Characterization of Shigella flexneri Serotype 6 Strains Isolated from Bangladesh and Identification of a New Phylogenetic Cluster.

A significant cause of shigellosis in Bangladesh and other developing countries is Shigella flexneri serotype 6. This serotype has been subtyped, on the basis of the absence or presence of a group-specific antigen, E1037, into S. flexneri 6a and 6b, respectively. Here, we provided rationales for the subclassification, using several phenotypic and molecular tools. A set of S. flexneri 6a and 6b strains isolated between 1997 and 2015 were characterized by analyzing their biochemical properties, plasmid profiles, virulence markers, pulsed-field gel electrophoresis (PFGE) results, and ribotype. Additionally, the genomic relatedness of these subserotypes was investigated with global isolates of serotype 6 using publicly available genomes. Both subserotypes of S. flexneri 6 agglutinated with monoclonal antiserum against S. flexneri (MASF) B and type VI-specific antiserum (MASF VI) and were PCR positive for O-antigen flippase-specific genes and virulence markers (ipaH, ial, sen, and sigA). Unlike S. flexneri 6a strains, S. flexneri 6b strains seroagglutinated with anti-E1037 antibodies, MASF IV-I. Notably, these two antigenically distinct subserotypes were clonally diverse, showing two distinct PFGE patterns following the digestion of chromosomal DNA with either XbaI or IceuI. In addition, hybridization of a 16S rRNA gene probe with HindIII-digested genomic DNA yielded two distinguishing ribotypes. Genomic comparison of S. flexneri subserotype 6a and 6b strains from Bangladesh indicated that, although these strains were in genomic synteny, the majority of them formed a unique phylogroup (PG-4) that was missing for the global isolates. This study supports the subserotyping and emphasizes the need for global monitoring of the S. flexneri subserotypes 6a and 6b. IMPORTANCE Shigella flexneri serotype 6 is one of the predominant serotypes among shigellosis cases in Bangladesh. Characterization of a novel subserotype of S. flexneri 6 (VI:E1037), agglutinated with type 6-specific antibody and anti-E1037, indicates a unique evolutionary ancestry. PFGE genotyping supports the finding that these two antigenically distinct subserotypes are clonally diverse. A phylogenetic study based on single-nucleotide polymorphism (SNP) data revealed that these two subserotypes were in genomic synteny, although their genomes were reduced. Interestingly, a majority of the S. flexneri 6 strains isolated from Bangladesh form a novel phylogenetic cluster. Therefore, this report underpins the global monitoring and tracking of the novel subserotype.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

Enteric Infections in Young Children are Associated with Environmental Enteropathy and Impaired Growth.

OBJECTIVE: To investigate the relationship between faecal contamination in child play spaces, enteric infections, environmental enteropathy (EE) and impaired growth among young children. METHODS: A prospective cohort study was conducted of 203 children 6-30 months of age in rural Bangladesh. Stool samples were analysed by quantitative PCR for Shigella, Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, Giardia intestinalis and Cryptosporidium spp. Four faecal markers of intestinal inflammation were also measured: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin. Child growth was measured at baseline and 9 months after enrolment. E. coli was measured in soil in child play spaces. RESULTS: Forty-seven percent of study children had three or more enteric pathogens in their stool. Thirty five percent (71/203) of children had Shigella, 30% (61/203) had ETEC, 73% (148/203) had C. jejuni, 79% (160/203) had Giardia intestinalis and none had Cryptosporidium. Children with ETEC had significantly higher calprotectin concentrations (Coefficient: 1.35, 95% Confidence Interval [CI]: 1.005, 1.82). Children with Shigella had a significantly higher odds of being stunted at our 9-month follow-up (OR: 2.01, 95% CI: 1.02, 3.93). Children with Giardia intestinalis had significantly higher E.coli counts in the soil collected from their play spaces (OR: 1.23, 95% CI: 1.02, 1.48). CONCLUSION: Enteric infections were significantly associated with EE and impaired growth in rural Bangladesh. These findings provide further evidence to support the hypothesis that contaminated soil in child play spaces can lead to enteric infections, many of which are likely subclinical, resulting in EE and impaired growth in young children.

Mouthing of Soil Contaminated Objects is Associated with Environmental Enteropathy in Young Children.

