they represent a new emerging clone. Due to the substantial differences in their genomic profiles, these strains have not originated from the existing subserotypes of serotype 1 by phage conversion; rather, a different mechanism may have resulted in the origin of these strains. Finally, it is important to emphasize that the provisional status of serotype 1c should be removed and it should be recognized as an accepted subserotype of serotype 1. ACKNOWLEDGMENTS We gratefully acknowledge N. I. A. Carlin, SBL Vaccin AB, Stockholm, Sweden, for supplying the monoclonal antibodies for S. flexneri serotype 1c. This study was funded by the U.S. Agency for International Development (USAID) under Cooperative Agreement HRN-A-00-9690005-00 and the Centre for Health and Population Research of ICDDR,B, which is supported by countries and agencies which share its concern for the health problems of developing countries. The present donors providing unrestricted support include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, The Netherlands, the Kingdom of Saudi Arabia, Sweden, Sri Lanka, Switzerland, and the United States. ICDDR,B acknowledges with gratitude the commitment of USAID and other donors to the Centre’s research effort. REFERENCES 1. Adams, M. M., G. E. Allison, and N. K. Verma. 2001. Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296. Microbiology 147:851–860. 2. Albert, M. J., N. A. Bhuiyan, K. A. Talukder, A. S. G. Faruque, S. Nahar, S. M. Faruque, M. Ansaruzzaman, and M. Rahman. 1997. Phenotypic and genotypic changes in Vibrio cholerae O139 Bengal. J. Clin. Microbiol. 35: 2588–2592. 3. Allison, G. E., and N. K. Verma. 2000. Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends Microbiol. 8:17–23. 4. Allison, G. E., D. Angeles, N. Tran-Dinh, and N. K. Verma. 2002. Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. J. Bacteriol. 184:1974–1987. 5. Ashraf, M. M., Z. U. Ahmed, and D. A. Sack. 1991. Unusual association of a plasmid with nalidixic acid resistance in an epidemic strain of Shigella dysenteriae type 1 from Asia. Can. J. Microbiol. 37:59–63. 6. Bradbury, W. C., A. D. Pearson, M. A. Marko, R. V. Congi, and J. L. Penner. 1984. Investigation of a Campylobacter jejuni outbreak by serotyping and chromosomal restriction endonuclease analysis. J. Clin. Microbiol. 19:342– 346. 7. Carlin, N. I. A., M. Rahman, D. A. Sack, A. Zaman, B. A. Kay, and A. A. Lindberg. 1989. Use of monoclonal antibodies to type Shigella flexneri in Bangladesh. J. Clin. Microbiol. 27:1163–1166. 8. Clemens, J. D., K. Kotloff, and B. A. Kay. 1999. Generic protocol to estimate the burden of Shigella diarrhoeaa and dysenteric mortality. WHO/VandB/ 99.26. World Health Organization, Geneva, Switzerland. 9. 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. 10. Edwards, P. R., and W. H. Ewing. 1972. In Identification of Enterobacteriaceae, p. 126–131. Burgess Publishing Company, Minneapolis, Minn.

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