Multidrug-resistant conjugative plasmid carrying mphA confers increased antimicrobial resistance in Shigella.

Shigellosis remains a common gastrointestinal disease mostly in children < 5 years of age in developing countries. Azithromycin (AZM), a macrolide, is currently the first-line treatment for shigellosis in Bangladesh; ciprofloxacin (CIP) and ceftriaxone (CRO) are also used frequently. We aimed to evaluate the current epidemiology of antimicrobial resistance (AMR) and mechanism(s) of increasing macrolide resistance in Shigella in Bangladesh. A total of 2407 clinical isolates of Shigella from 2009 to 2016 were studied. Over the study period, Shigella sonnei was gradually increasing and become predominant (55%) over Shigella flexneri (36%) by 2016. We used CLSI-guided epidemiological cut-off value (ECV) for AZM in Shigella to set resistance breakpoints (zone-diameter ≤ 15 mm for S. flexneri and ≤ 11 mm for S. sonnei). Between 2009 and 2016, AZM resistance increased from 22% to approximately 60%, CIP resistance increased by 40%, and CRO resistance increased from zero to 15%. The mphA gene was the key macrolide resistance factor in Shigella; a 63MDa conjugative middle-range plasmid was harboring AZM and CRO resistance factors. Our findings show that, especially after 2014, there has been a rapid increase in resistance to the three most effective antibiotics. The rapid spread of macrolide (AZM) resistance genes among Shigella are driven by horizontal gene transfer rather than direct lineage.

Increasing trend of antibiotic resistance in Shigella in Bangladesh: a plasmid-mediated transfer of mphA macrolide resistance gene.

Shigellosis remains a common gastrointestinal disease mostly in children <5 years of age in developing countries. Azithromycin (AZM), a macrolide, is currently the first-line treatment for shigellosis in Bangladesh; ciprofloxacin (CIP) and ceftriaxone (CRO) are also used frequently. We aimed to evaluate the current epidemiology of antimicrobial resistance (AMR) and mechanism(s) of increasing macrolide resistance in Shigella in Bangladesh. A total of 2407 clinical isolates of Shigella from 2009 to 2016 were studied. Over the study period, Shigella sonnei was gradually increasing and become predominant (55%) over Shigella flexneri (36%) by 2016. We used CLSI-guided epidemiological cut-off value (ECV) for AZM in Shigella to set resistance breakpoints (zone-diameter ≤ 15 mm for S. flexneri and ≤ 11 mm for S. sonnei). Between 2009 and 2016, AZM resistance increased from 22% to approximately 60%, CIP resistance increased by 40%, and CRO resistance increased from zero to 15%. The mphA gene was the key macrolide resistance factor in Shigella; a 63MDa conjugative middle-range plasmid was harboring AZM and CRO resistance factors. Our findings show that, especially after 2014, there has been a rapid increase in resistance to the three most effective antibiotics. The rapid spread of macrolide (AZM) resistance genes among Shigella are driven by horizontal gene transfer rather than direct lineage.

Presence of Functionally Active Cytolethal Distending Toxin Genes on a Conjugative Plasmid in a Clinical Isolate of Providencia rustigianii.

Providencia rustigianii is potentially enteropathogenic in humans. Recently, we identified a P. rustigianii strain carrying a part of the cdtB gene homologous to that of Providencia alcalifacines that produces an exotoxin called cytolethal distending toxin (CDT), encoded by three subunit genes (cdtA, cdtB, and cdtC). In this study, we analyzed the P. rustigianii strain for possible presence of the entire cdt gene cluster and its organization, location, and mobility, as well as expression of the toxin as a putative virulence factor of P. rustigianii. Nucleotide sequence analysis revealed the presence of the three cdt subunit genes in tandem, and over 94% homology to the corresponding genes carried by P. alcalifaciens both at nucleotide and amino acid sequence levels. The P. rustigianii strain produced biologically active CDT, which caused distension of eukaryotic cell lines with characteristic tropism of CHO and Caco-2 cells but not of Vero cells. S1-nuclease digested pulsed-field gel electrophoresis followed by Southern hybridization analysis demonstrated that the cdt genes in both P. rustigianii and P. alcalifaciens strains are located on large plasmids (140 to 170 kb). Subsequently, conjugation assays using a genetically marked derivative of the P. rustigianii strain showed that the plasmid carrying cdt genes in the P. rustigianii was transferable to cdt gene-negative recipient strains of P. rustigianii, Providencia rettgeri, and Escherichia coli. Our results demonstrated the presence of cdt genes in P. rustigianii for the first time, and further showed that the genes are located on a transferable plasmid, which can potentially spread to other bacterial species.

