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Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes

Department of Basic Sciences¹ and Department of Pathobiology and Population Medicine², College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762-6100, USA

Correspondence Mark L. Lawrence lawrence{at}cvm.msstate.edu

Received June 20, 2003
Accepted September 8, 2003

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

	Introduction

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Listeria monocytogenes is an important food-borne pathogen that can cause septicaemia, encephalitis, meningitis and gastroenteritis, particularly in children, the elderly and immunosuppressed individuals; it also causes miscarriage in pregnant women (Robinson et al., 2000). Despite being pathogenic at the species level, L. monocytogenes in fact comprises a diversity of strains with varying virulence. Whilst many L. monocytogenes strains have pathogenic potential and can result in disease and/or mortality, others have limited capability to establish infection and are relatively avirulent (Barbour et al., 1996, 2001; Erdenlig et al., 2000; Olier et al., 2002; Roche et al., 2003). The ability to distinguish virulent from avirulent strains of L. monocytogenes could potentially reduce unnecessary recalls of food products and help to prevent disease outbreaks.

Methods that have been developed for L. monocytogenes virulence assessment include mouse virulence assays and in vitro culture techniques. The mouse virulence assay provides an in vivo measurement of virulence and often serves as a reference standard for other methods (Pine et al., 1991; Nishibori et al., 1995; Erdenlig et al., 2000; Roche et al., 2001). In vitro culture techniques measure the ability of L. monocytogenes to cause cytopathogenic effects in the enterocyte-like cell line Caco-2 (Pine et al., 1991), to form plaques in the human adenocarcinoma cell line HT-29 (Roche et al., 2001) or to cause death in chicken embryos (Olier et al., 2002). Although they are somewhat slow and technically demanding, these methods have good predictive value.

Unfortunately, no reliable molecular method for prediction of L. monocytogenes virulence has been developed. PCR detection of known L. monocytogenes virulence genes, such as inlA, inlB, actA, hlyA, plcA and plcB, has not proven suitable for differentiation of virulent and avirulent isolates (Nishibori et al., 1995), as these genes are consistently found in L. monocytogenes (Jaradat et al., 2002). Genetic lineage analysis has some predictive value for L. monocytogenes pathogenicity; however, the correlation between genetic lineages and pathogenicity is not consistent for all strains (Piffaretti et al., 1989; Rasmussen et al., 1995; Wiedmann et al., 1997).

In the current study, we tested PCR primers that were derived from a panel of eight L. monocytogenes genes on 12 strains with known virulence and on a larger panel that included clinical, food and environmental isolates. We report evidence that virulent L. monocytogenes isolates contain genes that could potentially be used to differentiate virulent and avirulent isolates.

	Methods

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Bacterial strains and genomic DNA extraction.

In total, 52 Listeria strains and 15 other common Gram-positive and -negative species were examined (Table 1). Listeria strains were cultivated on tryptic soy agar plates with 5 % sheep blood or brain heart infusion (BHI) agar plates. Batch cultures were cultivated in BHI broth at 37 °C with rotary aeration. Genomic DNA was isolated from L. monocytogenes and other species by using a previously described technique (Liu et al., 2003).

Dot-blot hybridization.

A recombinant genomic DNA library of L. monocytogenes strain EGD was constructed in pGEM-3Zf(+) (Promega) as described previously (Liu & Yong, 1993; Liu, 1994). Cloned inserts were retrieved from the plasmid vector by using appropriate restriction enzymes (e.g. EcoRI and PstI or SmaI and HindIII) for use as probes. Each insert was screened against a panel of six L. monocytogenes strains, which consisted of three virulent strains (ATCC 19115, EGD and HCC7) and three avirulent strains (ATCC 15313^T, HCC23 and HCC25) by dot-blot hybridization (Liu, 1994; Liu & Yong, 1993). Approximately 300 clones were screened and clones that showed preferential binding to L. monocytogenes virulent strains were selected. Plasmid DNA from the selected clones was purified by using a QIAprep Spin Miniprep kit (Qiagen); DNA sequences of the inserts were determined by using a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems).

Mouse virulence assay.

