Displaying all 17 publications

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  1. Uyub AM, Azlan AA
    PMID: 11414414
    A total of 52 clinical strains of Helicobacter pylori were characterized on the basis of preformed enzyme production with API ZYM kits. Using the biotyping schemes as defined by Reina and Alomar (1989), Kung et al (1989) and Matsumoto et al (1996), 15.3% (8/52), 13.5% (7/52) and 11.5% (6/52) of the isolates were not biotypable, respectively. Two enzymes, valine arylamidase and cystine arylamidase could be additionally used to differentiate between isolates. Our isolates were either negative or positive for both the enzymes or positive only for cystine arylamidase. We propose the incorporation of these two enzymes into the Matsumoto et al (1996) biotyping scheme to biotype strains into additional enzyme biotypes.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  2. Loong SK, Khor CS, Jafar FL, AbuBakar S
    J Clin Lab Anal, 2016 Nov;30(6):1056-1060.
    PMID: 27184222 DOI: 10.1002/jcla.21980
    BACKGROUND: Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification.

    METHODS: One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method.

    RESULTS: Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates.

    CONCLUSION: The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools.

    Matched MeSH terms: Bacterial Typing Techniques/methods
  3. Ng HF, Ngeow YF
    Pathog Dis, 2020 11 11;78(8).
    PMID: 32945880 DOI: 10.1093/femspd/ftaa055
    The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  4. Neoh HM, Tan XE, Sapri HF, Tan TL
    Infect Genet Evol, 2019 10;74:103935.
    PMID: 31233781 DOI: 10.1016/j.meegid.2019.103935
    Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for bacteria typing. The method involves enzyme restriction of bacteria DNA, separation of the restricted DNA bands using a pulsed-field electrophoresis chamber, followed by clonal assignment of bacteria based on PFGE banding patterns. Various PFGE protocols have been developed for typing different bacteria, leading it to be one of the most widely used methods for phylogenetic studies, food safety surveillance, infection control and outbreak investigations. On the other hand, as PFGE is lengthy and labourious, several PCR-based typing methods can be used as alternatives for research purposes. Recently, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and whole genome sequencing (WGS) have also been proposed for bacteria typing. In fact, as WGS provides more information, such as antimicrobial resistance and virulence of the tested bacteria in comparison to PFGE, more and more laboratories are currently transitioning from PFGE to WGS for bacteria typing. Nevertheless, PFGE will remain an affordable and relevant technique for small laboratories and hospitals in years to come.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  5. Ahmed SA, Raabe CA, Cheah HL, Hoe CH, Rozhdestvensky TS, Tang TH
    Am J Trop Med Hyg, 2019 06;100(6):1328-1334.
    PMID: 30963989 DOI: 10.4269/ajtmh.18-0525
    The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  6. Ilina EN, Borovskaya AD, Serebryakova MV, Chelysheva VV, Momynaliev KT, Maier T, et al.
    Rapid Commun Mass Spectrom, 2010 Feb;24(3):328-34.
    PMID: 20049887 DOI: 10.1002/rcm.4394
    The characteristics of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based investigation of extremely variable bacteria such as Helicobacter pylori were studied. H. pylori possesses a very high natural variability. Accurate tools for species identification and epidemiological characterization could help the scientific community to better understand the transmission pathways and virulence mechanisms of these bacteria. Seventeen clinical as well as two laboratory strains of H. pylori were analyzed by the MALDI Biotyper method for rapid species identification. Mass spectra collected were found containing 7-13 significant peaks per sample, and only six protein signals were identical for more than half of the strains. Four of them could be assigned to ribosomal proteins RL32, RL33, RL34, and RL36. The reproducible peak with m/z 6948 was identified as a histidine-rich metal-binding polypeptide by tandem mass spectrometry (MS/MS). In spite of the evident protein heterogeneity of H. pylori the mass spectra collected for a particular strain under several cultivations were highly reproducible. Moreover, all clinical strains were perfectly identified as H. pylori species through comparative analysis using the MALDI Biotyper software (Bruker Daltonics, Germany) by pattern matching against a database containing mass spectra from different microbial strains (n = 3287) including H. pylori 26695 and J99. The results of this study allow the conclusion that the MALDI-TOF direct bacterial profiling is suited for H. pylori identification and could be supported by mass spectra fragmentation of the observed polypeptide if necessary.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  7. Podin Y, Kaestli M, McMahon N, Hennessy J, Ngian HU, Wong JS, et al.
    J Clin Microbiol, 2013 Sep;51(9):3076-8.
    PMID: 23784129 DOI: 10.1128/JCM.01290-13
    Misidentifications of Burkholderia pseudomallei as Burkholderia cepacia by Vitek 2 have occurred. Multidimensional scaling ordination of biochemical profiles of 217 Malaysian and Australian B. pseudomallei isolates found clustering of misidentified B. pseudomallei isolates from Malaysian Borneo. Specificity of B. pseudomallei identification in Vitek 2 and potentially other automated identification systems is regionally dependent.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  8. Liu Y, Lee MA, Ooi EE, Mavis Y, Tan AL, Quek HH
    J Clin Microbiol, 2003 Sep;41(9):4388-94.
    PMID: 12958274
    A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  9. Teh CS, Chua KH, Thong KL
    J Biomed Biotechnol, 2010;2010:817190.
    PMID: 20671932 DOI: 10.1155/2010/817190
    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.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  10. Shueh CS, Neela V, Hussin S, Hamat RA
    J Microbiol Methods, 2013 Aug;94(2):141-143.
    PMID: 23756145 DOI: 10.1016/j.mimet.2013.06.001
    We developed a time-saving and cost-efficient Pulsed Field Gel Electrophoresis (PFGE) method for the typing of Stenotrophomonas maltophilia by modifying the conventional procedures. Our modifications related to the cell suspension preparation, lysis of bacterial cells in plugs, washing steps, and consumption of restriction enzyme. Although few rapid PFGE protocols on Gram-negative bacteria are available, the use of comparatively large amounts of costly reagents prompted us to look for other alternative. Hence, by considering the speed, simplicity, and relatively low cost, the modified protocol may be of more practical value than other established protocols in investigating S. maltophilia nosocomial outbreaks.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  11. Amal MN, Zamri-Saad M, Siti-Zahrah A, Zulkafli AR, Nur-Nazifah M
    J Appl Microbiol, 2013 Jul;115(1):20-9.
    PMID: 23557382 DOI: 10.1111/jam.12210
    AIMS: The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques.

