Multiple Mechanisms Confer Resistance to Azithromycin in Shigella in Bangladesh: a Comprehensive Whole Genome-Based Approach.

Shigella is the second leading cause of diarrheal deaths worldwide. Azithromycin (AZM) is a potential treatment option for Shigella infection; however, the recent emergence of AZM resistance in Shigella threatens the current treatment strategy. Therefore, we conducted a comprehensive whole genome-based approach to identify the mechanism(s) of AZM resistance in Shigella. We performed antimicrobial susceptibility tests, polymerase chain reaction (PCR), Sanger (amplicon) sequencing, and whole genome-based bioinformatics approaches to conduct the study. Fifty-seven (38%) of the Shigella isolates examined were AZM resistant; Shigella sonnei exhibited the highest rate of resistance against AZM (80%). PCR amplification for 15 macrolide resistance genes (MRGs) followed by whole-genome analysis of 13 representative Shigella isolates identified two AZM-modifying genes, mph(A) (in all Shigella isolates resistant to AZM) and mph(E) (in 2 AZM-resistant Shigella isolates), as well as one 23S rRNA-methylating gene, erm(B) (41% of AZM-resistant Shigella isolates) and one efflux pump mediator gene, msr(E) [in the same two Shigella isolates that harbored the mph(E) gene]. This is the first report of msr(E) and mph(E) genes in Shigella. Moreover, we found that an IncFII-type plasmid predominates and can possess all four MRGs. We also detected two plasmid-borne resistance gene clusters: IS26-mph(A)-mrx(A)-mph(R)(A)-IS6100, which is linked to global dissemination of MRGs, and mph(E)-msr(E)-IS482-IS6, which is reported for the first time in Shigella. In conclusion, this study demonstrates that MRGs in association with pathogenic IS6 family insertion sequences generate resistance gene clusters that propagate through horizontal gene transfer (HGT) in Shigella. IMPORTANCE Shigella can frequently transform into a superbug due to uncontrolled and rogue administration of antibiotics and the emergence of HGT of antimicrobial resistance factors. The advent of AZM resistance in Shigella has become a serious concern in the treatment of shigellosis. However, there is an obvious scarcity of clinical data and research on genetic mechanisms that induce AZM resistance in Shigella, particularly in low- and middle-income countries. Therefore, this study is an approach to raise the alarm for the next lifeline. We show that two key MRGs [mph(A) and erm(B)] and the newly identified MRGs [mph(E) and msr(E)], with their origination in plasmid-borne pathogenic islands, are fundamental mechanisms of AZM resistance in Shigella in Bangladesh. Overall, this study predicts an abrupt decrease in the effectiveness of AZM against Shigella in the very near future and suggests prompt focus on seeking a more effective treatment alternative to AZM for shigellosis.

Draft Genome Sequences of Multidrug-Resistant Shigella Strains Isolated from Diarrheal Patients in Bangladesh.

The emergence of multidrug-resistant (MDR) Shigella strains has impaired the efficacy of first-line antimicrobials and exacerbated diarrhea-associated morbidity and mortality worldwide. We report the draft genome sequences of 11 MDR Shigella strains isolated from the stool specimens of diarrheal patients in Bangladesh.

Biofilms Comprise a Component of the Annual Cycle of Vibrio cholerae in the Bay of Bengal Estuary.

Vibrio cholerae, an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study of V. cholerae occurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. Toxigenic V. cholerae exhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenic V. cholerae, were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detect V. cholerae O1 cells throughout the year, as free-living cells, within clusters, or in association with plankton. V. cholerae O1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. Toxigenic V. cholerae O1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenic V. cholerae in the estuarine environment of Bangladesh.IMPORTANCEVibrio cholerae, the causative agent of cholera, is autochthonous in the estuarine aquatic environment. This study describes morphological changes in naturally occurring V. cholerae O1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh. V. cholerae O1 cells are predominantly coccoid within biofilms but are rod shaped as free-living cells and when attached to plankton or to particulate matter in interepidemic periods of the year. It is concluded that biofilms represent a stage of the annual life cycle of V. cholerae O1, the causative agent of cholera in Bangladesh.

