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Multiple-Locus Variable-Number Tandem Repeat Analysis of Vibrio cholerae in Comparison with Pulsed Field Gel Electrophoresis and Virulotyping
Cindy Shuan Ju Teh,Kek Heng Chua,Kwai Lin Thong
Journal of Biomedicine and Biotechnology , 2010, DOI: 10.1155/2010/817190
Abstract: 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 (<.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.
Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay
Kwai Thong, Susan Hoe, SD Puthucheary, Rohani Md Yasin
BMC Infectious Diseases , 2005, DOI: 10.1186/1471-2334-5-8
Abstract: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997–2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth.The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation.The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.Members of the genus Shigella, namely S. flexneri, S. dysenteriae, S. sonnei and S. boydii have caused and continue to be responsible for mortality and/or morbidity in high risk populations such as children under five years of age, senior citizens, toddlers in day-care centres, patients in custodial institutions, homosexual men and, war- and famine-engulfed people. Yearly episodes of shigellosis globally have been estimated to be 164.7 million and of these, 163.2 million were in developing countries and the remaining in industrialized nations. The mortality rate was approximately 0.7% [1]. A recent study by Lee & Puthucheary [2] on bacterial enteropathogens in childhood diarrhoea in a Malaysian urban hospital showed that Shigella spp. was the third most common bacteria isolated. S. flexneri and S. dysenteriae type 1 infections are usually characterized by frequent pa
Comparative Analysis of Salmonella typhi by rRNA Gene Restriction and Phage Typing
Kwai-Lin Thong,Martin Altwegg,Tikki Pang
Pakistan Journal of Biological Sciences , 2000,
Abstract: Phage typing and ribotyping were used to analyze 19 isolates of Salmonella typhi from sporadic cases and two outbreaks of typhoid fever in Malaysia in 1987 and 1990. The two outbreaks were associated with phage types D1 and E1 in Penang and Alor Setar respectively, and phage types D1, E1 and A (with 3 untypeable isolates) were present among the sporadic cases. Ribotyping detected 14 ribotypes among the 19 isolates thus establishing its higher discriminative capacity compared to phage typing. The outbreak isolates were more homogeneous by ribotyping and the data also suggested that one outbreak was propagated, multi-focal in nature (Penang) while another was a point- source traceable to a single event (Alor Setar).
Characterization of Multidrug Resistant ESBL-Producing Escherichia coli Isolates from Hospitals in Malaysia
King-Ting Lim,Rohani Yasin,Chew-Chieng Yeo,Savithri Puthucheary,Kwai-Lin Thong
Journal of Biomedicine and Biotechnology , 2009, DOI: 10.1155/2009/165637
Abstract: The emergence of Escherichia coli that produce extended spectrum -lactamases (ESBLs) and are multidrug resistant (MDR) poses antibiotic management problems. Forty-seven E. coli isolates from various public hospitals in Malaysia were studied. All isolates were sensitive to imipenem whereas 36 were MDR (resistant to 2 or more classes of antibiotics). PCR detection using gene-specific primers showed that 87.5% of the ESBL-producing E. coli harbored the TEM gene. Other ESBL-encoding genes detected were OXA, SHV, and CTX-M. Integron-encoded integrases were detected in 55.3% of isolates, with class 1 integron-encoded intI1 integrase being the majority. Amplification and sequence analysis of the 5CS region of the integrons showed known antibiotic resistance-encoding gene cassettes of various sizes that were inserted within the respective integrons. Conjugation and transformation experiments indicated that some of the antibiotic resistance genes were likely plasmid-encoded and transmissible. All 47 isolates were subtyped by PFGE and PCR-based fingerprinting using random amplified polymorphic DNA (RAPD), repetitive extragenic palindromes (REPs), and enterobacterial repetitive intergenic consensus (ERIC). These isolates were very diverse and heterogeneous. PFGE, ERIC, and REP-PCR methods were more discriminative than RAPD in subtyping the E. coli isolates.
