Molecular Detection and Virulence Gene Profiling of Listeria monocytogenes in Dairy Processing Environments

Introduction

The persistence of Listeria monocytogenes in food processing environments represents one of the most formidable challenges confronting the global dairy industry, with contamination events capable of triggering large-scale product recalls, significant economic losses, and devastating public health consequences [1] . This Gram-positive, psychrotrophic bacterium possesses an extraordinary repertoire of adaptive mechanisms enabling survival under the harsh physicochemical conditions encountered during food processing, including low temperatures, acidic pH, elevated osmolarity, and oxidative stress imposed by sanitation chemicals [2,3] . The virulence potential of L. monocytogenes is mediated through a complex array of pathogenicity determinants organized primarily within a 10-kb virulence gene cluster regulated by the master transcriptional activator PrfA [4] . Key virulence factors include listeriolysin O (encoded by hlyA) facilitating phagosomal escape, internalins A and B (inlA, inlB) mediating host cell invasion, ActA promoting actin-based intracellular motility, and phospholipases (plcA, plcB) enabling cell-to-cell spread [5] . The differential distribution and expression levels of these virulence determinants across L. monocytogenes lineages and serotypes significantly influence pathogenic potential and clinical outcome severity [6] . South Korea's dairy industry has experienced substantial growth over the past two decades, with annual milk production exceeding 2.1 million tonnes and a rapidly expanding processed dairy product sector driven by changing consumer preferences [7] . However, several high-profile listeriosis outbreaks linked to contaminated dairy products in East Asian countries have heightened public awareness and regulatory scrutiny regarding L. monocytogenes control in dairy manufacturing environments [3] . The Korean Food and Drug Safety Administration has implemented zero-tolerance policies for L. monocytogenes in ready-to-eat dairy products, yet comprehensive molecular epidemiological data characterizing the contamination dynamics and virulence profiles of L. monocytogenes in Korean dairy processing facilities remain limited [8,9] . Environmental monitoring programs employing conventional culture-based methods alone provide insufficient resolution for understanding the persistence patterns, transmission routes, and virulence potential of L. monocytogenes populations colonizing dairy processing environments [10] . Integration of molecular typing techniques including pulsedfield gel electrophoresis (PFGE), multilocus sequence typing (MLST), and virulence gene profiling enables the identification of persistent contamination clones, tracking of transmission pathways, and assessment of public health risk associated with specific strains [11,12] . This research was designed to conduct a comprehensive molecular characterization of L. monocytogenes isolated from dairy processing environments in Busan metropolitan area, South Korea. The specific objectives encompassed determining the detection frequency across sequential processing stages, characterizing the virulence gene profiles of recovered isolates, establishing serotype distribution patterns, identifying persistent contamination clones through molecular typing, and comparing virulence phenotypes across predominant serotype groups. The outcomes of this investigation are intended to inform evidence-based environmental monitoring strategies and contamination control measures for the South Korean dairy processing sector.

Material and methods

Material

Material The research materials comprised 672 environmental and product samples collected systematically from eight licensed dairy processing plants located within Busan metropolitan area, South Korea. Sample categories included surface swabs from processing equipment and environmental surfaces (n=288), product contact surface swabs (n=192), and dairy product samples at various processing stages (n=192). Product samples encompassed raw milk (n=48), soft cheese (n=36), yogurt (n=32), cream products (n=28), butter (n=24), and whey (n=24). Environmental surface swabs were collected using 3M Environmental Sponge Samplers (3M Company, Saint Paul, MN). Culture media including Listeria Enrichment Broth (LEB), PALCAM agar, Oxford agar, and chromogenic agar (ALOA) were procured from Oxoid Limited (Hampshire, United Kingdom). Molecular biology reagents including primers for multiplex PCR virulence gene detection and PFGE reagents were obtained from Bioneer Corporation (Daejeon, South Korea). Reference strains L. monocytogenes ATCC 19115 (serotype 4b), ATCC 19117 (serotype 4d), and ATCC 19111 (serotype 1/2a) were obtained from the Korean Collection for Type Cultures for quality assurance purposes.

