High Prevalence of Toxigenic and Nontoxigenic Clostridium difficile Strains in Malaysia.

Accumulating evidence shows a high prevalence of Clostridium difficile in Southeast Asia associated with a range of clinical presentations. However, severe infections are rarely reported. We investigated C. difficile infection (CDI) across four hospitals in Kuala Lumpur and Kota Bharu, Malaysia. Enzyme immunoassays for glutamate dehydrogenase (GDH) and toxin A or B were performed on diarrheal stool specimens collected from patients in 2015 and 2016. Specimens were also cultured and isolates of C. difficile characterized by PCR ribotyping and detection of toxin genes. In total, 437 specimens were collected and fecal toxin was detected in 3.0%. A further 16.2% of specimens were GDH positive and toxin negative. After culture, toxigenic strains were isolated from 10.3% and nontoxigenic strains from 12.4% of specimens. The most prevalent PCR ribotypes (RTs) were RT 017 (20.0%) and RT 043 (10.0%). The high prevalence of RT 017 and nontoxigenic strains in Malaysia and in neighboring Thailand and Indonesia suggests that they localize to the region of Southeast Asia, with an implication that they may mediate the burden of CDI in the region.

Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant Acinetobacter baumannii Isolate.

Acinetobacter baumannii has emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Imp(r)) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imp(s)) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imp(r) but not in the Imp(s) isolate. Notably, this finding is corroborated by an increase in the motility of the Imp(r) strain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage to A. baumannii and may contribute to clinical success.

Fatal case of amoebic liver abscess in a child.

We reported a case of amoebic liver abscess (ALA) in a 6-year-old Malaysian boy who presented with fever, lethargy, diarrhoea and right hypochondriac pain. On admission he was diagnosed with perforated acute appendicitis and a laparotomy was done. After surgery he developed acute respiratory distress. Ultrasonography, chest X-Ray and CT scan revealed two ALAs in the posterior segment of right lobe of liver, pleural effusion and collapsed consolidation of lungs bilaterally. Percutaneous liver abscesses drainage was done and intravenous Metronidazole was started. PCR carried out on the pus from the abscess was positive for Entamoeba histolytica. Patient however succumbed to the infection one week after admission.

Development of an instrument to measure medical students' perceptions of the assessment environment: initial validation.

INTRODUCTION: Assessment environment, synonymous with climate or atmosphere, is multifaceted. Although there are valid and reliable instruments for measuring the educational environment, there is no validated instrument for measuring the assessment environment in medical programs. This study aimed to develop an instrument for measuring students' perceptions of the assessment environment in an undergraduate medical program and to examine the psychometric properties of the new instrument. METHOD: The Assessment Environment Questionnaire (AEQ), a 40-item, four-point (1=Strongly Disagree to 4=Strongly Agree) Likert scale instrument designed by the authors, was administered to medical undergraduates from the authors' institution. The response rate was 626/794 (78.84%). To establish construct validity, exploratory factor analysis (EFA) with principal component analysis and varimax rotation was conducted. To examine the internal consistency reliability of the instrument, Cronbach's α was computed. Mean scores for the entire AEQ and for each factor/subscale were calculated. Mean AEQ scores of students from different academic years and sex were examined. RESULTS: Six hundred and eleven completed questionnaires were analysed. EFA extracted four factors: feedback mechanism (seven items), learning and performance (five items), information on assessment (five items), and assessment system/procedure (three items), which together explained 56.72% of the variance. Based on the four extracted factors/subscales, the AEQ was reduced to 20 items. Cronbach's α for the 20-item AEQ was 0.89, whereas Cronbach's α for the four factors/subscales ranged from 0.71 to 0.87. Mean score for the AEQ was 2.68/4.00. The factor/subscale of 'feedback mechanism' recorded the lowest mean (2.39/4.00), whereas the factor/subscale of 'assessment system/procedure' scored the highest mean (2.92/4.00). Significant differences were found among the AEQ scores of students from different academic years. CONCLUSIONS: The AEQ is a valid and reliable instrument. Initial validation supports its use to measure students' perceptions of the assessment environment in an undergraduate medical program.

Evolution of Influenza B Virus in Kuala Lumpur, Malaysia, between 1995 and 2008.

Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.

In vitro properties of designed antimicrobial peptides that exhibit potent antipneumococcal activity and produces synergism in combination with penicillin.

