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Genetic diversity and population structure of Vibrio cholerae (vol 37, pg 581, 1999)
Journal of Clinical Microbiology
... Diversity within toxigenic strains of V. cholerae has been demonstrated by multilocus enzyme electrophoresis (MLEE) (14,53), DNA sequence analysis of housekeeping genes (21,178) and genomic fingerprinting by ERIC-PCR (209). In order to understand the bacterial population structure, identification of the clonality of the isolates is critical. ...
... More detail discussion about bacterial population models is provided by Spratt and Maiden (176). In a large and diverse population, such as V. cholerae, a complex model showing a high degree of recombination in some branches and clonal lineages in others appears to be the best fit (14,53). ...
... However the focus has been on the epidemic clones (54,69). An extensive study of the genetic diversity and population structure of V. cholerae as an entire species was conducted by Beltran et al. (14). Using multilocus enzyme electrophoresis (MLEE) analysis of seventeen enzyme loci and a collection of 397 V. cholerae isolates, including 143 serogroup reference strains, it was observed that the genetic diversity of V. cholerae was high, with the mean diversity of each locus being 0.436, which was smaller than the diversity value of 0.627 for Salmonella enterica (164), but larger than the comparable value of 0.343 reported for Escherichia coli (163). ...
Thesis research directed by: Marine-Estuarine-Environmental Sciences. Title from t.p. of PDF. Thesis (Ph. D.) -- University of Maryland, College Park, 2004. Includes bibliographical references. Text.
... The polysaccharide biogenesis region is one of the most variable regions in the genome and lateral gene transfer has played an important role in its evolution. As a result, the serogroups in V. cholerae do not necessarily follow the evolution of its chromosomal backbone, as reflected in phylogenetic trees constructed in other studies (Beltran et al., 1999). For housekeeping genes such as those used for MLST, all of the genes are very closely related, indicating that the vast majority of housekeeping genes have a single lineage or phylogenetic branch for V. cholerae (Beltran et al., 1999; Stine et al., 2000; Garg et al., 2003). ...
... As a result, the serogroups in V. cholerae do not necessarily follow the evolution of its chromosomal backbone, as reflected in phylogenetic trees constructed in other studies (Beltran et al., 1999). For housekeeping genes such as those used for MLST, all of the genes are very closely related, indicating that the vast majority of housekeeping genes have a single lineage or phylogenetic branch for V. cholerae (Beltran et al., 1999; Stine et al., 2000; Garg et al., 2003). In contrast, gmd, a gene for O-antigen biogenesis in V. cholerae O139, has an allele that has greater similarity to E. coli than to Vibrio, consistent with lateral gene transfer (Garg et al., 2003). ...
Both NRT36S and A5 are NAG-ST-producing, serogroup O31 Vibrio cholerae. NRT36S is encapsulated and causes diarrhea when administered to volunteers; A5 is unencapsulated and does not colonize or cause illness in humans. The capsule/LPS (CPS/LPS) biogenesis regions in these two isolates were similar except that a 6.5-kb fragment in A5 has replaced a 10-kb fragment in NRT36S in the middle of the CPS/LPS gene cluster. Although the genes of the replaced region were homologous to genes from other CPS/LPS, they had little similarity to NRT36S and were not homologous to genes from other Vibrios. Data of this study highlight the apparent mobility within the CPS/LPS region that would provide a basis for the large number of observed V. cholerae serogroups and the emergence of novel epidemic strains.
... Токсинкорегулируемые пили адгезии (ТКПА) являются одним из основных факторов патогенности Vibrio cholerae и необходимы для колонизации холерными вибрионами тонкого кишечника [4]. Последовательность гена tcpA, кодирующего синтез основного белка ТКПА -пилина TcpA, отличается значительной вариабельностью у холерных вибрионов разных серогрупп, в том числе и у близкородственных холерных вибрионов О1 серогруппы классического и эльтор биоваров, которые имеют лишь 75 % идентичности аминокислотной последовательности кодируемого белка [1]. У холерных вибрионов не О1/не О139 серогрупп также выявлено несколько вариантов TcpA и кодирующих их генов [3,4]. ...
