## 1 Introduction  
_Listeria monocytogenes_ is an opportunistic foodborne pathogen responsible for listeriosis, a disease associated with high mortality rates [1]. Listeriosis is mainly caused by the consumption of contaminated, predominantly ready-to-eat, food [2]. Disinfection is the most common method used to eliminate _L. monocytogenes_ from the food-processing environment [3]. Quaternary ammonium compounds (QACs), such as benzalkonium chloride (BAC), are widely used as disinfectants in food-processing areas [4; 5; 6]. The presence of BAC in the environment is a growing public health concern, as BAC can select not only for biocide resistance but also for clinically relevant antibiotic resistance [7]. It is acknowledged that strains of _L. monocytogenes_ with a BAC minimum inhibitory concentration (MIC) of \(\geq\)10 mg/L harbor genetic determinants, such as _bcrABC_[8] and _qacH_[9], that are known to contribute to BAC resistance. Both transferable resistance genes are prevalent in the food industry [10; 11; 12]. The efflux systems encoded by these genes belong to the small multidrug resistance (SMR) protein family [8; 9]. These pumps are usually chromosomally encoded and affect a broad spectrum of antimicrobial compounds [13]. In some studies, the aforementioned acquired determinants are not detected in certain resistant strains [11; 14]. In these cases, resistance may be due to the overexpression of endogenous efflux pumps due to mutations in regulatory elements. This can occur due to exposure to QACs or the stress induced by these compounds [13]. Efflux pumps, such as MdrL (multidrug resistance _Listeria_) and Lde (_Listeria_ drug efflux), which belong to the major facilitator superfamily (MFS), and affect a different spectrum of antimicrobial compounds compared to SMR transporters, have been associated with _L. monocytogenes_ adaptation and resistance to BAC [5; 13; 15]. Although the BAC resistance genotype and phenotype may be unrelated in _L. monocytogenes_-forming biofilms [16], QACs used without subsequent effective rinsing [6; 17] may be present at residual sublethal concentrations after disinfection in industries, thereby providing a growth advantage to resistant bacteria. In fact, [PERSON] et al. [18] have shown that BAC resistance provides a growth advantage at residual QAC concentrations present after disinfection. Thus, resistance might be responsible for the persistence of _L. monocytogenes_ in the food industry [13]. Exposure of _L. monocytogenes_ to sub-inhibitory concentrations of BAC can differently influence the biofilm formation capacity of resistant (BAC-R) and sensitive (BAC-S) strains [19]. Previous results, using the crystal violet staining method [19], have shown that resistant strains growing in polystyrene microplates produced significantly more biofilm in the presence of sub-inhibitory concentrations of BAC than in the same conditions but without BAC [19]. Crystal violet staining for biofilm quantification in microtiter plates has limitations, as it stains both live and dead cells as well as some components present in the biofilm matrix (total biofilm biomass) and is, therefore, not applicable to viability measurements. Nevertheless, viability staining followed by confocal laser microscopy [20] confirmed the results of biofilm stimulation by BAC obtained with the crystal violet method [19]. As the explanation for the important problem of the persistence of _L. monocytogenes_ is still poorly understood [21], two other methods were used in this work to better understand how the strain susceptibility and the presence of BAC affect the resistance of biofilms: (i) The resazurin (RSZ)-based viability staining, which is often used to quantify metabolically active biofilm cells grown in microtiter plates [22], and represents a very simple and fast method to be used for high throughput screening of antimicrobials [23], and (ii) biofilm cell enumeration and biofilm reduction after BAC exposure of biofilms produced on stainless steel coupons (SSC) [16].