The virulence of 12 L. monocytogenes strains was assessed by using a mouse virulence assay (Erdenlig et al., 2000). Female, 6–8-week-old A/J mice (Jackson Laboratory) were housed (five mice per cage) and allowed to acclimatize for 1 week. For each L. monocytogenes strain, four groups of mice were inoculated intraperitoneally with 0.1 ml aliquots of appropriate dilutions of bacteria. One group of five mice was injected with 0.1 ml sterile saline and one group of three mice was not injected. On the fifteenth day after inoculation, all surviving mice were euthanized and one mouse per group was necropsied and cultured from the spleen for L. monocytogenes. The LD₅₀ for each strain was then calculated (Reed & Muench, 1938).

PCR.

Each reaction was performed in a 25 µl volume of reaction mixture that consisted of 0.5 U Taq DNA polymerase (Fisher Scientific), 50 µM dNTPs, 25 pmol each primer and 10 ng DNA. Cycling conditions consisted of 94 °C for 2 min, 25 cycles of 94 °C for 20 s, 60 °C for 20 s and 72 °C for 45 s, and finally 72 °C for 2 min.

	Results and Discussion

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
Identification of genes present in virulent L. monocytogenes

As a result of comparative screening against a panel of six L. monocytogenes strains of known virulence, two clones (lmo2–28 and lmo2–432) that hybridized predominantly with virulent isolates were selected from the recombinant DNA library of L. monocytogenes EGD (data not shown).

Clone lmo2–28 contained an insert of approximately 600 bp. DNA sequencing revealed that the first 458 bp corresponded to the genome sequence of L. monocytogenes EGD-e (serovar 1/2a) (GenBank accession no. AL591976) at positions 224 432–224 889. Therefore, the clone contained portions of two genes from the L. monocytogenes EGD-e chromosome: one encodes a protein similar to transcriptional regulators (lmo0833; bp 223 784–224 730) and the other encodes a protein of unknown function (lmo0834; bp 224 810–225 537) (Glaser et al., 2001) (Table 2). Neither of these genes has an orthologue in the genome of Listeria innocua strain CLIP 11262 (serovar 6a) (Glaser et al., 2001). PCR primers were designed to both genes.

The insert from clone lmo2–432 was approximately 900 bp in length. The first 420 bp of this insert was sequenced; it correlated to the genome sequence of L. monocytogenes EGD-e (GenBank accession no. AL591978) at positions 54 835–55 247. Therefore, this clone contained portions of a gene that encodes an unknown protein (lmo1188; bp 53 636–55 087) and a gene that encodes a putative transcriptional regulator (lmo1189; bp 55 085–55 679). lmo1188 has no orthologue in the L. innocua CLIP 11262 genome; on the other hand, lmo1189 does possess such an orthologue (Glaser et al., 2001). Therefore, primers were designed to lmo1188.

These findings suggest that transcriptional regulator genes might serve as useful virulence markers for L. monocytogenes. Therefore, nucleotide–nucleotide BLAST searches were conducted to compare the gene sequences of L. monocytogenes transcriptional regulators against the genome sequence of L. innocua (Glaser et al., 2001); three genes (lmo2672, lmo1116 and lmo1134), which had the lowest nucleotide identity with any sequences from the L. innocua genome, were selected from the published L. monocytogenes EGD-e transcriptional regulator list. In addition, as internalins play important roles in listerial internalization and virulence (Gaillard et al., 1991; Lingnau et al., 1995; Parida et al., 1998), we selected two genes (lmo2470 and lmo2821) that encode internalin-like proteins from the L. monocytogenes EGD-e genome sequence, which do not have orthologues in the L. innocua genome (Glaser et al., 2001).

Mouse virulence assay

Previous mouse virulence testing had shown that strains EGD, ATCC 19115 and HCC7 are virulent, but HCC23 and ATCC 15313^T are avirulent (Erdenlig et al., 2000). In the current study, three of these strains were tested as controls (EGD, ATCC 19115 and ATCC 15313^T) and nine additional strains were assessed (Table 3). Strains EGD and 874 were the most virulent of the strains tested (LD₅₀ < 10⁸); ATCC 19115, ATCC 19116, ATCC 19117, HCC8 and 1002 were the next most virulent strains (LD₅₀ < 10⁹). Strains ATCC 19112 and ATCC 19118 had intermediate virulence (LD₅₀ < 10¹⁰) and strains ATCC 19114, HCC25 and ATCC 15313^T were considered to be avirulent (LD₅₀ > 10¹⁰).

