VIRULENCE GENES OF LISTERIA MONOCYTOGENES ISOLATED FROM SOME READY-TO-EAT CHICKEN MEALS

Assiut University web-site: www.aun.edu.eg

VIRULENCE GENES OF LISTERIA MONOCYTOGENES ISOLATED FROM SOME READY-TO-EAT CHICKEN MEALS

SOHAILA, F.H. EL-HAWARY ¹, MOHAMED, H. MOHAMED ² and SAYED, H. AL-HABATY ³

^1,2 Department of Food Hygiene, Animal Health Research Institute, Assiut Provincial Lab.

³ Department of Bacteriology, Animal Health Research Institute, Assiut Provincial Lab.

Received: 30 September 2018;     Accepted: 28 October 2018

INTRODUCTION

Listeria monocytogenes has been recognized as an important opportunistic human pathogen since 1929 and as food borne pathogen since 1981 (Jeyasekaran et al., 1996). Ready-to-eat (RTE) meat products represent high risk to the consumers because they are usually cooked during manufacture and consumed without further heating, so cross contamination with food borne pathogens during the processing cannot be overcome (Goulet et al., 2008). The extended distribution throughout the food processing environment and asymptomatic human carriers (Wagner et al., 2005) and the psychrotrophic character of Listeria species appear to be the main causes of the prevalence in different kinds of refrigerated RTE meat products and contamination could occur either pre- or post-processing (Lianou and Sofos, 2007). Of the 20 RTE food categories evaluated by the Food and Drug Administration and the Food Safety and Inspection Service, deli meats were classified in the very high risk category to be the principal potential source of L. monocytogenes (FAO/WHO, 2004). In general, consumption of food contaminated with L. monocytogenes may cause listeriosis which may result in serious human illness with     symptoms      of     septicemia,      meningitis,

Corresponding author: MOHAMED, H. MOHAMED                 

E-mail address: sahlol72@yahoo.com

Present address: Department of Food Hygiene, Animal Health Research Institute, Assiut Provincial Lab.

encephalitis and gastroenteritis particularly in children, the elderly and immunosuppressed individuals. It may also cause miscarriage in pregnant women (Blum-Menezes et al., 2013).         L. monocytogenes had the second highest fatality rate (20%) and the highest hospitalization rate (90%) in virulence (Swaminathan, 2001). Multistate outbreaks of food borne listeriosis were recorded (Gottlieb et al., 2006).

In study conducted by Gusman et al. (2014), the prevalence of L. monocytogenes in examined samples of RTE foods was 1.97%, and the count of L. monocytogenes in all positive samples exceeded the limit of 100 colony forming units (CFUs) per gram. According to the data reported by the (EFSA) European Food Safety Authority (2015), prevalence rate of L. monocytogenes in RTE foods was 4.4%.

Multiple key virulence factors such as internalin (inlA), listeriolysin (hlyA), phosphatidylinositol phospholipase C (plcA), actin polymerization protein (actA) and invasive associated protein (iap) are important in L. monocytogenes pathogenesis (Furrer et al., 1991 and Portnoy et al., 1992). Therefore, detection of just one virulence associated gene by PCR is not always sufficient to identify      L. monocytogenes (Nishibori et al., 1995). In addition, it is plausible that some L. monocytogenes strain may lack one or more virulence determinants because of spontaneous mutations (Cooray et al., 1994).

L. monocytogenes is usually susceptible to a wide range of antibiotics, but in 1988 a multidrug-resistant strain was found in France (Poyart-Salmeron, 1990). Since then other strains resistant to one or more antibiotics have been recovered from food, the environment and from sporadic cases of human listeriosis (Conter et al., 2009). The occurrence of antibiotic resistance complicates therapy and lengthens convalescence from illness. Antibiotic use in clinical medicine (appropriate and otherwise) has contributed to the emergence of multidrug-resistant strains, but another contributor has been the use of antibiotics in animal feed as growth promoters (Harakeh et al., 2009). In 2013, based on the proposals issued by the European Food Safety Authority, the EU put forward and discussed with the member states a new legislation on the harmonized monitoring of antimicrobial resistance in Salmonella, Campylobacter and indicator bacteria in food-producing animals and food (EFSA, 2015), but there are relatively few epidemiological studies, and thus, only limited information on antibiotic resistance prevalence and spread concerning Listeria spp. Considering the high mortality rate of listeriosis in vulnerable populations, it is important to insure the effectiveness of antimicrobials and monitor the emergence of antimicrobial-resistant Listeria strains (Gómez et al., 2014).

The increase of RTE food consumption due to changes in the lifestyle and the ability of                 L. monocytogenes to attach to different surfaces forming biofilms and consequently its persistence in food environment necessitate periodically repeated surveys for determining the prevalence and the distribution of some virulence genes in                     L. monocytogenes isolated from RTE food and to evaluate the resistance genotype  of isolated             L. monocytogenes strains to selected antibiotics used for treating listeriosis.

MATERIALS AND METHODS

Collection of samples:

A total of 75samples of cooked RTE chicken meat (25 for each shawerma, burger and breast fillet) were collected during the period from October to December 2017 from different restaurants in Assiut province. The samples were collected hygienically in sterile plastic bags and transported to the laboratory in icebox within 2 to 4 h.