OBJECTIVE: To characterise childhood mouthing behaviours and to investigate the association between object-to-mouth and food-to-mouth contacts, diarrhoea prevalence and environmental enteropathy. METHODS: A prospective cohort study was conducted of 216 children ≤30 months of age in rural Bangladesh. Mouthing contacts with soil and food and objects with visible soil were assessed by 5-h structured observation. Stool was analysed for four faecal markers of intestinal inflammation: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin. RESULTS: Overall 82% of children were observed mouthing soil, objects with visible soil, or food with visible soil during the structured observation period. Sixty two percent of children were observed mouthing objects with visible soil, 63% were observed mouthing food with visible soil, and 18% were observed mouthing soil only. Children observed mouthing objects with visible soil had significantly elevated faecal calprotectin concentrations (206.81 µg/g, 95% confidence interval [CI]: 6.27, 407.36). There was also a marginally significant association between Escherichia coli counts in soil from a child's play space and the prevalence rate of diarrhoea (diarrhoea prevalence ratio: 2.03, 95% CI 0.97, 4.25). CONCLUSION: These findings provide further evidence to support the hypothesis that childhood mouthing behaviour in environments with faecal contamination can lead to environmental enteropathy in susceptible paediatric populations. Furthermore, these findings suggest that young children mouthing objects with soil, which occurred more frequently than soil directly (60% vs. 18%), was an important exposure route to faecal pathogens and a risk factor for environmental enteropathy.

Unsafe Child Feces Disposal is Associated with Environmental Enteropathy and Impaired Growth.

OBJECTIVE: To investigate the relationship between unsafe child feces disposal, environmental enteropathy, and impaired growth, we conducted a prospective cohort study of 216 young children in rural Bangladesh. STUDY DESIGN: Using a prospective cohort study design in rural Bangladesh, unsafe child feces disposal, using the Joint Monitoring Program definition, was assessed using 5-hour structured observation by trained study personnel as well as caregiver reports. Anthropometric measurements were collected at baseline and at a 9-month follow-up. Stool was analyzed for fecal markers of environmental enteropathy: alpha-1-antitrypsin, myeloperoxidase, neopterin (combined to form an environmental enteropathy disease activity score), and calprotectin. FINDINGS: Among 216 households with young children, 84% had an unsafe child feces disposal event during structured observation and 75% had caregiver reported events. There was no significant difference in observed unsafe child feces disposal events for households with or without an improved sanitation option (82% vs 85%, P = .72) or by child's age (P = .96). Children in households where caregivers reported unsafe child feces disposal had significantly higher environmental enteropathy scores (0.82-point difference, 95% CI 0.11-1.53), and significantly greater odds of being wasted (weight-for-height z score <-2 SDs) (9% vs 0%, P = .024). In addition, children in households with observed unsafe feces disposal had significantly reduced change in weight-for-age z-score (-0.34 [95% CI -0.68, -0.01] and weight-for-height z score (-0.52 [95% CI -0.98, -0.06]). CONCLUSION: Unsafe child feces disposal was significantly associated with environmental enteropathy and impaired growth in a pediatric population in rural Bangladesh. Interventions are needed to reduce this high-risk behavior to protect the health of susceptible pediatric populations.

Shigella Infections in Household Contacts of Pediatric Shigellosis Patients in Rural Bangladesh.

To examine rates of Shigella infections in household contacts of pediatric shigellosis patients, we followed contacts and controls prospectively for 1 week after the index patient obtained care. Household contacts of patients were 44 times more likely to develop a Shigella infection than were control contacts (odds ratio 44.7, 95% CI 5.5-361.6); 29 (94%) household contacts of shigellosis patients were infected with the same species and serotype as the index patient's. Pulsed-field gel electrophoresis showed that 14 (88%) of 16 with infected contacts had strains that were indistinguishable from or closely related to the index patient's strain. Latrine area fly counts were higher in patient households compared with control households, and 2 patient household water samples were positive for Shigella. We show high susceptibility of household contacts of shigellosis patients to Shigella infections and found environmental risk factors to be targeted in future interventions.

Liver-Directed AAV8 Booster Vaccine Expressing Plasmodium falciparum Antigen Following Adenovirus Vaccine Priming Elicits Sterile Protection in a Murine Model.

Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p < 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8+ T cells, especially resident memory CD8+ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.

FAS promoter polymorphisms and serum sFas level are associated with increased risk of nerve damage in Bangladeshi patients with Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is an autoimmune disorder of the peripheral nervous system triggered by molecular mimicry between pathogen lipopolysaccharides and host nerve gangliosides. Polymorphisms in the Fas receptor (FAS) and Fas ligand (FASL) genes may potentially alter the elimination of autoreactive immune cells and affect disease susceptibility or disease severity in GBS. We detected single nucleotide polymorphisms (SNPs) in FAS (-1377G/A and -670A/G) and FASL (-843C/T) in a prospective cohort of 300 patients with GBS and 300 healthy controls from the Bangladeshi population. Genotype distributions were not significantly different between patients with GBS and healthy controls. The FAS -670 AG heterozygous (P = 0.0005, OR = 2.5, 95% CI = 1.5-4.2) and GG homozygous (P = 0.0048, OR = 2.6, 95% CI = 1.3-5.0) genotypes were more common in patients with anti-GM1 antibodies than patients without anti-GM1 antibodies. The FAS -670 G allele was more prevalent in anti-GM1 antibody-positive than -negative patients (P = 0.0002, OR = 1.9, 95% CI = 1.4-2.7) and also in patients with the axonal subtype than demyelinating subtype (P < 0.0001, OR = 4.8, 95% CI = 2.3-10.1). The 1377G/-670G GG haplotype was significantly associated with the axonal subtype (P < 0.0001) and anti-ganglioside antibody-positivity (P = 0.0008) in GBS. Serum sFas (237.5 pg/mL vs. 159.5 pg/mL; P < 0.0001) and sFasL (225.1 pg/mL vs. 183.4 pg/mL; P = 0.0069) were elevated in patients with GBS compared to healthy controls, and among patients with high serum sFas was associated with severe GBS (P = 0.0406). In conclusion, this study indicates FAS-FASL promoter SNPs may promote the production of cross-reactive anti-ganglioside antibodies in GBS.

