Zamberi Sekawi

Predominance and Emergence of Clones of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus in Malaysia

ABSTRACT We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.

DNA vaccine constructs against enterovirus 71 elicit immune response in mice

BackgroundEnterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo.MethodsThe VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71.ResultsThe pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71.ConclusionThe promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

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 the polystyrene 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.

Inhibition of Growth of Highly Resistant Bacterial and Fungal Pathogens by a Natural Product

The continuous escalation of resistant bacteria against a wide range of antibiotics necessitates discovering novel unconventional sources of antibiotics. B. oleracea L (red cabbage) is health-promoting food with proven anticancer and anti-inflammatory activities. However, it has not been researched adequately for its antimicrobial activity on potential resistant pathogens. The methanol crude extract of B. oleracea L. was investigated for a possible anti-microbial activity. The screening method was conducted using disc diffusion assay against 22 pathogenic bacteria and fungi. It was followed by evaluation of the minimum inhibitory concentration (MIC). Moreover, the antibacterial and the antifungal activities were confirmed using the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC), respectively. Remarkable, antibacterial activity was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. Red cabbage is a rich source of phenolic compounds, anthocyanins being the most abundant class, which might explain its potent antimicrobial action. This extract is potentially novel for future antimicrobials, inexpensive, and readily available at a large scale for pharmaceutical companies for further investigation and processing.

The Prevalence of ESBLs Producing Klebsiella pneumoniae Isolates in Some Major Hospitals, Iran

Aims of this study were to investigate on antibiotic resistance and molecular epidemiology of K.pneumoniae producing ESBLs isolates of respiratory tract infections in some major hospitals in Iran. K.pneumonaie were obtained of patients with RTI. K. pneumoniae producing ESBLs detected by screening, confirming and PCR methods. During the 12-month period, a total of one hundred and thirteen of K.pneumoniae were found from RTI in three cities in different region of Iran which Sixty seven strains (59.2%) were ESBLs producer. In Ilam hospitals, seventeen strains (43.6%), in Milad hospital, thirty-seven strains (74%) and in Emam Reza hospital, thirteen strains (54.2%) were ESBLs producer. The findings showed that among sixty-seven K.pneumoniae producing ESBLs, Sixty-three strains (94%) were positive for blaSHV, eleven strains (16.4%) contained blaTEM and sixteen strains (23.9%) harbored blaCTX-M. Imipenem was found as an effectiveness antibiotic. In the current study, Majority of the ESBLs production had occurred in Milad hospital in Tehran (74%). In conclusion, spreading ESBL-producing strains is a concern, as it causes limitations to the antimicrobial agents for optimal treatment of patients.

Effect of organic acids on escherichia coli O157:H7 and staphylococcus aureus contaminated meat.

Appropriate and safe antibacterial agents able to decontaminate meat surfaces have long been big concern of meat industry. In an attempt to manage beef carcass contamination, spray wash treatments utilizing three concentrations (1, 1.5 and 2%) of acetic, lactic, propionic and formic acids were performed to evaluate their efficacy in reducing numbers of Escherichia coli O157:H7 and Staphylococcus aureus on meat tissues. The procured beef pieces of freshly slaughtered animals were decontaminated with hot water and then inoculated with E. coli O157:H7 and S. aureus individually which then were spray washed with organic acids separately. The total plate count of the treated samples showed that the populations of bacteria decreased after being exposed to organic acids. Spray wash of formic acid resulted in the highest reduction of both bacterial species on meat surface. Significantly, higher log reductions were obtained for S. aureus than E. coli O157:H7. It was concluded that organic acids are highly effective in decontaminating meat surfaces and organic acids are shown to be safe, simple, efficient, and cheap modality of meat decontamination which can be highly recommended for industrial scales.

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.

Methicillin-susceptible Staphylococcus aureus from clinical and community sources are genetically diverse.

Despite the association of methicillin-susceptible S. aureus (MSSA) with several life-threatening diseases, relatively little is known about their clinical epidemiology in Malaysia. We characterized MSSA isolates (n=252) obtained from clinical and community (carriage) sources based on spa sequencing and multilocus sequence typing (MLST). The prevalence of several important virulence genes was determined to further define the molecular characteristics of MSSA clones circulating in Malaysia. Among the 142 clinical and 110 community-acquired MSSA isolates, 98 different spa types were identified, corresponding to 8 different spa clonal clusters (spa-CCs). In addition, MLST analysis revealed 22 sequence types (STs) with 5 singletons corresponding to 12 MLST-CCs. Interestingly, spa-CC084/085 (MLST-CC15) (p=0.038), spa-non-founder 2 (MLST-ST188) (p=0.002), and spa-CC127 (MLST-CC1) (p=0.049) were identified significantly more often among clinical isolates. spa-CC3204 (MLST-CC121) (p=0.02) and spa-CC015 (MLST-CC45) (p=0.0002) were more common among community isolates. Five dominant MLST-CCs (CC8, CC121, CC1, CC45, and CC5) having clear counterparts among the major MRSA clones were also identified in this study. While the MSSA strains are usually genetically heterogeneous, a relatively high frequency (19/7.5%) of ST188 (t189) strains was found, with 57.8% of these strains carrying the Panton-Valentine leukocidin (PVL). Analysis of additional virulence genes showed a frequency of 36.5% and 36.9% for seg and sei and 0.8% and 6.3% for etb and tst genes, respectively. Arginine catabolic mobile element (ACME) was detected in 4 community isolates only. These represent the first isolates harbouring this gene in an Asian region. In conclusion, MSSA from the Malaysian community and their clinical counterparts are genetically diverse, but certain clones occur more often among clinical isolates than among carriage isolates and vice versa.

Comparative Characterisation of Genotypically Different Clones of MRSA in the Production of Biofilms

The ability to adhere and produce biofilms is characteristic of enhanced virulence among isolates of methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study is to find out whether these characteristics are consistently similar among isolates variations of MRSA. The study used 30 various isolates of MRSA belong to 13 spa types and 5 MLST types and determined the aggregation, the adherence, and the production of biofilms and slime for each isolate. The methods used to evaluate these characteristics were a modified Congo red agar assay (MCRA), a microtiter plate assay (MPA), high-magnification light microscopy, scanning electron microscopy (SEM), and PCR. The study found that isolates belonging to similar Spa, SCCmec, and ST types have similar abilities to produce biofilms; however, their ability to produce slime on CRA was found to be different. Moreover, isolates that have different Spa types showed high variation in their ability to produce biofilms. The results of light microscope revealed the isolates that produced strong and weak biofilms and formed similar aggregation on the glass surfaces. SEM results showed that all 30 MRSA isolates that were tested were 100% positive for biofilm formation, although to varying degrees. Further testing using PCR confirmed that 100% of the 30 isolates tested were positive for the presence of the icaADBC, fnbA, eno, ebps, clfA, and clfB genes. The prevalence of fib, cna, fnbB, and bbp in MRSA clones was 90, 93.33, 53.33, and 10%, respectively. This study indicate that differences in biofilm production capacities are caused by the differences in surface protein A (Spa) type and are not due to differences in MLST and SCCmec types.