Developed is the PCR assay for the detection of the structural genes of toxin co-regulated adhesion piluses - tcpA of different types. Determined are the universal primers, the usage of which provides for the detection of the stated above genes in V. cholerae of various serogroups. With the help of this PCR assay identified is a new variant of tcpA gene in toxigenic cholera vibrio of non-O1/non-O139 serogroup.
... Il faut garder à l'esprit dans ces comparaisons que les performances et le niveau de résolution d'une technique donnée peuvent varier quelque peu d'un laboratoire à l'autre.Duncan et al., 1994), Borrelia (étude portant sur 4 espèces du genre) (Boerlin et al., 1992), Campylobacter (étude portant sur C. jejuni et C. cob) (Aeschbacher & Piffaretti, 1989), Salmonella (étude portant sur 5 espèces du genre) (Boyd et al., 1996) (Selander et al., 1996).Toutefois, cette espèce est subdivisée en deux sous-groupes, chacun d'entre eux présentant une diversité génétique réduite (Duncan et al., 1994).Musser et al., 1991).(Duncan et al., 1994)(Helgason et al., 2000)(van derZee et al., 1997) (Boerlin et al., 1992) (Wise,Shimkets & McArthur, 1995) (Aeschbacher & Piffaretti, 1989)(Chooromoney et al., 1994) (Souza et al., 1999) (Musser et al., 1990)(ceUe étude (Selander et al., 1985) (Boerlin & Piffaretti, 1991) (Wasem, McCarthy & Murray, 1991) (Wasem et al., 1991) (O'Rourke & Stevens, 1993)(Caugant et al., 1988) (Caugant et al., 1987)(Selander, Li & Nelson, 1996) (Whittam, Ochman & Selander, 1983b) (Musser & Selander, 1986)(Takala et al., 1996)(Musser et al., 1991) (Beltran et al., 1999) (Ben Abderrazak et ai., 1999) (Note: La diversité génétique (H) est calculée selon la formule H=1-rxl(n/n-1) (Nei, 1978(a) Isolats d'au moins quatre espèces de Borrelia (B. burgdorferi, B. garinii,).)et ...
... The American continent has been free from cholera for more than a century, until an outbreak hit Peru in 1991 (Tauxe et al., 1994) (Beltran et al., 1999). Based on results of our analysis, and consistent with those of , it is concluded that ICEs isolated in America had an independent origin and evolution with respect to the ICEs circulating in Interestingly, from analysis of the conserved and variable regions of the ICEs that were discovered, it is concluded that all profiles indicate genetic hybrids, in which sequences belonging to R391, SXT, and unknown genetic regions are present in the same element. ...
... In the neighbour joining method (Fig. 5) [19], Escherichia coli[20], Salmonella spp. [21], Aeromonas salmonicida [22], Pisciricketssia salmonis [23], Flavobacterium psychrophilum [24], Vibrio vulnifi cus [25], Vibrio parahaemolyticus [26], Vibrio cholerae [27], Clostridium botulinum [28], and Renibacterium salmoninarum [29]. One of the primers OPY15 produced amplifi ed fragment common to all the strain with molecular weight 0.75 Kb referred to as unique band. ...
In the present study, DNA fingerprinting of eight strains of Flavobacterium columnare was done by random amplification of polymorphic DNA (RAPD) fingerprinting method. The strains were collected from Fish Health Management Division, Central Institute of Freshwater Aquaculture, Bhubaneswar, India. A total number of 160 primers were screened for RAPD-PCR, of which 10 primers yielded amplification with all the strains. The molecular weight of amplified bands varied from 0.29-2.63 Kb. The number of bands varied from 1 to 8. Unique band was seen with primer OPY-15 with molecular weight 0.75 Kb that can be used for epidemiological study. Genetic variability was investigated using NTSYS software. Highest genetic similarity was found between MS1 and MS3 followed by MS5 and MS7. Minimum genetic similarity was found between MS2 and MS8. Phylogenetic tree was constructed using UPGMA and neighbor joining methods.