Spleens from mice that died during the challenge contained viable L. monocytogenes that was recovered on BHI agar and confirmed by PCR. On the other hand, spleens from mice that survived the L. monocytogenes challenge by day 15 had no viable L. monocytogenes detectable on BHI agar (data not shown).

PCR

Results of PCR with oligonucleotide primers that were designed from the assembled panel of eight potential virulence genes (Table 2) correlated well with the mouse virulence results: avirulent strains ATCC 19114 and HCC25 (LD₅₀ > 10¹⁰) were non-reactive by PCR, whereas the other strains (LD₅₀ < 10¹⁰) were all PCR-positive for at least two of the primer sets tested (Table 4). In particular, primers derived from lmo2821 permitted identification of all virulent L. monocytogenes strains in our panel. PCR results also correlated with two previously tested strains (Erdenlig et al., 2000): HCC7, which is virulent by mouse assay, was PCR-positive for all primer sets tested, and HCC23, which is avirulent by mouse assay, was PCR-negative for all primer sets tested.

One notable exception was ATCC 15313^T, which was positive for seven of the eight genes tested by PCR, but was avirulent by mouse virulence assay. Strain ATCC 15313^T was originally isolated from an infected rabbit during an outbreak of listeriosis, but it later became avirulent after successive laboratory subculturing (Kathariou & Pine, 1991). It has been demonstrated that ATCC 15313^T does not express listeriolysin, a well-known virulence factor (Gaillard et al., 1986), and it has been proposed that this occurred spontaneously after its original isolation (Kathariou & Pine, 1991). Therefore, it would be logical that ATCC 15313^T, which is derived from a virulent L. monocytogenes isolate, would retain many of its virulence properties. In support of this, ATCC 15313^T has maintained its ability to cause cytopathogenic effects in Caco-2 monolayers (Kathariou & Pine, 1991).

The eight primer sets were then evaluated by PCR against a larger collection of bacterial strains (52 Listeria strains and 15 common Gram-positive and -negative species; Table 4). At least two PCR primer sets were positive for 19 of the 29 L. monocytogenes strains tested. The remaining 10 L. monocytogenes strains were not detected by any of the primers tested. The 19 isolates that were recognized were primarily clinical or food isolates, whereas the negative strains were primarily catfish isolates. The catfish isolates were obtained from healthy channel catfish during routine screening for potential food-borne pathogens. Therefore, they are considered to be environmental isolates that could be isolated from normal catfish as they enter the processing plant. The one PCR-negative strain that was not isolated from catfish was ATCC 19114, which is a human isolate. However, mouse assay confirmed that this strain is avirulent. All eight primer sets generated no PCR products with genomic DNA from other Listeria species or the other 15 species tested.

Putative internalin genes were detected in most L. monocytogenes strains in our panel, with lmo2821 being detected in 19 of 29 strains tested and lmo2470 being present in 18 of 29 strains. In particular, strain 874 was highly virulent in mice, yet it was PCR-positive for the internalin genes and negative for all transcriptional regulator genes in our panel. The putative transcriptional regulator genes lmo2672 and lmo1134 were detected in 17 of 29 strains. Genes lmo1116, lmo0834, lmo0833 and lmo1188 were detected in 15, 15, 14 and 13 of 29 strains, respectively.

In summary, our results provide evidence that virulent L. monocytogenes isolates contain genes that are not present in avirulent L. monocytogenes, and that detection of these genes has the potential to provide an alternative method for distinguishing virulent from avirulent isolates. Further testing on a larger panel of L. monocytogenes strains with known virulence will determine whether this technique has the potential to serve as a diagnostic assay.

	ACKNOWLEDGEMENTS

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This research was supported by Agricultural Research Service/US Department of Agriculture Agreement no. 58-6202-5-083. We thank Dr Catherine Donnelly of the Department of Nutrition and Food Sciences, University of Connecticut, and Dr Robert Mandrell of USDA-ARS, for providing Listeria isolates for this study. We also thank Drs Robert Read, Daniel Scruggs and Shane Burgess for their critical review of the manuscript. This paper is MAFES publication J10364.

	References

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