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Pulsed-field gel electrophoresis (PFGE) is commonly used in molecular epidemiology. However, this technique has never been used in studying intra-family spread of enteric diseases in Bangladesh. Our objective was to evaluate the intra-familial transmission of shigella infection using PFGE. Children of either sex, less than 10 years old, who were family contacts of shigella-infected index cases were the study population. PFGE was applied if the same serotypes/sub-serotypes of shigella were isolated from both the index case and the family contact children. In total, 227 index cases were studied. Shigella was isolated from 61 (27%) contact children on day 1 of enrolment, among which Shigella flexneri (41%) and S. boydii (41%) were dominant, followed by S. dysenteriae (10%), S. sonnei (3%), and shigella-like organisms (5%). Seventeen (28%) of the asymptomatic infections in contact children were caused by the same serotype of shigella as that found in the index case. The intra-familial shigella transmission rate was 8% (17/227). Of the 227 contact children, eight (4%) developed diarrhoea during a 10-day follow-up and shigella was isolated from five (2%) of these children, and three of them (S. flexneri 3a, 1b, and 3a) were identical to the strains from their respective index cases. Compared to children without asymptomatic carriage of shigella (2/166), the risk (odds ratio) of developing diarrhoea for the children with asymptomatic carriage of shigella identical to their cases (3/17) was 9·0 (95% CI 1·5–49·0, P=0·01). The attributable risk for symptomatic shigella infection by intra-familial transmission was 50%. Results of this study demonstrated that intra-familial transmission of shigella carries a higher risk for diarrhoea.

Received 12 April 2004; returned 13 June 2004; revised 2 August 2004; accepted 10 August 2004Objectives: 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 strainsassociated with epidemics in 1978, 1984 and 1994.Methods: The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemio-logical 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 andBangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae1, except for the presence of a new plasmid of 2.6MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strainsisolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly relatedto that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. Thecurrent ciprofloxacin-resistant strains belong to five subtypes (A3–A7), all of which were found inIndia, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealedthat amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated inBangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) fromciprofloxacin-resistant strains isolated in India.Conclusions: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxa-cin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in1978, 1984 and 1994.Keywords: pulsed-field gel electrophoresis, gyrA, QRDRs, epidemics

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. Shigellosis is one of the major diarrheal diseases afflicting humans in the developing and underdeveloped parts of the world, especially Bangladesh. Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody diarrhea and mucus in the stool. Shigellosis is endemic in Bangladesh, and it is estimated that dysentery accounts for 20% of deaths related to diarrhea among children (32). The genus Shigella, the causative agent of shigellosis, is comprised of four species, namely, S. flexneri, S. dysenteriae, S. boydii, and S. sonnei, and each of these species is further classified into 15 (including subtypes), 13, 18, and 1 serotypes, respectively, based on the O antigen component of lipopolysaccharide present on the outer membrane of the cell wall. S. flexneri and S. dysenteriae are the major concerns for developing countries. The manifestation of S. dysenteriae type 1 infection is more severe because of its capacity to produce Shiga toxin, which is exclusively produced by this type. Clinical infection can be transmitted by as few as 10 Shigella organisms (3), even without neutralization of gastric acid. At least three periods of epidemic outbreaks of dysentery due to S. dysenteriae 1 have been recorded previously in the Indian subcontinent, in 1972-1973, 1983-1984, and 1993-1994 (1, 9, 18, 24). Despite improvement of municipal water supplies and sanitation, shigellosis still occurs frequently. This raises important questions about the causes of shigellosis, its transmission, epidemiology, and the effectiveness of public health measures in overcoming this illness. Indiscriminate use of antibiotics in this region has resulted in the Shigella strains becoming resistant to multiple antibiotics. At present, most of the Shigella strains isolated from patients are resistant to ampicillin (AMP), sulfamethoxazole-trimethoprim (SXT), and nalidixic acid (2, 9, 15). The prevalence of the serotypes of S. flexneri in Bangladesh was described in a previous report (27), which showed that there was a temporal variation in the dominance of different subserotypes. The emergence of some atypical serotypes of S. flexneri has also been reported (27). There are no reports of outbreaks caused by the serotypes of S. dysenteriae other than serotype 1 in Bangladesh or any other part of the world. In the present study, a detailed epidemiological study of the prevalence of different S. dysenteriae serotypes and their susceptibility to commonly used antibiotics among hospital patients was conducted over the last 4 years to evaluate the present status of shigellosis caused by S. dysenteriae serotypes 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.

Twenty-one atypicalShigella flexneritype 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared withS. flexneriserotypes 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 theipaHgene, had thesengene (encodingShigellaenterotoxin 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 atypicalS. flexneritype 4 exist and strongly suggests that these are new subserotypes ofS. flexnerithat await further serological classification.

Although ciprofloxacin is one of the recommended drugs of choice for the treatment of gonorrhea, in vitro resistance to this drug has been observed in surveillance studies and case reports from many parts of the world, including Bangladesh. However, to our knowledge, there have been no prospective studies of the correlation between in vitro response to the drug and treatment outcome. Therefore, a prospective study of 217 female sex workers in Dhaka, Bangladesh, was conducted to examine the correlation between the in vitro response of Neisseria gonorrhoeae and the outcome of ciprofloxacin treatment. Overall, 37.8% of the gonococcal isolates recovered from female sex workers were resistant to ciprofloxacin, and there was a good correlation between in vitro resistance and treatment failure. These findings suggest that in vitro resistance to ciprofloxacin is predictive of clinical treatment failure in patients with gonorrhea.

Pulsed-field gel electrophoresis (PFGE) has been used successfully to discriminate between strains of many different bacterial species. In this study, digestion of bacterial DNA with the restriction endonuclease NotI and PFGE were evaluated for the typing of isolates of Shigella dysenteriae type 1, an important cause of epidemic dysentery. There were 27 isolates from four outbreaks of dysentery, and 44 isolates from endemic dysentery cases and a laboratory culture collection. The epidemic isolates yielded two types each with two subtypes, whereas the endemic isolates and culture collection yielded eight types with numerous subtypes. These findings suggest that S. dysenteriae 1 can be typed by PFGE.

Of 469 recently isolated Shigella flexneri strains, 452 agglutinated with Shigella flexneri -specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with S. flexneri 2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.

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.

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.

Shigella sonneiis a significant cause of diarrhoeal infection in both developing and industrialized countries. From 1999 to 2003, 445 strains ofShigella sonneiwere 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 (AmpRTetRSxtRStrR) and 4.2 % of strains were sensitive to all antibiotics tested. None of the strains were positive for theset1 gene, whereas 46 % were positive for thesengene. Forty-six per cent of the strains (stored at −70 °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 ofShigella sonneiisolated from patients in Bangladesh are diverse in their genetic pattern.

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.