Evidence of Virulent Multi-Drug Resistant and Biofilm-Forming Listeria Species Isolated from Various Sources in South Africa

Listeriosis is a foodborne disease caused by Listeria monocytogenes species and is known to cause severe complications, particularly in pregnant women, young children, the elderly, and immunocompromised individuals. The aim of this study was to investigate the presence of Listeria species in food and water using both biochemical and species-specific PCR analysis. L. monocytogenes isolates were further screened for the presence of various antibiotic resistance, virulence, and biofilm-forming determinants profiles using phenotypic and genotypic assays. A total of 207 samples (composed of meat, milk, vegetables, and water) were collected and analyzed for presence of L. monocytogenes using species specific PCR analysis. Out of 267 presumptive isolates, 53 (19.85%) were confirmed as the Listeria species, and these comprised 26 L. monocytogenes, 3 L. innocua, 2 L. welshimeri, and 1 L. thailandensis. The remaining 21 Listeria species were classified as uncultured Listeria, based on 16SrRNA sequence analysis results. A large proportion (76% to 100%) of the L. monocytogenes were resistant to erythromycin (76%), clindamycin (100%), gentamicin (100%), tetracycline (100%), novobiocin (100%), oxacillin (100%), nalidixic acid (100%), and kanamycin (100%). The isolates revealed various multi-drug resistant (MDR) phenotypes, with E-DA-GM-T-NO-OX-NA-K being the most predominant MDR phenotypes observed in the L. monocytogenes isolates. The virulence genes prfA, hlyA, actA, and plcB were detected in 100%, 68%, 56%, and 20% of the isolates, respectively. In addition, L. monocytogenes isolates were capable of forming strong biofilm at 4 °C (%) after 24 to 72 h incubation periods, moderate for 8% isolates at 48 h and 20% at 72 h (p < 0.05). Moreover, at 25 °C and 37 °C, small proportions of the isolates displayed moderate (8–20%) biofilm formation after 48 and 72 h incubation periods. Biofilm formation genes flaA and luxS were detected in 72% and 56% of the isolates, respectively. These findings suggest that proper hygiene measures must be enforced along the food chain to ensure food safety.


Introduction
Listeria species are Gram-positive bacteria that belong to Listeriacaeae family. They are widely distributed in nature and capable of causing listeriosis in humans and animals [1]. In terms of pathogenicity, L. monocytogenes and L. ivanovii are the most pathogenic species, and they are responsible for several outbreaks in humans [2]. Their ubiquitous nature allows them to strive in various environments, particularly water, soil, plants, animals, silage, biofilms contribute to an increase in antibiotic resistance, since biofilm-forming bacteria have been reported to be more resistant to antibiotics, when compared to their planktonic counterparts [34]. The biofilm structure (extracellular polymeric substance) enhances the ability of microbial cells to withstand and survive chemical compounds, including those used in food industry [35,36]. For this reason, understanding the pathogenicity of L. monocytogenes, through the determination of the presence of virulence genes that enable entry, proliferation, and spread in host cells, is critical [37][38][39]. Amongst them, the inlAB internalisation locus, Listeria pathogenicity island-1 (LIPI-1), hpt intracellular growth locus, InlB, prfA, plcA, hly, mpl, actA, and plcB, genes have been associated with virulence in L. monocytogenes [40][41][42][43][44][45]. Some studies have also reported a reciprocal association between antibiotic resistance and increased bacterial virulence and transmission [46,47]. Therefore, the aim of the current study was to investigate the occurrence, AMR, virulence, and biofilmforming potentials of Listeria sp. isolated from various sources in the North West province of South Africa.

Detection of Listeria spp.
Out of the 207 samples screened, a total of 267 isolates were presumptively positive for Listeria spp., based on the morphological and biochemical characteristics (Gram-positive rods, catalase-positive, beta hemolysis, H 2 S-negative, gas-negative, oxidase-negative, methyl red-positive, and esculin hydrolysis) of the isolates. Of these, 53 (19.85%) were confirmed positive for the genus Listeria spp. using PCR analysis. These constituted 14 (5.24%) from water samples, 36 (13.48%) from food samples and 3 (1.12%) from fecal samples. Based on species specific PCR assays, 25 (47.17%) isolates were confirmed as L. monocytogenes, 14 (26.41%) from water samples, 3 (1.12%) from vegetables, and 8 (15.09%) from meat/meat products) (Table 1). However, the 16S rRNA sequence BLAST search results revealed that of the 53 Listeria spp. isolates sequenced, 26 were identified as L. monocytogenes, while the others were 3 L. innocua, 2 L. welshimeri, 1 L. thailandensis, and 21 uncultured Listeria spp. A total of 25 L. monocytogenes isolate were subjected to antimicrobial susceptibility test against 12 antibiotics. The results revealed that 62.5% to 100% of L. monocytogenes isolates were resistant to most of the antibiotics, including ampicillin, trimethoprim and sulfamethoxazole, erythromycin, and norfloxacin. Twenty-four isolates were resistant to kanamycin. Most of the isolates were found to be resistant to a total of seven antibiotics (kanamycin, novobiocin, tetracycline, oxacillin, clindamycin, nalidixic acid, and gentamicin) tested. The isolates from water samples were more resistant than those isolated from food samples. Tables 2 and 3 showed the proportion of the AMR phenotypes of the isolates. Thirteen MAR phenotype patterns were observed in L. monocytogenes isolates, with E-DA-GM-T-NOV-OX-NA-K being the most frequently detected phenotype. The MAR indices ranged from 0.6 to 0.9, with a mean of 0.74 (Table 3).  = 3  1  1  3  3  3  3  3  3  3  3  3  3   Vryburg n = 3  0  1  2  2  3  3  0  3  3  3  3  3 Mafikeng

