Subtyping of Listeria monocytogenes Based on Nucleotide Polymorphism in the clpC, inlA, hlyA, and plcA Genes and Rapid Identification of L. monocytogenes Genetically Similar to Clinical Isolates

Abstract

To develop a new method for identification of Listeria monocytogenes genetically similar to clinical isolates, single-nucleotide polymorphism (SNP) typing and multi-locus sequence typing (MLST) of 126isolates of L. monocytogenes from clinical and environmental samples were performed based on sequence analysis of parts of four genes (hlyA, clpC, inlA, and plcA). Based on the sequences of the isolates in this study, SNP typing showed that hlyA, clpC, inlA, and plcA genes were categorized into 9, 14, 17, and 21 types, respectively. MLST showed that the isolates were grouped into 35 types including 12 types of clinical isolates. Out of those, four MLST types were found in food or environmental and clinical isolates. In particular, all clinical isolates with serotype 1/2a were grouped into the same hlyA SNP A5 type. A method using real-time PCR combined with Cycling Probe Technology was developed for rapid identification of SNP type of L. monocytogenes genetically similar to the clinical isolates. By using this method, the 1/2a clinical isolates showing MLST-2 were successfully identified with a specific primer set and a cycling probe designed on the basis of sequence of hlyA. Furthermore, clinical isolates of serotype 4b showing MLST-4 or -35 were successfully identified by a method using cycling probes based on sequences of clpC and inlA.Keywords: cycling probe technology, Listeria monocytogenes, real-time PCR, SNP typing *To whom correspondence should be addressed. Email: tmiyamot@agr.kyushu-u.ac.jp IntroductionListeria species are widely distributed in the environment. As they are found in soil and in mammals, they are often contaminants in various types of food, mainly meats and dairy products . Listeria monocytogenes is a significant food-borne pathogen and causes an infectious disease known as listeriosis. In the food industry, contamination of food by this bacterium may lead to serious problems since it can grow even at low temperatures and high salt concentrations during storage of readyto-eat foods such as unsterilized dairy products and raw vegetables.In Japan, although sporadic cases of listeriosis have been reported, no serious epidemic has occurred. However, L. monocytogenes is often detected in foodstuffs, which may lead to a potential outbreak of listeriosis (Okutani et al., 2004). Listeriosis may result in mortality for pregnant women, infants, immunocompromised individuals, and elderly individuals .For identification and subtyping of L. monocytogenes, several techniques, such as phenotypic typing (serological typing, phage typing, and multilocus enzyme electrophoresis) and molecular typing techniques (ribotyping, restriction enzyme analysis, PCR-based typing, and DNA sequencing) have been used in epidemiology (Gasanov et al., 2005). Furthermore, multilocus sequence typing (MLST) based on DNA sequences has extremely high and accurate discriminatory power (Gasanov et al., 2005). MLST has been applied to subtyping of L. monocytogenes (Cai et al., 2002;Salcedo et al., 2003). However, because it includes...

Review. Dairy farm management and production practices associated with the presence of Listeria monocytogenes in raw milk and beef

“…For molecular subtyping of L. monocytogenes, realtime PCR technology has been applied to single nucleotide polymorphism (SNP) typing based on MLST (Honjoh et al, 2008). However, rapid and reliable methods for SNP typing of L. monocytogenes are not available (Nightingale, 2010).…”

A New Rapid Real-Time PCR Method for Detection of Listeria monocytogenes Targeting the hlyA Gene

“…The amplification conditions were 10 sec preheating at 95℃, followed by 35 cycles of 5 sec denaturation at 95℃, 15 sec annealing at 57℃, and 10 sec elongation at 72℃. After amplification, 1 cycle of 1 min at 95℃, 30 sec at 57°C and then heating it to described (Honjoh, et al, 2008). The information of the newly isolated 66 isolates of L. monocytogenes is listed in Table 1.…”

“…Primer f2 − r2 were used to amplify 1070 − 1558 region and determine nucleotide sequence of base 1126 − 1527 of hlyA gene (Honjoh, et al, 2008). PCR was performed using thermal cyclers (model Dice; TaKaRa Bio Inc., Kyoto, Japan, or model PCR Express; Hybaid, Ashford, UK) according to the method described previously.…”

“…DNA sequencing and sequence analysis SLST analysis of the hlyA gene of L. monocytogenes was carried out as reported previously (Honjoh, et al, 2008). Primer f2 − r2 were used to amplify 1070 − 1558 region and determine nucleotide sequence of base 1126 − 1527 of hlyA gene (Honjoh, et al, 2008).…”

“…And the pathogen-specific gene, hlyA gene was chosen for specific detection primer design. We have been determined nucleotide sequences of hlyA gene and classified L. monocytogenes isolates by single-nucleotide polymorphism (SNP)-based single-locus sequence typing (SLST) and multi-locus sequence typing (MLST) (Honjoh, et al, 2008). In this study, nucleotide sequences of part of hlyA gene of newly isolated 66 isolates of L. monocytogenes were determined.…”