Salasawati Hussin

Simple, time saving pulsed-field gel electrophoresis protocol for the typing of Stenotrophomonas maltophilia

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.

Epidemiological Study on Staphylococcus aureus Isolates Reveals Inverse Relationship between Antibiotic Resistance and Virulence Repertoire

Acquisition of antibiotic resistance in Staphylococcus aureus has been associated with loss of pathogenic fitness and also virulence potential, where this phenomenon has been observed in specific clinical and laboratory S. aureus strains [1–4]. Interestingly, this phenomenon of “inverse relationship between antibiotic resistance and virulence” was also observed in a general population of both methicillin-resistant and -susceptible S. aureus (MRSA and MSSA), when a molecular epidemiological study was carried out at our teaching hospital in Kuala Lumpur, Malaysia.

Activated ADI pathway: the initiator of intermediate vancomycin resistance in Staphylococcus aureus

Comparative proteomic profiling between 2 vancomycin-intermediate Staphylococcus aureus (VISA) strains, Mu50Ω-vraSm and Mu50Ω-vraSm-graRm, and vancomycin-susceptible S. aureus (VSSA) strain Mu50Ω revealed upregulated levels of catabolic ornithine carbamoyltransferase (ArcB) of the arginine catabolism pathway in VISA strains. Subsequent analyses showed that the VISA strains have higher levels of cellular ATP and ammonia, which are by-products of arginine catabolism, and displayed thicker cell walls. We postulate that elevated cytoplasmic ammonia and ATP molecules, resulting from activated arginine catabolism upon acquisition of vraS and graR mutations, are important requirements facilitating cell wall biosynthesis, thereby contributing to thickened cell wall and consequently reduced vancomycin susceptibility in VISA strains.

Draft Genome Sequences of Four Nosocomial Methicillin-Resistant Staphylococcus aureus (MRSA) Strains (PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009) Representative of Dominant MRSA Pulsotypes Circulating in a Malaysian University Teaching Hospital

ABSTRACT Here, we report the draft genome sequences of four nosocomial methicillin-resistant Staphylococcus aureus strains (PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009) isolated from a university teaching hospital in Malaysia. Three of the strains belong to sequence type 239 (ST239), which has been associated with sustained hospital epidemics worldwide.

First report on the molecular epidemiology of Malaysian Staphylococcus epidermidis isolated from a University Teaching Hospital

BackgroundStaphylococcus epidermidis is a pathogen associated with nosocomial infections whose medical importance has increased due to progressively invasive medical procedures. In this study, we characterized the molecular epidemiology of S. epidermidis strains circulating in our university hospital situated in Kuala Lumpur, Malaysia.FindingsA total of 798 S. epidermidis were isolated from our university hospital, where 56.3% of the isolates were found to be cefoxitin (methicillin) resistant and also positive for the mecA gene. Staphylococcus Cassette Chromosome mec (SCCmec) typing revealed that 39.6% of the methicillin-resistant S. epidermidis (MRSE) were SCCmec-untypeable, with 54.6% harboring the cassette chromosome recombinase C (ccrC) gene. A total of 67 isolates from the neonatal intensive care unit (NICU) was selected for pulsed-field gel electrophoresis (PFGE) typing, where 13 pulsotypes were identified at a cut-off value of 80% similarity. No significant association was found between the PFGE pulsotypes, SCCmec types and antibiotic susceptibilities. Polymerase chain reaction (PCR) assays to detect biofilm-associated genes in the ica operon and also 4 staphylococcal toxin genes (cna, seh, PVL genes and tst-1) revealed that only 8.0% isolates had the complete operon, while cna was the most prevalent toxin gene detected amongst the isolates (35.8%).ConclusionTo our knowledge, this is the first report on the molecular epidemiology of Malaysian S. epidermidis. We found the strains to be low in virulence potential; nevertheless further studies have to be conducted to determine if this phenomenon translates into a better clinical outcome for patients.

Clonal distribution and possible microevolution of methicillin-resistant Staphylococcus aureus strains in a teaching hospital in Malaysia

OBJECTIVE To genotypically characterize methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from medical and surgical wards in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in 2009. METHODS MRSA strains were collected and molecularly typed by pulsed-field gel electrophoresis (PFGE). RESULTS PFGE typing on 180 MRSA isolated in UKMMC identified 5 pulsotypes (A-E) and 6 singletons, where pulsotypes B and C were suspected to be divergent clones originating from a single ancestor. This study also showed that most MRSA strains were isolated from swab (119 isolates), followed by blood (22 isolates), tracheal aspirate (11 isolates) and sputum (10 isolates). On the other hand, urine and bone isolates were less, which were 4 and 1 isolates, respectively. The distribution of different pulsotypes of MRSA among wards suggested that MRSA was communicated in surgical and medical wards in UKMMC, with pulsotype B MRSA as the dominant strain. Besides, it was found that most deceased patients were infected by pulsotype B MRSA, however, no particular pulsotype could be associated with patient age, underlying disease, or ward of admittance. CONCLUSIONS Five pulsotypes of MRSA and 6 singletons were identified, with pulsotype B MRSA as the endemic strains circulating in these wards, which is useful in establishment of preventive measures against MRSA transmission.

Antimicrobial resistance profiling and molecular typing of methicillin-resistant Staphylococcus aureus isolated from a Malaysian teaching hospital

The annual prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysia has been estimated to be 30 % to 40 % of all S. aureus infections. Nevertheless, data on the antimicrobial resistance and genetic diversity of Malaysian MRSAs remain few. In 2009, we collected 318 MRSA strains from various wards of our teaching hospital located in Kuala Lumpur, the capital city of Malaysia, and performed antimicrobial susceptibility testing on these strains. The strains were then molecularly characterized via staphylococcal cassette chromosome (SCC) mec and virulence gene (cna, sea, seb, sec, sed, see, seg, seh, sei, eta, etb, Panton-Valentine leukocidin and toxic shock syndrome toxin-1) typing; a subset of 49 strains isolated from the intensive care unit was also typed using PFGE. Most strains were found to be resistant to ciprofloxacin (92.5 %), erythromycin (93.4 %) and gentamicin (86.8 %). The majority (72.0 %) of strains were found to harbour SCCmec type III-SCCmercury with the presence of ccrC, and carried the sea+cna gene combination (49.3 %), with cna as the most prevalent virulence gene (94.0 %) detected. We identified four PFGE clusters, with pulsotype C (n=19) as the dominant example in the intensive care unit, where this pulsotype was found to be associated with carriage of SCCmec type III and the sea gene (P=0.05 and P=0.02, respectively). In summary, the dominant MRSA circulating in our hospital in 2009 was a clone that was ciprofloxacin, erythromycin and gentamicin resistant, carried SCCmec type III-SCCmercury with ccrC and also harboured the sea+cna virulence genes. This clone also appears to be the dominant MRSA circulating in major hospitals in Kuala Lumpur.