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

Diphtheria anti-toxoid antibody levels among pre-clinical students and staff in an institute of higher learning in Malaysia: are they protected?

Introduction: Little is known about the sero-prevalence of diphtheria anti-toxoid antibody levels among medical students in Malaysia. They too, just like other health care workers (HCWs) are at risk of contracting and transmitting diphtheria. Fortunately, this can be prevented by giving a specific vaccine: the diphtheria, tetanus and pertussis (DTP) vaccine. Nonetheless, data from local or regional surveys are needed before any decision is made by the respective authorities. General objective: We studied the epidemiology of diphtheria anti-toxoid antibody levels and vaccination history amongst medical students and staff in Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Specific objectives: We determined the level of diphtheria anti-toxoid antibodies amongst pre-clinical students and staff. Methodology: A total of 152 sera were collected from subjects aged 19 to 63, and diphtheria anti-toxoid levels were measured by an enzyme-linked immunosorbent assay. Results: One hundred and fifty-two (94.4%) blood samples out of 161 participants were successfully withdrawn, which comprised 105 (69.1%) and 47 (30.9%) medical students and staff, respectively. A total of 77.6% and the other 22.4% of the subjects had full and basic protection, respectively. Higher levels were predominant amongst males and they were 1.3 times more protected than females in 20-29 year-old group (85.1% vs 66.2%; odd ratios 1.25 [95% CI 1.03-1.50]; P=0.03). No significant difference in the levels of immunity among subjects for ethnicity and academic position (P>0.05). Recommendations: Level of full protection against diphtheria toxin should be clearly defined by broad population based studies using several comparable detection methods. Medical students and staff with basic protection should be closely monitored or should be given a booster dose for those who are at high risk of acquiring the disease. Thus, a standard degree of coverage should be clearly determined for health workers to prevent a potential outbreak. Conclusion: Students and staff possess immunity towards diptheria toxin however the level of full protective antibody is yet to be determined in future

Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus

Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR

Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times