This study was aimed to measure the basic knowledge on food safety and food handling practices am... more This study was aimed to measure the basic knowledge on food safety and food handling practices among migrant food handlers as these information is scarce in Malaysia. A cross-sectional study was conducted face-to-face amongst 383 migrant food handlers from three major cities in Peninsular Malaysia through questionnaire. Socio-demographic information of all respondents was collected. Questions on food safety knowledge (i.e. food cleanliness and hygiene, symptom of foodborne illnesses and foodborne pathogens) and food handling practices were assessed. The compiled data were analyzed by using the Statistical Packages for Social Science (SPSS) 16.0. Overall, migrant food handlers had poor level of knowledge on food safety with an average food handling practice. Significant effects were observed between respondents’ food safety knowledge and socio-demography (country of origin and educational level) and two factors namely; respondents’ nationality and attendance at food training programs showed significant associations with their food handling practices. Multiple logistic regression analyses revealed that attendance at food training programs was a significant and independent predictor of the respondent’s food handling practice. The study’s findings highlighted issues with regards to the extent of knowledge acquisition on food safety and hygiene by migrant food handlers. Therefore, this warrants improvements not only in the better delivery methods of training modules but also tight enforcement of attendance at the programs by the respective authorities.
The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids
Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often ... more Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.
Vibrio parahaemolyticus, an important human pathogen, is associated with gastroenteritis and tran... more Vibrio parahaemolyticus, an important human pathogen, is associated with gastroenteritis and transmitted through partially cooked seafood. It has become a major concern in the production and trade of marine food products. The prevalence of potentially virulent and pathogenic V. parahaemolyticus in raw seafood is of public health significance. Here we describe the genome sequence of a V. parahaemolyticus isolate of crustacean origin which was cultured from prawns in 2008 in Selangor, Malaysia (isolate PCV08-7). The next generation sequencing and analysis revealed that the genome of isolate PCV08-7 has closest similarity to that of V. parahaemolyticus RIMD2210633. However, there are certain unique features of the PCV08-7 genome such as the absence of TDH-related hemolysin (TRH), and the presence of HU-alpha insertion. The genome of isolate PCV08-7 encodes a thermostable direct hemolysin (TDH), an important virulence factor that classifies PCV08-7 isolate to be a serovariant of O3:K6 strain. Apart from these, we observed that there is certain pattern of genetic rearrangements that makes V. parahaemolyticus PCV08-7 a non-pandemic clone. We present detailed genome statistics and important genetic features of this bacterium and discuss how its survival, adaptation and virulence in marine and terrestrial hosts can be understood through the genomic blueprint and that the availability of genome sequence entailing this important Malaysian isolate would likely enhance our understanding of the epidemiology, evolution and transmission of foodborne Vibrios in Malaysia and elsewhere.
Chlorination disinfection by-products in drinking water have often been associated with the occur... more Chlorination disinfection by-products in drinking water have often been associated with the occurrence of bladder cancer and rectal cancer. Ozonation is one of the safe alternatives for water treatment. In this study, ozone for ozonation was produced based on Dielectric Barrier Discharge (DBD) and the efficacy of ozone on complete disinfection of microorganisms in well water was investigated at laboratory scale. The efficacy of ozonation was also compared with chlorination on well water disinfection. Well water in Bachok was chosen in this study because chlorination is still widely used by the residents in the district as a means of disinfection of the well water for daily consumption. The data showed that the complete inactivation of microorganisms in well water was achieved after 120 seconds of ozonation, same as the time required by chorination.
Salmonella Typhimurium is an important nontyphoidal Salmonella serovar associated with foodborne ... more Salmonella Typhimurium is an important nontyphoidal Salmonella serovar associated with foodborne diseases in many parts of the world. This organism is the major causative agent of nontyphoidal salmonellosis in Malaysia. We aimed to investigate the genetic profiles of the strains isolated from clinical, zoonotic, and dietary sources in Malaysia using multilocus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). By focusing on the 5 common variable number tandem repeat (VNTR) loci, we found that PFGE (D ＝ 0.99) was more discriminative than MLVA (D ＝ 0.76). The low MLVA score might be because of a lack of VNTR loci STTR6 (81.0z) and STTR10pl (76.2z). Both subtyping methods suggested that our S. Typhimurium strains were largely endemic with limited genetic variation. Furthermore, we observed that biphasic S. Typhimurium strains were dominant (99z) and multidrug resistance was prevalent (50z) within our sample pool. The most frequently observed phenotypes were resistance to compound sulfonamides (49z), tetracycline (51z), and streptomycin (52z). In this study, we documented the genetic relationship, antimicrobial resistance characteristics, and flagellar-phase dominance among S. Typhimurium strains found in Malaysia.
