Mohd Nasir Mohd Desa

Penicillin susceptibility and molecular characteristics of clinical isolates of Streptococcus pneumoniae at the University of Malaya Medical Center, Kuala Lumpur, Malaysia

OBJECTIVE To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center. METHODS From March 1999 to July 2000, 100 clinical isolates of Streptococcus pneumoniae were obtained from 93 patients of various ages and from various body sites. The minimum inhibitory concentrations (MICs) for penicillin and ceftriaxone were determined by E test, and results were interpreted according to guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Fifty isolates were further serotyped, and analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) of the penicillin-binding protein (pbp) 2b and 2x genes. RESULTS The majority of the isolates were from respiratory sites. Thirty-one isolates showed decreased susceptibility to penicillin (PRSP), and many of these also showed decreased susceptibility to ceftriaxone. Twelve serogroup/types (SGTs) were present, with 19F being the most common. PFGE analysis identified two dominant profiles, consisting mainly of PRSPs that had common serotypes (19F) and pbp gene patterns within their respective groups, although PCR-RFLP analysis showed different patterns of pbp genes among the PRSPs as compared to penicillin-susceptible strains, which had a uniform pattern. CONCLUSION PRSPs were genetically related as shown by PFGE and serotype. The consistency of pbp gene patterns, observed among many of the PRSPs within their respective PFGE profiles, supported their relatedness as established by PFGE.

Double Gene Targeting Multiplex Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products

Beef, buffalo, and pork adulteration in the food chain is an emerging and sensitive issue. Current molecular techniques to authenticate these species depend on polymerase chain reaction (PCR) assays involving long and single targets which break down under natural decomposition and/or processing treatments. This novel multiplex polymerase chain reaction-restriction fragment length polymorphism assay targeted two different gene sites for each of the bovine, buffalo, and porcine materials. This authentication ensured better security, first through a complementation approach because it is highly unlikely that both sites will be missing under compromised states, and second through molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120, 138, and 146 bp) were stable under extreme boiling and autoclaving treatments. Target specificity and authenticity were ensured through cross-amplification reaction and restriction digestion of PCR products with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter products revealed rampant substitution of beef with buffalo but purity in porcine materials.

Group B streptococcal bacteremia in a major teaching hospital in Malaysia: a case series of eighteen patients

BACKGROUND Group B Streptococcus (GBS) is a leading cause of infections such as meningitis and septicemia in neonates and pregnant women; however the significance of invasive GBS disease has not been clearly defined in non-pregnant adults. METHODS We reviewed the hospital records of 18 cases with GBS bacteremia who attended the Universiti Kebangsaan Malaysia Medical Centre from June 2010 to October 2011. We analyzed the clinical findings of both bacteremic adults and neonates and compared them to previous studies of GBS bacteremia. Serotyping was done by latex agglutination test using 10 distinct antisera (Ia, Ib, and II-IX). RESULTS During the period of 1 year and 4 months, there were 18 patients with GBS bacteremia. Five cases occurred in neonates, one in a parturient woman, and 12 in other adults. All neonates with bacteremia were males and two of them were premature. Septicemia was the most common clinical presentation in neonates. They were treated with intravenous (IV) penicillin G and gentamicin. The adults included nine men (69%) and four women (31%). Their mean age was 60 years and all patients had more than two underlying conditions. The most common clinical syndrome was pneumonia (n=6, 46.5%). The others were peritonitis (n=3, 23.1%), primary bacteremia (n=2, 15.5%), septic arthritis (n=2, 15.5%), skin and soft tissue infection (n=1, 7.7%), meningitis (n=1, 8%), urinary tract infection (n=1, 8%), and intravascular device infection (n=1, 7.7%). Cardiovascular diseases (n=7, 53.8%) were the most common underlying conditions, and diabetes mellitus (n=5, 38.5%) was second. The other co-morbid conditions were hyperlipidemia (n=3, 23.1%), renal disease (n=3, 23.1%), liver disease and/or alcohol abuse (n=3, 23.1%), autoimmune disease or immunosuppressive condition (n=2, 15.5%), malignancy (n=2, 15.5%), respiratory disease (n=1, 8%), and postpartum condition (n=1, 8%), as well as miscellaneous conditions including intravenous drug abuse, HIV infection, and trauma (n=2, 15.5%). Polymicrobial bacteremia was found in five (45.4%) cases and Staphylococcus aureus was the most common concurrent bacterial isolate. Of the 18 GBS isolates in both adults and neonates, serotype Ia was predominant (38.9%), followed by VI (27.8%), V (11.1%), and III (5.5%); the remaining 16.7% were non-typeable. CONCLUSIONS GBS bacteremia is a significant problem and is associated with serious underlying disease, which may result in a high rate of mortality, not only in neonates and pregnant women, but also in non-pregnant adults.