Variable Carbon Catabolism among Salmonella enterica Serovar Typhi Isolates
Lay Ching Chai, Boon Hong Kong, Omar Ismail Elemfareji, Kwai Lin Thong
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036201
Abstract: Background Salmonella enterica serovar Typhi (S. Typhi) is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever) and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study. Methodology/Principal Findings To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas oftyphoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates) was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested. Conclusion The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen.
Characterization of Shigella sonnei in Malaysia, an increasingly prevalent etiologic agent of local shigellosis cases
Xiu Pei Koh, Chien Shun Chiou, Noni Ajam, Haruo Watanabe, Norazah Ahmad, Kwai Lin Thong
BMC Infectious Diseases , 2012, DOI: 10.1186/1471-2334-12-122
Abstract: Forty non-repeat clinical strains of S. sonnei isolated during the years 1997–2000, and 2007–2009 were studied. The strains were isolated from stools of patients in different hospitals from different regions in Malaysia. These epidemiologically unrelated strains were characterized using biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and MLVA.The two biotypes identified in this study were biotype a (n?=?29, 73%) and biotype g (n?=?11, 27%). All the 40 strains were sensitive to kanamycin, ceftriaxone and ciprofloxacin. Highest resistance rate was observed for streptomycin (67.5%), followed by tetracycline (40%) and trimethoprim-sulfamethoxazole (37.5%). All the S. sonnei biotype g strains had a core resistance type of streptomycin - trimethoprim-sulfamethoxazole - tetracycline whereas the 29 biotype a strains were subtyped into eight resistotypes. All the strains were equally distinguishable by PFGE and MLVA. Overall, PFGE analysis indicated that S. sonnei biotype a strains were genetically more diverse than biotype g strains. Cluster analysis by MLVA was better in grouping the strains according to biotypes, was reflective of the epidemiological information and was equally discriminative as PFGE.The S. sonnei strains circulating in Malaysia throughout the period studied were derived from different clones given their heterogeneous nature. MLVA based on seven selected VNTR loci was rapid, reproducible and highly discriminative and therefore may complement PFGE for routine subtyping of S. sonnei.
Mimotopes of heat shock proteins of Salmonella enterica serovar Typhi identified from phage-displayed peptide library
Yuen Hawk Leong,1 Shamala Devi,2 Thong Kwai Lin2*
Journal of Infection in Developing Countries , 2008,
Abstract: Background: Heat shock proteins (HSPs) are known to be involved in the pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. The objective of this study was to apply a phage display library to identify mimotopes of two HSPs, HSP90 and DnaK in S. Typhi.Methodology: A 12-mer random peptide library expressed on the surface of the filamentous phage, M13, was used to select the mimotopes of two S. Typhi heat shock proteins by biopanning with monoclonal antibodies (mAbs), DnaK and HSP90. The immunogenicity of the selected peptides was determined through binding affinity with polyclonal antibodies from pooled typhoid-confirmed patients’ sera and purified HSPs mAb using Western blotting and ELISA.Results: Five rounds of biopanning resulted in enrichment of phage clones expressing the binding motifs TDxSTRP and FPSHYWLYPPPT, respectively. The selected peptides showed strong immunoreactivity with patients’ sera. Thus, monoclonal antibodies against HSP and patient sera can select common mimotopes from the random peptide library.Conclusion: These findings may provide fundamental information for further studies on diagnostic application or vaccine design against this aetiologic agent of typhoid fever.