Background: Hypermethylation in promoter regions of genes might lead to altered gene functions an... more Background: Hypermethylation in promoter regions of genes might lead to altered gene functions and result in malignant cellular transformation. Thus, biomarker identification for hypermethylated genes would be very useful for early diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). The objectives of this study were to screen and validate differentially hypermethylated genes in OSCC and correlate the hypermethylation-induced genes with demographic, clinocopathological characteristics and survival rate of OSCC. Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study. Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients’ age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference. Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential biomarkers for OSCC as diagnostic, prognostic and therapeutic targets in the future.
Vibrio vulnificus is a highly invasive human pathogen that exists naturally in estuarine environm... more Vibrio vulnificus is a highly invasive human pathogen that exists naturally in estuarine environment and coastal waters. In this study, we used different PCR assays to detect V. vulnificus in 260 seafood and 80 seawater samples. V. vulnificus was present in about 34 (13%) of the 260 seafood samples and 18 (23%) of the 80 seawater samples. Repetitive extragenic palindromic PCR (REP-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) were applied to subtype the V. vulnificus isolates. Twenty-five REP profiles and 45 ERIC profiles were observed, and the isolates were categorized into 9 and 10 distinct clusters at the similarity of 80%, by REP-PCR and ERIC-PCR, respectively. ERIC-PCR is more discriminative than REP-PCR in subtyping V. vulnificus, demonstrating high genetic diversity among the isolates.
Background: Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigen... more Background: Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigenic E. coli (VTEC), has caused significant economic losses in the pig farming industry worldwide. However, there is limited information on VTEC in Malaysia. The objective of this study was to characterize pathogenic E. coli isolated from post-weaning piglets and growers with respect to their antibiograms, carriage of extended-spectrum beta-lactamases, pathotypes, production of hemolysins and fimbrial adhesins, serotypes, and genotypes. Results: PCR detection of virulence factors associated with different E. coli pathotypes (ETEC, EPEC, EHEC, and VTEC) revealed that VTEC was the only pathotype identified from six swine farms located at north-western Peninsular Malaysia. A low prevalence rate of VTEC was found among the swine samples (n = 7/345) and all 7 VTEC isolates were multidrug resistant. Five of these isolates from different hosts raised in the same pen were likely to be of the same clone as they shared identical sero-pathotypes (O139:H1, VT2e/α-hly/F18), resistance profiles and DNA fingerprinting profiles. Two other serotypes, O130: H26 (n = 1) and O168: H21 (n = 1) carrying virulence factors were also identified. O168: H21 is possibly a new serotype as this has not been previously reported. Conclusions: The occurrence of VTEC with infrequently encountered serotypes that are multidrug resistant and harbouring virulence factors may be of public health concern. The detection of possible clones in this study also showed that the combination of different typing tools including phenotyping and genotyping methods is useful for molecular epidemiologic surveillance and studies.
Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study w... more Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.
Salmonellosis is an important zoonotic disease with worldwide distribution. Reptile-associated Sa... more Salmonellosis is an important zoonotic disease with worldwide distribution. Reptile-associated Salmonellosis in humans is an increasing public health concern. This study was conducted to determine the prevalence and antimicrobial susceptibility of Salmonella isolated from snakes. Cloacal swab samples were used for isolation by conventional culture, biochemical and serological test. Confirmations of Salmonella were determined by polymerase chain reaction (PCR) using genus specific primers for invA genes. A total of 42 snakes were screened for the presence of Salmonella, 16 (38%) were positive for Salmonella. Among those positive for Salmonella serovars, 11 were from the wild snakes while 5 were captive snakes. No significant difference was found in the prevalence of Salmonella between wild and captive snakes (p-value = 0.096). The serovars identified were Salmonella Typhimurium (n=2), S. Corvallis (n=2), S. Poona (n=1) and S. Mbandaka (n=2), while the rest untypable S. enterica (n=9). The resistance to antibiotics observed are as follows; cephalexin (12.5%), cephalothin (12.5%) and amoxicillin-clavulanic acid (6.25%). Interestingly all Salmonella isolates were sensitive to chloramphenicol, gentamycin, enrofloxacin, sulphamethazole-trimethoprim and tetracycline. To avoid Salmonella transmission, veterinarians and reptile keepers should take hygienic precautions to minimise reptile-associated salmonellosis.
