A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species

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.

Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species

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.

ermA, ermC , tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia.

The objective of this study was to determine the expression and transferability of tetracycline and erythromycin resistance among 188 MRSA strains from a Malaysian tertiary hospital. The minimum inhibitory concentrations (MICs) for oxacillin, erythromycin, tetracycline and ciprofloxacin ranged from 4 to 512 μg/ml, 0.25 to 256 μg/ml, 0.5 to 256 μg/ml and 0.5 to 512 μg/ml, respectively. Tetracycline-resistant strains showed co-resistance towards ciprofloxacin and erythromycin. There was a significant increase (P<0.05) of high-level tetracycline (≥MIC 256 μg/ml) and erythromycin (≥MIC 128 μg/ml) resistant strains in between the years 2003 and 2008. All erythromycin-resistant strains harboured ermA or ermC gene and all tetracycline-resistant strains harboured tetM or tetK gene. The blaZ was detected in all MRSA strains, whereas ermA, tetM, ermC, tetK and msrA genes were detected in 157 (84%), 92 (49%), 40 (21%), 39 (21%) and 4 (2%) MRSA strains, respectively. The blaZ, tetM, ermC and tetK genes were plasmid-encoded, with ermC gene being easily transmissible. Tn5801-like transposon was present in 78 tetM-positive strains. ermA and tetM genes were the most prevalent erythromycin and tetracycline resistance determinants, respectively, in MRSA strains. The association of resistance genes with mobile genetic elements possibly enhances the spread of resistant traits in MRSA.

Application of amplified ribosomal DNA restriction analysis in identification of Acinetobacter baumannii from a Tertiary Teaching Hospital, Malaysia

Acinetobacter baumannii, genomic species 3 and 13TU are being increasingly reported as the most important Acinetobacter species that cause infections in hospitalized patients. These Acinetobacter species are grouped in the Acinetobacter calcoaceticus- Acinetobacter baumannii (Acb) complex. Differentiation of the species in the Acb-complex is limited by phenotypic methods. Therefore, in this study, amplified ribosomal DNA restriction analysis (ARDRA) was applied to confirm the identity A. baumannii strains as well as to differentiate between the subspecies. One hundred and eighty-five strains from Intensive Care Unit, Universiti Malaya Medical Center (UMMC) were successfully identified as A. baumannii by ARDRA. Acinetobacter genomic species 13TU and 15TU were identified in 3 and 1 strains, respectively. ARDRA provides an accurate, rapid and definitive approach towards the identification of the species level in the genus Acinetobacter. This paper reports the first application ARDRA in genospecies identification of Acinetobacter in Malaysia.

Determination of toxinotypes of environmental Clostridium perfringens by Polymerase Chain Reaction

Toxinotype of Clostridium perfringens (CP) isolates collected from the Bernam River, Selangor River and Tengi Canal between April 2007 and January 2008 were determined by Polymerase Chain Reaction (PCR) using published primers. All the 147 isolates were toxinotype Type A, harbouring the alpha toxin gene. In addition, 5 of the isolates also had the enterotoxin (CPE) gene.

Application of ribosomal RNA gene restriction patterns analysis and pulsed-field gel electrophoresis in distinguishing Salmonella weltevreden isolates in Malaysia.

A representative sample of 20 isolates of Salmonella weltevreden strains from stool cultures of patients admitted at the University Hospital, Kuala Lumpur, Malaysia were analyzed. All the strains were susceptible to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, tetracycline, trimethoprim, gentamicin and co-trimoxazole. Ribosomal RNA gene restriction pattern analysis of PstI-digested DNA gave three ribotypes while pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested DNA gave ten distinct profiles. PFGE was more discriminative than ribotyping in distinguishing the strains. The majority of the strains analyzed were very closely related with similarity coefficient values ranging from 0.8 to 1.0. Both PFGE and ribotyping could distinguish one of the strains which was obtained from a patient following a bone marrow transplant for beta-thalassemia major, indicating that this particular strain was unrelated to the rest of the strains from patients with acute gastroenteritis.

Role of molecular typing in an outbreak of Salmonella paratyphi A.

During the post monsoon season of 1996 an outbreak of human Salmonellosis caused by Salmonella serovar-paratyphi A occurred in New Delhi and had continued for over 2 months. A total of 36 clinically diagnosed enteric-fever cases were reported during this outbreak. The isolates were compared following their characterisation by biotyping, antibiogram-analysis, plasmid-profiling and IS200 probing, to study the relatedness in order to delineate a common source. The study included representative strains from both outbreak (15) and sporadic (7) cases for comparative analysis. Biotyping, antibiogram, whole cell protein-analysis and plasmid-profiling could not discriminate sporadic cases from outbreak strains, suggesting that a single clone/type (PT-1) may be prevalent in our region. In contrast, molecular-typing using IS200-probing revealed 2 clonally related strains circulating during the outbreak, as compared to the unrelated sporadic strains which exhibited considerable genetic diversity. Molecular analysis by IS200-probing, helped to assign an index case which provided a history of later outbreaks, since paratyphi A was repeatedly cultured in later outbreaks also. The study also suggests that genetic rearrangements can occur during the emergence of outbreaks. It reaffirmed the usefulness of IS200-probing in epidemiological investigations of Salmonella enterica serovars.