Comparative genomics of closely related Salmonella enterica serovar Typhi strains reveals genome dynamics and the acquisition of novel pathogenic elements

BACKGROUND: Typhoid fever is an infectious disease of global importance that is caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi). This disease causes an estimated 200,000 deaths per year and remains a serious global health threat. S. Typhi is strictly a human pathogen, and some recovered individuals become long-term carriers who continue to shed the bacteria in their faeces, thus becoming main reservoirs of infection. RESULTS: A comparative genomics analysis combined with a phylogenomic analysis revealed that the strains from the outbreak and carrier were closely related with microvariations and possibly derived from a common ancestor. Additionally, the comparative genomics analysis with all of the other completely sequenced S. Typhi genomes revealed that strains BL196 and CR0044 exhibit unusual genomic variations despite S. Typhi being generally regarded as highly clonal. The two genomes shared distinct chromosomal architectures and uncommon genome features; notably, the presence of a ~10 kb novel genomic island containing uncharacterised virulence-related genes, and zot in particular. Variations were also detected in the T6SS system and genes that were related to SPI-10, insertion sequences, CRISPRs and nsSNPs among the studied genomes. Interestingly, the carrier strain CR0044 harboured far more genetic polymorphisms (83% mutant nsSNPs) compared with the closely related BL196 outbreak strain. Notably, the two highly related virulence-determinant genes, rpoS and tviE, were mutated in strains BL196 and CR0044, respectively, which revealed that the mutation in rpoS is stabilising, while that in tviE is destabilising. These microvariations provide novel insight into the optimisation of genes by the pathogens. However, the sporadic strain was found to be far more conserved compared with the others. CONCLUSIONS: The uncommon genomic variations in the two closely related BL196 and CR0044 strains suggests that S. Typhi is more diverse than previously thought. Our study has demonstrated that the pathogen is continually acquiring new genes through horizontal gene transfer in the process of host adaptation, providing novel insight into its unusual genomic dynamics. The understanding of these strains and virulence factors, and particularly the strain that is associated with the large outbreak and the less studied asymptomatic Typhi carrier in the population, will have important impact on disease control

Outbreak-associated Vibrio cholerae Genotypes with Identical Pulsotypes, Malaysia, 2009

A cholera outbreak in Terengganu, Malaysia, in November 2009 was caused by 2 El Tor Vibrio cholerae variants resistant to typical antimicrobial drugs. Evidence of replacement of treatable V. cholerae infection in the region with antimicrobial-resistant strains calls for increased surveillance and prevention measures

Genotypic and phenotypic characterization of Escherichia coli isolated from indigenous individuals in Malaysia

Objective(s): The occurrence of asymptomatic verocytotoxin (VT)-producing Escherichia coli (VTEC) infections among humans in recent years is posing a high risk to public health. Thus, the role of asymptomatic human carriers as a source of dissemination should not be underestimated. This study aimed to elucidate the phenotypic and genotypic characteristics of E. coli in the stool samples collected from indigenous individuals in Malaysia. Materials and Methods: E. coli strains (n=108) were isolated from stool samples obtained from 41 indigenous individuals. All strains were subjected to Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) typing and confirmation of VTEC variants. Non-duplicate strains were selected based on REP-PCR profiles and further subjected to antimicrobial susceptibility test (AST). The genotypic and phenotypic characteristics of the strains were then correlated with the demographic data of the subjects. Results: A total of 66 REP-PCR profiles grouped in 53 clusters (F=85%) were obtained. Four genetically distinct strains were confirmed as VTEC (eaeA-positive). The predominant resistance was against ampicillin (34.2%), followed by trimethoprim-sulfamethoxazole (32.9%), ampicillin-sulbactam (5.5%), and ciprofloxacin (1.4%). All isolates were sensitive to amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, and meropenem. Conclusion: Genetically diverse E. coli and VTEC strains were found to colonize the intestines of the indigenous populations. This study is important for the prospective surveillance of E. coli among the indigenous individuals in Malaysia, especially in asymptomatic VTEC infection and antimicrobial resistance phenomenon

Comparative genomic analysis of Vibrio Cholerae and its colonization factors / Cindy Teh Shuan Ju.