    METHODS AND RESULTS: A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP-PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP-PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

    CONCLUSIONS: Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

    SIGNIFICANCE AND IMPACT OF THE STUDY: Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.

    Matched MeSH terms: Bacterial Typing Techniques/methods
  12. Lim BK, Thong KL
    J Infect Dev Ctries, 2009 Jul 01;3(6):420-8.
    PMID: 19762954
    BACKGROUND: Differentiation of Salmonella enterica into its serogroups is important for epidemiological study. The objective of the study was to apply a multiplex PCR targeting serogroups A, B, C1, D, E and Vi-positive strains of Salmonella enterica commonly found in Malaysia. A separate H-typing multiplex PCR which identified flagellar antigen "a", "b" or "d" was also optimized to confirm clinical serotypes, S. Paratyphi A and S. Typhi.

    METHODOLOGY: Sixty-seven laboratory Salmonella enterica strains were tested. Six sets of primers targeting defined regions of the O antigen synthesis genes (rfb gene cluster) and Vi antigen gene (viaB) were selected and combined into a multiplex PCR for O-grouping. Four primers (H-for, Ha-rev, Hb-rev and Hd-rev) were used in the second step multiplex PCR for H-typing. The optimized mPCR assays were further evaluated with 58 blind-coded Salmonella strains.

    RESULTS: The multiplex PCR results obtained showed 100% concordance to the conventionally typed serogroups. Validation with 58 blind coded Salmonella strains yield 100% accuracy and specificity.

    CONCLUSION: Based on this study, PCR serogrouping proved to be a rapid, alternative method for further differentiation of Salmonella enterica.

