The O139 serogroup of Vibrio cholerae comprises diverse clones of epidemic and nonepidemic strains derived from multiple V. cholerae O1 or non-O1 progenitors

Sixty-four representative strains of Vibrio cholerae O139 were analyzed, to re-examine the origin of this serogroup. Ribotyping differentiated the strains into 3 HindIII and 7 BglI ribotypes. One HindIII and 5 BglI ribotypes were shared by all toxigenic O139 strains. Of 6 nontoxigenic O139 strains, 3 shared ribotypes with the toxigenic strains, carried genes encoding toxin coregulated pilus, and were susceptible to the cholera toxin-converting bacteriophage CTXPhi. The remaining 3 strains belonged to 2 different ribotypes distinct from toxigenic O139 strains and were resistant to CTXPhi and JA-1, an O139-specific lytic bacteriophage. Polymerase chain reaction amplicons corresponding to the gmhD gene carried by these 3 strains also differed from those of the toxigenic O139 strains but were identical to those of 15 environmental non-O1-non-O139 strains. Thus, the O139 antigen is present in different lineages, and this serogroup appears to comprise epidemic and nonepidemic strains derived separately from different progenitors.     

Induction of the Lysogenic Phage Encoding Cholera Toxin in Naturally Occurring Strains of Toxigenic Vibrio cholerae O1 and O139

In toxigenic Vibrio cholerae, the CTX genetic element which carries the genes for cholera toxin (CT) is the genome of a lysogenic bacteriophage (CTXΦ). Clinical and environmental strains of V. cholerae O1 or O139 and stools that were culture positive for cholera were analyzed to study the induction and transmission of CTXΦ. To our knowledge, this is the first report of the examination of CTXΦ in clinical materials and in naturally occurring strains. DNA probe analysis revealed that 4.25% (6 of 141) of the isolated V. cholerae strains spontaneously produced a detectable level of extracellular CTXΦ particles in the culture supernatants whereas another 34.04% (48 of 141) produced CTXΦ particles when induced with mitomycin C. CTXΦ isolated from 10 clinical or environmental strains infected a CT-negative recipient strain, CVD103, both inside the intestines of infant mice and under laboratory conditions. All culture-positive stools analyzed were negative for the presence of CTXΦ both in the DNA probe assay and by in vivo assay for the infection of the recipient strain in infant mice. These results suggested that naturally occurring strains of toxigenic V. cholerae are inducible lysogens of CTXΦ but that cholera pathogenesis in humans is not associated with the excretion of CTXΦ particles in stools, indicating that induction of the phage may not occur efficiently inside the human intestine. However, in view of the efficient transmission of the phage under conditions conducive to the expression of toxin-coregulated pili, it appears that propagation of CTXΦ in the natural habitat may involve both environmental and host factors.     

Development of a Multiplex PCR-Based Rapid Typing Method for Enterohemorrhagic Escherichia coli O157 Strains

Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx₁, stx₂, and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks.Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is a food-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans (18). Since its initial identification as a food-borne pathogen in 1982, EHEC O157 has been implicated in numerous outbreaks and sporadic cases, mainly in industrialized countries (8). To prevent and control EHEC O157 infections, rapid detection of outbreaks and the identification of contamination sources are crucial. Thus, suitable tools for epidemiologic studies and systematic surveillance, such as rapid and efficient strain-typing systems, are needed.Among the currently available methods for molecular typing of EHEC O157 strains, pulsed-field gel electrophoresis (PFGE) has the highest discrimination power and is widely used for epidemiologic studies and the surveillance of O157:H7 infections (1, 12, 19, 21). However, PFGE requires strong technical skills and 1 or more days to generate the results. It is also difficult to obtain consistently reproducible results among different laboratories, which hinders interlaboratory data comparisons. Other strain-typing methods for EHEC O157, including PCR-restriction fragment length polymorphism (15, 16), polymorphic amplified typing sequences (5, 6), and multiple-locus variable-number tandem repeat analysis (9), have also been developed. Although these methods have their own advantages, they require special techniques and/or equipment and are time-consuming.Previously, we determined the whole-genome sequence of E. coli O157:H7 strain RIMD 0509952 (referred to as EHEC O157 Sakai) and identified a total of 98 copies of insertion sequence (IS) elements in the genome (2). Among these, IS629 and ISEc8 predominated, with 23 copies of IS629 and 11 copies of ISEc8 being identified. Using whole-genome PCR scanning (WGPScanning) and microarray analyses of eight EHEC O157 clinical isolates (10, 11), we identified numerous genomic segments that carried structural polymorphisms (ranging from several hundred base pairs to a few thousand base pairs) among the eight EHEC O157 strains and EHEC O157 Sakai. Recently, we analyzed all of these segments and found insertions/deletions of IS629 and ISEc8 in the generation of these structural polymorphisms. In particular, the genomic locations of IS629 varied significantly between the strains; we identified a total of 77 genomic loci into which IS629 was inserted in some of the nine strains that we examined (13).In the present study, based on the variable genomic location of IS629 among EHEC O157 strains, we have developed a simple and rapid multiplex PCR-based, strain-typing method for EHEC O157 strains, which we term the O157 IS-printing method.     

Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli

Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5–5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.     

Inference of the impact of insertion sequence (IS) elements on bacterial genome diversification through analysis of small-size structural polymorphisms in Escherichia coli O157 genomes

Mobile genetic elements play important roles in the evolution and diversification of bacterial genomes. In enterohemorrhagic Escherichia coli O157, a major factor that affects genomic diversity is prophages, which generate most of the large-size structural polymorphisms (LSSPs) observed in O157 genomes. Here, we describe the results of a systematic analysis of numerous small-size structural polymorphisms (SSSPs) that were detected by comparing the genomes of eight clinical isolates with a sequenced strain, O157 Sakai. Most of the SSSPs were generated by genetic events associated with only two insertion sequence (IS) elements, IS629 and ISEc8, and a number of genes that were inactivated or deleted by these events were identified. Simple excisions of IS629 and small deletions (footprints) formed by the excision of IS629, both of which are rarely described in bacteria, were also detected. In addition, the distribution of IS elements was highly biased toward prophages, prophage-like integrative elements, and plasmids. Based on these and our previous results, we conclude that, in addition to prophages, these two IS elements are major contributors to the genomic diversification of O157 strains and that LSSPs have been generated mainly by bacteriophages and SSSPs by IS elements. We also suggest that IS elements possibly play a role in the inactivation and immobilization of incoming phages and plasmids. Taken together, our results reveal the true impact of IS elements on the diversification of bacterial genomes and highlight their novel role in genome evolution.Insertion sequence (IS) elements are the smallest autonomously mobile genetic elements (generally 700–2500 base pairs [bp] in size) and are widely distributed in both eukaryotic and bacterial genomes. Most bacterial IS elements are of the DNA type (Chandler and Mahillon 2002). They only encode transposases (TPases) and thus are phenotypically cryptic by themselves, but the insertion/transposition of IS elements can result in gene inactivation and/or affect the expression of adjacent genes. IS elements can also induce a variety of genomic rearrangements, including deletions, inversions, and duplications (Wei et al. 2003; Kothapalli et al. 2005; Iguchi et al. 2006). Thus, it is generally assumed that IS elements play important roles in bacterial genome evolution. During long-term experimental evolution of Escherichia coli strain B in vitro (Papadopoulos et al. 1999; Schneider et al. 2000), several IS-mediated mutations were detected; some of these mutations contributed to adaptation to or improved fitness in the medium used for the experiment (glucose minimal medium). Several comparative genome analyses have also revealed that IS elements induce large genomic inversions and translocations in some bacterial species (Jin et al. 2002; Parkhill et al. 2003; Yang et al. 2005; Chain et al. 2006; Rohmer et al. 2007; Naito et al. 2008; Salzberg et al. 2008). However, it remains largely unknown how and to what extent IS elements affect genome diversification among closely related bacterial strains or species.IS elements are classified into about 20 families based on various features, such as the sequences of the TPases and the terminal inverted repeats. The mechanism of transposition differs between IS families, but most IS elements transpose through either the nonreplicative (cut-and-paste) or the replicative (copy-and-paste) mechanisms (Chandler and Mahillon 2002). The IS3 family is one of the largest IS families, and its molecular mechanism of transposition has been well characterized. In several IS3 family members, such as IS2, IS3, IS150, and IS911, transposition has been shown to be achieved through the formation of a “figure eight” intermediate (Lewis and Grindley 1997; Sekine et al. 1999; Haas and Rak 2002; Rousseau et al. 2002; Ohtsubo et al. 2004). One study revealed that in IS911, a circular form that integrates into a new insertion site is replicatively generated through the formation of a “figure eight” intermediate (Duval-Valentin et al. 2004). On the other hand, very little is known about the fate of the donor DNA in IS transpositions, although some kind of genomic alteration could occur in the chromosomal regions that serve as the donor DNA. A detailed structural comparison of closely related genomes that contain a number of active copies of IS3 family members may shed some light on this unresolved scientific issue.E. coli comprise a wide variety of strains with different phenotypes. Most are commensal inhabitants of vertebrates and rarely cause diseases, except in immunocompromised hosts. However, several E. coli clones or lineages have acquired specific sets of virulence-related genes that cause a range of diseases in humans. Among the various types of pathogenic strains, enterohemorrhagic E. coli (EHEC) O157:H7, which causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, is one of the most important food-borne pathogens in many industrialized countries (Mead and Griffin 1998).The chromosome of the sequenced O157:H7 strain RIMD 0509952 (referred to as O157 Sakai) contains 1.4 Mb of sequences that are not present in the benign laboratory E. coli strain K-12. These Sakai-specific sequences, or S-loops, are scattered throughout a 4.1-Mb chromosome backbone that is shared with K-12 (Hayashi et al. 2001). Most of the large Sakai-specific sequences are prophages and prophage-like integrative elements (Sp1-Sp18 and SpLE1-SpLE6, respectively), and many of the virulence-related genes in O157 have been introduced into the O157 genome by these genetic elements or a large plasmid, pO157 (Makino et al. 1998; Hayashi et al. 2001). K-12 also contains 11 prophages and prophage-like elements, implying that bacteriophages are major contributors to the genomic diversification of E. coli (Ohnishi et al. 2001; Hayashi et al. 2008). The O157 Sakai genome is also rich in IS elements; 25 types of IS elements (98 copies in total) have been identified, and 37 of the 98 copies are apparently intact (Hayashi et al. 2001). These intact IS elements, including IS629, an IS3 family member (19 copies), ISEc8 (eight copies), IS677 (three copies), IS1-Nuxi and IS609 (two copies each), and three other types (one copy each), may also contribute to genomic diversification in the O157 lineage.We previously compared the genomic structures and compositions of eight O157 clinical strains isolated in Japan in 1998 with those of O157 Sakai by whole genome PCR scanning (WGPS), a long-range PCR-based method for fine comparison of genome structures (Ohnishi et al. 2002), and comparative genomic hybridization (CGH) using an O157 oligo DNA microarray (Ogura et al. 2006). The results of these analyses and the data from another laboratory (Wick et al. 2005) revealed that an unexpectedly high degree of genomic diversity exists in the O157 lineage. Among the numerous structural polymorphisms identified, the longer polymorphisms were exclusively observed in regions harboring prophages, indicating that prophages are a major contributor to genomic diversity within the O157 lineage (Ohnishi et al. 2002). This conclusion is supported by the results of genomic comparisons of O157 substrains obtained from long-term subculturing in vitro (Iguchi et al. 2006). In our WGPS analysis of the eight O157 strains, we also identified numerous genomic segments that exhibited small size differences, ranging from a few hundred bases to several kilobases. However, it is not known how these small-size structural polymorphisms (referred to as SSSPs) are generated.In the present study, we systematically analyzed SSSP-containing genomic segments found in the eight O157 strains. Most were generated by genetic events associated with two IS elements, IS629 and ISEc8, and we identified several genes that were inactivated or deleted by such events. We also found simple excisions of IS629 and small deletions that were generated upon the excision of IS629, both of which have rarely been described in bacteria. These results indicate that these two IS elements should be regarded as major factors contributing to the genomic diversification of O157. The distribution of IS elements in O157 is highly biased toward prophages, prophage-like integrative elements, and plasmids, suggesting that these elements may play a role in the inactivation and immobilization of incoming phages and plasmids.     

