Clonogrouping, a Rapid Multiplex PCR Method for Identification of Major Clones of Listeria monocytogenes

Abstract

f Three multiplex PCR assays were developed to identify the 11 most common Listeria monocytogenes clones in clinical and food samples; 270 (95.7%) of 282 strains of serogroups IVb, IIb, IIa, and IIc were identified accurately. This novel tool is a rapid and efficient alternative to multilocus sequence typing for identification of L. monocytogenes clones. Human and food isolates of the food-borne pathogen Listeria monocytogenes belong predominantly to two phylogenetic lineages (1-3). Lineage I isolates belong mostly to serotypes 4b and 1/2b, whereas lineage II isolates mostly belong to serotypes 1/2a and 1/2c. A multiplex PCR assay (4) allows identification of PCR serogroups IVb (serotypes 4b and rare variants 4d and 4e), IIa (serotypes 1/2a and 3a), IIb (serotypes 1/2b, 3b, and 7), IIc (serotypes 1/2c and 3c), and L (serotypes 4a, 4ab, and 4c). Unfortunately, serogrouping has limited discriminatory power, thus providing limited resolution for epidemiological typing. Multilocus sequence typing (MLST) can discriminate isolates within the same serogroup (5). The large-scale application of MLST led to the identification of numerically predominant and internationally widespread clonal complexes (CCs), which are considered clones, i.e., sets of isolates descending from a single common ancestor (5-9). Most major clones have been involved in outbreaks of listeriosis (7,(10)(11)(12). Rapid identification of MLST clones is important for epidemiological surveillance and outbreak investigation. However, MLST is time-consuming and is too expensive for routine use in most laboratories involved in L. monocytogenes typing. The objective of this work was to enable rapid affordable identification of major clones of L. monocytogenes by multiplex PCR.Clone-specific target genes for PCR were identified based on comparative analyses of 104 genomes that were representative of the diversity of clones of L. monocytogenes (our unpublished results). We determined the pan-genome, i.e., the full complement of protein-coding gene families of the species, as described previously (13). Gene families specific for major clones, lineages, or serogroups were identified and represented putative target genes for PCR identification. Among these, target genes that were of sufficient size and were not present on mobile genetic elements were selected. Primers were then carefully designed (see Table S1 in the supplemental material), using Primer3 (14), to produce amplification products with different sizes and to amplify DNA at a unique annealing temperature. Target genes were grouped into three "clonogrouping" multiplex PCR assays, which were intended to be used downstream of the widely used PCR serogrouping assay. First, the IVb clonogrouping PCR assay was designed to identify serogroup IVb clones CC1, CC2, CC4, and CC6 or any other serogroup IVb isolate. Second, the IIb PCR assay was designed to identify serogroup IIb clones CC3 and CC5 or any other serogroup IIb isolate. Third, the IIaIIc PCR assay was designed to identify either serogroup IIa clones...

Genomic Diversity of Common Sequence Types of Listeria monocytogenes Isolated from Ready-to-Eat Products of Animal Origin in South Africa

“…The absence of LIPI3 in two samples of lineage II was expected as the sequence types have been reported to lack it LIPI [1]. The LIPI4 is a newly described Listeria pathogenicity island [56] which encodes for the genes that annotated as a cellobiose family PTS system [55], and it was not identified in any of the strains identified in this study. The LIPI4 appears to be strongly associated with ST4 isolates [1]; hence, it was not observed in the current study.…”

Prevalence and molecular characterization of Listeria spp. and Listeria monocytogenes isolated from fish, shrimp, and cooked ready-to-eat (RTE) aquatic products in Iran

“…in raw fish and shrimp samples, recently reviewed in Jami, Ghanbari, Zunabovic, Domig, and Kneifel (2014). In these reports, the rate of L. monocytogenes contamination in raw fish and shrimp samples ranged from 43.3% (Chen et al, 2010) to 0% (Kuzmanovi c et al, 2011;Minami et al, 2010;Wang, Chern, Li, Yan, & Hsieh, 2012).…”

“…Many studies have reported the incidence of L. monocytogenes in fruits and vegetables (Hadjilouka, Andritsos, Paramithiotis, Mataragas, & Drosinos, 2014;Montero et al, 2015), dairy products (Dalzini et al, 2015;Karthikeyan, Gunasekaran, & Rajendhran, 2015;Soni, Singh, Singh, & Dubey, 2013), meat (Hadjilouka et al, 2014;Kovacevic, Mesak, & Allen, 2012;Montero et al, 2015), fish (Chou & Wang, 2006;Kovacevic et al, 2012;Wang et al, 2013), seafood products (Boerlin, Boerlin-Petzold, Bannerman, Bille, & Jemmi, 1997;Li et al, 2015;Miya, Takahashi, Ishikawa, Fujii, & Kimura, 2010;Momtaz & Yadollahi, 2013), and food processing environments (Chen, Pyla, Kim, Silva, & Jung, 2010;Li et al, 2015). However, the occurrence of L. monocytogenes in seafood has received less attention in Iran and very limited information is available on the prevalence and contamination levels of Listeria spp.…”

“…However, classical serotyping by agglutination method has several disadvantages, such as the high cost of antisera and presence of nontypeable isolates. To overcome these problems, several molecular techniques have been suggested (Chenal-Francisque et al, 2015;Doumith, Buchrieser, Glaser, Jacquet, & Martin, 2004;Kerouanton et al, 2010) for the rapid identification of L. monocytogenes genoserogroups. These approaches are more sensitive, specific, and cost effective for the discrimination of L. monocytogenes genotypes, and could be used as first-level characterization of L. monocytogenes, especially in epidemiological investigations and food safety monitoring programs.…”

MALDI-TOF mass spectrometry-based identification of Listeria species in surveillance: A prospective study

“…A validation set of 386 Listeria strains representative of Listeria genus and L. monocytogenes diversity (Chenal-Francisque et al, 2015) was first analyzed (Table 1). A particular focus was given to L. monocytogenes (n=353), to fully cover the diversity of this species, which is the main common pathogenic Listeria isolated in clinical and food microbiological laboratories and responsible for human listeriosis.…”