Prevalence of virulence genes and cytolethal distending toxin production in Campylobacter jejuni isolates from diarrheal patients in Bangladesh.

From 300 stool samples, 58 Campylobacter strains were isolated by standard microbiological and biochemical methods. Of these, 40 strains were identified as Campylobacter jejuni and 5 as Campylobacter coli. The presence of flaA (100%), cadF (100%), racR (100%), dnaJ (100%), pldA (100%), ciaB (95%), virB11 (0%), ceuE (82.5%), cdtA (97.5%), cdtB (97.5%), cdtC (97.5%), and wlaN (7.5%) genes was detected in C. jejuni by PCR. All C. jejuni strains but one produced cytolethal distending toxin in a HeLa cell assay.

A novel serovar of Shigella dysenteriae from patients with diarrhoea in Bangladesh.

Every year, around 3 % of isolates from patients with diarrhoea at Dhaka Hospital, ICDDR,B, are identified as Shigella-like organisms (SLOs) based on their activity in biochemical tests. These isolates do not react with any of the current Shigella antisera including all existing and provisional serotypes. Among these SLOs, a unique cluster of seven isolates with an identical plasmid profile was found and these isolates were further characterized by phenotypic and genotypic techniques. All were nonlactose fermenters, with an identical biochemical pattern typical of Shigella dysenteriae. They were classified as invasive since they harboured the 140 MDa invasive plasmid, were able to bind Congo red, produced keratoconjunctivitis in the guinea pig eye, and were positive by PCR for the ipaH gene and Shigella enterotoxin 2 [ShET-2] gene. All isolates were resistant to ampicillin, tetracycline and sulfamethoxazole-trimethoprim but were susceptible to mecillinam, nalidixic acid, ceftriaxone and ciprofloxacin. Six of the isolates were identical in DNA pattern by PFGE with the seventh exhibiting a closely related pattern; both patterns were distinguishable from all other Shigella and Escherichia coli patterns. An antiserum prepared against one of the isolates reacted with all isolates and did not cross-react with other Shigella and E. coli serotype reference strains. It is therefore proposed that these isolates represent a new provisional serovar of S. dysenteriae, type strain KIVI 162.

Antibiotic resistance and genetic diversity of Shigella sonnei isolated from patients with diarrhoea between 1999 and 2003 in Bangladesh.

Shigella sonnei is a significant cause of diarrhoeal infection in both developing and industrialized countries. From 1999 to 2003, 445 strains of Shigella sonnei were isolated from patients admitted to the diarrhoea treatment centre of the International Center for Diarrhoeal Disease Research, Bangladesh. More than 60% of the isolates were resistant to nalidixic acid, 89% to sulfamethoxazole-trimethoprim and 9.5% to ampicillin. In addition, 4% of strains were resistant to multiple antibiotics (AmpR TetR SxtR StrR) and 4.2% of strains were sensitive to all antibiotics tested. None of the strains were positive for the set1 gene, whereas 46% were positive for the sen gene. Forty-six per cent of the strains (stored at -70 degrees C) harboured the 120 MDa invasive plasmid and representative strains produced keratoconjunctivitis in the guinea pig eye. In addition, three plasmids of approximately 5, 1.8 and 1.4 MDa were found to be present in more than 90% of the strains. A self-transmissible, middle-ranged plasmid (35-80 MDa) carrying the multiple antibiotic resistance gene was found in some strains. PFGE analysis of the strains identified five unique types with many subtypes, which were characterized into four unique types by ribotyping analysis. It can be concluded that endemic strains of Shigella sonnei isolated from patients in Bangladesh are diverse in their genetic pattern.

Toxin(s), other than cholera toxin, produced by environmental non O1 non O139 Vibrio cholerae.

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Isolation and characterization of provisional serovar Shigella boydii E16553 from diarrhoeal patients in Bangladesh.