    Matched MeSH terms: Bacterial Typing Techniques/methods*
  13. Shuan Ju Teh C, Lin Thong K, Tein Ngoi S, Ahmad N, Balakrish Nair G, Ramamurthy T
    J Gen Appl Microbiol, 2009 Dec;55(6):419-25.
    PMID: 20118606
    A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.
    Matched MeSH terms: Bacterial Typing Techniques/methods
  14. Teh CSJ, Lau MY, Chong CW, Ngoi ST, Chua KH, Lee WS, et al.
    J Microbiol Methods, 2021 04;183:106184.
    PMID: 33662480 DOI: 10.1016/j.mimet.2021.106184
    Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 103 -104 CFU/mL (or g) and a high PCR efficiency with different targets (R2 > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  15. Lim KT, Hanifah YA, Mohd Yusof MY, Thong KL
    Trop Biomed, 2012 Jun;29(2):212-9.
    PMID: 22735842 MyJurnal
    Staphylococcus aureus is a persistent human pathogen responsible for a variety of infections ranging from soft-tissue infections to bacteremia. It produces a variety of virulence factors which are responsible for specific acute staphylococcal toxaemia syndromes. The objective of this study was to determine the prevalence of a repertoire of toxin genes among Malaysian MRSA strains and their genetic diversity by PCR-RFLP of coa gene. One hundred eighty-eight strains (2003, 2004, 2007 and 2008) of methicillin-resistant S. aureus (MRSA) were screened for 20 genes encoding for extracellular virulence determinant (sea, seb, sec, sed, see, seg, seh, sei, sej, tst, eta, etb, etd) and adhesins (cna, etb, fnbA, fnbB, hlg, ica, sdrE). The genetic relatedness of these strains was determined by PCR-RFLP of coa gene and agr grouping. Majority of the strains were tested positive for efb and fnbA (96% each), ica (78%) and hlg (59%) genes. A total of 101 strains were positive for at least one type of staphylococcal enterotoxin genes with sea being the predominant. Genes for seb, sed, see, seh, sej, eta and etb were not detected in any of the MRSA strains. The prevalence of sea, sec and ica among strains isolated in 2008 was increased significantly (p< 0.05) compared to 2003. Most of the strains were of agr type I (97.5%) followed by agr type II (1.2%) and agr type III (0.6%). All sea, sei and tst gene-positive strains were of agr type I. The only etd positive strain was agr type III. PCR-RFLP of coa produced 47 different patterns. The number of strains with virulence factors (sea, sec and ica) had increased over the years. No direct correlation between PCR-RFLP- coa profiles and virulotypes was observed.
    Matched MeSH terms: Bacterial Typing Techniques/methods
  16. Ghaznavi-Rad E, Goering RV, Nor Shamsudin M, Weng PL, Sekawi Z, Tavakol M, et al.
    Eur J Clin Microbiol Infect Dis, 2011 Nov;30(11):1365-9.
    PMID: 21479532 DOI: 10.1007/s10096-011-1230-1
    The usefulness of mec-associated dru typing in the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Malaysia was investigated and compared with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa and SCCmec typing. The isolates studied included all MRSA types in Malaysia. Multilocus sequence type ST188 and ST1 isolates were highly clonal by all typing methods. However, the dru typing of ST239 isolates produced the clearest discrimination between SCCmec IIIa and III isolates, yielding more subtypes than any other method. Evaluation of the discriminatory power for each method identified dru typing and PFGE as the most discriminatory, with Simpson's index of diversity (SID) values over 89%, including an isolate which was non-typeable by spa, but dru-typed as dt13j. The discriminatory ability of dru typing, especially with closely related MRSA ST239 strains (e.g., Brazilian and Hungarian), underscores its utility as a tool for the epidemiological investigation of MRSA.
    Matched MeSH terms: Bacterial Typing Techniques/methods*
  17. Tiong V, Thong KL, Yusof MY, Hanifah YA, Sam JI, Hassan H
    Jpn J Infect Dis, 2010 Sep;63(5):317-22.
    PMID: 20858996
    The genetic diversity and antimicrobial resistance rates of clinical Salmonella isolates (2007-2008) at the University of Malaya Medical Centre, Kuala Lumpur, were investigated and the genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic (REP)-PCR. XbaI-PFGE analysis generated 57 profiles (Dice coefficient, F=0.08-1.00), whereas REP-PCR using the REP primer generated only 35 (F=0.34-1.00). PFGE was therefore the more discriminative and reproducible method for assessing the genetic diversity of salmonellae. The antibiograms of 78 Salmonella isolates were assessed against 19 antimicrobials using the disk diffusion method. Twenty serotypes were identified, with the most common being S. Enteritidis (18%) followed by S. Typhimurium (14%), S. Paratyphi B var Java (9%), S. Weltevreden (9%), and S. Corvallis (9%). A total of 38 resistant profiles were defined, with 53.8% of the isolates being resistant to three or more antimicrobials. The highest resistance rates were observed for cephalothin (55.1%), tetracycline (47.4%), and nalidixic acid (35.9%). The presence of multidrug-resistant Salmonella strains is a cause for concern as it may limit the treatment of severe salmonellosis. One multidrug-resistant S. Enteritidis strain was a putative extended-spectrum beta-lactamase producer, based on a double disk diffusion analysis, and was resistant to ceftriaxone (MIC>32 microg/mL). The data generated by this study will contribute towards epidemiological monitoring and investigations of Salmonella infections in Malaysia.
    Matched MeSH terms: Bacterial Typing Techniques/methods
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