Genotyping of Salmonella enterica serovar Typhimurium isolates by multilocus variable number of tandem repeat high-resolution melting analysis (MLV-HRMA).

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most important virulent foodborne pathogens in industrialized countries. The ability to type bacterial strains is essential for surveillance, investigation of outbreaks, and epidemiological studies. Multilocus variable number tandem repeat combined with high-resolution melting analysis (MLV-HRMA) is a fast, cost-efficient, and easy sample genotyping method. In this study, MLV-HRMA and multilocus variable number tandem repeat analysis (MLVA) were used to differentiate between the allelic variants in 5 tandem repeat (TR) loci in 117 Salmonella Typhimurium isolates derived from various farms, slaughterhouses, market, and humans in Thailand. Both MLV-HRMA and MLVA analyses resulted in the identification of a total of 43 different genotypes, but slight differences were observed in cluster analysis results between the 2 methods. The unweighted pair-group method with arithmetic mean-based cluster analysis showed the same core clades; some small differences in the placement of sister-clades and subgrouping were observed due to the inability to reliably type the polymorphic STTR3 locus in the MLV-HRMA. The results of this study show that the MLV-HRMA, following the selection of suitable TR loci, is a relatively reliable and rapid screening method capable of differentiating between Salmonella Typhimurium isolates on the basis of allelic diversity at TR loci. As such, MLV-HRMA can be potentially used to investigate and track sources of contamination in order to effectively control Salmonella contamination in the food supply chain.

Development of new multilocus variable number of tandem repeat analysis (MLVA) for Listeria innocua and its application in a food processing plant.

Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Microbiological evaluation of water during the 2011 flood crisis in Thailand.

In 2011, a severe flood occurred in Thailand, covering nearly half the country in water for several months. The contamination of floodwater and subsequent contamination of water for human consumption could have potentially led to a widespread health crisis. However, to date, no study has been conducted to determine the safety of the waters used for human consumption in Thailand during the severe flood. Therefore, we conducted microbiological analysis of 4 kinds of water (floodwater, river water, tap water, and filtered tap water) collected from industrial and residential areas that were damaged due to flooding. Higher net levels of bacteria were found in water with a higher turbidity. No clear trend was observed in the pH value of all 4 water samples. The level of total bacterial contamination in the water samples was estimated by real-time quantitative polymerase chain reaction (PCR). Eleven of the 12 tap water samples and all of the filtered tap water samples had a total bacterial load that exceeded the Thai water quality standards. One of the tap water samples and one of the filtered tap water samples were found to be positive for Shigella sp., although none of the floodwater samples showed detectable levels of this pathogen as determined by PCR analysis. One of the samples of floodwater was also found to be positive for Leptospira sp., but none of the tap water or filtered tap water samples were positive. Most of the tap water samples and all filtered tap water samples were found to be contaminated with Vibrio cholerae. Bacterial contamination in water samples was also analyzed by denaturing gradient gel electrophoresis (DGGE) analysis. These results revealed that several microorganisms were transferred via floodwater to different areas in the central part of Thailand and cross-contaminated between floodwater and water for human consumption.