Aims: The aim of the study was to isolate and characterise bacteriophages specific to Pseudomonas aeruginosa carrying virulence genes. Methodology and results: Four clinical strains of P. aeruginosa CL1, CL2, CL3 and CL4 were obtained from Queen Elizabeth Hospital, Kota Kinabalu, Sabah. The bacterial strains were screened for virulence genes exoS, toxA and oprI and biofilm production. Six P. aeruginosa specific bacteriophages, namely PAtk1, PAtk2, PAtk3, PAtk4, PAtk5 and PAtk6, were isolated from Tasik Kejuruteraan, Universiti Kebangsaan Malaysia. These bacteriophages were screened for lytic spectrum against P. aeruginosa and two species of Enterobacteriaceae (Escherechia coli and Salmonella Typhi). PCR results showed that all strains possessed exoS, toxA and oprI genes except CL2 that lacked exoS. Nevertheless, it was CL2 that produced the highest biofilm density. Further, based on Transmission Electron Microscopy, PAtk15 and PAtk6 were classified into the family Myoviridae and Siphoviridae, respectively. Among all six isolated phages, only PAtk4 and PAtk6 showed the broadest lytic spectrum in which lytic activity was observed against all clinical P. aeruginosa strains. Conclusion, significance and impact of study: In this study we reported the isolation of six bacteriophages from Myoviridae and Siphoviridae that are specific to P. aeruginosa possessing exoS, toxA and oprI genes. Bacteriophages Patk4 and PAtk6 were able to infect all four strains of P. aeruginosa, making these phages potential agents in combating infections by the bacterium.

Aims: To develop a real-time polymerase chain reaction system Vi-qPCR in the detection of Salmonella enterica serovar Typhi (S. Typhi), targeting the vexC gene encoding for Vi antigen (capsular polysaccharide antigen) and to evaluate its sensitivity and specificity performance using pure cultures of S. Typhi and other enteric pathogens. Methodology and results: Microbiological, biochemical and serotyping tests were conducted to determine the phenotypic characteristics of S. Typhi and other enteric pathogens in our collection. Primers were designed using Primer3 software and their in-silico specificity were analysed using Basic Local Alignment System Tool (BLAST). Optimisation of PCR annealing temperature was done prior to assessment of sensitivity and specificity performance against artificial serially diluted seeded stools. The primers were found to be 100% specific in the detection of S. Typhi towards 32 tested clinical strains. Verification of gene amplification by comparing the nucleotide sequences against reference genes in the GenBank database revealed high specificity to S. Typhi. Statistical analysis indicates that this method results in 100% sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Moreover, Vi-qPCR allows the detection of S. Typhi as low as 13 +/- 1.4 CFU/g of stool sample. Conclusion, significance and impact of study: A rapid and sensitive method for detection of Salmonella enterica serovar Typhi (S. Typhi) is desired as a diagnostic tool to improve typhoid management. The Vi-qPCR represent a promising non-invasive diagnostic tool for medical microbiology laboratories as a method for the detection of S. Typhi in both pure culture and stool specimens especially in chronic asymptomatic carriers where shedding of S. Typhi is intermittent and sometimes occurs in low level.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen with multiple antibiotic resistance that causes morbidity and mortality worldwide. Multidrug-resistant (MDR) MRSA with increased resistance to currently available antibiotics has challenged the world to develop new therapeutic agents. Stigmasterol and lupeol, from the plant Phyllanthus columnaris, exhibit antibacterial activities against MRSA. The aim of this study was to utilise next-generation sequencing (NGS) to provide further insight into the novel transcriptional response of MRSA exposed to stigmasterol and lupeol. Methods: Time-kill analysis of one MRSA reference strain (ATCC 43300) and three clinical isolates (WM3, BM1 and KJ7) for both compounds was first performed to provide the bacteriostatic/bactericidal profile. Then, MRSA ATCC 43300 strain treated with both compounds was interrogated by NGS. Results: Both stigmasterol and lupeol possessed bacteriostatic properties against all MRSA tested; however, lupeol exhibited both bacteriostatic and bactericidal properties within the same minimum inhibitory concentration and minimum bactericidal concentration values against BM1 (12.5 mg/mL). Transcriptome profiling of MRSA ATCC 43300 revealed significant modulation of gene expression with multiple desirable targets by both compounds, which caused a reduction in the translation processes leading to inhibition of protein synthesis and prevention of bacterial growth. Conclusions: This study highlights the potential of both stigmasterol and lupeol as new promising anti-MRSA agents. (C) 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.