Listeria monocytogenes in Ruminants at an Abattoir: Prevalence, Virulence Characteristics, Serotypes and Antibiotic Resistance in Eastern Türkiye

Abstract

Listeria monocytogenes is a pathogenic bacteria that causes listeriosis in both animals and humans. The aim of this study was to determine at the abattoir the prevalence of L. monocytogenes in healthy cattle, sheep and goats. The serotype was also examined along with the antibiotic resistance and clonal relationships with virulence genes. The samples were examined for the isolation of L. monocytogenes according to Food and Drug Administration/Bacteriological Analytical Manual and International Organization for Standardization methods. The isolated species were identified via matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry-MALDI-TOF MS and confirmed the isolates found using polymerase chain reaction, (PCR) after which they were serotyped with PCR and tested for antibiotic resistance against 16 antimicrobial agents using the disc diffusion method. The genetic similarity analysis (pulsed-field gel electrophoresis (PFGE) was performed. One abattoir located in Van state, Türkiye was selected and 600 samples were obtained by surface swabbing of 20 cattle, 80 sheep and 50 goat carcasses at two steps of slaughtering. Comparisons using a chi-square test of the prevalence of L. monocytogenes isolates from two visits, the four carcass surface parts, three animal species and two slaughter processes were made. Samples were evaluated for the presence of L. monocytogenes and 21 of the 150 carcasses (14%) were contaminated with L. monocytogenes. The highest prevalence of L. monocytogenes was found in cattle carcass (25%), followed by goat carcass (18%) and sheep carcass  (8.75%). The most prevalent virulence factor genes prs, iap, hlyA, inlA and plcA were and detected in all isolates. The inlB identified in 15 (71.4%) isolates, followed by prfA 1 (4.7%). All of strains were encoded 1/4b (4d,4e) serotype gene. Resistance was observed most frequently to clindamycin (19.04%) and less frequently to erythromycin (9.5%), ampicillin (9.5%), penicillin G (9.5%), and trimethoprim-sulfamethoxazole (9.5%). Pulsed-field gel electrophoresis typing was used to identify a total of 21 L. monocytogenes isolates. Thirteen pulsotypes were identified from the isolates. L. monocytogenes was detected in the carcass of twenty-one animals, during slaughtering before washing (fifteen carcass), and after end washing (six carcasses). There were no significant differences in L. monocytogenes prevalence among three animal types, four carcass sites, two visits and two slaughter processes. The frequency of isolates are low, but the data suggest the potential public health risk that these carcasses represent as reservoirs of L. monocytogenes and cross-contamination occurs at different stages (animals, parts of carcass, stages of slaughtering). Therefore, for hygienic meat production, HACCP systems could be implimented effectively.

Keywords: Antimicrobial Resistance; Listeria monocytogenes; Serotype; PFGE; Virulence.

Introduction

Foodborne pathogens continue to pose significant threats to food safety and international trade. Worldwide, foodborne infections present a major public health concern (1). One thousand eight hundred seventy-six cases of listeriosis were recorded worldwide in 2020; this translates to an infection rate of 0.42 per 100,000 people in the European Union
(2). After consuming pathogen-contaminated foods, severe health implications for listeriosis, ranging from an invasive to a non-invasive sickness, develop. People with cancer, diabetes, or acquired immunodeficiency syndrome (AIDS) are more likely to get invasive listeriosis. Newborns, the elderly and pregnant women are also more likely to get this type of listeriosis. In instances of invasive listeriosis, severe symptoms (gastroenteritis, meningitis, pneumonia, septicemia, and stillbirth/abortion) develop. In healthy individuals, noninvasive listeriosis and less severe symptoms (fever, diarrhea, headache, and muscular discomfort) are often noticed (3, 4).
An intracellular pathogen, L. monocytogenes has the ability to infect numerous animal species, including ruminants (cattle, sheep, and goats). It has been isolated mainly from the outer surfaces of cattle, sheep, goats, and poultry, although various reports have also detected its presence in muscle tissue, although at low proportions (5, 6). L.  monocytogenes possesses an array of virulence factors contributing to its survival, host  invasion, immune system evasion, and infectivity. Listeriolysin O (LLO, encoded by hlyA),
a virulence regulator (encoded by prfA), actin (encoded by actA), invasion-associated protein (encoded by iap), phosphatidylinositolphospholipase C (PI-PLC, encoded by plcA, plcB, and plcC), and a metalloprotease (encoded by mpl) are all components of Listeria pathogenic 1 (LIPI-1), while LIPI-2 contains the internalins (encoded by inlA, inlC, and inlJ) (7, 8).
For L. monocytogenes subtyping, antibodies that bind to the somatic (O)  lipopolysaccharides and flagellin antigens on the bacterial outer membrane are used to  identify the first level of subtyping. Thirteen serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7) have been found in L. monocytogenes that are categorized into four distinct lineages (I–IV) (9, 10). Lineage I and II (1/2a, 1/2b, and 4b) serotypes account for the vast majority of worldwide listeriosis cases. Most listeriosis epidemics are caused by contaminants 1/2a, 1/2b, 1/2c, and 4b in food or the food processing environment. When it comes to human listeriosis, genetic isolates from the lineage I strains are more frequently found, while those from the animal lineage III strains are more frequently found in animal specimens (11).
Zoonoses present the worrisome problem of transfer of antimicrobial-resistant organisms from animals to humans in an interconnected globalized world (12). L. monocytogenes has three mobile genetic elements for the emergence of antibiotic resistance: self-transferable plasmids, plasmids that can be mobilized and  conjugative transposons that can be transferred. Efflux pumps have been reported to contribute to antimicrobial resistance in Listeria (13). The prevalence of antibiotic-resistant strains of L. monocytogenes has increased in recent years, particularly in the case of  isolates collected from the food supply (14). Multidrug-resistant L. monocytogenes, notably in ready-to-eat foods, serves as a public health indicator, especially among those at high risk. In order to avoid an increase in medication resistance, it is critical to raise public awareness about the significance of food safety rules and the drugs administered to  humans and animals (15). Food animal and product contamination is a major concern since it is difficult to regulate and involves a wide range of elements. Animal handling activities, such as slaughtering and processing, as well as environmental elements such as fauna and water sources and animal dung disposal are examples of human factors (16).
There is a little data available with respect to the prevalence of L. monocytogenes in  Türkiye. Since food-borne outbreaks of listeriosis have not reached epidemic levels in Türkiye yet, the disease hasn't been regularly recorded there (17). The aim of this study was to identify L. monocytogenes from randomly selected carcass samples of healthy  ruminant animals (cow, sheep, goat) using matrix assisted laser desorption/ionization time-of-flight mass spectrometry-MALDITOF MS, to investigate the antibiotic resistance  phenotype, virulence genes, serotypes of the isolates and to detect their genetic similarities using pulsed field gel electrophoresis (PFGE).