In previous studies with strains of the Shigella dysenteriae provisional serovars E22383 and E23507 from diarrhoeal stools from patients in Bangladesh, two strains of Shigella species were identified as Shigella boydii provisional serovar E16553 by a reference laboratory. Further tests with an antiserum to an international type strain of the provisional serovar E16553 identified an additional 15 isolates. None of the isolates reacted with antisera to the established Shigella serovars or any other provisional serovars reported so far and all showed biochemical reactions typical of S. boydii. All of the isolates harboured the 140 MDa invasion plasmid, had the ipaH gene and produced keratoconjunctivitis in the guinea pig eye. All isolates were susceptible to ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and mecillinam but eight strains were resistant to tetracycline. A single PFGE type (type A) was shown for all 17 clinical isolates, indicating a common source of origin. The pulsotype of the Bangladeshi isolates was closely related to that of a Japanese strain but was different from that of the type strain. On the basis of these biochemical, serological and virulence markers, and diverse geographical origin, it is recommended that the provisional status of serovar E16553 be changed and that it be included in the international serotyping classification scheme as S. boydii 19.

Fluoroquinolone resistance linked to both gyrA and parC mutations in the quinolone resistance-determining region of Shigella dysenteriae type 1.

We examined the quinolone resistance-determining region (QRDR) of gyrA, gyrB, and parC of recently isolated fluoroquinolone-resistant S. dysenteriae type 1 strains from south Asia and compared data with fluoroquinolone-susceptible strains associated with previous epidemics of 1978, 1984, and 1994. In fluoroquinolone-resistant strains, double mutations (Ser83-->Leu, Asp87-->Asn or Gly) and a single mutation (Ser80-->Ile) were detected in the QRDRs of gyrA and parC, respectively.

Phenotypic and genotypic characterization of serologically atypical strains of Shigella flexneri type 4 isolated in Dhaka, Bangladesh.

Twenty-one atypical Shigella flexneri type 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared with S. flexneri serotypes 4a and 4b. The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b. Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars. Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole. All of the strains harbored the 140-MDa plasmid, had the ipaH gene, had the sen gene (encoding Shigella enterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties. All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains. Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker. Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b. This study concluded that two different clones of atypical S. flexneri type 4 exist and strongly suggests that these are new subserotypes of S. flexneri that await further serological classification.

Temporal shifts in the dominance of serotypes of Shigella dysenteriae from 1999 to 2002 in Dhaka, Bangladesh.

A total of 358 Shigella dysenteriae strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrheal Disease Research, Bangladesh, between the years 1999 and 2002 were included in this study. S. dysenteriae type 1, the dominant serotype in 1999 (76.4%), declined to 6.5% in 2002. On the other hand, S. dysenteriae types 2 to 12 were isolated with increasing frequencies of 19, 67, 73.5, and 87% in 1999, 2000, 2001, and 2002, respectively. Of these, types 2 and 4 were the most dominant serotypes, accounting for more than 18.7 and 28.5% of the total isolates, respectively. There was no isolation of serotypes 5, 7, 8, and 13 during this period. Twenty-eight (7.8%) of the isolates were atypical and agglutinated only with the polyvalent antiserum of S. dysenteriae. More than 98% of type 1 strains isolated between 1999 and 2001 were resistant to ampicillin, sulfamethoxazole-trimethoprim, and nalidixic acid. Among other serotypes of S. dysenteriae, Nal(r) type 2 strains were isolated in 2001 and 2002. Although heterogeneous plasmid profiles were obtained depending on the presence or absence of a single plasmid, core plasmids were defined for particular serotypes. On the other hand, the same plasmid profile was found to be shared by different serotypes. Interestingly, plasmid patterns of types 2 and 4 were almost identical except that a middle-range plasmid of 70 to 60 MDa was present in type 4 in addition to the core plasmids. All the strains harboring the 140-MDa plasmid were positive for the ipaH gene, had Congo red binding abilities, and were positive by the Sereny test, demonstrating their invasive properties.

Phenotypic and genotypic characterization of provisional serotype Shigella flexneri 1c and clonal relationships with 1a and 1b strains isolated in Bangladesh.

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.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

OBJECTIVES: The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. METHODS: The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. RESULTS: Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. CONCLUSIONS: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.