Listeria monocytogenes is a Gram-positive bacterial pathogen and the causative agent of listeriosis, a severe foodborne infection. L. monocytogenes is notorious for its ability to persist in food processing environments (FPEs) via a variety of adaptive traits. Even though traits such as cold tolerance, biofilm formation and sanitizer resistance have been extensively investigated for their roles in persistence of L. monocytogenes in FPEs, much less is known about resistance to bacteriophages. Previous studies explored phage resistance mechanisms in laboratory-created mutants but it is imperative to investigate phage resistance that is naturally exhibited in FPE-derived strains. Here, we integrated the analysis of whole genome sequence data from a panel of serotype 1/2a strains of sequence types 321 and 391 from turkey processing plants, with the determination of cell surface substituents required for phage adsorption and phage infection assays with the four wide-host-range phages A511, P100, 20422-1 and 805405-1. Using a specific set of recombinant phage protein probes, we discovered that phage-resistant strains lacked one or both of the serogroup 1/2-specific wall teichoic acid carbohydrate decorations, N-acetylglucosamine and rhamnose. Furthermore, these phage-resistant strains harbored substitutions in lmo1080, lmo1081, and lmo2550, which mediate carbohydrate decoration of the wall teichoic acids.

Listeria monocytogenes is a bacterial foodborne pathogen and the causative agent of the disease listeriosis, which though uncommon can result in severe symptoms such as meningitis, septicemia, stillbirths, and abortions and has a high case fatality rate. This pathogen can infect humans and other animals, resulting in massive health and economic impacts in the United States and globally. Listeriosis is treated with antimicrobials, typically a combination of a beta-lactam and an aminoglycoside, and L. monocytogenes has remained largely susceptible to the drugs of choice. However, there are several reports of antimicrobial resistance (AMR) in both L. monocytogenes and other Listeria species. Given the dire health outcomes associated with listeriosis, the prospect of antimicrobial-resistant L. monocytogenes is highly problematic for human and animal health. Developing effective tools for the control and elimination of L. monocytogenes, including strains with antimicrobial resistance, is of the utmost importance to prevent further dissemination of AMR in this pathogen. One tool that has shown great promise in combating antibiotic-resistant pathogens is the use of bacteriophages (phages), which are natural bacterial predators and horizontal gene transfer agents. Although native phages can be effective at killing antibiotic-resistant pathogens, limited host ranges and evolved resistance to phages can compromise their use in the efforts to mitigate the global AMR challenge. However, recent advances can allow the use of CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) to selectively target pathogens and their AMR determinants. Employment of CRISPR-Cas systems for phage amendment can overcome previous limitations in using phages as biocontrol and allow for the effective control of L. monocytogenes and its AMR determinants.

Listeria monocytogenes presents a significant concern for the food industry due to its ability to persist in the food processing environment. One of the factors contributing to its persistence is decreased sensitivity to disinfectants. Our objective was to assess the diversity of L. monocytogenes sensitivity to food industry disinfectants by testing the response of 1,671 L . monocytogenes isolates to quaternary ammonium compounds (QACs) and 414 isolates to peracetic acid (PAA) using broth microdilution and growth curve analysis assays, respectively, and to categorize the isolates into sensitive and tolerant. A high phenotype-genotype concordance (95%) regarding tolerance to QACs was obtained by screening the genomes for the presence of QAC tolerance-associated genes bcrABC , emrE , emrC, and qacH . Based on this high concordance, we assessed the QAC genes’ dissemination among publicly available L. monocytogenes genomes ( n = 39,196). Overall, QAC genes were found in 23% and 28% of the L. monocytogenes collection in this study and in the global data set, respectively. bcrABC and qacH were the most prevalent genes, with bcrABC being the most detected QAC gene in the USA, while qacH dominated in Europe. No significant differences ( P > 0.05) in the PAA tolerance were detected among isolates belonging to different lineages, serogroups, clonal complexes, or isolation sources, highlighting limited variation in the L. monocytogenes sensitivity to this disinfectant. The present work represents the largest testing of L. monocytogenes sensitivity to important food industry disinfectants at the phenotypic and genomic level, revealing diversity in the tolerance to QACs while all isolates showed similar sensitivity to PAA. IMPORTANCE: Contamination of Listeria monocytogenes within food processing environments is of great concern to the food industry due to challenges in eradicating the isolates once they become established and persistent in the environment. Genetic markers associated with increased tolerance to certain disinfectants have been identified, which alongside other biotic and abiotic factors can favor the persistence of L. monocytogenes in the food production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis, this study significantly enhances the understanding of the L. monocytogenes tolerance to quaternary ammonium compounds (QACs) and the genetic determinants associated with the increased tolerance. We provide a global overview of the QAC genes prevalence among public L. monocytogenes sequences and their distribution among clonal complexes, isolation sources, and geographical locations. Additionally, our comprehensive screening of the peracetic acid (PAA) sensitivity shows that this disinfectant can be used in the food industry as the lack of variation in sensitivity indicates reliable effect and no apparent possibility for the emergence of tolerance.

Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease.

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for the severe disease listeriosis and notorious for its ability to persist in food processing plants, leading to contamination of processed, ready-to-eat foods. L. monocytogenes persistence in various food processing environments (FPEs) has been extensively investigated by various subtyping tools, with increasing use of whole genome sequencing (WGS). However, major knowledge gaps remain. There is a need for facility-specific molecular signatures not only for adequate attribution of L. monocytogenes to a specific FPE but also for improved understanding of the ecology and evolution of L. monocytogenes in the food processing ecosystem. Furthermore, multiple strains can be recovered from a single FPE sample, but their diversity can be underestimated with common molecular subtyping tools. In this study we investigated a panel of 54 L. monocytogenes strains from four turkey processing plants in the United States. A combination of WGS and phenotypic assays was employed to assess strain persistence as well as identify facility-specific molecular signatures. Comparative analysis of allelic variation across the whole genome revealed that allelic profiles have the potential to be specific to individual processing plants. Certain allelic profiles remained associated with individual plants even when closely-related strains from other sources were included in the analysis. Furthermore, for certain sequence types (STs) based on the seven-locus multilocus sequence typing scheme, presence and location of premature stop codons in inlA, inlB length, prophage sequences, and the sequence content of a genomic hotspot could serve as plant-specific signatures. Interestingly, the analysis of different isolates from the same environmental sample revealed major differences not only in serotype and ST, but even in the sequence content of strains of the same ST. This study highlights the potential for WGS data to be deployed for identification of facility-specific signatures, thus facilitating the tracking of strain movement through the food chain. Furthermore, deployment of WGS for intra-sample strain analysis allows for a more complete environmental surveillance of L. monocytogenes in food processing facilities, reducing the risk of failing to detect strains that may be clinically relevant and potentially novel.

Listeria monocytogenes is a facultative intracellular foodborne pathogen and the causative agent of the severe disease listeriosis. It is found ubiquitously in the environment and exhibits innate resistance to certain antimicrobials, but acquired antimicrobial resistance remains relatively uncommon. Given the potentially dire health outcomes associated with listeriosis, acquisition of antimicrobial resistance (AMR) by this pathogen is of considerable public health concern. AMR in L. monocytogenes has been surveyed frequently in strains of clinical and food origin, but much less commonly in wildlife. We analyzed 158 strains of L. monocytogenes and 27 of non-pathogenic Listeria spp. isolated from wild black bears ( Ursus americanus ) for resistance to a panel of antimicrobials. AMR was uncommon and noted mostly for tetracycline. Tetracycline resistance was more common in Listeria innocua than in L. monocytogenes . All tetracycline-resistant L. monocytogenes strains belonged to sequence type ST1039 and harbored the Tn 916 -like tet (M) transposon Tn 916.1039 in a conserved chromosomal location. In contrast, three different tetracycline resistance elements, i.e., the tet (M) elements Tn 5801.UAM and Tn 5801.551 and the tet (S) element Tn 6000.205 , were identified among tetracycline-resistant strains of L. innocua . The greater prevalence and diversity of tetracycline resistance elements among bear-derived non-pathogenic Listeria strains suggest the potential of the latter to serve as reservoirs for retention and diversification of AMR determinants in this wildlife host and warrant their further monitoring and study. IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria . Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears ( Ursus americanus ) from the United States, including 158 and 27 L . monocytogenes and L. innocua , respectively. Tetracycline resistance was the most prevalent resistance trait. In L . monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn 916 -like element Tn 916.1039 . In contrast, three distinct, novel tetracycline resistance elements (Tn 5801.UAM , Tn5801.551 , and Tn 6000.205 ) were identified in L. innocua . Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria .