Evolution, distribution and genetics of atypical Vibrio cholerae – A review

Vibrio cholerae is the etiological agent of cholera, a severe diarrheal disease, which can occur as either an epidemic or sporadic disease. Cholera pandemic-causing V. cholerae O1 and O139 serogroups originated from the Indian subcontinent and spread globally and millions of lives are lost each year, mainly in developing and underdeveloped countries due to this disease. V. cholerae O1 is further classified as classical and El Tor biotype which can produce biotype specific cholera toxin (CT). Since 1961, the current seventh pandemic El Tor strains replaced the sixth pandemic strains resulting in the classical biotype strain that produces classical CT. The ongoing evolution of Atypical El Tor V. cholerae srains encoding classical CT is of global concern. The severity in the pathophysiology of these Atypical El Tor strains is significantly higher than El Tor or classical strains. Pathogenesis of V. cholerae is a complex process that involves coordinated expression of different sets of virulence-associated genes to cause disease. We are yet to understand the complete virulence profile of V. cholerae, including direct and indirect expression of genes involved in its survival and stress adaptation in the host. In recent years, whole genome sequencing has paved the way for better understanding of the evolution and strain distribution, outbreak identification and pathogen surveillance for the implementation of direct infection control measures in the clinic against many infectious pathogens including V. cholerae. This review provides a synopsis of recent studies that have contributed to the understanding of the evolution, distribution and genetics of the seventh pandemic Atypical El Tor V. cholerae strains.</p

Core and Accessory Genome Comparison of Australian and International Strains of O157 Shiga Toxin-Producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australian STEC epidemiology differs from many other countries, as severe outbreaks and HUS cases appear to be more often associated with non-O157 serogroups. It is not known why Australian strains of O157 STEC might differ in virulence to international strains. Here we investigate the reduced virulence of Australian strains. Multiple genetic analyses were performed, including SNP-typing, to compare the core genomes of the Australian to the international isolates, and accessory genome analysis to determine any significant differences in gene presence/absence that could be associated with their phenotypic differences in virulence. The most distinct difference between the isolates was the absence of the stx2a gene in all Australian isolates, with few other notable differences observed in the core and accessory genomes of the O157 STEC isolates analyzed in this study. The presence of stx1a in most Australian isolates was another notable observation. Acquisition of stx2a seems to coincide with the emergence of highly pathogenic STEC. Due to the lack of other notable genotypic differences observed between Australian and international isolates characterized as highly pathogenic, this may be further evidence that the absence of stx2a in Australian O157 STEC could be a significant characteristic defining its mild virulence. Further work investigating the driving force(s) behind Stx prophage loss and acquisition is needed to determine if this potential exists in Australian O157 isolates.</p

Genomic and Evolutionary Insights into Australian Toxigenic Vibrio cholerae O1 Strains

Vibrio cholerae O1 is the causative agent of cholera, a severe diarrheal disease which can cause death if left untreated. In this study, a collection of clinical and environmental V. cholerae serogroup O1 isolates from Australia (1977 to 1987) (from local cases and cases acquired through international travel) and publicly available international isolates were characterized for genotypic features (virulence genes, mobile genetic elements [MGEs], and antimicrobial resistance gene profiles). Whole-genome sequencing (WGS) was used to investigate and compare the genetic relatedness between the 44 Australian and nine travel-associated isolates and the 60 publicly available international V. cholerae sequences representing pre-seventh-pandemic (pre-7PET) isolates and different waves of 7PET isolates. In this study, 36 (81%) Australian clinical and aquatic isolates harbored the cholera toxin-producing genes located in the CTX bacteriophage region. All the Australian environmental and clinical isolates lacked the seventh-pandemic virulence-associated genomic islands (VSP-I and -II). In silico multilocus sequence typing (MLST) classified all nine internationally acquired isolates as sequence type 69 (ST69), 36 clinical and aquatic isolates as ST70, and eight isolates from Australia as ST71. Most of the nontoxigenic clinical and aquatic isolates of ST71 had diverse genetic variations compared to ST70 Australian strains. The antimicrobial resistance-associated genes gyrA, parC, and parE had no mutations in all the environmental and clinical isolates from Australia. The SXT genetic element and class 1 integron gene sequences were not detected in Australian strains. Moreover, in this study, a Bayesian evolutionary study suggests that two distinct lineages of ST71 (new set of strains) and ST70 strains were prevalent around similar times in Australia, in;1973 and 1969. IMPORTANCE Australia has its own indigenous V. cholerae strains, both toxigenic and nontoxigenic, that are associated with disease. Exotic strains are also detected in Australian patients returning from overseas travel. The clinical and aquatic V. cholerae O1 toxin gene-positive isolates from Australia responsible for cases in 1977 to 1987 were linked to acquisition from Queensland waterways but until now had not been characterized genetically. It is important to determine the genetic relatedness of Australian strains to international strains to assist in understanding their origin. This is the first extensive study to provide sequences and genomic analysis focused on toxigenic O1 V. cholerae clinical and environmental strains from Australia and its possible evolutionary relationship with other publicly available pre-7PET and 7PET V. cholerae strains. It is important to understand the population genetics of Australian V. cholerae from a public health perspective to assist in devising control measures and management plans for reducing V. cholerae exposure in Australia, given previous Australian disease clusters.</p

