Whole-Genome Sequencing-Based Characterization of 100 Listeria monocytogenes Isolates Collected from Food Processing Environments over a Four-Year Period

Abstract

Listeria monocytogenes is frequently found in foods and processing facilities, where it can persist, creating concerns for the food industry. Its ability to survive under a wide range of environmental conditions enhances the potential for cross-contamination of the final food products, leading to possible outbreaks of listeriosis. In this study, whole-genome sequencing (WGS) was applied as a tool to characterize and track 100 L. monocytogenes isolates collected from three food processing environments. These WGS data from environmental and food isolates were analyzed to (i) assess the genomic diversity of L. monocytogenes, (ii) identify possible source(s) of contamination, cross-contamination routes, and persistence, (iii) detect absence/presence of antimicrobial resistance-encoding genes, (iv) assess virulence genotypes, and (v) explore in vivo pathogenicity of selected L. monocytogenes isolates carrying different virulence genotypes. The predominant L. monocytogenes sublineages (SLs) identified were SL101 (21%), SL9 (17%), SL121 (12%), and SL5 (12%). Benzalkonium chloride (BC) tolerance-encoding genes were found in 62% of these isolates, a value that increased to 73% among putative persistent subgroups. The most prevalent gene was emrC followed by bcrABC, qacH-Tn6188, and qacC. The L. monocytogenes major virulence factor inlA was truncated in 31% of the isolates, and only one environmental isolate (L. monocytogenes CFS086) harbored all major virulence factors, including Listeria pathogenicity island 4 (LIPI-4), which has been shown to confer hypervirulence. A zebrafish embryo infection model showed a low (3%) embryo survival rate for all putatively hypervirulent L. monocytogenes isolates assayed. Higher embryo survival rates were observed following infection with unknown virulence potential (20%) and putatively hypovirulent (53 to 83%) L. monocytogenes isolates showing predicted pathogenic phenotypes inferred from virulence genotypes. IMPORTANCE This study extends current understanding of the genetic diversity among L. monocytogenes from various food products and food processing environments. Application of WGS-based strategies facilitated tracking of this pathogen of importance to human health along the production chain while providing insights into the pathogenic potential for some of the L. monocytogenes isolates recovered. These analyses enabled the grouping of selected isolates into three putative virulence categories according to their genotypes along with informing selection for phenotypic assessment of their pathogenicity using the zebrafish embryo infection model. It has also facilitated the identification of those isolates with genes conferring tolerance to commercially used biocides. Findings from this study highlight the potential for the application of WGS as a proactive tool to support food safety controls as applied to L. monocytogenes.

Does Shiga Toxin-Producing Escherichia coli and Listeria monocytogenes Contribute Significantly to the Burden of Antimicrobial Resistance in Uruguay?

“…Listeria monocytogenes is intrinsically resistant to fosfomycin due to the lack of expression of transport systems through the membrane. Also, the presence of fosX gene could explain another resistance mechanism in L. monocytogenes, since it was globally present in all strains analyzed here as well as in all the 100 studied by Hurley et al (36). The FosX protein catalyzes the hydration of fosfomycin breaking the oxirane ring (37).…”

<p>Genomic and Phenotypic Diversity of <em>Listeria monocytogenes</em> Causing Pregnancy-Associated Listeriosis from Zhejiang Province, China, 2016–2018</p>

“…Detection of genetic-related clone with no evident epidemiological association highlighted that the application of a WGS-based analysis as a powerful surveillance tool. 3 It also demonstrated that some L. monocytogenes isolates, such as Lmo5272 and S12003 ( Figure 1) probably exist in this area for a long period, suggesting a more active surveillance system is warranted.…”

“…Genome assembly was performed as described previously. 3 Multilocus sequencing typing (MLST) and antimicrobial resistance genes analysis were performed using online tools (http:// www.genomicepidemiology.org/).…”

“…1 It is well documented that epidemic and sporadic L. monocytogenes infections are usually associated with contaminated food. [2][3][4][5][6][7] Human invasive infection by L. monocytogenes leads to relatively rare but serious food-borne diseases mainly affecting elderly people, immunocompromised individuals and pregnant women. 8 Clinical manifestations include sepsis and infection of the central nervous system, which can lead to lifelong sequelae or even high mortality.…”

Ecogenetics of antibiotic resistance in Listeria monocytogenes

“…Disinfectants contribute to the persistence of L. monocytogenes in food processing and other environments with frequent use of biocides. From 10% to 80% of L. monocytogenes strains isolated from food and food processing environments are tolerant to biocides (Hurley et al, 2019). Resistance to benzalkonium chloride (BAC) might be due to the overexpression of the chromosomal efflux pump MdrL, which is also able to extrude some antibiotics (such as macrolides or third-generation cephalosporins), ethidium bromide, and heavy metals (Jiang et al, 2019;Mata, Baquero, & Pérez-Diaz, 2000;Romanova, Wolffs, Brovko, & Griffiths, 2006).…”