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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]