Development of New Multilocus Variable Number of Tandem Repeat Analysis (MLVA) for Listeria innocua and Its Application in a Food Processing Plant

Abstract

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.

A review of Listeria monocytogenes from meat and meat products: Epidemiology, virulence factors, antimicrobial resistance and diagnosis

“…Once L. monocytogenes enters the processing facilities it is unlikely to be eradicated if proper monitoring programmes are not intensive as its survival is influenced by several complex factors (Carpentier & Cerf 2011 ). The contributing factors to survival of L. monocytogenes include the ability to proliferate under harsh stress conditions such as low temperature, pH and osmotic stress (Takahashi et al 2014 ), resistance to sanitation agents and formation of biofilms (Carpentier & Cerf 2011 ). Meat-processing facilities use cold storage (4 °C) to reduce the proliferation of bacteria on meat.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…An assessment of the capacity of MLVA‐CE to identify Listeria innocua , an important hygiene indicator bacterium, revealed that the method detected 60 L. innocua strains, which were classified into 11 patterns. These results indicate that the MLVA‐PCR‐CE approach is an effective method of tracking contamination sources (Takahashi and others ).…”

Identification of Listeria species and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) Typing of Listeria innocua and Listeria monocytogenes Isolates from Cattle Farms and Beef and Beef-Based Products from Retail Outlets in Mpumalanga and North West Provinces, South Africa

“…Multilocus Variable-number Tandem Repeat Analysis (MLVA) is a Polymerase Chain Reaction (PCR) based typing method that characterizes bacteria through the detection of tandem repeats at several specific loci in the genome of bacteria [ 37 ]. This method types bacterial strains by exploiting the fact that the number of repeat units in the variable number of tandem repeats region varies from strain to strain [ 38 ]. Among other typing methods, MLVA is considered a rapid, robust, and convenient bacterial subtyping method with reliable discriminatory power for genotyping bacterial isolates [ 36 , 39 , 40 ].…”

“…Murphy et al [ 29 ] developed an MLVA protocol for typing L. monocytogenes isolates from salmon and other related food products. Takahashi et al [ 38 ] developed an MLVA protocol for typing L. innocua isolates originating from a food processing facility with the intention of identifying the contamination routes. Miya et al [ 41 ] also used this method to type L. monocytogenes serotype 4b isolates that had been previously characterized by PFGE, EcoRI ribotyping, and MLST and concluded that MLVA highly discriminated the isolates.…”