Evidence for microRNA-mediated regulation of steroidogenesis by hypoxia.

Environmental hypoxia can occur in both natural and occupational environments. Over the recent years, the ability of hypoxia to cause endocrine disruption via perturbations in steroid synthesis (steroidogenesis) has become increasingly clear. To further understand the molecular mechanism underlying hypoxia-induced endocrine disruption, the steroid-producing human cell line H295R was used to identify microRNAs (miRNAs) affecting steroidogenic gene expression under hypoxia. Hypoxic treatment of H295R cells resulted in the downregulation of seven steroidogenic genes and one of these, CYP19A1 (aromatase), was shown to be regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). Using bioinformatic and luciferase reporter analyses, miR-98 was identified to be a CYP19A1-targeting miRNA from a subset of HIF-1-inducible miRNAs. Gain- and loss-of-function analysis suggested that under hypoxia, the increased expression of miR-98 led to the downregulation of CYP19A1 mRNA and protein expression and that it may have contributed to a reduction in estradiol (E2) production. Intriguingly, luciferase reporter assays using deletion constructs of a proximal 5'-flanking region of miR-98 did not reveal a hypoxia-responsive element (HRE)-containing promoter. Overall, this study provided evidence for the role of miRNAs in regulating steroidogenesis and novel insights into the molecular mechanisms of hypoxia-induced endocrine disruption.

Regulation of CYP11B1 and CYP11B2 steroidogenic genes by hypoxia-inducible miR-10b in H295R cells.

Although numerous studies have shown that hypoxia affects cortisol and aldosterone production in vivo, the underlying molecular mechanisms regulating the steroidogenic genes of these steroid hormones are still poorly known. MicroRNAs are post-transcriptional regulators that control diverse biological processes and this study describes the identification and validation of the hypoxia-inducible microRNA, miR-10b, as a negative regulator of the CYP11B1 and CYP11B2 steroidogenic genes in H295R human adrenocortical cells. Using the human TaqMan Low Density miRNA Arrays, we determined the miRNA expression patterns in H295R cells under normoxic and hypoxic conditions, and in cells overexpressing the human HIF-1α. Computer analysis using three in silico algorithms predicted that the hypoxia-inducible miR-10b molecule targets CYP11B1 and CYP11B2 mRNAs. Gene transfection studies of luciferase constructs containing the 3'-untranslated region of CYP11B1 or CYP11B2, combined with miRNA overexpression and knockdown experiments provide compelling evidence that CYP11B1 and CYP11B2 mRNAs are likely targets of miR-10b.

Cholera between 1991 and 1997 in Mexico was associated with infection by classical, El Tor, and El Tor variants of Vibrio cholerae.

Vibrio cholerae O1 biotype El Tor (ET), the cause of the current 7th pandemic, has recently been replaced in Asia and Africa by an altered ET biotype possessing cholera toxin (CTX) of the classical (CL) biotype that originally caused the first six pandemics before becoming extinct in the 1980s. Until recently, the ET prototype was the biotype circulating in Peru; a detailed understanding of the evolutionary trend of V. cholerae causing endemic cholera in Latin America is lacking. The present retrospective microbiological, molecular, and phylogenetic study of V. cholerae isolates recovered in Mexico (n = 91; 1983 to 1997) shows the existence of the pre-1991 CL biotype and the ET and CL biotypes together with the altered ET biotype in both epidemic and endemic cholera between 1991 and 1997. According to sero- and biotyping data, the altered ET, which has shown predominance in Mexico since 1991, emerged locally from ET and CL progenitors that were found coexisting until 1997. In Latin America, ET and CL variants shared a variable number of phenotypic markers, while the altered ET strains had genes encoding the CL CTX (CTX(CL)) prophage, ctxB(CL) and rstR(CL), in addition to resident rstR(ET), as the underlying regional signature. The distinct regional fingerprints for ET in Mexico and Peru and their divergence from ET in Asia and Africa, as confirmed by subclustering patterns in a pulsed-field gel electrophoresis (NotI)-based dendrogram, suggest that the Mexico epidemic in 1991 may have been a local event and not an extension of the epidemics occurring in Asia and South America. Finally, the CL biotype reservoir in Mexico is unprecedented and must have contributed to the changing epidemiology of global cholera in ways that need to be understood.