Isolation and identification of L. monocytogenes:

Listeria monocytogenes was isolated from RTE chicken meat samples following the procedure recommended by the International Organization for Standardization (ISO11290 -1, 2017). Briefly, a 25 g meat sample was aseptically homogenized in 225 ml pre-enrichment half-Fraser broth (CM0895, Oxoid Ltd) supplemented with half-Fraser supplement (SR0166E, Oxoid Ltd) in Stomacher bags (Seward Ltd, West Sussex, UK) for 30 s using a Stomacher circulator (Easy Mix, AES Laboratoire, Bruz, France), followed by incubation at 30ºC for 24 h. Then 0.1 ml half-Fraser broth was added to 10 ml Fraser broth containing Fraser supplement and incubated at 37ºC for 48 h. At the end of incubation, a loopful of Fraser broth was streaked on chromogenic Listeria agar (ALOA) supplemented with Brilliance Listeria Differential Supplement (SR0228E, Oxoid Ltd) and incubated at 35ºC for 24 to 48 h.  L. monocytogenes appear as green–blue colonies surrounded by an opaque halo. For biochemical identification of L. monocytogenes, five suspect colonies from each plate were streaked on TSA (M290, Oxoid Ltd) supplemented by (0.6%) yeast extract (LP0021) and incubated at 37ºC for 18–24 h.

Biochemical confirmation of L. monocytogenes:

Suspected colonies were verified by Gram staining, catalase, oxidase, haemolysis and CAMP tests, motility, Methyl Red-Voges Proskauer (MR-VP) reactions, nitrate reduction and the production  of acids from rhamnose, xylose and mannitol for the identification as described by ISO11290 -1 (2017).

PCR assay for identification of 16S rRNA, virulence genes and resistance genes of                 L. monocytogenes:-

The isolated L. monocytogenes strains were sent to the Reference laboratory for veterinary Quality Control of poultry production in Animal Health Research Institute, Dokki, Giza, Egypt, for identification of 16S rRNA, virulence genes and resistance genes of L. monocytogenes as follow:

DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 20 µl of proteinase K and 200 µl of lysis buffer at 56°C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer:

Primers used were supplied from Metabion (Germany) are listed in table (1).

PCR amplification:

Primers were utilized in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler.

Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the products was loaded in each gel slot. Gelpilot100 bp and 100 bp plus Ladders (Qiagen, Germany, GmbH) and generuler 100 bp ladder (Fermentas, Germany) were used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table 1: Primers sequences, target genes, amplicon sizes and cycling conditions used in PCR assayes for L. monocytogenes.

RESULTS

Table 2: Isolation rate of Listeria monocytogenes from some ready-to-eat chicken samples.

Table 3: PCR results of the specific gene and different virulence genes of isolated Listeria monocytogenes

inlA gene (internalin A gene)

inlB gene (internalin B gene)

hly gene (listeriolysin O gene)

iap gene(invasion- associated protein)

plcA gene (Phospholipase gene)

prfA gene (Pleiotropic regulatory factor)

Table 4: PCR results of resistance genes of isolated Listeria monocytogenes

mefA gene ( macrolides resistance gene)

Kan gene (Kanamycin resistance gene)

Aad6 gene (aminoglycosides resistance gene)

tetM gene (tetracycline resistance gene)

Cat gene (chloramphenicol resistance gene)

Figure 1: Agarose gel electrophoresis of PCR of 16S rRNAgene (1200bp) in isolated Listeria monocytogenes.

Lane L    : 100 -1500bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes for 16S rRNAgene.

Lane Neg.: Control negative Listeria monocytogenes for 16S rRNAgene.

Lanes: 1—4 are positive Listeria monocytogenes for 16S rRNAgene.

Figure 2: Agarose gel electrophoresis of PCR of inlB gene (343bp) and inlA gene (800bp) in isolated Listeria monocytogenes.

Lane L    : 100 -1000bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes forinlB gene and inlA gene.

Lane Neg.: Control negative Listeria monocytogenes for inlB gene and inlA gene.

Lanes: 1—4 are positive Listeria monocytogenes for inlB gene and inlA gene.

Figure 3: Agarose gel electrophoresis of PCR of hly gene (174bp) and iap gene (131bp) in isolated Listeria monocytogenes.

Lane L    : 100 -600bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes forhly gene and iap gene.

Lane Neg.: Control negative Listeria monocytogenes for hly gene and iap gene

Lanes: 1—4 are negative Listeria monocytogenes for hly gene and iap gene.

Figure 4: Agarose gel electrophoresis of PCR of plcA gene (1484bp) and prfA gene (1052bp) in isolated Listeria monocytogenes.

Lane L    : 100 -1500bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes forplcA gene andprfA gene.

Lane Neg.: Control negative Listeria monocytogenes for plcA gene andprfA gene.

Lanes: 1—4 are negative Listeria monocytogenes for plcA gene andprfA gene.

Figure 5: Agarose gel electrophoresis of PCR of mefA gene (345bp) in isolated Listeria monocytogenes.

Lane L    : 100 -600bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes for mefA gene.

Lane Neg.: Control negative Listeria monocytogenes for mefA gene.

Lanes: 1—4 are positive Listeria monocytogenes for mefA gene.

Figure 6: Agarose gel electrophoresis of PCR of Kan gene (621bp) and Aad6gene (978bp) in isolated Listeria monocytogenes.

Lane L    : 100 -1000bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes forKan gene andAad6 gene.

Lane Neg.: Control negative Listeria monocytogenes for Kan gene andAad6 gene.

Lanes: Left 1—4 are negative Listeria monocytogenes for Kan gene & Right 1-4 are positive Listeria monocytogenes for Aad6 gene.

Figure 7: Agarose gel electrophoresis of PCR of tetM gene (405bp) and Cat gene (384bp) in isolated Listeria monocytogenes.

Lane L    : 100 -600bp ladder as molecular size DNA marker.

Lane Pos: Control positive Listeria monocytogenes fortetMgene andCat gene.

Lane Neg.: Control negative Listeria monocytogenes for tetMgene and<e