Environmental prevalence of toxigenic Vibrio cholerae O1 in Bangladesh coincides with V. cholerae non-O1 non-O139 genetic variants which overproduce autoinducer-2.

Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera.

Prevalence, Serovar, and Antimicrobial Resistance of Nontyphoidal Salmonella in Vegetable, Fruit, and Water Samples in Ho Chi Minh City, Vietnam.

In this study, we investigated the prevalence, serovar distribution, and antimicrobial resistance pattern of Salmonella isolates from vegetable, fruit, and water samples in Ho Chi Minh City, Vietnam. Salmonella was detected in 75% (30/40), 57.1% (12/21), 17.5% (28/160), and 2.5% (1/40) of river water, irrigation water, vegetable, and ice water samples, respectively. However, no Salmonella was isolated from 160 fruit and 40 tap water samples examined. A total of 102 isolates obtained from 71 samples belonged to 34 different serovars, of which Salmonella Rissen was the most prevalent, followed by Salmonella London, Salmonella Hvittingfoss, and Salmonella Weltevreden. Certain Salmonella serovars such as Newport, Rissen, and Weltevreden were isolated from both vegetable and water samples. Antimicrobial resistance was most commonly observed against tetracycline (35.3%), followed by chloramphenicol (34.3%), ampicillin (31.4%), trimethoprim/sulfamethoxazole (23.5%), and nalidixic acid (10.8%). Of 102 isolates analyzed, 52 (51%) showed resistance to at least 1 antimicrobial class whereas 27 (26.5%) showed multidrug resistant (MDR) phenotype, being resistant to at least three different classes of antimicrobials. Determination of the presence and type of ß-lactamase genes showed the cooccurrence of blaTEM-1 and blaCMY-2 in one Salmonella Agona isolate from a river water sample. Taken together, these data indicated that both environmental water and vegetables were contaminated with Salmonella, including MDR strains, and that environmental water used in irrigation might have been the source of Salmonella contamination in the vegetables.

In silico comparative genomics of SARS-CoV-2 to determine the source and diversity of the pathogen in Bangladesh.

The COVID19 pandemic caused by SARS-CoV-2 virus has severely affected most countries of the world including Bangladesh. We conducted comparative analysis of publicly available whole-genome sequences of 64 SARS-CoV-2 isolates in Bangladesh and 371 isolates from another 27 countries to predict possible transmission routes of COVID19 to Bangladesh and genomic variations among the viruses. Phylogenetic analysis indicated that the pathogen was imported in Bangladesh from multiple countries. The viruses found in the southern district of Chattogram were closely related to strains from Saudi Arabia whereas those in Dhaka were similar to that of United Kingdom and France. The 64 SARS-CoV-2 sequences from Bangladesh belonged to three clusters. Compared to the ancestral SARS-CoV-2 sequence reported from China, the isolates in Bangladesh had a total of 180 mutations in the coding region of the genome, and 110 of these were missense. Among these, 99 missense mutations (90%) were predicted to destabilize protein structures. Remarkably, a mutation that leads to an I300F change in the nsp2 protein and a mutation leading to D614G change in the spike protein were prevalent in SARS-CoV-2 genomic sequences, and might have influenced the epidemiological properties of the virus in Bangladesh.

Vibrio cholerae strains with inactivated cqsS gene overproduce autoinducer-2 which enhances resuscitation of dormant environmental V. cholerae.

BACKGROUND: Toxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters. METHODS: Environmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays. RESULTS: Two of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water. SIGNIFICANCE: V. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae.

Toxigenic properties and stx phage characterization of Escherichia coli O157 isolated from animal sources in a developing country setting.

BACKGROUND: In many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh. RESULTS: We analysed 47 stx2 positive E. coli O157 of food/animal origin for stx2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q933 and Q21) and stx2 upstream region. Of the 47 isolates, 46 were positive for both stx2a and stx2d while the remaining isolate was positive for stx2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx2 promoter. Additionally, all isolates were negative for antiterminator Q933while a majority of isolates were positive for Q21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability. CONCLUSION: Despite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh.

