| Summary: | <i>Listeria monocytogenes</i> is an important pathogen that has been implicated in foodborne illnesses and the recall of products such as fruit and vegetables. This study determines the prevalence of virulence-associated genes and serogroups and evaluates the effects of different growth media and environmental conditions on biofilm formation by <i>L. monocytogenes</i>. Eighteen <i>L. monocytogenes</i> isolates from Hass avocados sold at markets in Guadalajara, Mexico, were characterized by virulence-associated genes and serogroup detection with PCR. All isolates harbored 88.8% <i>actA</i>, 88.8% <i>plcA</i>, 83.3% <i>mpl</i>, 77.7% <i>inlB</i>, 77.7% <i>hly</i>, 66.6% <i>prfA</i>, 55.5% <i>plcB</i>, and 33.3% <i>inlA</i>. The results showed that 38.8% of isolates harbored virulence genes belonging to <i>Listeria</i> pathogenicity island 1 (LIPI-1). PCR revealed that the most prevalent serogroup was serogroup III (1/2b, 3b, and 7 (<i>n</i> = 18, 66.65%)), followed by serogroup IV (4b, 4d–4e (<i>n</i> = 5, 27.7%)) and serogroup I (1/2a–3a (<i>n</i> = 1, 5.5%)). The assessment of the ability to develop biofilms using a crystal violet staining method revealed that <i>L. monocytogenes</i> responded to supplement medium TSBA, 1/10 diluted TSBA, and TSB in comparison with 1/10 diluted TSB (<i>p</i> < 0.05) on polystyrene at 240 h (<i>p</i> < 0.05). In particular, the biofilm formation by <i>L. monocytogenes</i> (7.78 ± 0.03–8.82 ± 0.03 log<sub>10</sub> CFU/cm<sup>2</sup>) was significantly different in terms of TSBA on polypropylene type B (PP) (<i>p</i> < 0.05). In addition, visualization by epifluorescence microscopy, scanning electron microscopy (SEM), and treatment (DNase I and proteinase K) revealed the metabolically active cells and extracellular polymeric substances of biofilms on PP. <i>L. monocytogenes</i> has the ability to develop biofilms that harbor virulence-associated genes, which represent a serious threat to human health and food safety.
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