Background: Typhoid fever is an infectious disease of global importance that is caused by Salmone... more 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.
The present study aims to develop a system which consists of four pairs of primers that specifica... more The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.
Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the pres... more Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (104 cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H2S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.
Introduction. Acinetobacter calcoaceticus-baumannii complex (ACB complex) is a leading opportunis... more Introduction. Acinetobacter calcoaceticus-baumannii complex (ACB complex) is a leading opportunistic pathogen in intensive care units (ICUs). Effective control of spread requires understanding of its epidemiological relatedness. This study aims to determine the genetic relatedness and antibiotic susceptibilities of ACB complex in an ICU in Malaysia. Methodology. Pulsed field gel electrophoresis (PFGE), E-test, and disk diffusion were used for isolates characterization. Results. During the study period (December 2011 to June 2012), 1023 patients were admitted to the ICU and 44 ACB complex (blood, 𝑛 = 21, and blind bronchial aspirates, 𝑛 = 23) were recovered from 38 ICU patients. Six isolates were from non-ICU patients. Of the 44 ICU isolates, 88.6% exhibited multidrug-resistant (MDR) patterns. There was high degree of resistance, with minimum inhibitory concentration90 (MIC90) of >32 𝜇g/mL for carbapenems and ≥256 𝜇g/mL for amikacin, ampicillin/sulbactam, and cefoperazone/sulbactam. Isolates from the main PFGE cluster were highly resistant. There was evidence of dissemination in non-ICU wards. Conclusion. High number of clonally relatedMDR ACB complex was found.While the ICU is a likely reservoir facilitating transmission, importation from other wards may be important contributor. Early identification of strain relatedness and implementation of infection control measures are necessary to prevent further spread.
Kosakonia radicincitans (formerly known as Enterobacter radicincitans), an endophytic bacterium w... more Kosakonia radicincitans (formerly known as Enterobacter radicincitans), an endophytic bacterium was isolated from the symptomatic tissues of bacterial wilt diseased banana (Musa spp.) plant in Malaysia. The total genome size of K. radicincitans UMEnt01/12 is 5 783 769 bp with 5463 coding sequences (CDS), 75 tRNAs, and 9 rRNAs. The annotated draft genome of the K. radicincitans UMEnt01/12 strain might shed light on its role as a bacterial wilt-associated bacterium.
The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern... more The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinoloneresistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes.Mutations were detected in QRDR of gyrA (𝑛 = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (𝑛 = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 𝜇g/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which ismore prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening formutations at theQRDRs of gyrase and topoisomerase IV genes in Salmonella.This assaymarkedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.
Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health... more Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 105 CFU/ml, while PCR displayed a limit of 5.9 x 107 CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 104 CFU/g, whereas PCR was 3.6 x 105 CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
Nosocomial infection caused by Acinetobacter baumannii is of great concern due to its increasing ... more Nosocomial infection caused by Acinetobacter baumannii is of great concern due to its increasing resistance to most antimicrobials. In this study, 54 nonrepeat isolates of A. baumannii from the main tertiary hospital in Terengganu, Malaysia, were analyzed for their antibiograms and genotypes. Out of the 54 isolates, 39 (72.2%) were multidrug resistant (MDR) and resistant to carbapenems whereas 14 (25.9%) were categorized as extensive drug resistant (XDR) with additional resistance to polymyxin B, the drug of “last resort.” Pulsed-field gel electrophoresis analyses showed that the polymyxin-resistant isolates were genetically diverse while the carbapenem-resistant isolates were clonally related. The 14 XDR isolates were further investigated for mutations in genes known to mediate polymyxin resistance, namely, pmrCAB, and the lipopolysaccharide biosynthesis genes, lpxA, lpxC, lpxD, and lpsB. All 14 isolates had a P102H mutation in pmrA with no mutation detected in pmrC and pmrB. No mutation was detected in lpxA but each polymyxin-resistant isolate had 2–4 amino acid substitutions in lpxD and 1-2 substitutions in lpxC. Eight resistant isolates also displayed a unique H181Y mutation in lpsB.The extent of polymyxin resistance is of concern and the novel mutations discovered here warrant further investigations.