... Genetic differences have been identified in the chromosomal regions encoding the capsule of the two serogroups, suggesting that this region was recently acquired in V. cholerae O139. 97 Examining the genetic diversity of 143 V. cholerae serogroups, Beltrán et al. 108 concluded that based on MLEE patterns, new cholera clones probably arise from the modification of epidemic lineages. As it has been shown that the V. cholerae genome is more susceptible to genetic alterations than other species 99,107 , there is a distinct possibility that new pandemic serogroups of V. cholerae may emerge. ...
... Intrigued by the emergence of the pathogenic O139 strain in India, increased interests have been focused on gaining a full understanding of the biology of the non-O1 groups (Dalsgaard et al., 1995;Beltran et al., 1999). Most environmental non-O1 strains are thought not to produce cholera toxin. ...
Vibrio cholerae is the causative agent of the severe dehydrating diarrheal disease cholera. This bacterium has been detected in many estuaries around the world and the United States. In this study we examine the abundance and distribution of V. cholerae in recreational beach waters and tributaries of Southern California. Water samples were taken from 11 beach locations adjacent to freshwater runoff sources between February 8th and March 1st, 1999. Water samples were also taken from rivers, creeks and coastal wetlands along the Southern California coast between May 19th and June 28th, 1999. In addition to the detection of V. cholerae, environmental parameters including temperature, salinity, coliphage counts, viable heterotrophic plate counts and total bacterial direct counts were also determined to understand the relationships between the presence of V. cholerae and environmental conditions. A direct colony hybridization method using an oligonucleotide probe specific for the 16S–23S intergenic spacer region of V. cholerae, detected V. cholerae in 3 of the 11 beach samples with the highest concentration (60.9 per liter) at the mouth of Malibu Lagoon. V. cholerae and coliphage were not correlated for beach samples, indicating that the presence of V. cholerae is independent of sewage pollution. V. cholerae were detected in all samples taken from rivers, creeks and wetlands of coastal Southern California where salinities were between 1 to 34 parts per thousand (ppt), but was not found at a freshwater sampling site in upper San Juan Creek. The highest density of V. cholerae was found in San Diego Creek with a concentration of 4.25105 CFU/L. The geographical distribution of V. cholerae was inversely correlated with salinity. High concentrations of V. cholerae were more frequently detected in waters with lower (but above 0) salinity. The results of this study provide insight into the ecology of this aquatic species and are potentially important to the understanding of the epidemiology of cholera on a global scale.
... These observations indicate O antigen switching in strains of Y. enterocolitica as suggested recently by MLST [49]. Such observations have however been reported in other bacteria also [24,41,50]. Thus, given the enormous discriminatory power of genotyping techniques such observations also emphasize the need to discuss threadbare , the question of suitability of widely used typing techniques like serotyping. ...
Genetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.
All loci were polymorphic and generated 62 electrophoretic types (ETs) and 12 restriction types (RTs). The mean genetic diversity (H) of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98) was more discriminatory and clustered Y. enterocolitica biovar 1A strains into four groups, while MLRT (DI = 0.77) identified two distinct groups. BURST (Based Upon Related Sequence Types) analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change.
MLEE revealed greater genetic diversity among strains of Y. enterocolitica biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.
... It is isolated from muds and waters and also from molluscs and crustaceans (Nair et al., 1988Nair et al., , 1991). The environmental isolates are much more variable than those of the sixth and seventh pandemics (Beltran et al., 1999; Farfan et al., 2000), which can now be seen to be closely related clones with human pathogenic properties. The VPI was initially thought to be present only in the epidemic V. cholerae isolates, but a number of studies have shown that it is also present in some environmental isolates, with recovery of novel tcpA alleles (Novais et al., 1999; Nandi et al., 2000; Mukhopadhyay et al., 2001; Boyd & Waldor, 2002; Li et al., 2002 Li et al., , 2003). ...
