Kwai Lin Thong

Genetic Diversity of Clinical and Environmental Strains of Salmonella enterica Serotype Weltevreden Isolated in Malaysia

ABSTRACT The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.

Loop-Mediated Isothermal Amplification Assay for the Rapid Detection ofStaphylococcus 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.

Genotypic Characterization of Salmonella typhi by Amplified Fragment Length Polymorphism Fingerprinting Provides Increased Discrimination as Compared to Pulsed-Field Gel Electrophoresis and Ribotyping

Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six S. typhi isolates from diverse geographic areas (Malaysia, Indonesia, India, Chile, Papua New Guinea and Switzerland) gave unique, heterogeneous profiles when typed by AFLP, a result which was consistent with ribotyping and PFGE analysis. In a further study of selected S. typhi isolates from Papua New Guinea which caused fatal and non-fatal disease previously shown to be clonally related by PFGE, AFLP discriminated between these isolates but did not indicate a linkage between genotype with virulence. We conclude that AFLP (discriminatory index=0.88) has a higher discriminatory power for strain differentiation among S. typhi than ribotyping (DI=0.63) and PFGE (DI=0.74).

Variations in motility and biofilm formation of Salmonella enterica serovar Typhi

BackgroundSalmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only. The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely unknown. The main objective of this study was to explore and investigate the motility and biofilm forming behaviour among S. Typhi strains of diverse background.ResultsSwim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S. Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated swimming motility with featureless or bull’s eye morphology. Interestingly, S. Typhi strains of blood-borne origin exhibited significantly higher swimming motility (P < 0.05) than stool-borne strains suggesting that swimming motility may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains.ConclusionIn summary, both swimming and swarming motility are conserved among S. Typhi strains but there was variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of this pathogen.

Colorimetric detection of DNA hybridization based on a dual platform of gold nanoparticles and graphene oxide

The unique property of gold nanoparticles (Au NP) to induce colour change and the versatility of graphene oxides (GO) in surface modification makes them ideal in the application of colorimetric biosensor. Thus we developed a label free optical method to detect DNA hybridization through a visually observed colour change. The Au NP is conjugated to a DNA probe and is allowed to hybridize with the DNA target to the GO thus causing a change in colour from pinkish-red to purplish blue. Spectrophometry analysis gave a wavelength shift of 22 nm with 1 µM of DNA target. Sensitivity testing using serially diluted DNA conjugated GO showed that the optimum detection was at 63 nM of DNA target with the limit at 8 nM. This proves the possibility for the detection of DNA hybridization through the use of dual nanoparticle system by visual observation.

Utility of multilocus variable-number tandem-repeat analysis as a molecular tool for phylogenetic analysis of Shigella sonnei.

ABSTRACT A panel of 916 isolates, including 703 closely related IST1 isolates, were characterized by inter-IS1 spacer typing (IST), pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) to evaluate the utility of MLVA as a molecular tool for the phylogenetic analysis of Shigella sonnei. The global phylogenetic patterns determined by IST, PFGE, and MLVA were concordant. MLVA was carried out using 26 VNTR loci with a range of degrees of variability. MLVA data for the 703 IST1 isolates revealed that diversification among the closely related isolates was attributed mainly to four highly variable loci. The phylogenetic pattern for the closely related isolates determined using MLVA profiles of 8 highly variable loci was in agreement with that determined using the 26-locus profiles. A clustering analysis using the profiles of 18 loci with limited variability established clear phylogenetic relationships among IST clonal groups. Accordingly, MLVA is a useful tool for the phylogenetic analysis of S. sonnei. Combined VNTR loci with higher variability are useful markers for resolving closely related isolates, whereas combined loci with lower variability are suitable for establishing clear phylogenetic relationships between strains or clones that have evolved over a longer timescale.

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims:  To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation.

Pathogenic and Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular Malaysia

Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.

Multidrug-resistant strains of Salmonella enterica serotype typhi are genetically homogenous and coexist with antibiotic-sensitive strains as distinct, independent clones

OBJECTIVE The goal of this study was to report the molecular analysis of antibiotic-sensitive and multidrug-resistant (MDR) strains of Salmonella typhi, using pulsed-field gel electrophoresis (PFGE), with a particular emphasis on the coexistence of these strains in a typhoid-endemic region of Karachi, Pakistan. METHODS One hundred isolates of S. typhi in humans (50 MDR and 50 antibiotic-sensitive isolates) from sporadic cases of typhoid fever were analyzed by Vi-phage typing, antibiograms and PFGE. RESULTS The MDR S. typhi strains were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Analysis by PFGE showed that 50 MDR isolates of S. typhi had a single, homogenous PFGE profile, which was distinctly different from that of 50 antibiotic-sensitive isolates obtained in the same time frame from the same area. This latter group of isolates showed much greater diversity of PFGE profiles, as has been observed in other endemic regions. CONCLUSIONS Multidrug-resistant and antibiotic-susceptible strains of S. typhi can coexist in endemic areas as epidemiologically independent pathogens and are not in competition for continued persistence and transmission.

RNA-seq analysis of Macrobrachium rosenbergii hepatopancreas in response to Vibrio parahaemolyticus infection

BackgroundThe Malaysian giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean worldwide. However, production of this prawn is facing a serious threat from Vibriosis disease caused by Vibrio species such as Vibrio parahaemolyticus. Unfortunately, the mechanisms involved in the immune response of this species to bacterial infection are not fully understood. We therefore used a high-throughput deep sequencing technology to investigate the transcriptome and comparative expression profiles of the hepatopancreas from this freshwater prawn infected with V. parahaemolyticus to gain an increased understanding of the molecular mechanisms underlying the species’ immune response to this pathogenic bacteria.ResultA total of 59,122,940 raw reads were obtained from the control group, and 58,385,094 reads from the Vibrio-infected group. Via de novo assembly by Trinity assembler, 59,050 control unigenes and 73,946 Vibrio-infected group unigenes were obtained. By clustering unigenes from both libraries, a total of 64,411 standard unigenes were produced. The standard unigenes were annotated against the NCBI non-redundant, Swiss-Prot, Kyoto Encyclopaedia of Genes and Genome pathway (KEGG) and Orthologous Groups of Proteins (COG) databases, with 19,799 (30.73%), 16,832 (26.13%), 14,706 (22.83%) and 7,856 (12.19%) hits respectively, giving a final total of 22,455 significant hits (34.86% of all unigenes). A Gene Ontology (GO) analysis search using the Blast2GO program resulted in 6,007 unigenes (9.32%) being categorized into 55 functional groups. A differential gene expression analysis produced a total of 14,569 unigenes aberrantly expressed, with 11,446 unigenes significantly up-regulated and 3,103 unigenes significantly down-regulated. The differentially expressed immune genes fall under various processes of the animal immune system.ConclusionThis study provided an insight into the antibacterial mechanism in M. rosenbergii and the role of differentially expressed immune genes in response to V. parahaemolyticus infection. Furthermore, this study has generated an abundant list of transcript from M.rosenbergii which will provide a fundamental basis for future genomics research in this field.