Molecular prediction of the O157: H-negative phenotype prevalent in Australian Shiga toxin-producing Escherichia coli cases improves concordance of in silico serotyping with phenotypic motility

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australia is unique in its STEC epidemiology, as severe cases are typically associated with non-O157 serogroups, and locally acquired O157 isolates are H-negative/nonmotile. The H-negative phenotype and reduced severity of disease compared to that associated with H7/motile strains are distinct features of Australian O157 strains, but the molecular mechanism behind this phenotype has not been reported. Accurate characterization of the H-negative phenotype is important in epidemiological surveillance of STEC. Serotyping is moving away from phenotype-based methods, as next generation sequencing allows rapid extrapolation of serotype through in silico detection of the O-antigen processing genes, wzx, wzy, wzm, and wzt, and the H-antigen gene, fliC. The detection and genotyping of fliC alone is unable to determine the motility of the strain. Typically, most Australian O157:H-negative strains carry an H7 genotype yet phenotypically are nonmotile; thus, many are mischaracterized as H7 strains by in silico serotyping tools. Comparative genomic analysis of flagellar genes between Australian and international isolates was performed and an insertion at nucleotide (nt) 125 in the flgF gene was identified in H-negative isolates. Chi-square results showed that this insertion was significantly associated with the H-negative phenotype (P 0.0001). Phylogenetic analysis was also completed and showed that the Australian H-negative isolates with the insertion in flgF represent a clade within the O157 serogroup, distinct from O157:H7 serotypes. This study provides a genetic target for inferring the nonmotile phenotype of Australian O157 STEC, which increases the predictive value of in silico serotyping.</p

Comparative genomics identifies distinct lineages of <i>S</i>. Enteritidis from Queensland, Australia

<div><p><i>Salmonella enterica</i> is a major cause of gastroenteritis and foodborne illness in Australia where notification rates in the state of Queensland are the highest in the country. <i>S</i>. Enteritidis is among the five most common serotypes reported in Queensland and it is a priority for epidemiological surveillance due to concerns regarding its emergence in Australia. Using whole genome sequencing, we have analysed the genomic epidemiology of 217 <i>S</i>. Enteritidis isolates from Queensland, and observed that they fall into three distinct clades, which we have differentiated as Clades A, B and C. Phage types and MLST sequence types differed between the clades and comparative genomic analysis has shown that each has a unique profile of prophage and genomic islands. Several of the phage regions present in the <i>S</i>. Enteritidis reference strain P125109 were absent in Clades A and C, and these clades also had difference in the presence of pathogenicity islands, containing complete SPI-6 and SPI-19 regions, while P125109 does not. Antimicrobial resistance markers were found in 39 isolates, all but one of which belonged to Clade B. Phylogenetic analysis of the Queensland isolates in the context of 170 international strains showed that Queensland Clade B isolates group together with the previously identified global clade, while the other two clades are distinct and appear largely restricted to Australia. Locally sourced environmental isolates included in this analysis all belonged to Clades A and C, which is consistent with the theory that these clades are a source of locally acquired infection, while Clade B isolates are mostly travel related.</p></div

Maximum-likelihood phylogeny of QLD <i>S</i>. Enteritidis isolates, <i>S</i>. Typhimurium LT2 and <i>S</i>. Gallinarum 287/91 rooted to <i>S</i>. Berta.

<p>Isolates cluster into three clades as indicated. The scale bar shows substitutions per site. Sequence type and Phage type are indicated by colour as shown in the legend. The presence and absence of regions of difference (RODs) and prophages are also indicated in the following order: φSE10, φSE12, φSE12A, φSE14, φSE20, RE-2010, ROD4, ROD9, ROD13, ROD17, ROD21, ROD25, ROD28, ROD34, ROD37, ROD42, with a white box indicating absence and a black box indicating presence. Blue stars, red triangles and blue boxes indicate partial presence of the prophage.</p

Heatmap showing the presence and absence of acquired antimicrobial resistance genes and point mutations in <i>gyrA</i>.

<p>Blue boxes indicate absence and red boxes indicate presence of genes or point mutations.</p

Culture-independent detection of chlorhexidine resistance genes qacA/B and smr in bacterial DNA recovered from body sites treated with chlorhexidine-containing dressings

Purpose. Dressings containing chlorhexidine gluconate (CHG) are increasingly used in clinical environments for prevention of infection at central venous catheter insertion sites. Increased tolerance to this biocide in staphylococci is primarily associated with the presence of qacA/B and smr genes. Methodology. We used a culture-independent method to assess the prevalence of these genes in 78 DNA specimens recovered from the skin of 43 patients at catheter insertion sites in the arm that were covered with CHG dressings. Results. Of the 78 DNA specimens analysed, 52 (67 %) possessed qacA/B and 14 (18 %) possessed smr; all samples positive for smr were also positive for qacA/B. These prevalence rates were not statistically greater than those observed in a subsample of specimens taken from non-CHG treated contralateral arms and non-CHG-dressing exposed arms. A statistically greater proportion of specimens with greater than 72 h exposure to CHG dressings were qac-positive (P=0.04), suggesting that the patients were contaminated with bacteria or DNA containing qacA/B during their hospital stay. The presence of qac genes was not positively associated with the presence of DNA specific for Staphylococcus epidermidis and Staphylococcus aureus in these specimens. Conclusion. Our results show that CHG genes are highly prevalent on hospital patients’ skin, even in the absence of viable bacteria