The Vibrio pathogenicity island (VPI) encodes the toxin-coregulated pilus and other virulence factors for Vibrio cholerae to colonize the human intestine to cause cholera. We assessed the level of genetic variation of VPI in nine nonpandemic isolates, and compared them with the sixth and seventh pandemic strains by sequencing c. 5 kb each from the start, middle and end regions of the VPI. Variation is similar among the three regions at around 2%, except for the tcpA gene, which has a much higher level of variation (23%). Numerous recombination segments were identified with sizes up to 2177 bp. Nearly all VPI genes sequenced have a ratio of synonymous to nonsynonymous substitutions considerably lower than that for housekeeping genes, suggesting that VPI genes are under positive selection pressure for change. The tagA gene was deleted or damaged in six isolates, which is likely to affect the efficiency of colonization of the human intestine. Two genes, orf2 and acfD, previously found to be translated differently in the sixth and seventh pandemic strains, were determined to be mutant in the seventh and sixth pandemic strains, respectively. These findings enhance our understanding of variation in the VPI, and of the pathogenic potential of VPI-positive environmental isolates.
... According to MEE data, V. mimicus is a heterogeneous genetic group of microorganisms. The mean genetic diversity per locus was 0.431, comparable to the value of 0.436 found by Beltran et al. (2) for V. cholerae strains. These degrees of variation were less than the 0.52 reported for the Escherichia coli reference collection (12). ...
In this study, we demonstrated that analyzed strains ofVibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that
the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal
intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical
strains were analyzed.
... The present estimate of MGD is 0.339. Our previous estimated MGD was 0.326 using nearly half the strains of the present study. Chen et al. (1991) found 0.311 and); P1 (peptidase leucyl-leucyl-leucine); P2 (peptidase leucyl glycil glycine) with the addition of the locus LAP: leucyl leucyl aminopeptidase (Pasteur et al. 1990). Beltran et al. (1999) from a large sample of American non- O1 isolates found a higher diversity of 0.436. A possible source of variation in MGD seems to be the choice of loci. Our choice was aimed at finding loci diagnostic of the species V. cholerae with low genetic diversity GD and this may have biased in part the MGD. This diversity is considerable and is ...
Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.
... Each of the loci examined had a variable number of observed alleles: 9 for dnaE, 20 for lap, 11 for rstR, 11 for gmd, 2 for recA, 8 for pgm, 4 for gyrB, 7 for cat, and 5 for chi. The most variable, lap with 20 alleles, was expected because it is a highly variable locus when analyzed with multilocus enzyme electrophoresis (25). The most common allele was present in 91% of isolates (n=87) for dnaE, 77% (n=86) for lap, 79% (n=90) for rstR, 82% (n=87) for gmd, 99% (n=96) for recA, 90% (n=94) for pgm, 97% (n=92) for gyrB, 93% (n=88) for cat, and 94% (n=89) for chi. ...
Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. cholerae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics.
... Because such fragments are ordinarily incorporated into the recipient chromosome by homologous recombination , transformation usually mediates gene exchange between taxonomically closely related organisms and especially between strains of the same species. For genetically diverse species such as V. cholerae363738394041, fragments acquired by transformation can be expected to affect fitness through the acquisition of genes that are present in the donor but missing or defective in the recipient, and whose presence in the recipient would confer a selective advantage [42]. One such gene class, which varies between V. cholerae serogroups, is the O-antigen–encoding sequences, which, based on antigenic differences, exceed 200 in number [4] . ...
The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.
... A change in serogroup may have a selective advantage, permitting the pathogen to escape immune detection. In Vibrio cholerae, several distinct O-antigens (O1, O37, O139, O27, O53 and O65) have been found among the genetically related strains that have been associated with epidemic disease and that carry the gene for cholera toxin (Bik et al., 1995; Comstock et al., 1996; Stroeher et al., 1997; Beltran et al., 1999; Li et al., 2002). The genes at the ends of the transferred segment have been suggested to be regions of enhanced recombination, although the breakpoints were not precisely defined (Bik et al., 1995; Li et al., 2002; Blokesch & Schoolnik, 2007). ...
