Genotypic and Phenotypic Characterization of Listeria Monocytogenes Reveals Challenges to Sanitization Efficacy in Brazilian Food Industries

Introduction: Listeria monocytogenes is a critical hygiene indicator in food processing environments (FPEs) and a lethal pathogen. Industrial sanitation eliminates most microbiota; however, suboptimal efficacy exerts strong selective pressure in clean niches, threatening food safety. Persistent, biofilm‑forming, stress‑tolerant strains predominate, frequently combining enhanced virulence and environmental adaptability. This study characterized the genotypic (serogroup, virulence, biofilm‑associated genes) and phenotypic (biofilm formation ability and antimicrobial resistance profile) profiles of L. monocytogenes isolated from food contact surfaces after standard industrial sanitation.

Methods: Seventeen isolates came from industrial polystyrene and stainless-steel surfaces across three facilities (one poultry, one pork, and one dairy plant) after the pre-operational hygiene stage, following ISO 11290. Phenotypic characterization assessed susceptibility to 12 antimicrobials from 10 classes and adhesion on polystyrene microplates (CLSI, 2021; Stepanović, 2000). Genotypic analysis included multiplex PCR serogrouping (I, III, IV) and detection of virulence genes (inlA, inlB, inlC, hlyA, actA) alongside genes for biofilm formation, regulation, and stress adaptation (prfA, flaA, agrABCD, luxS).

Results: Isolates belonged to serogroups I (1/2a), III (1/2b), and IV (4b), with I predominating in poultry and IV in pork, comprising 10 pork, 6 poultry, and 1 dairy isolates. All were resistant to clindamycin, and 64.7% to penicillin, yielding two profiles, with combined clindamycin-penicillin resistance dominating. All carried the full virulence gene set (hlyA, inlA, inlB, inlC, prfA) plus the agrABCD system and luxS, while actA was restricted to pork isolates. Despite this biofilm‑associated genotype, all strains exhibited weak polystyrene adhesion.

Conclusions: The repeated isolation of L. monocytogenes serogroups 4b and 1/2a with multivirulence characteristics and adhesion capacity demonstrates the selection and survival of high‑risk strains after industrial cleaning. Widespread clindamycin resistance and dominant penicillin co‑resistance amplify this public health alert. Revising sanitation validation protocols to include sessile‑cell assays is imperative to eliminate these persistent pathogens, mitigating cross‑contamination and food safety risks under a One Health perspective.