Biomedical subjects
Publications and source records attributed to Shah M Faruque.
We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.
A live oral Vibrio cholerae O1 El Tor vaccine, Peru-15 was tested in a double-blind, randomized placebo controlled study for safety and immunogenicity in Phase I and Phase II studies in 240 Bangladeshi children aged 9 months-5 years of age. Two different doses (2x10(7) and 2x10(8)cfu) were tested. Vaccination did not elicit adverse events and the strain was genetically stable. Vibriocidal antibody responses developed in 42/50 (84%) toddlers (2-5 years) and 35/50 (70%) of younger children (9-23 months) and overall 77/100 (77%) who received the high dose. LPS-IgA-antibody responses were seen in 60% of toddlers and 34% of infants; 40% responded with IgA antibodies to cholera toxin. The responses to the reduced dose was lower. These studies demonstrate that Peru-15 at a dose of 2x10(8)cfu is safe and immunogenic in children in Bangladesh.
The factors that enhance the waterborne spread of bacterial epidemics and sustain the epidemic strain in nature are unclear. Although the epidemic diarrheal disease cholera is known to be transmitted by water contaminated with pathogenic Vibrio cholerae, routine isolation of pathogenic strains from aquatic environments is challenging. Here, we show that conditionally viable environmental cells (CVEC) of pathogenic V. cholerae that resist cultivation by conventional techniques exist in surface water as aggregates (biofilms) of partially dormant cells. Such CVEC can be recovered as fully virulent bacteria by inoculating the water into rabbit intestines. Furthermore, when V. cholerae shed in stools of cholera patients are inoculated in environmental water samples in the laboratory, the cells exhibit characteristics similar to CVEC, suggesting that CVEC are the infectious form of V. cholerae in water and that CVEC in nature may have been derived from human cholera stools. We also observed that stools from cholera patients contain a heterogeneous mixture of biofilm-like aggregates and free-swimming planktonic cells of V. cholerae. Estimation of the relative infectivity of these different forms of V. cholerae cells suggested that the enhanced infectivity of V. cholerae shed in human stools is largely due to the presence of clumps of cells that disperse in vivo, providing a high dose of the pathogen. The results of this study support a model of cholera transmission in which in vivo-formed biofilms contribute to enhanced infectivity and environmental persistence of pathogenic V. cholerae.
Cholera is a waterborne diarrheal disease that continues to plague the developing world. Individuals become infected by consuming water from reservoirs contaminated by virulent strains of the bacterium Vibrio cholerae. Epidemiological and environmental observations of a cholera outbreak in Dhaka, Bangladesh, suggest that lytic bacteriophage specific for V. cholerae may limit the severity of cholera outbreaks by killing bacteria present in the reservoir and in infected individuals. To quantify this idea and generate testable hypotheses, we analyzed a mathematical model that combines the epidemiology of cholera with the population dynamics of the bacteria and phage. Under biologically reasonable conditions, we found that vibriophage can ameliorate cholera outbreaks. If phage predation limits bacterial density before an outbreak, a transient reduction in phage density can disrupt that limitation, and subsequent bacterial growth can initiate a cholera outbreak. The severity of the outbreak depends on the density of phage remaining in the reservoir. If the outbreak is initiated instead by a rise in bacterial density, the introduction of phage can reduce the severity of the outbreak and promote its decline. In both situations, the magnitude of the phage effect depends mainly on vibrio growth and phage mortality rates; the lower the rates, the greater the effect. Our analysis also suggests that either bacteria in the environmental reservoir are hyperinfectious or most victims ingest bacteria amplified in food or drinking water contaminated by environmental water carrying few viable V. cholerae. Our theoretical results make a number of empirically testable predictions.
Predicting cholera epidemics through monitoring the environment for the presence of pathogenic Vibrio cholerae is complicated by the presence in water of a large number of mostly nonpathogenic V. cholerae strains. V. cholerae strains causing recent cholera epidemics in Bangladesh carry the sulfamethoxazole-trimethoprim (SXT) element, which encodes resistance to several antibiotics. Here, we show that the use of a culture medium containing streptomycin, sulfamethoxazole, and trimethoprim (the antibiotic selection technique [AST]) can significantly enhance the isolation of environmental V. cholerae O1 with epidemic potential (P<.001). The AST was also used to monitor the recent emergence and spread of a new multiple-antibiotic-resistant strain of V. cholerae in Bangladesh. The results of this study support the hypothesis that pre-epidemic amplification of pathogenic V. cholerae occurs in the human host and leads to the start of an epidemic cycle dominated by a single clone of V. cholerae that spreads rapidly through environmental waters.
Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.
Factors that enhance the transmission of pathogens are poorly understood. We show that Vibrio cholerae shed in human 'rice-water' stools have a 10-fold lower oral infectious dose in an animal model than in vitro grown V. cholerae, which may aid in transmission during outbreaks. Furthermore, we identify a bacterial factor contributing to this enhanced infectivity: The achievement of a transient motile but chemotaxis-defective state upon shedding from humans. Rice-water stool V. cholerae have reduced levels of CheW-1, which is essential for chemotaxis, and were consequently shown to have a chemotaxis defect when tested in capillary assays. Through mutational analyses, such a state is known to enhance the infectivity of V. cholerae. This is the first report of a pathogen altering its chemotactic state in response to human infection in order to enhance its transmission.
The incidence of aetiology-specific diarrhoea and the pathogenicity of infectious agents in a birth cohort (n=252) in rural Bangladesh were determined. Stool specimens or rectal swabs were collected from diarrhoeal cases over two years and routinely on a monthly basis. Stool samples from children with diarrhoea were compared with stool samples from children without diarrhoea to calculate rates of isolation and pathogenicity of agents. In total, 1750 stool specimens from diarrhoea patients and 5679 stool specimens from children without diarrhoea were tested. An infectious agent was identified in 58% of the stool specimens from diarrhoea patients and 21.6% of the stool specimens from children without diarrhoea. The most commonly-isolated pathogens from all specimens were enterotoxigenic Escherichia coli (ETEC), enteroadherent E. coli, Shigella, Campylobacter jejuni, Giardia, and rotavirus. ETEC (ST and LT-ST toxin), enterotoxigenic Bacteroides fragilis, Shigella, and rotavirus were associated more with disease than with asymptomatic infections. Aetiology-specific infections were associated with acute episodes. The isolated enteropathogens were essentially the same as those found in other tropical rural settings. Enterotoxigenic B. fragilis was also identified as a pathogen. Ongoing vaccine efforts focusing on Shigella, rotavirus, and ETEC would be useful.
BACKGROUND: A live oral Vibrio cholerae O1 El Tor vaccine candidate, Peru-15, was studied for safety, immunogenicity, and excretion in phase 1 (inpatient) and phase 2 (outpatient) studies of Bangladeshi adults.METHODs. The study was conducted among adults, by use of a double-blind, randomized, placebo-controlled design. A single dose of Peru-15 (approximately 2 x 108 cfu) or placebo (buffer only) was given in standard bicarbonate and ascorbic acid buffer.RESULTS. Study treatment did not elicit any major adverse events in the volunteers, during either the inpatient or the outpatient phases, and there were no reports of diarrhea. V. cholerae was isolated from the stool of only 1 volunteer and was found to be genetically identical to the vaccine strain. Vibriocidal antibody responses were seen in 30 (75%) of 40 vaccine recipients and in 3 (10%) of 30 placebo recipients. Peripheral blood immunoglobulin (Ig) A and IgM antibody-secreting cell responses to lipopolysaccharide were seen in the majority of vaccine recipients (response rate, 78%--88%). Seroconversion for lipopolysaccharide-specific IgA antibodies was seen in 88% of vaccine recipients. The response in vaccine recipients was significantly higher than that in placebo recipients, in all of the immunological assays (P=.036 to <.001). A lower immunological response against cholera toxin B subunit was detected.CONCLUSIONS. The safety and immunogenicity of this Peru-15 vaccine candidate indicates the usefulness of future studies in Bangladesh, where cholera is endemic.