A persistent antimicrobial resistance pattern and limited methicillin-resistance-associated genotype in a short-term Staphylococcus aureus carriage isolated from a student population

The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton-Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n=9) and V (n=1). MLST was resolved for only three MRSAs, ST508 (n=1), ST88 (n=1) and ST96 (n=1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria.

Growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of Candida glabrata are affected by different glucose concentrations

Glucose is an important fuel source to support many living organisms. Its importance in the physiological fitness and pathogenicity of Candida glabrata, an emerging human fungal pathogen has not been extensively studied. The present study aimed to investigate the effects of glucose on the growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of C. glabrata. In addition, its effect on the expression of a putative high affinity glucose sensor gene, SNF3 was also investigated. Glucose concentrations were found to exert effects on the physiological responses of C. glabrata. The growth rate of the species correlated positively to the amount of glucose. In addition, low glucose environments were found to induce C. glabrata to form biofilm and resist amphotericin B. Conversely, high glucose environments promoted oxidative stress resistance of C. glabrata. The expression of CgSNF3 was found to be significantly up-regulated in low glucose environments. The expression of SNF3 gene in clinical isolates was found to be higher compared to ATCC laboratory strains in low glucose concentrations, which may explain the better survivability of clinical isolates in the low glucose environment. These observations demonstrated the impact of glucose in directing the physiology and virulence fitness of C. glabrata through the possible modulation by SNF3 as a glucose sensor, which in turn aids the species to adapt, survive and thrive in hostile host environment.

Determination of phenotypes and pneumococcal surface protein A family types of Streptococcus pneumoniae from Malaysian healthy children

BACKGROUND There is limited information about pneumococcal carriage among healthy children in Malaysia. Therefore, this study was conducted to determine the prevalence rate, serotype distribution, susceptibility pattern, and pneumococcal surface protein A (PspA) family types of Streptococcus pneumoniae isolates in the nasal carriage of children 5 years old or younger in three day care centers in Kuala Lumpur, Malaysia. METHODS Nasal swabs were collected from 195 healthy children, age 5 years or younger, from June to December 2010. S pneumoniae was identified by phenotypic and genotypic methods. The serotyping was performed using Pneumotest kit (Statens Serum Institut, Copenhagen, Denmark) and the susceptibility pattern was determined by using the E-test method (AB Biodisk, Solna, Sweden). PspA family typing was done using polymerase chain reaction. RESULTS S pneumoniae was found in the nasal carriage of 35.4% of children (69 of 195) and penicillin resistance was found in 23.2% (16 of 69). Among the 69 isolates, multidrug-resistant S pneumoniae (MDRSP) was present in 20.3%. All 16 penicillin-resistant S pneumoniae (PRSP) isolates were resistant to erythromycin and 14 PRSPs (87.5%) were resistant to co-trimoxazole. The six most common serotypes were 6A, 23F, 19A, 6B, 19F, and 15C, which were found in 87% of all isolates. Of the 69 isolates, 24.6% belonged to PspA family 1, 71.0% to PspA family 2, and 4.3% to PspA family 3. CONCLUSION Twenty-eight of the isolates (40.6%) belonged to serotypes included in the pneumococcal polysaccharide vaccines (PCV) 7 and 10, whereas 48 (69.5%) were included in PCV13. The high rate of PRSP and MDRSP supports the need for continuing surveillance of pneumococcal carriage. The major PspA families were 1 and 2 (95.7%), thus making them suitable candidates for future vaccines.