Vibrio cholerae, the causative agent of cholera, is endemic in Malaysia and is spread through the ingestion of contaminated food or water. The aims of this study were to develop a multiple PCR assay for differentiation of V. cholerae from other Vibrio species; to compare the efficiency of PCR assay with conventional biochemical tests and API 20E.; to characterize the strains based on their biotypes, serogroups, and virulotype by using a multiplex PCR assay; to determine the genetic relatedness of strains by using genotyping methods such as RAPD-PCR, ERIC-PCR, REP-PCR, MLVA, PFGE, MLST and MVLST; to study the virulence factors which cause colonization in different serogroups of V. cholerae; and the influence of host environment for colonization. Four pairs of primers were designed for differential detection of sister groups of Vibrio species Strains tested were differentiated into V. cholerae (493/338 bp), V. parahaemolyticus (493/409 bp), V. vulnificus (493/656 bp), Vibrio species (493 bp), and non-Vibrio (no amplification) based on pntA and gyrB genes. This multiplex PCR assay was more sensitivie and specific than API 20E identification assay. Another multiplex PCR assay based on ompW, hlyA, orf complex, toxR, ctxA, tcpI for V. cholerae biotyping, serogrouping and virulotyping was developed and tested on 43 V. cholerae strains. A total of 22 El Tor O1 and one O139 V. cholerae that harboured all virulence genes were identified. One El Tor O1 V. cholerae presented identical virulotype to 17 other non-O1/non-O139 V. cholerae, while the tcpI gene was detected in two non-O1/non-O139 V. cholerae. The 43 strains were also subtyped into 38, 40, 35, 30, 35, 38, 29 and 27 profiles by RAPD-PCR, ERIC-PCR, REPPCR, VCR-PCR, PFGE, MLVA, MLST and MVLST, respectively with discriminatory power ranging from 0.910 to 0.996. Overall, genetic diversity of non-O1/non-O139 V. cholerae strains was high while some of the O1 strains were indistinguishable. However, iv the unrelated strains which shared the same profiles were distinguished based on the combined analyses of the eight genotyping methods. However, each method possesses its own limitations. MLST and MVLST gave precise description of point mutation but were expensive. Overall, MLVA developed in this study remains the most suitable genotyping methods based on discriminatory ability, ease of operation, cost, timeline and data management. However, a combination of several genotyping methods may overcome the inefficiency of each single method and therefore able to distinguish unrelated strains. Finally, clinical and environmental O1 strains could colonize the mouse intestines but prolonged colonization was only observed with environmental strains which showed upregulated expression of rtxA and hlyA genes. The tcpI+ non-O1/non-O139 V. cholerae strain was a more efficient colonizer while the non-toxigenic O1 V. cholerae could colonize the mouse intestine once the virulence genes were favourably enriched and ‘turnon’ in the host environment. In conclusion, this study provided alternative approaches for rapid differentiation of V. cholerae from other pathogenic Vibrio species, as well as to biotype, serogroup and virulotype the V. cholerae strains. Regardless of serogroups, year, source and location of isolation, all the unrelated strains were distinguishable and therefore suggests a high diversity of V. cholerae population in Malaysia. Different traits of strains posses different colonization ability and tcpI gene might be the key regulator for colonization in non-O1/non-O139 V. cholerae. However, colonization in non-toxigenic O1 V. cholerae might be facilitated once the virulence genes were ‘enriched’ in the host environment

Multiple-Locus Variable-Number Tandem Repeat Analysis of Vibrio cholerae in Comparison with Pulsed Field Gel Electrophoresis and Virulotyping

Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F=0.63) while PFGE generated 35 pulsotypes (F=0.71). Simpson's index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D=0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P<.01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation

Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields

Research Article Multiple-Locus Variable-Number Tandem Repeat Analysis of Vibrio cholerae in Comparison with Pulsed Field Gel Electrophoresis and Virulotyping

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F = 0.63) while PFGE generated 35 pulsotypes (F = 0.71). Simpson’s index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D = 0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P &lt;.01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation. 1

Molecular characterization of extended-spectrum β-lactamase-producing Klebsiella pneumoniae from a Malaysian hospital

Multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae associated with nosocomial infections have caused serious problems in antibiotic management with limited therapeutic choices. This study aimed to determine the genotypic and phenotypic characteristics of K. pneumoniae strains isolated from a tertiary hospital in Malaysia. Ninety-seven clinical K. pneumoniae strains were analyzed for antimicrobial susceptibility, all of which were sensitive to amikacin and colistin (except one strain), while 31.9 % and 27.8 % were MDR and ESBL producers, respectively. PCR and DNA sequencing of the amplicons indicated that the majority of MDR strains (26/27) were positive for blaTEM, followed by blaSHV (24/27), blaCTX-M-1 group (23/27), blaCTX-M-9 group (2/27), and mcr-1 (1/27). Thirty-seven strains were hypervirulent and PCR detection of virulence genes showed 38.1 %, 22.7 %, and 16.5 % of the strains were positive for K1, wabG, and uge genes, respectively. Genotyping by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) showed that these strains were genetically diverse and heterogeneous. Sequence types, ST23, ST22, and ST412 were the predominant genotypes. This is the first report of colistin-resistant K. pneumoniae among clinical strains associated with mcr-1 plasmid in Malaysia. The findings in this study have contributed to the effort in combating the increase in antimicrobial resistance by providing better understanding of genotypic characteristics and resistance mechanisms of the organisms. © 2019, Sociedade Brasileira de Microbiologia