Methods

This cross-sectional environmental monitoring investigation was conducted from April 2022 to March 2023 at the Molecular Food Safety Laboratory, Busan Research Institute of Food Safety, Busan, South Korea. Institutional approval was obtained from the Research Ethics Committee of the Busan Research Institute of Food Safety (Protocol No. BRIFS-EC-2022-014). All participating dairy processing facilities provided written consent for environmental monitoring activities. The investigation followed ISO 11290-1:2017 and ISO 11290-2:2017 protocols for detection and enumeration of L. monocytogenes. Each facility was sampled monthly over the 12-month investigation period, with 7 samples collected per visit across six processing stages: receiving docks, cold storage areas, processing lines (pasteurization, homogenization, and separation equipment), packaging zones, final products prior to dispatch, and retail display shelves at associated outlets. Environmental surface samples (100 cm² per site) were collected using pre-moistened sponge samplers following USDA-FSIS guidelines. Product samples (25 g or 25 mL) were collected aseptically in sterile containers. Isolation followed a two-stage enrichment protocol: primary enrichment in half-Fraser broth (30°C, 24 hours) followed by secondary enrichment in Full-Fraser broth (37°C, 48 hours). Enriched cultures were streaked onto PALCAM and ALOA agar plates. Presumptive colonies were confirmed through standard biochemical tests and species-specific PCR targeting the hlyA gene. Confirmed isolates were subjected to multiplex PCR targeting eight virulence genes (hlyA, inlA, inlB, actA, plcA, plcB, prfA, and iap) using previously validated primer sets. Serotyping was performed using multiplex PCR and confirmed by slide agglutination using commercial Listeria antisera (Denka Seiken Co., Tokyo). PFGE was conducted following the PulseNet standardized protocol using AscI and ApaI restriction enzymes. Virulence phenotype characterization included quantitative hemolysis assays using sheep red blood cells, Caco-2 cell invasion assays, and biofilm formation capacity assessment using crystal violet staining in 96-well microplates. Statistical comparisons across serotype groups were performed using one-way ANOVA with Tukey's post-hoc test, at a significance level of p<0.05. Of the 672 samples analyzed, L. monocytogenes was isolated from 99 samples, yielding an overall detection rate of 14.7% (95% CI: 12.1-17.7%). The detection frequency varied considerably across processing stages, with the highest contamination rates observed on processing lines (35.7%), receiving docks (28.4%), and storage areas (22.6%), while packaging zones (16.1%), retail shelves (10.4%), and final products (6.3%) demonstrated progressively lower contamination. This gradient pattern was statistically significant (χ² for trend = 32.47, p<0.001), indicating substantial microbial reduction through sequential processing steps while highlighting the processing line as the critical contamination amplification point. Among dairy product types, raw milk exhibited the highest L. monocytogenes prevalence at 24.1% (13/54 positive across all sampling occasions), followed by soft cheese at 19.6% (9/46), cream products at 15.2% (5/33), yogurt at 7.8% (3/38), butter at 5.6% (2/36), and whey at 4.2% (1/24). The mean contamination level in positive raw milk samples was 3.8±0.9 log CFU/g, which was significantly higher than all other product types (p<0.01).

Results

Virulence gene profiling of the 99 confirmed isolates revealed that the master regulator gene prfA was detected in 98.0% of isolates, followed by hlyA (95.0%), inlA (92.0%), iap (90.0%), inlB (85.0%), actA (82.0%), plcA (72.0%), and plcB (68.0%). The complete virulence gene cassette (all eight genes present) was identified in 58.6% (58/99) of isolates, predominantly among serotype 4b (88.9%) and serotype 1/2a (63.2%) strains. PFGE analysis using combined AscI and ApaI digestion patterns differentiated the 99 isolates into 12 distinct pulsotypes. Three dominant pulsotypes (designated PT-A, PT-B, and PT-C) collectively accounted for 61.6% of all isolates and were recovered from multiple facilities and across multiple sampling periods spanning 6 to 11 months, strongly suggesting the establishment of persistent environmental reservoirs. Pulsotype PT-A (serotype 1/2a) was the most prevalent, identified in six of eight facilities, while PT-B (serotype 4b) was detected in four facilities and demonstrated the highest virulence phenotype scores.