Distribution of CBP genes in Streptococcus pneumoniae isolates in relation to vaccine types, penicillin susceptibility and clinical site

Choline-binding proteins (CBP) have been associated with the pathogenesis of Streptococcus pneumoniae. We screened, using PCR, for the presence of genes (cbpA, D, E, G) encoding these proteins in 34 isolates of pneumococci of known serotypes and penicillin susceptibility from invasive and non-invasive disease. All isolates harboured cbpD and cbpE whereas cbpA and cbpG were found in 47% and 59% respectively; the latter were more frequent in vaccine-associated types and together accounted for 77% of these isolates. No association was observed with penicillin susceptibility but 85% of non-invasive isolates were positive for these genes.

Phylogenetic and Transcripts Profiling of Glucose Sensing Related Genes in Candida glabrata

Background: The sensing mechanism of glucose in Saccharomyces cerevisiae is well studied. However, such information is scarcely found in other yeast species such as Candida glabrata. Objectives: This study aimed to identify the glucose sensing pathway related genes of C. glabrata and to analyze the regulation pattern of these genes in response to different surrounding glucose concentrations through the quantitative real time polymerase chain reaction (qRT-PCR). Materials and Methods: Phylogenetic analysis was carried out on predicted amino acid sequences of C. glabrata and S. cerevisiae to compare their degree of similarity. In addition, the growth of C. glabrata in response to different amounts of glucose (0%, 0.01%, 0.1%, 1% and 2%) was evaluated via the spot dilution assay on prepared agar medium. Besides, the SNF3 and RGT2, which act as putative glucose sensors, and the RGT1 and MIG1, which act as putative transcriptional regulators and selected downstream hexose transporters (HXTs), were analysed through qRT-PCR analysis for the gene expression level under different glucose concentrations. Results: Comparative analysis of predicted amino acids in the phylogenetic tree showed high similarity between C. glabrata and S cerevisiae. Besides, C. glabrata demonstrated the capability to grow in glucose levels as low as 0.01% in the spot dilution assay. In qRT-PCR analysis, differential expressions were observed in selected genes when C. glabrata was subjected to different glucose concentrations. Conclusions: The constructed phylogenetic tree suggests the close evolutionary relationship between C. glabrata and S. cerevisiae. The capability of C. glabrata to grow in extremely low glucose environments and the differential expression of selected glucose-sensing related genes suggested the possible role of these genes in modulating the growth of C. glabrata in response to different glucose concentrations. This study helps deepen our understanding of the glucose sensing mechanism in C. glabrata and serves to provide fundamental data that may assist in unveiling this mechanism as a potential drug target.

Genotypic characterization of Streptococcus pneumoniae serotype 19F in Malaysia

Streptococcus pneumoniae is an epidemiologically important bacterial pathogen. Recently, we reported the antibiotic susceptibility patterns of a limited collection of pneumococcal isolates in Malaysia with a high prevalence of erythromycin resistant strains. In the present study, 55 of the pneumococcal isolates of serotype 19F were further analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The generated genotypic patterns were then correlated with the antibiograms previously reported. Forty-seven different PFGE profiles (PTs) were obtained, showing that the isolates were genetically diverse. MLST identified 16 sequence types (STs) with ST-236 being predominant (58.2%), followed by ST-81 (10.3%). Among the ST-236 isolates, 22 were erythromycin resistant S. pneumoniae (ERSP) and 15 were trimethoprim/sulfamethoxazole (TMP/SMX) resistant, while among ST-81, four isolates were ERSP and two were TMP/SMX resistant. The high prevalence of erythromycin resistant serotype 19F isolates of ST-236 in this study has also been reported in other North and South East Asian countries.

Draft Genome Sequence of Methicillin-Resistant Staphylococcus aureus KT/Y21, a Sequence Type 772 (ST772) Strain Isolated from a Pediatric Blood Sample in Terengganu, Malaysia

ABSTRACT Here, we report the draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) strain, KT/Y21, isolated from a blood sample of a pediatric patient. This strain belongs to sequence type 772 (ST772), harbors the staphylococcal cassette chromosome mec element (SCCmec) type V, and is positive for the Panton-Valentine leukocidin (PVL) pathogenic determinant.