Background: Promoter hypermethylation leads to altered gene functions and may result in malignant... more Background: Promoter hypermethylation leads to altered gene functions and may result in malignant cellular transformation. Thus, identification of biomarkers for hypermethylated genes could be useful for diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). Objectives: To screen hypermethylated genes with a microarray approach and to validate selected hypermethylated genes with the methylation-specific polymerase chain reaction (MSPCR). Materials and Methods: Genome-wide analysis of normal oral mucosa and OSCC tissues was conducted using the Illumina methylation microarray. The specified differential genes were selected and hypermethylation status was further verified with an independent cohort sample of OSCC samples. Candidate genes were screened using microarray assay and run by MSPCR analysis. Results: TP73, PIK3R5, and CELSR3 demonstrated high percentages of differential hypermethylation status. Conclusions: Our microarray screening and MSPCR approaches revealed that the signature candidates of differentially hypermethylated genes may possibly become potential biomarkers which would be useful for diagnostic, prognostic and therapeutic targets of OSCC in the near future.
We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could s... more We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt =31.12).We have also detected 46 generic E. coli from50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1.Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years.The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.
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Papers by Thong Kwai Lin
migrant food handlers as these information is scarce in Malaysia. A cross-sectional study was conducted
face-to-face amongst 383 migrant food handlers from three major cities in Peninsular Malaysia through
questionnaire. Socio-demographic information of all respondents was collected. Questions on food safety
knowledge (i.e. food cleanliness and hygiene, symptom of foodborne illnesses and foodborne pathogens)
and food handling practices were assessed. The compiled data were analyzed by using the Statistical
Packages for Social Science (SPSS) 16.0. Overall, migrant food handlers had poor level of knowledge on
food safety with an average food handling practice. Significant effects were observed between respondents’
food safety knowledge and socio-demography (country of origin and educational level) and
two factors namely; respondents’ nationality and attendance at food training programs showed significant
associations with their food handling practices. Multiple logistic regression analyses revealed that
attendance at food training programs was a significant and independent predictor of the respondent’s
food handling practice. The study’s findings highlighted issues with regards to the extent of knowledge
acquisition on food safety and hygiene by migrant food handlers. Therefore, this warrants improvements
not only in the better delivery methods of training modules but also tight enforcement of attendance at
the programs by the respective authorities.
non-pandemic clone. We present detailed genome statistics and important genetic features of this bacterium and discuss how its survival, adaptation and virulence in marine and terrestrial hosts can be understood through the genomic blueprint and that the availability of genome sequence entailing this important Malaysian isolate would likely enhance
our understanding of the epidemiology, evolution and transmission of foodborne Vibrios in Malaysia and elsewhere.
disinfection. Well water in Bachok was chosen in this study because chlorination is still widely used by the residents in the district as a means of disinfection of the well water for daily consumption. The data showed that the complete inactivation of microorganisms in well water was achieved after 120 seconds of ozonation, same as the time required by chorination.
sample pool. The most frequently observed phenotypes were resistance to compound sulfonamides (49z), tetracycline (51z), and streptomycin (52z). In this study, we documented the genetic relationship, antimicrobial resistance characteristics, and flagellar-phase dominance among S. Typhimurium strains found in Malaysia.
Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study.
Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients’ age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference.
Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential biomarkers for OSCC as diagnostic, prognostic and therapeutic targets in the future.
this study, we used different PCR assays to detect V. vulnificus in 260 seafood and 80 seawater samples. V. vulnificus was present in about 34 (13%) of the 260 seafood samples and 18 (23%) of the 80 seawater samples. Repetitive extragenic palindromic PCR (REP-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) were applied to subtype the V. vulnificus isolates. Twenty-five REP profiles and 45 ERIC profiles were observed, and the isolates were categorized into 9 and 10 distinct clusters at the similarity of 80%, by REP-PCR and ERIC-PCR, respectively. ERIC-PCR is more discriminative than REP-PCR in subtyping V. vulnificus, demonstrating high genetic diversity among the isolates.