Characterization of a serologically atypical Shigella flexneri Z isolated from diarrheal patients in Bangladesh and a proposed serological scheme for Shigella flexneri.

BACKGROUND: Atypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. Despite being a provisional serotype, continuous isolation of these strains in diverse geographical regions implicated a great necessity to study the overall characteristics of these strains. Therefore, we extensively characterized S. flexneri Z strains using various phenotypic and molecular tools. METHOD: Of 3569 S. flexneri isolated between 1997 and 2015, 95 strains were identified as S. flexneri Z using a panel of polyvalent absorbed antisera and monoclonal antisera of S. flexneri (MASF). Of them, randomly selected 65 strains were molecular O-serotyped using multiplex PCR and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and PFGE) to determine relationship with other subserotypes of S. flexneri. RESULTS: All these atypical S. flexneri Z strains were agglutinated with MASF B and IV-I antisera. Concordantly, these strains were positive to opt-gene, responsible for MASF IV-I sero-positive phenotype. However, molecular O-serotyping of all 65 strains could not differentiate between Z and Yb giving similar amplification products (wzx1-5 and opt). Contrarily, MASF based serotypic scheme distinguished among Z and Yb as well as Ya. All these S. flexneri Z showed typical biochemical reaction of S. flexneri, harboured a 140 MDa virulence plasmid and virulence markers namely ipaH, ial, sen, sigA and sepA genes. Along with the virulence plasmid, small plasmids (2.6, 1.8 and 1.6 MDa) were present as core plasmid. Moreover, a middle ranged plasmid and a 4.0 MDa sized plasmid were observed in 65% and 20% strains, respectively. Analysis of PFGE on XbaI-digested chromosomal DNA of Bangladeshi strains showed that S. flexneri Z had a close relatedness with Ya and Yb but completely different than the strains of Xa, Xb, 2a and 2b. This observation was found to be unequivocal while the overall result of biotyping, plasmid profile, and virulence factors was compared. Therefore, we conclude that these atypical serotype Z isolated in Bangladesh had a clonal relationship with Ya and Yb of Bangladesh and the opt gene played an important role in serotypic switching among them. Current serotyping scheme of S. flexneri strains fails to place many such atypical strains (1c, 1c+6, 1d, type 4, and 4c) including S. flexneri Z isolated from different parts of the world. Therefore, an updated serotyping scheme for identification of subserotypes of S. flexneri has been proposed to avoid multiple naming of the same subserotype having similar agglutination pattern.

Vaccinomics Approach for Designing Potential Peptide Vaccine by Targeting Shigella spp. Serine Protease Autotransporter Subfamily Protein SigA.

Shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of 165 million people worldwide per year. Casein-degrading serine protease autotransporter of enterobacteriaceae (SPATE) subfamily protein SigA, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type-2 (HEp-2) and is shown to be highly immunogenic. In the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic SigA of Shigella spp. At first, 44 SigA proteins from different variants of S. flexneri, S. dysenteriae, S. boydii, and S. sonnei were assessed to find the most antigenic protein. We retrieved 12 peptides based on the highest score for human leukocyte antigen (HLA) supertypes analysed by NetCTL. Initially, these peptides were assessed for the affinity with MHC class I and class II alleles, and four potential core epitopes VTARAGLGY, FHTVTVNTL, HTTWTLTGY, and IELAGTLTL were selected. From these, FHTVTVNTL and IELAGTLTL peptides were shown to have 100% conservancy. Finally, IELAGTLTL was shown to have the highest population coverage (83.86%) among the whole world population. In vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. METHODS: A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). RESULTS: Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 µg/ml and 32µg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. CONCLUSION: Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Fluoroquinolone resistance mechanisms of Shigella flexneri isolated in Bangladesh.

OBJECTIVE: To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China. METHODS: A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004-2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). RESULTS: Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (CipR) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6-32 mg/L, 8-32 mg/L, and 8-24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser83Leu, Asp87Asn/Gly) and single mutation in parC gene (Ser80Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp87Asn) and China (Asp87Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness. CONCLUSIONS: Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.

Changing trends in the prevalence of Shigella species: emergence of multi-drug resistant Shigella sonnei biotype g in Bangladesh.

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.