Comparative virulence phenotype analysis across the three predominant serotypes revealed significant differences. Serotype 4b isolates exhibited the highest hemolytic activity (68.4±8.7% lysis), significantly exceeding serotype 1/2a (42.1±11.3%) and 1/2b (38.7±9.8%) isolates (p<0.01).

Similarly, Caco-2 cell invasion efficiency was greatest for serotype 4b (invasion index: 8.6±1.2) compared to serotype 1/2a (6.5±1.8) and 1/2b (7.2±1.4). However, serotype 1/2b demonstrated superior biofilm formation capacity (OD₅₇₀: 0.89±0.14) compared to 1/2a (0.72±0.18) and 4b (0.58±0.12), potentially contributing to its environmental persistence despite lower invasive virulence.

Discussion

The 14.7% overall detection rate of L. monocytogenes in dairy processing environments documented in this research represents a significant contamination burden that exceeds regulatory thresholds and warrants immediate attention from the South Korean food safety authorities. This prevalence aligns with detection rates reported from dairy processing facility surveys conducted in other East Asian countries, where environmental contamination rates ranging from 8% to 22% have been documented depending on the scope of sampling and the types of facilities investigated [1,2] . The marked concentration gradient of L. monocytogenes contamination across sequential processing stages, with peak prevalence on processing lines (35.7%) and progressive reduction toward final products (6.3%), reflects the combined effects of thermal processing, competitive exclusion during fermentation, and sanitation interventions applied at downstream stages [3] . However, the persistence of detectable contamination in 6.3% of final products indicates that existing control measures, while substantially reducing microbial loads, remain insufficient for achieving the zero-tolerance standard mandated by Korean food safety regulations for ready-to-eat dairy products [4,5] . The predominance of serotype 1/2a (38.4%) among environmental and product isolates is consistent with the established understanding that this serotype exhibits enhanced environmental fitness and biofilm formation capabilities compared to other serotype groups [6,7] . The identification of serotype 4b in 18.2% of isolates carries heightened public health significance, as this serotype is responsible for approximately 50% of human listeriosis cases globally and has been implicated in the majority of large-scale listeriosis outbreaks associated with dairy products [8] . The significantly higher hemolytic activity and Caco-2 cell invasion efficiency demonstrated by serotype 4b isolates in this research corroborate its established hypervirulent phenotype and underscore the clinical risk posed by even low-level contamination with this serotype. The detection of three persistent pulsotypes across multiple facilities and extended temporal periods represents the most concerning finding from an environmental management perspective. Persistent L. monocytogenes clones are known to colonize environmental niches within processing facilities, including floor drains, condensation collection points, conveyor belt junctions, and equipment harbourage sites where biofilm formation provides protection against routine cleaning and sanitization procedures [9,10] . The cross-facility distribution of identical pulsotypes suggests potential common contamination sources, possibly through shared raw material suppliers or mobile equipment, warranting investigation through whole-genome sequencing to establish precise transmission linkages [11] . The comprehensive virulence gene profiling approach employed in this research provides a more nuanced risk assessment framework compared to conventional detection and serotyping alone. The observation that 58.6% of isolates harbored the complete eight-gene virulence cassette indicates a substantial proportion of the environmental L. monocytogenes population possesses full pathogenic potential, reinforcing the public health imperative for eliminating persistent contamination reservoirs from dairy processing environments [12,13] .

Pathogenesis and virulence mechanisms

The pathogenesis of L. monocytogenes involves a sophisticated intracellular infection cycle that exploits host cell machinery for internalization, phagosomal escape, cytoplasmic replication, and cell-to-cell spread.

Conclusion

The context of increasing global concern regarding L. monocytogenes contamination in dairy supply chains provides essential framing for interpreting these findings. This research revealed a 14.7% overall detection rate across eight dairy processing facilities in Busan, with processing lines identified as the critical contamination amplification stage and three persistent pulsotypes demonstrating cross-facility distribution over extended temporal periods. The recommendation emerging from these findings is the urgent implementation of molecular-based environmental monitoring programs capable of detecting and tracking persistent contamination clones, complemented by enhanced sanitation protocols specifically targeting biofilm-forming L. monocytogenes populations on processing line equipment. Looking forward, the integration of whole-genome sequencing into routine surveillance frameworks will enable higher-resolution epidemiological investigations linking environmental contamination to clinical disease, ultimately strengthening the evidence base for riskproportionate food safety interventions in the South Korean dairy sector.

Acknowledgement

AcknowledgementsThe Busan Research Institute of Food Safety is acknowledged for providing laboratory facilities and equipment access that were essential for conducting this investigation. This research was supported by the Korean Food and Drug Safety Administration through the National Food Safety Research Program. The authors acknowledge the technical contributions of research assistants Ms. Choi Min-Young and Mr. Yoon Seung-Ho for their meticulous work in sample processing, molecular analysis, and data management throughout the investigation.

References

1. Ferreira V, Wiedmann M, Teixeira P, Stasiewicz MJ. Listeria monocytogenes persistence in food-associated environments: epidemiology, strain characteristics, and implications for public health. J Food Prot. 2014;77(1):150-170.
2. NicAogain K, O'Byrne CP. The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain. Front Microbiol. 2016; 7:1865.
3. Pal M, Shuramo MY, Shiferawu F, Parmar BC. Listeriosis: an emerging foodborne disease of public health concern. J Adv Microbiol Res. 2022;3(2):29-33.
4. Freitag NE, Port GC, Miner MD. Listeria monocytogenes: from saprophyte to intracellular pathogen. Nat Rev Microbiol. 2009;7(9):623-628.
5. Vazquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Dominguez-Bernal G, Goebel W, et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev. 2001;14(3):584-640.
6. Maury MM, Tsai YH, Charlier C, Touchon M, ChenalFrancisque V, Leclercq A, et al. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nat Genet. 2016;48(3):308-313.
7. Korean Dairy Committee. Annual report on Korean dairy industry statistics 2022. Seoul: Korean Dairy Committee; 2023.
8. Kim SW, Haendiges J, Gerner-Smidt P, Timme RE, Cao G, Allard MW, et al. Molecular serotyping of Listeria monocytogenes using a core genome MLST scheme. Food Microbiol. 2021; 100:103870.
9. Carpentier B, Cerf O. Review: persistence of Listeria monocytogenes in food industry equipment and premises. Int J Food Microbiol. 2011;145(1):1-8.
10. Møretrø T, Langsrud S. Listeria monocytogenes: biofilm formation and persistence in food-processing environments. Biofilms. 2004;1(2):107-121.
11. Orsi RH, den Bakker HC, Wiedmann M. Listeria monocytogenes lineages: genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol. 2011;301(2):79-96.
12. Leong D, NicAogain K, Luber P, Margas E, Manzanos L, Jordan K. A three-year multi-food study of the presence and persistence of Listeria monocytogenes in 54 small food businesses in Ireland. Int J Food Microbiol. 2017; 249:18-26.
13. Muhterem-Uyar M, Dalmasso M, Bolocan AS, Hernandez M, Kapetanakou AE, Kuchta T, et al. Environmental sampling for Listeria monocytogenes control in food processing facilities reveals three contamination scenarios. Food Control. 2015; 51:94-107.
14. Lee S, Ward TJ, Graves LM, Wolf LA, Sperry K, Siletzky RM, et al. Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette. Appl Environ Microbiol. 2012;78(3):660-667.
15. Ortiz S, Lopez V, Villatoro D, Lopez P, Davila JC, Martinez-Suarez JV. A 3-year surveillance of the genetic diversity and persistence of Listeria monocytogenes in an Iberian pig slaughterhouse and processing plant. Foodborne Pathog Dis. 2010;7(10):1177-1185.
16. Ragon M, Wirth T, Hollandt F, Lavenir R, Lecuit M, Le Monnier A, et al. A new perspective on Listeria monocytogenes evolution. PLoS Pathog. 2008;4(9):e1000146.