Phage predation of Vibrio cholerae has recently been reported to be a factor that influences seasonal epidemics of cholera in Bangladesh. To understand more about this phenomenon, we studied the dynamics of the V. cholerae-phage interaction during a recent epidemic in Dhaka. Because the outbreak strain causing this epidemic was resistant to multiple antibiotics, including streptomycin, we used a selective medium containing streptomycin to monitor accurately the abundance of this strain in the environment. The changing prevalence in the environment of the epidemic V. cholerae O1 strain and a particular lytic cholera phage (JSF4) to which it was sensitive was measured every 48-72 h for 17 weeks. We also monitored the incidence of phage excretion in stools of 387 cholera patients during the epidemic. The peak of the epidemic was preceded by high V. cholerae prevalence in the environment and was followed by high JSF4 phage levels as the epidemic ended. The buildup to the phage peak in the environment coincided with increasing excretion of the same phage in the stools of cholera patients. These results suggest that patients toward the end of the epidemic ingested both JSF4 phage and the outbreak V. cholerae strain. Host-mediated phage amplification during the cholera epidemic likely contributed to increased environmental phage abundance, decreased load of environmental V. cholerae and, hence, the collapse of the epidemic. Thus, in vivo phage amplification in patients and subsequent phage predation in the environment may explain the self-limiting nature of seasonal cholera epidemics in Bangladesh.
Non-O1, non-O139 Vibrio cholerae can cause gastroenteritis and extraintestinal infections, but, unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content of non-O1, non-O139 strains and their phylogenetic relationship to other pathogenic V. cholerae. Comparative genomic microarray analysis of four pathogenic non-O1, non-O139 strains indicates that these strains are quite divergent from O1 and O139 strains. Genomic sequence analysis of a non-O1, non-O139 strain (AM-19226) that appeared particularly pathogenic in experimental animals suggests that this strain carries a type III secretion system (TTSS) that is related to the TTSS2 gene cluster found in a pandemic clone of Vibrio parahaemolyticus. The genes for this V. cholerae TTSS system appear to be present in many clinical and environmental non-O1, non-O139 strains, including at least one clone that is globally distributed. We hypothesize that the TTSS present in some pathogenic strains of non-O1, non-O139 V. cholerae may be involved in the virulence and environmental fitness of these strains.
The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.
Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.
Understanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from specimens collected during the early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. cholerae colonization factor, was significantly more highly expressed in early than in late stages of infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators toxRS and tcpPH was uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines.
KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.
Sixteen of the 18 Vibrio cholerae rough strains isolated from hospitalised diarrhoea patients were found to contain O1 serotype-specific (wbe) genes and all currently known virulence genes. Expression of the regulatory element ToxR was evident in these strains. Cholera toxin production ability of the rough strains was found to be higher (c. three- to five-fold) as compared to the smooth counterparts and this was transcriptionally regulated. Strains exhibiting the rough phenotype were more amenable to the uptake of CTXphi, which led us to consider that the rough phenotype could play a role in the generation of genetic diversity among V. cholerae strains.
To understand the evolutionary events and possible selection mechanisms involved in the emergence of pathogenic Vibrio cholerae, we analyzed diverse strains of V. cholerae isolated from environmental waters in Bangladesh by direct enrichment in the intestines of adult rabbits and by conventional laboratory culture. Strains isolated by conventional culture were mostly (99.2%) negative for the major virulence gene clusters encoding toxin-coregulated pilus (TCP) and cholera toxin (CT) and were nonpathogenic in animal models. In contrast, all strains selected in rabbits were competent for colonizing infant mice, and 56.8% of these strains carried genes encoding TCP alone or both TCP and CT. Ribotypes of toxigenic O1 and O139 strains from the environment were similar to pandemic strains, whereas ribotypes of non-O1 non-O139 strains and TCP(-) nontoxigenic O1 strains diverged widely from the seventh pandemic O1 and the O139 strains. Results of this study suggest that (i) the environmental V. cholerae population in a cholera-endemic area is highly heterogeneous, (ii) selection in the mammalian intestine can cause enrichment of environmental strains with virulence potential, (iii) pathogenicity of V. cholerae involves more virulence genes than currently appreciated, and (iv) most environmental V. cholerae strains are unlikely to attain a pandemic potential by acquisition of TCP and CT genes alone. Because most of the recorded cholera pandemics originated in the Ganges Delta region, this ecological setting presumably favors extensive genetic exchange among V. cholerae strains and thus promotes the rare, multiple-gene transfer events needed to assemble the critical combination of genes required for pandemic spread.
Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.