CHARACTERISATION, GENETIC TYPING AND ANTIMICROBIAL RESISTANCE OF LISTERIA MONOCYTOGENES FROM FOOD CHAIN OF KOSOVO

Abstract

Listeria monocytogenes (L. monocytogenes) is one of the main pathogens, psychrotrophic, known for its ability to not only survive but also grow at a wide range of temperatures. This pathogen is transmitted by the consumption of contaminated food, and it causes a disease called listeriosis. This study provides a genetic characterization, including phylogenetic lineages, molecular serotyping and clonal complexes of L. monocytogenes strains isolated from different food products within the food chain of the Republic of Kosovo. Given the limited data on antibiotic resistance among L. monocytogenes isolates in Kosovo, this study also aimed to assess the circulating CCs and sequence types (STs) and their antibiotic resistance profiles using a panel of 18 antibiotics. These samples were categorized as follows: 648 from ready-to-eat (RTE) food products, 281 from food products typically consumed cooked (FPCC), 60 from raw materials (RM), and 6 from environmental sources. The overall of L. monocytogenes contamination across these samples was determined to be 11.76% (117 out of 995). Specifically, contamination rates were 6.33% (41 out of 648) in RTE products, 14.95% (42 out of 281) in FPCC, 55.00% (33 out of 60) in RM, and 16.66% (1 out of 6) in environmental samples. Sequence typing and antimicrobial resistance were performed on 114 isolates. L. monocytogenes isolates were detected in the collected samples through the official EN ISO 11290-1:2017 method with two-phase enrichment. DNA was extracted from pure isolates using the specific kit for Gram-positive bacteria - Mericon DNA Bacteria Plus Kit (Qiagen). Strains were typed as follows: The concentration of DNA extracts was adjusted to between 0.1 and 1 ng/µL, then a real-time multiplex PCR analysis was performed using Taqman® PCR probes from TIB Molbio. Three different real-time PCR thermocyclers were used to perform the tests: A Mic-IV real-time PCR thermocycler from Bio Molecular Systems (Upper Coomera, Australia), a RotorGene Q real-time PCR thermocycler from QIAGEN (Hilden, Germany), and a QuantStudio 5 real-time PCR thermocycler from Thermo Fisher Scientific (Waltham, USA). The following target genes were used for molecular serotyping lmo0737, lmo1118, ORF2819, ORF2110, prs, and plca. Clonal complexes (CCs) were identified by amplifying CC-specific genomic regions according to ANSES procedures Geno-Listeria. Seven housekeeping loci (abcZ, bglA, cat, dap, dat, ldh, and lhkA) were selected for the characterization of L. monocytogenes isolates by MLST. Antibiotic susceptibility testing performed using the Sensititre GPN3F panel revealed significant variability in resistance patterns. All L. monocytogenes isolates underwent comprehensive molecular analyses, including serotyping and clonal complex (CC) identification using real-time PCR and multilocus sequence typing (MLST). The isolates were classified into four molecular serotypes: IIa (34.19%), IIb (3.48%), IIc (32.48%), and IVb (29.91%), representing two major lineages: Lineage I (33.33%) and Lineage II (66.66%). In total, 14 distinct CCs were identified among the 41 RTE isolates, with CC29 (7 isolates), CC2 (6 isolates), and CC6 (6 isolates) being the most prevalent. By contrast, CC9 was notably dominant in both FPCC (21 isolates) and RM (14 isolates). Importantly, 30 isolates were identified as belonging to CC1, CC2, CC4, or CC6, clonal complexes that are frequently linked to severe human infections. Among the 114 isolates tested, 21 distinct sequence types (STs) were identified via MLST. CC9-ST9 was the most abundant in meat products, accounting for 38.75% of isolates, while CC29-ST29 was predominant in dairy products, representing 24.0% of isolates. Resistance was observed against several antibiotics, including levofloxacin (22.8%), gentamicin and rifampin (17.5%), quinupristin/dalfopristin (14.9%), erythromycin (11.4%), penicillin (7.89%), tetracycline (1.75%), and streptomycin (0.88%). Furthermore, 27 distinct multiple antibiotic resistance (MAR) phenotypes were identified among the isolates, with resistance ranging from 3 to 12 antibiotics. Alarmingly, the antimicrobial resistance index (ARI) for certain food categories, such as meat and meat products (MMP, ARI =0.22) and fish meat products (FMP, ARI = 0.26), exceeded the permissible threshold of 0.2 defined by the Krumperman model. Notably, 34 isolates (29.8%) exhibited MAR indices above this critical threshold. The discovery of multidrug-resistant L. monocytogenes strains, including those associated with high-risk sequence types, poses a significant threat to food safety and public health. These findings highlight the urgent need for enhanced surveillance systems to monitor the occurrence and spread of antimicrobial-resistant (AMR) L. monocytogenes within the food chain. Proactive measures, including stricter regulations and routine monitoring, are essential to mitigate the risks posed by AMR pathogens and to safeguard public health in Kosovo.