In Gram-negative bacteria, the O-antigen-encoding genes may be transferred between lineages, although mechanisms are not fully understood. To assess possible lateral gene transfer (LGT), 21 Argentinean Vibrio cholerae O-group 1 (O1) isolates were examined using multilocus sequence typing (MLST) to determine the genetic relatedness of housekeeping genes and genes from the O1 gene cluster. MSLT analysis revealed that 4.4% of the nucleotides in the seven housekeeping loci were variable, with six distinct genetic lineages identified among O1 isolates. In contrast, MLST analysis of the eight loci from the O1 serogroup region revealed that 0.24% of the 4943 nucleotides were variable. A putative breakpoint was identified in the JUMPstart sequence. Nine conserved nucleotides differed by a single nucleotide from a DNA uptake signal sequence (USS) also found in Pastuerellaceae. Our data indicate that genes in the O1 biogenesis region are closely related even in distinct genetic lineages, indicative of LGT, with a putative DNA USS identified at the defined boundary for the DNA exchange.
Vibrio cholerae is a ubiquitous aquatic microbe in temperate and tropical coastal areas. It is a diverse species, with many isolates that are harmless to humans, while others are highly pathogenic. Most notable among them are strains belonging to the pandemic O1/O139 serogroup lineage, which contains the causative agents of cholera. The environmental selective regimes that led to this diversity are key to understanding how pathogens evolve in environmental reservoirs. A local population of V. cholerae and its close relative Vibrio metoecus from a coastal pond and lagoon system was extensively sampled during two consecutive months across four size fractions (480 isolates). In stark contrast to previous studies, the observed population was highly clonal, with 60% of V. cholerae isolates falling into one of five clonal complexes, which varied in abundance in the short temporal scale sampled. V. cholerae clonal complexes had significantly different distributions across size fractions and the two environments sampled, the pond and the lagoon. Sequencing the genomes of 20 isolates representing these five V. cholerae clonal complexes revealed different evolutionary trajectories, with considerable variations in gene content with potential ecological significance. Showing genotypic differentiation and differential spatial distribution, the dominant clonal complexes are likely ecologically divergent. Temporal variation in the relative abundance of these complexes suggests that transient blooms of specific clones could dominate local diversity.
IMPORTANCE: Vibrio cholerae is commonly found in coastal areas worldwide, with only a single group of this bacterium capable of causing severe cholera outbreaks. However, the potential to evolve the ability to cause disease exists in many strains of this species in its aquatic reservoir. Understanding how pathogenic bacteria evolve requires the study of their natural environments. By extensive sampling in a geographically restricted location in the United States, we found that most cells of a V. cholerae population belong to only a small number of strains. Analysis of their genome composition and spatial distribution indicates differential environmental adaptations between these strains. Other strains exist in smaller numbers, and the population was found to be temporally varied. This suggests frequent bloom and collapse cycles on a time scale of weeks. These population dynamics make it possible that more virulent strains could stochastically rise to large numbers, allowing for infection to occur.