Genome sequencing and analysis of Salmonella enterica serovar Typhi strain CR0063 representing a carrier individual during an outbreak of typhoid fever in Kelantan, Malaysia
Ramani Baddam, Narender Kumar, Sabiha Shaik, Tiruvayipati Suma, Soo Tein Ngoi, Kwai-Lin Thong, Niyaz Ahmed
Gut Pathogens , 2012, DOI: 10.1186/1757-4749-4-20
Abstract: Salmonella enterica serovar Typhi, the aetiologic agent of typhoid fever is still posing a major health problem for the developing world, as about 16 million new cases are reported each year [1]. S. Typhi causes systemic infections (typhoid fever) as well as chronic infections (asymptomatic carriers) in humans, the latter serve as the source of infection [2]. The transmission of S. Typhi is primarily through faecal-oral route and a significant number of infected individuals become chronic asymptomatic carriers and keep shedding S. Typhi in faeces for decades [3]. This results in endemicity of S. Typhi in regions of the world with underdeveloped sanitation and community hygiene [4].Carrier identification becomes extremely important as some of the ancestral haplotypes were observed in recent isolates suggesting their persistence in these asymptomatic carriers [5]. Traditional methods such as culturing of bacteria from faecal samples are not fool proof as the carriers shed bacteria intermittently. Serological tests to detect specific antibodies such as anti-H and anti-O are unable to differentiate between carriers and individuals who have recovered from the infection [6]. Especially, in areas endemic for S. Typhi, due to high background levels of these antibodies, serological tests cannot be adopted for the identification of a carrier [7]. Thus, there is an urgent need for inexpensive and efficient detection methods for the establishment of carrier state, perhaps based on genomic markers.The genetic typing tools such as PFGE, AFLP, ribotyping etc. can resolve limited genetic variation occurring within specific sites, and therefore are incapable of differentiating highly clonal strains such as outbreak related strains from the ones not associated with the outbreak (carrier isolates) [8-10]. High-throughput sequencing technologies have already been employed as a high resolution molecular epidemiologic tool to discern microevolution of highly related strains [11].In this
Antibacterial Evaluation of Some Schiff Bases Derived from 2-Acetylpyridine and Their Metal Complexes
Nura Suleiman Gwaram,Hapipah Mohd Ali,Hamid Khaledi,Mahmood Ameen Abdulla,A. Hamid A. Hadi,Thong Kwai Lin,Chai Lay Ching,Cher Lin Ooi
Molecules , 2012, DOI: 10.3390/molecules17055952
Abstract: A series of Schiff bases derived from 2-acetylpyridne and their metal complexes were characterized by elemental analysis, NMR, FT-IR and UV-Vis spectral studies. The complexes were screened for anti-bacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumanni (AC), Klebsiella pneumonie (KB) and Pseudomonas aeruginosa (PA) using the disc diffusion and micro broth dilution assays. Based on the overall results, the complexes showed the highest activities against MRSA while a weak antibacterial activity was observed against A. baumanii and P. aeruginosa.
Prevalence and Genetic Characterization of Carbapenem- and Polymyxin-Resistant Acinetobacter baumannii Isolated from a Tertiary Hospital in Terengganu, Malaysia
Soo-Sum Lean,Zarizal Suhaili,Salwani Ismail,Nor Iza A. Rahman,Norlela Othman,Fatimah Haslina Abdullah,Zakaria Jusoh,Chew Chieng Yeo,Kwai-Lin Thong
ISRN Microbiology , 2014, DOI: 10.1155/2014/953417
Abstract: 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. 1. Introduction Acinetobacter baumannii is a Gram-negative bacterium increasingly found in the hospital environment due to its ability to survive for long periods on inanimate objects [1, 2]. Nosocomial A. baumannii isolates are mostly multidrug resistant (MDR) (i.e., resistant towards more than three classes of antibiotics) [3]. However, extensive drug resistant (XDR) isolates, which are resistant to all but one or two classes of antibiotics, and even pandrug resistant (PDR) isolates that are resistant to all classes of antibiotics, are developing at an alarming rate [4]. Since A. baumannii has the ability to colonize both viable and damaged tissues and is also resistant towards nearly all antimicrobials, it has become a cause of great concern. Carbapenems are among the very few antibiotics left that can be used for the treatment of A. baumannii infections. Nevertheless, the efficacy of carbapenems is increasingly compromised by the rapid emergence of carbapenem-hydrolysing -lactamase enzymes. Several types of class D -lactamases including OXA-23, OXA-24, OXA-58, and intrinsic OXA-51-like enzymes are known to be important contributors to carbapenem resistance [5]. The rapid development of carbapenem-resistant MDR A. baumannii has led to the use of
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