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.

Environmental Spread of New Delhi Metallo-ß-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh.

Resistance to carbapenem antibiotics through the production of New Delhi metallo-ß-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the blaNDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among blaNDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including blaCTX-M-1 (80%), blaCTX-M-15 (63%), blaTEM (76%), blaSHV (33%), blaCMY-2 (16%), blaOXA-48-like (2%), blaOXA-1 (53%), and blaOXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying blaNDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community.IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the blaNDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

Quorum Regulated Resistance of Vibrio cholerae against Environmental Bacteriophages.

Predation by bacteriophages can significantly influence the population structure of bacterial communities. Vibrio cholerae the causative agent of cholera epidemics interacts with numerous phages in the aquatic ecosystem, and in the intestine of cholera patients. Seasonal epidemics of cholera reportedly collapse due to predation of the pathogen by phages. However, it is not clear how sufficient number of the bacteria survive to seed the environment in the subsequent epidemic season. We found that bacterial cell density-dependent gene expression termed "quorum sensing" which is regulated by signal molecules called autoinducers (AIs) can protect V. cholerae against predatory phages. V. cholerae mutant strains carrying inactivated AI synthase genes were significantly more susceptible to multiple phages compared to the parent bacteria. Likewise when mixed cultures of phage and bacteria were supplemented with exogenous autoinducers CAI-1 or AI-2 produced by recombinant strains carrying cloned AI synthase genes, increased survival of V. cholerae and a decrease in phage titer was observed. Mutational analyses suggested that the observed effects of autoinducers are mediated in part through the quorum sensing-dependent production of haemaglutinin protease, and partly through downregulation of phage receptors. These results have implication in developing strategies for phage mediated control of cholera.

Safe distances between groundwater-based water wells and pit latrines at different hydrogeological conditions in the Ganges Atrai floodplains of Bangladesh.

BACKGROUND: Groundwater drawn from shallow tubewells in Bangladesh is often polluted by nearby pit latrines, which are commonly used toilets in rural and sub-urban areas of the country. METHODS: To determine the minimum safe distance of a tubewell from a pit latrine in different hydrogeological conditions of Bangladesh, 20 monitoring wells were installed at three study sites (Manda, Mohanpur and Bagmara) with the vertical and horizontal distances ranging from 18-47 to 2-15 m, respectively. Water samples were collected three times in three seasons and tested for faecal coliforms (FC) and faecal streptococci (FS) as indicators of contamination. Soil samples were analysed for texture, bulk density and hydraulic conductivity following standard procedures. Sediment samples were collected to prepare lithological logs. RESULTS: When the shallow aquifers at one of the three sites (Mohanpur) were overlained by 18-23-m-thick aquitards, the groundwater of the monitoring wells was found contaminated with a lateral and vertical distances of 2 and 31 m, respectively. However, where the aquitard was only 9 m thick, contamination was found up to lateral and vertical distances of 4.5 and 40.5 m, respectively. The soil textures of all the sites were mainly composed of loam and sandy loam. The hydraulic conductivities in the first aquifer at Manda, Mohanpur and Bagmara were 5.2-7.3, 8.2 and 1.4-15.7 m/h, respectively. CONCLUSIONS: The results showed that the safe distance from the tubewell to the pit latrine varied from site to site depending on the horizontal and vertical distances of the tubewell as well as hydrogeological conditions of a particular area.

Faecal contamination of commuters' hands in main vehicle stations in Dhaka city, Bangladesh.

BACKGROUND: Faecal-oral carriage via hands is an important transmission pathway for diarrhoeal pathogens. The level of faecal contamination of commuters' hands in Dhaka, Bangladesh, was examined in this study. METHODS: A total of 900 hand washing samples, including both left and right hands, were collected during one year to cover three different seasons in Bangladesh: winter, summer and rainy seasons. Standard membrane filtration technique was used to quantify total coliforms (TC), faecal coliforms (FC), faecal streptococci (FS), Escherichia coli (EC) and Clostridium perfringens (CP). RESULTS: The hands of the commuters were contaminated with TC, FC, FS, CP and EC. The TC, FC, FS, CP and EC counts were 1.95, 1.65, 4.04, 1.54 and1.46 log10 colony forming units (cfu) in the left hand; and 2.13, 1.82, 4.11, 1.52 and 1.61 log10 cfu in the right hand, respectively. There were no statistically significant differences in counts of left and right hands. The highest counts were observed for FS in all seasons. CONCLUSIONS: This evidence based study may be used to provide interventions to reduce the contamination of commuters' hands through washing with detergent and, thus, help to prevent the spread of infectious diseases.

Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence.

Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study.

BACKGROUND: Shigellosis is an acute, severe bacterial colitis that, in high-income countries, is typically associated with travel to high-risk regions (Africa, Asia, and Latin America). Since the 1970s, shigellosis has also been reported as a sexually transmitted infection in men who have sex with men (MSM), in whom transmission is an important component of shigellosis epidemiology in high-income nations. We aimed to use sophisticated subtyping and international sampling to determine factors driving shigellosis emergence in MSM linked to an outbreak in the UK. METHODS: We did a large-scale, cross-sectional genomic epidemiological study of shigellosis cases collected from 29 countries between December, 1995, and June 8, 2014. Focusing on an ongoing epidemic in the UK, we collected and whole-genome sequenced clinical isolates of Shigella flexneri serotype 3a from high-risk and low-risk regions, including cases associated with travel and sex between men. We examined relationships between geographical, demographic, and clinical patient data with the isolate antimicrobial susceptibility, genetic data, and inferred evolutionary relationships. FINDINGS: We obtained 331 clinical isolates of S flexneri serotype 3a, including 275 from low-risk regions (44 from individuals who travelled to high-risk regions), 52 from high-risk regions, and four outgroup samples (ie, closely related, but genetically distinct isolates used to determine the root of the phylogenetic tree). We identified a recently emerged lineage of S flexneri 3a that has spread intercontinentally in less than 20 years throughout regions traditionally at low risk for shigellosis via sexual transmission in MSM. The lineage had acquired multiple antimicrobial resistance determinants, and prevailing sublineages were strongly associated with resistance to the macrolide azithromycin. Eight (4%) of 206 isolates from the MSM-associated lineage were obtained from patients who had previously provided an isolate; these serial isolations indicated atypical infection patterns (eg, reinfection). INTERPRETATION: We identified transmission-facilitating behaviours and atypical course(s) of infection as precipitating factors in shigellosis-affected MSM. The intercontinental spread of antimicrobial-resistant shigella through established transmission routes emphasises the need for new approaches to tackle the public health challenge of sexually transmitted infections in MSM. FUNDING: Wellcome Trust (grant number 098051).

Phenotypic and genotypic characteristics of Vibrio cholerae O1 isolated from the Sierra Leone cholera outbreak in 2012.

BACKGROUND: This study describes phenotypic, genotypic and antibiotic susceptibility patterns of the strains isolated from the 2012 Sierra Leone cholera outbreak. Rectal swabs were collected from patients and cultured for Vibrio cholerae O1. METHODS: The isolates were subjected to multiplex PCR, mismatch amplification mutation assay (MAMA) PCR, pulsed field gel electrophoresis (PFGE), and antibiotic sensitivity tests using disk diffusion and minimum inhibitory concentration (MIC) E-test following standard procedures. RESULTS: Out of 17 rectal swabs tested, 15 yielded V. cholerae O1 biotype El Tor, serotype Ogawa. All the strains belonged to 'altered' variants as MAMA PCR result showed the presence of classical cholera toxin B. PFGE result revealed four pulse types. Using antibiotic disk diffusion, all the isolates were resistant to erythromycin, chloramphenicol, furazolidone, and trimethoprim/sulfamethoxazole (SXT) except SL1 which was sensitive to chloramphenicol and SXT. All the isolates were sensitive to nalidixic acid, tetracycline, doxycycline, azithromycin, and ciprofloxacin except SL2 which was resistant to nalidixic acid. However, variable sensitivity patterns were observed for kanamycin. The ranges of MIC were 0.125-0.50 mg/l, 0.003-0.023 mg/l and 0.38-0.75 mg/l for azithromycin, ciprofloxacin and tetracycline, respectively. CONCLUSIONS: This study demonstrates that altered variants of V. cholerae O1 of four clonal types were responsible for the 2012 outbreak of cholera in Sierra Leone.

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.

RS1 satellite phage promotes diversity of toxigenic Vibrio cholerae by driving CTX prophage loss and elimination of lysogenic immunity.

In El Tor biotype strains of toxigenic Vibrio cholerae, the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.