Vibrio cholerae is an estuarine bacterium associated with a single peak of cholera (March�May) in coastal
villages of Bangladesh.For anunknownreason, however, cholera occurs in a unique dual peak (March�May and September�November) pattern in the city of Dhakathat is bordered by a heavily polluted freshwater river system and flood embankment. In August 2007, extreme flooding was accompanied by an unusually severe diarrhea outbreak in Dhaka that resulted in a record high illness. This study was aimed to understand the unusual outbreak and if it was related to the circulation of a new V. cholerae clone. Nineteen V. cholerae isolated during the peak of the 2007 outbreak were subjected to extensive phenotypic and molecular analyses, including multilocus genetic screening by polymerase chain reaction (PCR), sequence-typing of the ctxB gene, and pulsed-field gel electrophoresis (PFGE). Factors associated with the unusual incidence of cholera were determined and analysis of the disease severity was done. Overall, microbiological and molecular data confirmed that the hypervirulent V. cholerae was O1 biotype El Tor (ET) that possessed cholera toxin (CT) of the classical biotype. The PFGE (NotI) and dendrogram clustering confirmed that the strains were clonal and related to the pre-2007 variant ET from Dhaka and Matlab and resembled one of two distinct clones of the variant ETconfirmed to be present in the estuarine ecosystem of Bangladesh. Results of the analyses of both diarrheal case data for three consecutive years (2006-�2008) and regional droclimatology over three decades (1980�2009) clearly indicate that the pattern of cholera occurring in Dhaka, and not seen at other endemic sites, was associated with flood waters transmitting the infectious clone circulating via the fecal-oral route during and between the dual seasonal cholera peaks in Dhaka. Circular river systems and flood embankment likely facilitate transmission of infectious V. cholerae throughout the year that leads to both sudden and off-season outbreaks in the densely populated urban ecosystem of Dhaka. Clonal recycling of hybrid ElTor with increasing virulence in a changing climate and in a region with a growing urban population represents a serious public health concern for Bangladesh.
Vibrio cholerae, etiologic agent of cholera, is transmitted to humans by ingestion of contaminated food or water. Even though serogroups O1 and O139 are the ones usually associated to epidemic cholera, isolates from other serogroups also cause gastroenteritis and extraintestinal infections. During the period 2003-2005, presence of V. cholerae in stools was investigated in children with diarrhea that seaked assistance at the Niño Jesús Hospital in Tucumán. Thirty four isolates of V. cholerae non-O1, non-O139 were recovered. We characterized the isolates studying its virulence factors by PCR, antimicrobial susceptibility patterns and genetic diversity by pulsed-field gel electrophoresis. Eight virulence patterns were obtained although no isolate was positive for the cholera toxin or the thermostable toxin. Four isolates were positive for the type three secretion system. The 17.6% of the isolates were resistant or intermediate to ampicillin and 5.9% were resistant to trimethoprim-sulfamethoxazole. By Sfil-PFGE, all isolates were genetically very diverse, as 27 different patterns were identified in 29 typeable isolates by pulsed-field gel electrophoresis. Although it has a low incidence, V. cholerae continues to be a causative agent of diarrhea in children, who are affected by a variety of circulating strains of V. cholerae non-O1, non-O139.
The genes encoding cholera toxin (ctxA andctxB) are encoded in the genome of CTXφ, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXφ, we examined genome diversity in CTXφs derived from a variety of epidemic and
nonepidemic Vibriosp. natural isolates. Among these were three V. choleraestrains that contained CTX prophage sequences but not thectxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXφ replicative
form, with the exception of missingctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5′ ofctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXφ precursor that lacked
ctxABsimultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional
CTXφs, two CTXφ-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicusisolates. Comparative nucleotide sequence analyses revealed that the CTXφs derived from classical and El Tor V. choleraeisolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the twoV. cholerae O1 biotypes independently acquired distinct CTXφs.
Many of the genes responsible for the virulence of bacterial pathogens are carried by mobile genetic elements that can be transferred horizontally between different bacterial lineages. Horizontal transfer of virulence-factor genes has played a profound role in the evolution of bacterial pathogens, but it is poorly understood why these genes are so often mobile. Here, I present a hypothetical selective mechanism maintaining virulence-factor genes on horizontally transmissible genetic elements. For virulence factors that are secreted extracellularly, selection within hosts may favour mutant 'cheater' strains of the pathogen that do not produce the virulence factor themselves but still benefit from factors produced by other members of the pathogen population within a host. Using simple mathematical models, I show that if this occurs then selection for infectious transmission between hosts favours pathogen strains that can reintroduce functional copies of virulence-factor genes into cheaters via horizontal transfer, forcing them to produce the virulence factor. Horizontal gene transfer is thus a novel mechanism for the evolution of cooperation. I discuss predictions of this hypothesis that can be tested empirically and its implications for the evolution of pathogen virulence.