Results: PCR detection of virulence factors associated with different E. coli pathotypes (ETEC, EPEC, EHEC, and VTEC) revealed that VTEC was the only pathotype identified from six swine farms located at north-western Peninsular Malaysia. A low prevalence rate of VTEC was found among the swine samples (n = 7/345) and all 7 VTEC isolates were multidrug resistant. Five of these isolates from different hosts raised in the same pen were likely to be of the same clone as they shared identical sero-pathotypes (O139:H1, VT2e/α-hly/F18), resistance profiles and DNA fingerprinting profiles. Two other serotypes, O130: H26 (n = 1) and O168: H21 (n = 1) carrying virulence factors were also identified. O168: H21 is possibly a new serotype as this has not been previously reported.
Conclusions: The occurrence of VTEC with infrequently encountered serotypes that are multidrug resistant and harbouring virulence factors may be of public health concern. The detection of possible clones in this study also showed that the combination of different typing tools including phenotyping and genotyping methods is useful for molecular epidemiologic surveillance and studies.
used for isolation by conventional culture, biochemical and serological test. Confirmations of Salmonella were determined by polymerase chain reaction (PCR) using genus specific primers for invA genes. A total of 42 snakes were screened for the presence of Salmonella, 16 (38%) were positive for Salmonella. Among those positive for Salmonella serovars, 11 were from the wild snakes while 5 were captive snakes. No significant difference was found in the prevalence of Salmonella between wild and captive snakes (p-value = 0.096). The serovars identified were Salmonella Typhimurium (n=2), S. Corvallis (n=2), S. Poona (n=1) and S. Mbandaka (n=2), while the rest untypable S. enterica (n=9). The resistance to antibiotics observed are as follows; cephalexin (12.5%), cephalothin (12.5%) and amoxicillin-clavulanic acid (6.25%). Interestingly all Salmonella isolates were sensitive to chloramphenicol, gentamycin, enrofloxacin, sulphamethazole-trimethoprim and
tetracycline. To avoid Salmonella transmission, veterinarians and reptile keepers should take hygienic precautions to minimise reptile-associated salmonellosis.
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.
produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A
and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.
care units (ICUs). Effective control of spread requires understanding of its epidemiological relatedness. This study aims to
determine the genetic relatedness and antibiotic susceptibilities of ACB complex in an ICU in Malaysia. Methodology. Pulsed field
gel electrophoresis (PFGE), E-test, and disk diffusion were used for isolates characterization. Results. During the study period
(December 2011 to June 2012), 1023 patients were admitted to the ICU and 44 ACB complex (blood, 𝑛 = 21, and blind bronchial
aspirates, 𝑛 = 23) were recovered from 38 ICU patients. Six isolates were from non-ICU patients. Of the 44 ICU isolates, 88.6%
exhibited multidrug-resistant (MDR) patterns. There was high degree of resistance, with minimum inhibitory concentration90
(MIC90) of >32 𝜇g/mL for carbapenems and ≥256 𝜇g/mL for amikacin, ampicillin/sulbactam, and cefoperazone/sulbactam. Isolates
from the main PFGE cluster were highly resistant. There was evidence of dissemination in non-ICU wards. Conclusion. High
number of clonally relatedMDR ACB complex was found.While the ICU is a likely reservoir facilitating transmission, importation
from other wards may be important contributor. Early identification of strain relatedness and implementation of infection control
measures are necessary to prevent further spread.
method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 105 CFU/ml, while PCR displayed a limit of 5.9 x 107 CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 104 CFU/g, whereas PCR was 3.6 x 105 CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
Objectives: To screen hypermethylated genes with a microarray approach and to validate selected hypermethylated genes with the methylation-specific polymerase chain reaction (MSPCR).
Materials and Methods: Genome-wide analysis of normal oral mucosa and OSCC tissues was conducted using the Illumina methylation microarray. The specified differential genes were selected and hypermethylation status was further verified with an independent cohort sample of OSCC samples. Candidate genes were screened using microarray assay and run by MSPCR analysis.
Results: TP73, PIK3R5, and CELSR3 demonstrated high percentages of differential hypermethylation status.
Conclusions: Our microarray screening and MSPCR approaches revealed that the signature candidates of differentially hypermethylated genes may possibly become potential biomarkers which would be useful for diagnostic, prognostic and therapeutic targets of OSCC in the near future.
VT1 to VT2 was 6 : 1.Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years.The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.