Antimicrobial peptides (AMPs) represent a promising class of novel antimicrobial agents owing to their potent antimicrobial activity. In this study, two lead peptides from unrelated classes of AMPs were systematically hybridized into a series of five hybrid peptides (DM1-DM5) with conserved N- and C-termini. This approach allows sequence bridging of two highly dissimilar AMPs and enables sequence-activity relationship be detailed down to single amino acid level. Presence of specific amino acids and physicochemical properties were used to describe the antipneumococcal activity of these hybrids. Results obtained suggested that cell wall and/or membrane targeting could be the principal mechanism exerted by the hybrids leading to microbial cell killing. Moreover, the pneumocidal rate was greater than penicillin (PEN). Combination treatment with both DMs and PEN produced synergism. The hybrids were also broad spectrum against multiple common clinical bacteria. Sequence analysis showed that presence of specific residues has a major role in affecting the antimicrobial and cell toxicity of the hybrids than physicochemical properties. Future studies should continue to investigate the mechanisms of actions, in vivo therapeutic potential, and improve rational peptide design based on the current strategy.

Activity of Novel Synthetic Peptides against Candida albicans.

Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8-16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64-128 mg/L to 16-64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited.

Draft Genome Sequence of Dematiaceous Coelomycete Pyrenochaeta sp. Strain UM 256, Isolated from Skin Scraping.

Pyrenochaeta, classified under the order Pleosporales, is known to cause diseases in plants and humans. Here, we report a draft genome sequence of a Pyrenochaeta sp. isolated from a skin scraping, with an estimated genome size of 39.4 Mb. Genes associated with the synthesis of proteases, toxins, plant cell wall degradation, and multidrug resistance were found.

Draft Genome Sequence of Herpotrichiellaceae sp. UM 238 Isolated from Human Skin Scraping.

Herpotrichiellaceae spp. are known to be opportunistic human pathogens. Here, we report the ~28.46-Mb draft genome of Herpotrichiellaceae sp. UM 238, isolated from human skin scraping. The UM 238 genome was found to contain many classes of protective genes that are responsible for fungal adaptation under adverse environmental conditions.

Draft Genome Sequence of the First Isolate of Extensively Drug-Resistant (XDR) Mycobacterium tuberculosis in Malaysia.

The emergence of the global threat of extensively drug-resistant (XDR) Mycobacterium tuberculosis reveals weaknesses in tuberculosis management and diagnostic services. We report the draft genome sequence of the first extensively drug-resistant Mycobacterium tuberculosis strain isolated in Malaysia. The sequence was also compared against a reference strain to elucidate the polymorphism that is related to their extensive resistance.

Ceftriaxone resistance and genes encoding extended-spectrum ß-lactamase among non-typhoidal Salmonella species from a tertiary care hospital in Kuala Lumpur, Malaysia.

The prevalence of ceftriaxone resistance and the associated genes encoding extended-spectrum ß-lactamase (ESBL) was determined in 149 non-duplicate non-typhoidal Salmonella isolated in 2008-2009 from patients in a tertiary care hospital in Kuala Lumpur, Malaysia. The resistance rate to ceftriaxone was 2.7% (2/74) in 2008, 4.0% (3/75) in 2009, and 3.4% (5/149) overall. CTX-M ESBL genes were detected in 2 of the 5 ceftriaxone-resistant isolates. The prevalence of ceftriaxone resistance, although low, is a concern because it limits therapeutic options. Continued surveillance of ceftriaxone resistance is important to monitor its trends.

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp.

BACKGROUND: Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules. METHODOLOGY/PRINCIPAL FINDINGS: Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C(12). The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown. CONCLUSIONS/SIGNIFICANCE: These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.

Genome sequence of an unclassified pleosporales species isolated from human nasopharyngeal aspirate.

Pleosporales is the largest order in the fungal class Dothideomycetes. We report the 36,814,818-bp draft genome sequence and gene annotation of UM1110, a Pleosporales isolate associated with unclassified genera that is potentially a new fungal species. Analysis of the genome sequence led to the finding of genes associated with fungal adhesive proteins, secreted proteases, allergens, and pseudohyphal development.

In vitro activity of tigecycline against Acinetobacter baumannii isolates from a teaching hospital in Malaysia.

