Identification and Characterisation of Bacteria Isolated (During a Cholera Outbreak) in Limbang, Sarawak

Amirah Zakirah, Ja’afar (2021) Identification and Characterisation of Bacteria Isolated (During a Cholera Outbreak) in Limbang, Sarawak. UNSPECIFIED thesis, UNSPECIFIED.

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Abstract

Cholera epidemics have occurred in Malaysia since 1991 till 2003 which can be proved from the records by the Infectious Diseases Division of the Ministry of Health. Additionally, from 1994 to 2003, Sarawak saw a series of cholera epidemics. Cholera outbreaks in Malaysia are primarily caused by the El Tor O1 Vibrio cholerae serogroup. The aims of this study were to identify bacterial species from clinical and environmental samples (n=28) from Limbang, Sarawak by collaboration with Sarawak Government Hospital by the approach of active cased detection (ACD) and passive case detection (PCD), and to detect the toxin genes of the V. cholerae from the isolates. Apart from that, to determine the genetic relatedness of the isolates, to detect antibiotic susceptibility, and to determine the isolates' minimal biofilm eradication concentration (MBEC). All the isolates were sub-cultured in alkaline peptone water (APW). The boiled-cell method was used for DNA extraction. The total DNA extracted was amplified by polymerase chain reaction (PCR). In this work, two forms of PCR were used: 16S rRNA PCR and multiplex PCR. The results obtained from the study found out that 16 out of 28 (57.14 %) samples were confirmed to be V. cholerae species. Four primers specific for V. cholerae were used in multiplex PCR (O1 type, O139 type, ctxA and ctxAB) to confirm the species type and the toxin genes. All samples shown positive for V. cholerae O1 serotype and 100% positive to all genes for the identification of ctxA and ctxAB genes. Following that, all 16 isolates were subjected to molecular typing using the random amplified polymorphic DNA (RAPD) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR techniques. Antimicrobial testing was also performed using the disk-diffusion method and the MBEC method with chloramphenicol antibiotic. Sixteen confirmed V. cholerae O1 were successfully fingerprinted and demonstrated to be strain discriminating. Although all isolates were sensitive to Norfloxacin, they exhibited six distinct forms of antimicrobial resistance. Finally, all isolates were eradicated at concentrations of 50 mg/mL and 100 mg/mL. Multiplex PCR was demonstrated in this work to be useful for research purposes in the field of molecular genetics including cholera outbreaks. Then, both RAPD-PCR and ERIC-PCR are suitable for profiling, and Norfloxacin can be utilised for future therapy because it shown 100 % susceptibility to all isolates in this research. Keywords: Vibrio cholerae O1, ctxAB genes, polymerase chain reaction, antibiotic, MBEC

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