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

The cholera toxin genes of Vibrio cholerae are encoded by the filamentous phage, CTXphi. Chromosomal CTXphi 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 RS1phi) by using the CTXphi morphogenesis genes. RS1phi is a satellite phage that further controls expression and dissemination of CTXphi. Here we describe a CTXphi-independent mechanism for production of RS1phi. A nontoxigenic environmental V. cholerae strain (55V71) was identified that supports production of RS1phi. However, newly infected CTX-negative strains did not produce RS1phi, indicating that additional 55V71 genes were involved in production of RS1phi. 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-1phi. The replicative form DNA of KSF-1phi was cloned into pUC18, and the resulting construct pKSF-1.1 supported the production of RS1phi particles by CTX-negative V. cholerae strains. RS1phi particles produced in this way infect recipient V. cholerae strains by a mechanism that is independent of the CTXphi receptor, the toxin-coregulated pilus. Thus, KSF-1phi is capable of facilitating the transfer of the RS1 element to strains that do not express toxin coregulated pilus. Given that RS1phi can enhance coproduction of CTXphi particles, KSF-1phi-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.     

Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.     

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.     

Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin, which is encoded by a lysogenic filamentous bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor that is also the receptor for CTXPhi. The genes involved in the biosynthesis of TCP reside in a pathogenicity island, which has been reported to correspond to the genome of another filamentous phage (designated VPIPhi) and to encode functions necessary for the production of infectious VPIPhi particles. We examined 46 V. cholerae strains having diverse origins and carrying different genetic variants of the TCP island for the production of the VPIPhi and CTXPhi in different culture conditions, including induction of prophages with mitomycin C and UV irradiation. Although 9 of 10 V. cholerae O139 strains and 12 of 15 toxigenic El Tor strains tested produced extracellular CTXPhi, none of the 46 TCP-positive strains produced detectable VPIPhi in repeated assays, which detected as few as 10 particles of a control CTX phage per ml. These results contradict the previous report regarding VPIPhi-mediated horizontal transfer of the TCP genes and suggest that the TCP island is unable to support the production of phage particles. Further studies are necessary to understand the mechanism of horizontal transfer of the TCP island.     

Analysis of Clinical and Environmental Strains of Nontoxigenic Vibrio cholerae for Susceptibility to CTXΦ: Molecular Basis for Origination of New Strains with Epidemic Potential

Toxigenic Vibrio cholerae strains are lysogens of CTXΦ, a filamentous phage which encodes cholera toxin. The receptor for CTXΦ for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXΦ attachment sequence attRS, as well as for susceptibility of the strains to CTXΦ, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXΦ, using a genetically marked derivative of the phage CTX-KmΦ, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXΦ. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXΦ, leading to the origination of potential new epidemic clones.