M. Kamruzzaman

New Variants of Vibrio cholerae O1 Biotype El Tor with Attributes of the Classical Biotype from Hospitalized Patients with Acute Diarrhea in Bangladesh

ABSTRACT The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.

Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area

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.

Prevalence of the Pandemic Genotype of Vibrio parahaemolyticus in Dhaka, Bangladesh, and Significance of Its Distribution across Different Serotypes

ABSTRACT Sixty-six strains of Vibrio parahaemolyticus belonging to 14 serotypes were isolated from hospitalized patients in Dhaka, Bangladesh, from January 1998 to December 2000. Among these, 48 strains belonging to four serotypes had the pandemic genotype and possessed the tdh gene. A marker (open reading frame ORF8) for a filamentous phage previously thought to correspond to the pandemic genotype was found to have a poor correlation with the pandemic genotype.

Isolation of Shigella dysenteriae Type 1 and S. flexneri Strains from Surface Waters in Bangladesh: Comparative Molecular Analysis of Environmental Shigella Isolates versus Clinical Strains

ABSTRACT Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemics spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.

Reemergence of Epidemic Vibrio cholerae O139, Bangladesh

During March and April 2002, a resurgence of Vibrio cholerae O139 occurred in Dhaka and adjoining areas of Bangladesh with an estimated 30,000 cases of cholera. Patients infected with O139 strains were much older than those infected with O1 strains (p<0.001). The reemerged O139 strains belong to a single ribotype corresponding to one of two ribotypes that caused the initial O139 outbreak in 1993. Unlike the strains of 1993, the recent strains are susceptible to trimethoprim, sulphamethoxazole, and streptomycin but resistant to nalidixic acid. The new O139 strains carry a copy of the Calcutta type CTXCalc prophage in addition to the CTXET prophage carried by the previous strains. Thus, the O139 strains continue to evolve, and the adult population continues to be more susceptible to O139 cholera, which suggests a lack of adequate immunity against this serogroup. These findings emphasize the need for continuous monitoring of the new epidemic strains.

Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae

The factors that enhance the waterborne spread of bacterial epidemics and sustain the pathogens in nature are unclear. The epidemic diarrheal disease cholera caused by Vibrio cholerae spreads through water contaminated with the pathogen. However, the bacteria exist in water mostly as clumps of cells, which resist cultivation by standard techniques but revive into fully virulent form in the intestinal milieu. These conditionally viable environmental cells (CVEC), alternatively called viable but nonculturable cells, presumably play a crucial role in cholera epidemiology. However, the precise mechanism causing the transition of V. cholerae to the CVEC form and this forms significance in the biology of the pathogen are unknown. Here we show that this process involves biofilm formation that is dependent on quorum sensing, a regulatory response that is controlled by cell density. V. cholerae strains carrying mutations in genes required for quorum sensing and biofilm formation displayed altered CVEC formation in environmental water following intestinal infections. Analysis of naturally occurring V. cholerae CVEC showed that organisms that adopt this quiescent physiological state typically exist as clumps of cells that comprise a single clone closely related to isolates causing the most recent local cholera epidemic. These results support a model of cholera transmission in which in vivo-formed biofilms convert to CVEC upon the introduction of cholera stools into environmental water. Our data further suggest that a temporary loss of quorum sensing due to dilution of extracellular autoinducers confers a selective advantage to communities of V. cholerae by blocking quorum-mediated regulatory responses that would break down biofilms and thus interfere with CVEC formation.

RS1 Element of Vibrio cholerae Can Propagate Horizontally as a Filamentous Phage Exploiting the Morphogenesis Genes of CTXΦ

ABSTRACT In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXΦ morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXΦ is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXΦ genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Kmr) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Kmr transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmΦ). Analysis of V. cholerae strains for susceptibility to RS1-KmΦ showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX−) O1 and O139 strains which carried genes encoding the CTXΦ receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXΦ, the RS1Φ genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmΦ which also harbored the CTXΦ genome produced a detectable level of extracellular RS1-KmΦ. This suggested that the core genes of CTXΦ are also required for the morphogenesis of RS1Φ. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXΦ.

Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island

ABSTRACT The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXΦ), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXΦ. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXΦ, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXΦ, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.

Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples

Cholera epidemics have long been known to spread through water contaminated with human fecal material containing the toxigenic bacterium Vibrio cholerae. However, detection of V. cholerae in water is complicated by the existence of a dormant state in which the organism remains viable, but resists cultivation on routine bacteriological media. Growth in the mammalian intestine has been reported to trigger “resuscitation” of such dormant cells, and these studies have prompted the search for resuscitation factors. Although some positive reports have emerged from these investigations, the precise molecular signals that activate dormant V. cholerae have remained elusive. Quorum-sensing autoinducers are small molecules that ordinarily regulate bacterial gene expression in response to cell density or interspecies bacterial interactions. We have found that isolation of pathogenic clones of V. cholerae from surface waters in Bangladesh is dramatically improved by using enrichment media containing autoinducers either expressed from cloned synthase genes or prepared by chemical synthesis. These results may contribute to averting future disasters by providing a strategy for early detection of V. cholerae in surface waters that have been contaminated with the stools of cholera patients or asymptomatic infected human carriers.

CTXΦ-independent production of the RS1 satellite phage by Vibrio cholerae

The cholera toxin genes of Vibrio cholerae are encoded by the filamentous phage, CTXΦ. Chromosomal CTXΦ prophage DNA is often found flanked by copies of a related genetic element designated RS1, and RS1 DNA can be packaged into filamentous phage particles (designated RS1Φ) by using the CTXΦ morphogenesis genes. RS1Φ is a satellite phage that further controls expression and dissemination of CTXΦ. Here we describe a CTXΦ-independent mechanism for production of RS1Φ. A nontoxigenic environmental V. cholerae strain (55V71) was identified that supports production of RS1Φ. However, newly infected CTX-negative strains did not produce RS1Φ, indicating that additional 55V71 genes were involved in production of RS1Φ. Analysis of nucleic acids from phage preparations of 55V71 revealed a 7.5-kb single-stranded DNA, whose corresponding replicative form was found in plasmid preparations. This DNA likely corresponds to the genome of a new filamentous phage, which we have designated KSF-1Φ. The replicative form DNA of KSF-1Φ was cloned into pUC18, and the resulting construct pKSF-1.1 supported the production of RS1Φ particles by CTX-negative V. cholerae strains. RS1Φ particles produced in this way infect recipient V. cholerae strains by a mechanism that is independent of the CTXΦ receptor, the toxin-coregulated pilus. Thus, KSF-1Φ is capable of facilitating the transfer of the RS1 element to strains that do not express toxin coregulated pilus. Given that RS1Φ can enhance coproduction of CTXΦ particles, KSF-1Φ-mediated dissemination of RS1 may indirectly promote the spread of toxin genes among V. cholerae strains. This study also shows that filamentous phages can package diverse DNA elements and thus may play a role in horizontal transfer of more genes than previously appreciated.