Changing genotypes of cholera toxin (CT) of Vibrio cholerae O139 in Bangladesh and description of three new CT genotypes.

We determined the genotype of cholera toxin by amplifying and sequencing the B-subunit in a sequential collection of 90 strains of Vibrio cholerae O139 isolated over the past 13 years since its first description in 1992. Representative strains isolated during 1993-1997 harboured ctxB of El Tor type (genotype 3). Twenty-six strains isolated during 1999, 2001, 2005 and three strains isolated in 1998, 2000 and 2002 were identified to belong to new ctxB genotypes 4 and 5, respectively. Genotype 5 was similar to genotype 1 except at position 28 (D-->A). The genotype 6 was similar to genotype 4 except at position 34 (H-->P). The implication of switch in terms of function of the toxin and its impact on human disease is unclear. How this change has influenced their prevalence relative to that of V. cholerae O1 in human infection is also not clear. The other common virulence gene clusters including the Vibrio pathogenicity island-1, Vibrio seventh pandemic island (VSP)-I and VSP-II of V. cholerae O139 did not show any remarkable difference from that of the O1 El Tor strains. Overall, the majority of the O139 strains tested in this study were similar to the El Tor strains but had altered ctxB genotype. This change and the impact that it causes to the epidemiology of cholera caused by O139 should be closely monitored.

Peruvian Vibrio cholerae O1 El Tor strains possess a distinct region in the Vibrio seventh pandemic island-II that differentiates them from the prototype seventh pandemic El Tor strains.

A collection of environmental and clinical strains of Vibrio cholerae O1 isolated from the beginning of the Latin American epidemic of cholera in 1991 to 2003 from multiple locations in Peru were characterized and compared with V. cholerae O1 El Tor strains of the seventh pandemic from the rest of the world (Asia, Africa, Australia and Europe) using a multilocus virulence gene profiling strategy and DNA sequencing. Peruvian strains differed from El Tor strains from the rest of the world by the failure of PCR to amplify genes VC0512, VC0513, VC0514 and VC0515 in the Vibrio seventh pandemic island-II (VSP-II) gene cluster. Sequencing of the VSP-II gene cluster and its flanking regions in one Peruvian strain (PERU-130) confirmed the PCR results, indicating that the Peruvian strain had low DNA homology (46.6 %) compared to the reference strain N16961 within the VSP-II region encompassing genes VC0511 to VC0515. Based on these differences in VSP-II, and based on the overall similarity between the pulsotypes of the Peruvian strains and the El Tor reference strain N16961, we concluded that the Peruvian, Eurasian and African strains belonged to the same clonal complex, and that the Peruvian strains represented variants that had independently evolved for a relatively short time. Since these ORFs in VSP-II of Peruvian strains are unique and conserved, they could form the basis for tracking the origin of the Peruvian strains and therefore of the Latin American pandemic.

Multilocus genetic analysis reveals that the Australian strains of Vibrio cholerae O1 are similar to the pre-seventh pandemic strains of the El Tor biotype.

Episodes of cholera stemming from indigenous Vibrio cholerae strains in Australia are mainly associated with environmental sources. In the present study, 10 V. cholerae O1 strains of Australian origin were characterized. All of the strains were serogroup O1 and their conventional phenotypic traits categorized them as belonging to the El Tor biotype. Genetic screening of 12 genomic regions that are associated with virulence in V. cholerae showed variable results. Analysis of the ctxAB gene showed that the Australian environmental reservoir contains both toxigenic and non-toxigenic V. cholerae strains. DNA sequencing revealed that all of the toxigenic V. cholerae strains examined were of ctxB genotype 2. Whole genome PFGE analysis revealed that the environmental toxigenic V. cholerae O1 strains were more diverse than the non-toxigenic environmental O1 strains, and the absence of genes that make up the Vibrio seventh pandemic island-I and -II in all of the strains indicates their pre-seventh pandemic ancestry.

Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor.

A mismatch amplification mutation PCR assay was developed and validated for rapid detection of the biotype specific cholera toxin B subunit of V. cholerae O1. This assay will enable easy monitoring of the spread of a new emerging variant of the El Tor biotype of V. cholerae O1.

O3:K6 serotype of Vibrio parahaemolyticus identical to the global pandemic clone associated with diarrhea in Peru.

OBJECTIVES: To determine if the Vibrio parahaemolyticus O3:K6 global pandemic clone has spread into Peru. METHODS: A collection of 100 V. parahaemolyticus strains isolated from diarrhea cases in Peru were serotyped for O:K antigens and genotyped for the presence of the species-specific toxR gene and for the tdh and trh genes. In addition, the group-specific PCR (GS-PCR) and PCR for the presence of the open reading frame ORF8 of the filamentous phage f237 was performed to determine the pandemic status of the strains. RESULTS: Fifty strains of V. parahaemolyticus in this collection were identified as pandemic strains. Forty-six ORF8 and GS-PCR positive strains were identical to the global pandemic clone O3:K6, while four strains that also possessed the pandemic genotype and were ORF8 and GS-PCR positive belonged to serotypes O3:K68, O3:K58 and OUT (untypable):K6. One of the O3:K6 strains was isolated in 1996, indicating that the pandemic strain was present in Peru at about the same time that it caused the first outbreak in Calcutta in February 1996. CONCLUSIONS: Based on this first report in Peru of such strains, we recommend including V. parahaemolyticus in the differential diagnosis of the etiologic agents for diarrhea in this part of the world.

Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji.

Five strains of Vibrio cholerae O1, one each from an Australian and a New Zealand tourist with gastrointestinal illness returning from an island resort in Fiji and the remaining three from water sources located in the same resort, were extensively characterized. Conventional phenotypic traits that are used for biotyping of O1 V. cholerae categorized all five strains as belonging to the El Tor biotype. Genetic screening of 11 regions that are associated with virulence in V. cholerae showed variable results. The absence of genes comprising Vibrio seventh pandemic island-I (VSP-I) and VSP-II in all the strains indicated that these strains were very similar to the pre-seventh pandemic V. cholerae O1 El Tor strains. The ctxAB genes were absent in all strains whereas orfU and zot were present in four strains, indicating that the strains were non-toxigenic. Four strains carried a truncated CTX prophage. Although epidemiological and molecular studies suggested that these strains did not cause cholera amongst tourists at the resort, their similarity to pre-seventh pandemic strains, their prior association with gastrointestinal illness and their presence in the island resort setting warrant more attention.

Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.

The Matlab variants of Vibrio cholerae O1, defined as hybrids between the classical and El Tor biotypes, were first isolated from hospitalized patients with acute secretory diarrhoea in Matlab, a rural area of Bangladesh. These variants could not be categorized as classical or El Tor biotypes by phenotypic and genotypic tests, and had representative traits of both the biotypes. A number of virulence-associated genes and/or gene clusters were screened by PCR and DNA sequencing. El Tor-specific gene clusters, Vibrio seventh-pandemic islands (VSP)-I and -II and repeat toxin (RTX) were present in the genome of these variants, indicating their El Tor lineage, whereas the nucleotide-sequence-derived CtxB amino acid sequence of these strains grouped them under the classical biotype. Matlab variants possessed all the necessary genes to initiate pandemics. The genetic relatedness of Matlab variants to the V. cholerae strains recently isolated in Mozambique is another important observation of this study, which underscores the epidemiological significance of Matlab variants.

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh.

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.

Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh.

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.