We report a set of oligonucleotide primers and amplification conditions for the polymerase chain reaction to detect the ctx operon of Vibrio cholerae. The results of this assay on strains of V. cholerae and related organisms were identical with those obtained by the DNA colony hybridization test with the polynucleotide probe. The detection limit of this system was 1 pg of chromosomal DNA or broth culture containing three viable cells. The polymerase chain reaction method could directly detect the ctx operon sequences in rice-water stool samples from patients with cholera.
A novel Vibrio cholerae insertion sequence element, designated IS1004, was characterized and used for DNA fingerprinting of Vibrio spp. IS1004 comprises 628 bp and contains an open reading frame whose product shows a large degree of sequence identity with the IS200-encoded transposase. IS1004 was present in one to eight copies in most of the V. cholerae strains analyzed. The IS1004-generated fingerprints of epidemic V. cholerae strains with serotype O1 were closely related, although it was possible to distinguish between the two biotypes, classical and El Tor. Non-O1 serotype strains generally showed heterogeneous patterns unrelated to those of the epidemic O1 strains. Several strains were observed with identical or related fingerprint patterns but expressed different serotypes. Conversely, strains with different fingerprint patterns but identical serotypes were also found. These observations indicate that the gene clusters coding for distinct O antigens may be transferred horizontally between V. cholerae strains. Two examples of non-O1 strains with a fingerprint resembling that of epidemic O1 strains were found; they were the O139 Bengal strain and an O37 strain. The O139 Bengal strain is closely related to the El Tor biotype. The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio. Our study, however, shows that the O37 Sudan strain is genetically closely related to classical O1 strains. Similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes. Thus, O37 Sudan represents a second example of an epidemic V. cholerae strain carrying non-O1 antigens. This study underlines the importance of genotypic methods for the differentiation of V. cholerae strains and for recognition of strains with epidemic potential.
Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.
Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3.
Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V. cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993). The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S. Gulf Coast, and 5 O139 Bengal isolates. Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates. O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites. The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide. Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.
Sequences of the icd gene, encoding isocitrate dehydrogenase (IDH), were obtained for 33 strains representing the major phylogenetic lineages of Escherichia coli and Salmonella enterica. Evolutionary relationships of the strains based on variation in icd are generally similar to those previously obtained for several other housekeeping and for invasion genes, but the sequences of S. enterica subspecies V strains are unusual in being almost intermediate between those of the other S. enterica subspecies and E. coli. For S. enterica, the ratio of synonymous (silent) to nonsynonymous (replacement) nucleotide substitutions between pairs of strains was larger than comparable values for 12 other housekeeping and invasion genes, reflecting unusually strong purifying selection against amino acid replacement in the IDH enzyme. All amino acids involved in the catalytic activity and conformational changes of IDH are strictly conserved within and between species. In E. coli, the level of variation at the 3' end of the gene is elevated by the presence in some strains of a 165-bp replacement sequence supplied by the integration of either lambdoid phage 21 or defective prophage element e14. The 72 members of the E. coli Reference Collection (ECOR) and five additional E. coli strains were surveyed for the presence of phage 21 (as prophage) by PCR amplification of a phage 21-specific fragment in and adjacent to the host icd, and the sequence of the phage 21 segment extending from the 3' end of icd through the integrase gene (int) was determined in nine strains of E. coli. Phage 21 was found in 39% of E. coli strains, and its distribution among the ECOR strains is nonrandom. In two ECOR strains, the phage 21 int gene is interrupted by a 1,313-bp insertion element that has 99.3% nucleotide sequence identity with IS3411 of E. coli. The phylogenetic relationships of phage 21 strains derived from sequences of two different genomic regions were strongly incongruent, providing evidence of frequent recombination.