The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.

Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniaeinduced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.

Lectins, also known as sugar binding proteins, play an essential role in the initiation of bacterial infections and biofilm production. To date, several lectins of Gram-negative bacteria such as Pseudomonas aeruginosa, Burkholderia cenocepacia, Ralstonia solanacearum and Chromobacterium violaceum have been identified. There are no published reports on the presence of lectins in Burkholderia pseudomallei, the causative agent of melioidosis. The aim of this study was to identify possible lectin genes of B. pseudomallei and generate recombinant proteins for assessment of hemagglutinating activity. Seven hypothetical lectins of B. pseudomallei were retrieved from the UniProt database. Four lectin domains, i.e., ricin B, C-type, H-type and Bulb-type lectins were identified. In silico analysis using a ligand binding site prediction server (3DLigandSite) predicted the presence of Nacetylglucosamine and calcium binding sites in two C-type lectins. Four recombinant proteins with the molecular weights of 11.7, 30.2, 36.2 and 46.4 kDa were expressed from the cloned genes; however none of them expressed any hemagglutinating activity. Further characterization of B. pseudomallei lectins may be able to provide insights into bacterial-host interaction that are required to initiate infections.

Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.

The increased frequency of antibiotic resistance is known to be associated with the dissemination of integrons in the Enterobacteriaceae. This study determined the prevalence and type of integrons amongst 160 extended-spectrum beta-lactamase producing enterobacterial isolates kept in our culture collection. Integrons were detected in 98(61.3%) isolates, including 28(62.2%) Escherichia coli, 34(64.2%) Klebsiella spp., 27(61.4%), Enterobacter spp. and 9(50.0%) Citrobacter spp. investigated in this study. Restriction analysis of the integron gene fragments revealed that class I integron was the principal integron detected in 92(57.5%) of our isolates. Class II integron was detected in 6(3.8%) of our isolates, while no class III integron was detected in this study. The high rates of integron prevalence particularly of the class I integron in the E. coli and Klebsiella spp. concur with previous studies in other geographical regions. The higher (>50%) integron prevalence of Citrobacter and Enterobacter isolates comparing to previous studies suggests the potential of these isolates as sources for dissemination of resistance determinants. The finding in this study serves as a basis for further study on the antibiotic resistance mechanisms of enterobacterial species in this teaching hospital.

Abstract. Melioidosis has been recognized as an important cause of sepsis in the tropics. The disease caused by an environmental saprophyte Burkholderia pseudomallei, affects mostly adults with underlying immunocompromised conditions. In this study, the enzymatic profiles of 91 clinical and 9 environmental isolates of B. pseudomallei were evaluated using the APIZYM system, in addition to assessment of protease, phospholipase C and sialidase activities using agar plate methods and other assays. The activity of 10 enzymes - alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were detected in >75% of the clinical isolates. The majority of B. pseudomallei isolates in this study exhibited protease and phospholipase activities. No sialidase activity was detected. Five Burkholderia thailandensis isolates had similar APIZYM profiles as B. pseudomallei clinical isolates except for the lower detection rate for N-acetyl-β-glucosaminidase. The subtle differences in the number of enzymes secreted and the levels of enzymatic activities of phenotypically identical clinical and environmental strains of B. pseudomallei give weight to the fact that the causative agent of melioidodis originates from the environment.

This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.

Limited information is available on human exposure to Bartonella infection, i.e., Bartonella henselae (causative agent of cat scratch disease) and Bartonella quintana (causative agent of trench fever) in West Malaysia. This study reports a review of serological findings obtained from patients attending to a teaching hospital in Klang Valley, Malaysia. An indirect immunofluorescence assay (IFA) was used to determine IgG and IgM antibody titers against B. henselae and B. quintana. In a pilot study conducted between 2013-2015, IgG antibodies against Bartonella spp. (either B. quintana and B. henselae) were detected in 14 (36.8%) of 38 patients who were clinically suspected of rickettsial infections, while IgM antibody was detected in 4 (10.5%) patients. This has prompted us to investigate the serologic responses of patients who were clinically suspected of other febrile causes besides rickettsial infection. Of the 59 serum samples analysed in a follow-up investigation, Bartonella IgG antibodies were detected from 7 (11.9%) patients, of which 5 (27.8%) and 2 (18.2%) patients were clinically suspected of rickettsial infection (n=18) and dengue (n=11), respectively. None of the sera obtained from the leptospirosis (n=10), legionellosis (n=10) and mycoplasma infection (n=10) groups were seropositive to Bartonella spp. The review of Bartonella serological findings in this study highlights that Bartonella infection is not uncommon and should be considered as one of the causes for febrile illness in Malaysia.

Rickettsioses are a common health problem in many geographical areas, including rural areas in Southeast Asia. Co-infection of rickettsioses and malaria has been reported in Africa, where common reservoir and vectors are available. In this study, blood samples of Malaysian patients microscopically positive (n=148) and negative (n=88) for malaria parasites (Plasmodium knowlesi, Plasmodium malariae, Plasmodium falciparum, and Plasmodium vivax) were screened for the presence of rickettsial DNA, using PCR assays targeting specific genes. A partial fragment of rickettsial ompB gene was successfully amplified and sequenced from a patient microscopically positive for Plasmodium spp. and PCR-positive for P. vivax. BLAST analysis of the ompB sequence demonstrated the highest sequence similarity (99.7% similarity, 408/409nt) with Rickettsia sp. RF2125 (Genbank accession no. JX183538) and 91.4% (374/409 nt) similarity with Rickettsia felis URRWXCal2 (Genbank accession no. CP000053). This study reports rickettsial infection in a malaria patient for the first time in the Southeast Asia region.

Candida glabrata has been reported as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. This study was aimed to determine the genetic diversity, antifungal susceptibility and enzymatic profiles of C. glabrata isolated from vaginal and blood samples in the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre. A random amplified polymorphic DNA (RAPD) analysis method, using M13 and (GTG)5 primers, was used for strain differentiation of C. glabrata isolates. Antifungal susceptibility testing of C. glabrata isolates was determined using E-test against amphotericin B, caspofungin, fluconazole and voriconazole and microbroth dilution method against clotrimazole. The enzymic profiles of C. glabrata were determined using APIZYM semi-quantitation kit and egg-yolk agar method. A total of 14 RAPD patterns were identified amongst C. glabrata isolates investigated this study. Susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole was noted. Approximately one third of the isolates demonstrated resistance to clotrimazole (MIC>1 μg/ml). A single isolate of C. glabrata was resistant to caspofungin (MIC:1.5 μg/ml). Enzymatic activities of acid and alkaline phosphatase, aminopeptidases, esterase and lipase and phospholipase were detected in the C. glabrata isolates. The genetic diversity and antifungal susceptibility profiles of C. glabrata isolates were presented in this study. Continued surveillance and monitoring of the incidence and antifungal resistance in C. glabrata isolates is necessary.