Vibrio parahaemolyticus is a Gram-negative bacterium naturally present in coastal water. V. parahaemolyticus grows preferentially during warm periods when the sea surface temperature increases (T > 15 °C) (Brauge et al., 2024). It is a human pathogen that induces gastro-enteritis associated to seafood consumption and that resulted in several outbreaks (Fearnley et al., 2024; Yan et al., 2024). Chen et al. (2023) characterized epidemiological data of V. parahaemolyticus outbreaks in Zhejiang in China between 2010 and 2022. They reported that twenty-nine outbreaks were related with meat, vegetables, beans, eggs and fruit products, which are not the usual vector of V. parahaemolyticus. Moreover, 110 outbreaks were associated to cooked seafood which are normally free of V. parahaemolyticus. These unusual sources were most probably due to cross-contamination phenomena. Indeed, Martinez-Urtaza et al. (2016) reported a cross-contamination by V. parahaemolyticus of cooked shrimps after a contact with contaminated ice on a cruise boat in Spain resulting in 100 infections. In South Korea, a gastroenteritis outbreak affected 237 individuals, with V. parahaemolyticus detected in 34 of them. Epidemiological investigations revealed cross-contamination involving squid, eggs, and pancakes used in the preparation of kimbap. This contamination was likely due to the use of the same knife and cutting board for handling these different ingredients, suggesting a transfer of the pathogen from inert surfaces to food (Jung, 2018).

Cross-contamination of food from inert surfaces, such as aquaculture tank, processing areas or tools can occur due to the presence of biofilms on these surfaces. Biofilms are multicellular structures surrounded by an extracellular matrix, which provides them an enhanced resistance to environmental stress and promotes the persistence of bacteria under hostile conditions. V. parahaemolyticus is able to form biofilm on various surfaces including marine organisms, such as mussel (Ashrafudoulla et al., 2019), prawn or crab shells (Han et al., 2016), which can be naturally contaminated with the presence of V. parahaemolyticus in aquaculture tank water (Narayanan et al., 2020). This ability to form biofilm on seafood is an open door to contaminate agri-food plant since V. parahaemolyticus is also able to form biofilm on industrial surfaces such as stainless steel (Mougin et al., 2019) or polypropylene (Su et al., 2025), which are both widely used in the food processing industry.

To limit the biofilm proliferation and the risk of cross-contamination, aquaculture and industrial professionals implemented several prophylactic measures, such as surface disinfection. Several disinfectants prevent biofilm formation in food processing environment. Among them, quaternary ammonium (QACs) are widely used in food industry and the commonly used QACs is the benzalkonium chloride (BAC) (Buffet-Bataillon et al., 2012). QACs are cationic detergents which disturb with the bacteria cytoplasmic membrane (Gerba, 2015), leading to cellular lysis. However, the overuse of treatment promotes the emergence of antimicrobial resistant bacteria, which are able to resist to antibiotics and disinfectant compounds. Disinfectant resistance is mainly conferred by the presence of efflux pumps that are classed in six families: resistance nodulation cell division (RND), major facilitator (MF), multidrug and toxic compound extrusion (MATE), small multidrug resistance (SMR), ATP binding cassette (ABC), and proteobacterial antimicrobial compound efflux (PACE) (Hassan et al., 2018; Matsuo et al., 2013). Several efflux pumps coded by antimicrobial resistance genes (ARG) have already been detected in V. parahaemolyticus, such as qacE and qacEΔ1 which belong to the SMR family and confer resistance to QACs (Kazama et al., 1999; Slipski et al., 2021). Additionally, qacEΔ1 gene is mainly borne by genetic mobile element (MGE) belonging to mobilome such as plasmid or integron (Li et al., 2015). Indeed, qacEΔ1 is classically associated with the 3′ region of class 1 integrons (Mazel, 2006), and it is detected in many Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii (Wang et al., 2008). This gene has been reported chromosomally located, for example in Salmonella genomic island 1-PmJN16 found in Proteus mirabilis (Bie et al., 2018), as well as on plasmids in Vibrio species such as Vibrio cholerae and V. parahaemolyticus (Ceccarelli et al., 2006; Li et al., 2015). In addition, some efflux pumps and membrane component, such as vmeA-Z and vopC genes respectively, are also present in V. parahaemolyticus genome. These efflux pumps belong to the RND family and confer resistance to multiple antimicrobial compounds (Matsuo et al., 2007, Matsuo et al., 2013).

In addition to genetic resistance mechanisms, several studies have shown that in response to environmental stress, V. parahaemolyticus can enter VBNC (Viable But NonCulturable) state. This transient state allows the bacterium to survive under unfavorable conditions without being detectable by standard culture-based methods, as it loses its ability to grow on culture media while maintaining residual metabolic activity and virulence potential (Wong and Wang, 2004). The VBNC state poses a significant challenge in agri-food environments, particularly in processing facilities, as it can lead to false-negative results during surface sampling, thereby undermining the effectiveness of environmental monitoring and control plans. In the VBNC state, bacteria undergo major transcriptomic changes but can continue to express stress response genes and efflux pump genes, as demonstrated in E. coli (Ye et al., 2020). Moreover, VBNC cells are able to release intact DNA, including plasmids, into the environment (Yin et al., 2023), thereby persisting undetected while contributing to the horizontal transfer of ARGs. To date, most of these mechanisms have been described in E. coli, whereas little is known about their occurrence in biofilms. Current research on VBNC in V. parahaemolyticus has mainly focused on identifying environmental factors that trigger this state. Mougin et al. (2019) showed that moderate thermal stress conditions, such as brine baths at 8 °C for 48 h, were capable of inducing the VBNC state in V. parahaemolyticus biofilms. A recent study conducted by Wang et al. (2025) showed that sodium hypochlorite treatment on planktonic V. parahaemolyticus is also in capacity to induce VBNC state. These studies confirmed that this mechanism could be triggered by environmental stresses, suggesting a high adaptive plasticity of the species. More recently, Mougin et al. (2024) described that the presence of residual BAC, a commonly used disinfectant, led to increased biomass production and a significant reduction in metabolic activity in a weak biofilm-forming strain of V. parahaemolyticus after 24 h of exposure to various residual BAC concentrations. Using flow cytometry, the authors also observed an increase in the proportion of damaged cells. Although these findings provide new insights into the effects of low BAC concentrations, they are based on only two strains and only detected injured cells, which could not be definitively classified as VBNC.

However, the variability of V. parahaemolyticus biofilms for resistance to QACs, their ability to enter VBNC states and the relationship of these two points are still undetermined. In this context, the present study aimed (i) to characterize the resistome of 39 V. parahaemolyticus strains, with a particular focus on antimicrobial resistance genes (ARGs) related to disinfectant resistance, and (ii) to assess the influence of qacEΔ1 on biofilm formation and the ability to enter the VBNC state under BAC concentrations representative of industrial use conditions. This approach will contribute to a better understanding of the mechanisms underlying resistance and persistence of V. parahaemolyticus in response to disinfectant exposure.