Detection of cholera toxin (ctxA and ctxAB) genes in Vibrio cholerae isolated from clinical and environmental samples in Limbang Sarawak by multiplex polymerase chain reaction (PCR)

Aims@#Cholera epidemics have been occurred in Malaysia since 1991 till 2003 which can be proved from the records by the Infectious Diseases Division of the Ministry of Health. Moreover, there were also course of cholera epidemics from the year 1994 to 2003 which had been happened in Sarawak. Cholera outbreaks in Malaysia mostly caused by the El Tor O1 Vibrio cholerae serogroup. The aims of this study were to detect the presence of V. cholerae in clinical and environmental samples (n=28) from Limbang, Sarawak by collaboration with Sarawak Government Hospital and to detect the toxin genes from the isolates.@*Methodology and results@#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). Two types of PCR were used in this study which are 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. @*Conclusion, significance and impact of study@#From this study, it showed that multiplex PCR can be used for research purposes in molecular genetics field involving cholera outbreak.

A preliminary study on detection of periodontal pathogens from saliva samples of selected Sarawakian

Aims@#The oral cavity has the most complex microbiota after the stomach. A disturbed oral equilibrium can lead to the onset and development of periodontal disease. The known causative agents are the red complex bacteria (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola). This study was carried out to provide insights on the prevalence of periodontal pathogens in Sarawakian oral cavity since the data at present is still lacking.@*Methodology and results@#A total of two millilitres (2 mL) of saliva samples were collected from twenty-seven (n=27) individuals (21 gingivitis, 6 healthy) between aged 18 until 30 years old, from Sarawak General Hospital. DNA extraction for the saliva samples was done by using phenol-chloroform method. Then, 16S rRNA PCR was performed followed by species-specific PCR for red complex bacteria detection. Statistical data was analysed using GraphPad Prism 8.4.1 software. As a result, 14% of gingivitis-affected female subjects were found with all the member of red complex species. Co-occurrence of red complex species was observed but no significant difference was found. An alarming presence of red complex bacteria particularly T. forsythia was detected in 57% of gingivitis subject as compared to the other red complex species. @*Conclusion, significance and impact of study@#The risk of acquiring periodontal disease increases by having at least one of the red complex species in the oral environment. Therefore, the rapid molecular detection of red complex bacteria in this study is useful for risk assessment of periodontal disease and proper species-targeted treatment to patients especially Sarawakian in general as the result has shed lights to the fairly poor oral status of volunteers.

Effects of Sarawak local salts and commercial sodium chloride on biofilm formation of Vibrio cholerae

Aims@#Bacterial biofilms can be defined as a community of microorganisms in which cells adhere to one another on a surface and are embedded in a protective matrix of lipids, nucleic acids, proteins and polysaccharides. Biofilm produced by Vibrio cholerae represents a significant threat to food safety, as they can lead to the transmission of diseases. Hence, the purpose of this study is to review the effect of different types of sodium chloride on minimum biofilm eradication concentration (MBEC) and morphology of biofilm formation of Vibrio cholerae. @*Methodology and results@#In this study, V. cholerae biofilm was treated with four different types of sodium chloride; ‘Bario’ salt, ‘Bakelalan’ salt, commercial sodium chloride and laboratory sodium chloride. By using MBEC test, the concentration of sodium chloride needed to eradicate the biofilm of V. cholerae was determined. Based on the result obtained, commercial sodium chloride and laboratory sodium chloride showed the highest anti-biofilm activity against the biofilm of V. cholerae at 500 mg/mL concentration while no complete eradication of V. cholerae biofilm was achieved when treated with Sarawak local salts (‘Bario’ salt and ‘Bakelalan’ salt). However, noticeable inhibitions of bacterial growth were seen at the highest concentration of local salts. @*Conclusion, significance and impact of study@#Commercial sodium chloride and laboratory sodium chloride showed a better anti-biofilm activity towards the V. cholerae biofilm formation as compared to the local salts. Thus, commercial sodium chloride and laboratory sodium chloride can be an effective anti-biofilm agent to mitigate the biofilm formation of V. cholerae. Further studies can be done to determine the MBEC values of other pathogenic bacteria against commercial and laboratory sodium chloride.

The effect of mouthwash on the DNA yield and quality of oral bacteria

Aims@#The application of mouthwash is one of the oral hygiene treatments that commonly use after tooth brushing to control the bacterial colonization from overgrowth. This research is focused on investigating the effect of mouthwash on oral microbiome by analyzing the quality and yield of DNA obtained before and after using mouthwash and also to compare the bacterial abundance via 16S rRNA PCR detection.@*Methodology and results@#The DNA was extracted from the saliva samples before and after using mouthwash using Phenol-Chloroform extraction method. The DNA extract was then evaluated using Nano Drop ND-1000 UV/VIS Spectrophotometer to determine the DNA quality and DNA yield. After that, the 16S rRNA gene was amplified via PCR for bacterial detection in the saliva using 27 F and 1492 R primers set, and the PCR products were observed on 1.5% gel electrophoresis. Statistical analysis was performed by using Graphpad Prism 7.03 software. For DNA yield, there was significantly higher yield observed after mouthwash usage with 80% of the samples was found to yield more DNA. To assess DNA quality, absorbance ratio of A260/A280 and A260/A230 were used. The DNA quality was seen to be similar for both A260/A280 and A260/A230 absorbance ratio even after the usage of mouthwash. The amplification of 16S rRNA gene was successful and 1500 bp expected band size was observed.@*Conclusion, significance and impact of study@#This study demonstrated the usage of mouthwash is useful to increase the DNA yield as compared to without using mouthwash. However in terms of quality, no difference is seen. This result can be used to provide insight on mouthwash usage for saliva sampling in a non-invasive manner.