The In vitro susceptibility of clinical and environmental isolates of Acinetobacter baumannii to tigecycline and other antibiotics was determined by disk diffusion method. The E-test was used to determine the minimum inhibitory concentration (MIC). The growth curves of tigecycline treated environmental and clinical strains were established. Fifty-seven percent and 75% of the clinical and environmental isolates were MDR strains, respectively. Ninety-five percent of the clinical isolates were susceptible to tigecycline and 5% showed intermediate resistance with MIC ranging between 0.032 and 3 mg/l. Tigecycline susceptible and intermediate resistance among the environmental isolates were 40% and 60%, respectively, with a significantly lower MIC range of 0.5-4 mg/l. The bacterial growth curves demonstrated the higher ability of the environmental strains to tolerate the antibiotic effects than the clinical strains. The relatively high resistance profile among the environmental isolate suggests an insidious emergence of tigecycline resistance amongst A. baumannii. Strict infection control procedures are imperative to prevent the dissemination of tigecycline-resistant A. baumannii strains in the hospital environment.

Draft genome sequence of Daldinia eschscholzii isolated from blood culture.

Daldinia eschscholzii is an invasive endophyte that is most commonly found in plant tissues rich in secondary metabolites. We report the draft genome sequence of D. eschscholzii isolated from blood culture. The draft genome is 35,494,957 bp in length, with 42,898,665 reads, 61,449 contigs, and a G+C content of 46.8%. The genome was found to contain a high abundance of genes associated with plant cell wall degradation enzymes, mycotoxin production, and antifungal drug resistance.

Sequencing of Cladosporium sphaerospermum, a Dematiaceous fungus isolated from blood culture.

Cladosporium sphaerospermum is one of the most widely distributed allergens causing serious problems in patients with respiratory tract disease. We report the 26,644,473-bp draft genome sequence and gene annotation of C. sphaerospermum UM843. Analysis of the genome sequence led to the finding of genes associated with C. sphaerospermum's melanin biosynthesis, allergens, and antifungal drug resistance.

Proteinase, phospholipase, biofilm forming abilities and antifungal susceptibilities of Malaysian Candida isolates from blood cultures.

This study was conducted to determine the proteinase, phospholipase, and biofilm forming abilities of Candida isolates in blood cultures of specimens from patients at the University Malaya Medical Center, Kuala Lumpur, Malaysia. Proteinase and phospholipase activities were detected in 93.7% and 73.3%, respectively, of 15 Candida albicans isolates. Amongst the 26 non-C. albicans Candida isolates, proteinase and phospholipase activities were detected in 88.5% and 7.7% of the isolates, respectively. There was no significant difference in the expression levels of proteinase amongst the Candida isolates studied (P = 0.272), but the phospholipase activity of C. albicans was significantly higher than that of the non-C. albicans Candida isolates (P = 0.003). There was no significant difference in the biofilm forming abilities of C. albicans and non-C. albicans Candida isolates on the polystyrene microtiter wells (P = 0.379). In addition, the findings of this study demonstrate increased resistance of Candida isolates in biofilms to amphotericin and fluconazole, as compared to their planktonic counterparts.

Macrorestriction analysis and antimicrobial susceptibility profiling of Salmonella enterica at a University Teaching Hospital, Kuala Lumpur.

The genetic diversity and antimicrobial resistance rates of clinical Salmonella isolates (2007-2008) at the University of Malaya Medical Centre, Kuala Lumpur, were investigated and the genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic (REP)-PCR. XbaI-PFGE analysis generated 57 profiles (Dice coefficient, F=0.08-1.00), whereas REP-PCR using the REP primer generated only 35 (F=0.34-1.00). PFGE was therefore the more discriminative and reproducible method for assessing the genetic diversity of salmonellae. The antibiograms of 78 Salmonella isolates were assessed against 19 antimicrobials using the disk diffusion method. Twenty serotypes were identified, with the most common being S. Enteritidis (18%) followed by S. Typhimurium (14%), S. Paratyphi B var Java (9%), S. Weltevreden (9%), and S. Corvallis (9%). A total of 38 resistant profiles were defined, with 53.8% of the isolates being resistant to three or more antimicrobials. The highest resistance rates were observed for cephalothin (55.1%), tetracycline (47.4%), and nalidixic acid (35.9%). The presence of multidrug-resistant Salmonella strains is a cause for concern as it may limit the treatment of severe salmonellosis. One multidrug-resistant S. Enteritidis strain was a putative extended-spectrum beta-lactamase producer, based on a double disk diffusion analysis, and was resistant to ceftriaxone (MIC>32 microg/mL). The data generated by this study will contribute towards epidemiological monitoring and investigations of Salmonella infections in Malaysia.