Proportion of Virulence Genes Detection in L. monocytogenes
Out of eight virulence genes, five (prfA, actA, hlyA, inlC, inlJ) were detected in 25 L. monocytes by PCR. The prfA was the most frequently (100%) detected gene in all L. monocytogenes isolates when compared to the hlyA (68%, n = 17) and actA (56%, n = 14) genes. Only five isolates harbored the plcA gene. Out of 25 L. monocytogenes isolates, 3 from food samples carried 8 virulence genes. In general, 5 virulence genes (actA, hlyA, inlC, inlJ, prfA) were most often detected in this study ( Figure S1). The microtiter plate assay was employed to assess the ability of L. monocytogenes isolates to form biofilm at different temperatures (4, 25, and 37 °C) over 24, 48, and 72 h

Phenotypic Assessment of Biofilm Formation
The microtiter plate assay was employed to assess the ability of L. monocytogenes isolates to form biofilm at different temperatures (4, 25, and 37 • C) over 24, 48, and 72 h periods. Based on biofilm formation patterns, the isolates were grouped either as nonadherent, weakly, moderately, or strongly adherent strains ( Table 4). The results showed that all the isolates (n = 25) exhibited strong biofilm attachments, following 24, 48, and 72 h incubation periods at 4 • C. At 25 • C, 2 (8%) L. monocytogenes isolates produced moderate biofilm, while 23 (92%) formed a strong biofilm attachment, following 24 and 72 h incubation periods. Strong biofilm formation was demonstrated by 20 (80%) isolates at 37 • C after a 48 h incubation period. At 37 • C, the production of biofilm was strong after a 24 h incubation period, and the biofilm production of some isolates decreased after 72 h (p < 0.05), as shown in (Appendix A) Figures A1-A3.

Biofilm Formation Genes
PCR was utilized to determine the presence of genes associated with biofilm formation. Out of 25 L. monocytogenes isolates, 14 were positives for luxS and flaA (56% and 72%, respectively). A total of 9 (36%) isolates from food samples carried luxS gene followed by 5 (20%) from water and food samples harboring flaA gene. A total of 9 isolates (5 from food samples and 4 from water samples) were positive for both flaA and luxS genes ( Figure S2).

Discussion
L. monocytogenes is capable of causing a sporadic disease, termed listeriosis, in humans and animals that results in very high hospitalization and fatalities [48]. Listeriosis is amongst the most problematic food-borne diseases in several countries, and it is associated with contamination at all stages in the food production process [49][50][51][52]. This pathogen is a psychrophile and can grow at refrigerated temperatures [53][54][55]. Due to this feature, it is very difficult to inactivate and curb the proliferation of L. monocytogenes cells in RTE food. This, therefore, indicates the need for continuous monitoring of this pathogen, especially in the food industry [56]. The main objective of this study was to assess the presence of Listeria sp. from various sources that are linked to the food chain, especially after South Africa recorded the world's most severe outbreak of listeriosis in 2017 [57]. The results obtained in this study show that the occurrence of Listeria spp. was higher in food products (13.48%) than water (5.24%) and cattle feces (1.12%) samples. L. monocytogenes was detected in water and food. Interestingly, L. monocytogenes was most often detected in meat and meat products (eight; 15.09%), when compared to vegetables (three; 5.66%). These findings are a cause for concern, given the fact that listeriosis outbreaks have been associated with meat and poultry products, such as hot dogs [58], RTE pork meats [59], and turkey meat products [60]. In addition, L. monocytogenes was most frequently isolated in packing houses that processed frozen (41.3%), fresh-cut vegetables (7.9%) [61,62] and vegetables (9.5%) [63], mushrooms (1.6%) [64], and prepackaged salads and canned vegetables (5.4%) [65]. Food products tested in this study, such as cucumber and lettuce, may have been contaminated with L. monocytogenes as a result of soil contact. This pathogen may be introduced into food processing plants by human carriers (workers) and animals through faecal shedding [66,67]. The risks that these bacteria (L. monocytogenes) provide to public health are highlighted by their ability to survive for years, or even decades, in food processing facilities, as well as their ability to assist in food contamination during processing, handling, and packaging and the ease of foodborne transmission to consumers. Moreover, the detection of L. monocytogenes, including other species, such as L. ivanovii, L. grayi, and L. innocua, in wastewater effluents in a treatment facility in South Africa clearly indicates the potential to contaminate the environment. This may also increase the routes of transmission and dissemination of L. monocytogenes to foods [68].
Another objective of this study was to determine the antimicrobial resistant profiles of L. monocytogenes isolated from different sources within the food chain. In this study, large proportions of L. monocytogenes isolates were resistant to the majority of the antibiotics (62.5% to 100%). These results are consistent with another study, which reported high (90.74%) resistant L. monocytogenes isolates against nalidixic acid [69]. MAR (Multi-antibiotic resistance is a bacterium that is resistant to several antibiotics) were most prominent with nalidixic acid/cloxacillin (35.2%), nalidixic acid/colistin (31.5%), and cloxacillin/colistin/nalidixic acid (29.6%) [70]. However, a low (5.6%) AMR pattern was shown in chloramphenicol/nitrofurantin/cotrimoxazole [61]. In this study, the MAR phenotype E-DA-GM-T-NOV-OX-NA-K appeared in three different locations on different samples, which could be due to the use of antibiotics in those area. The continuous misuse of antibiotics led to the development of antibiotic resistance, specifically multi-drug resistant (MDR, which is an isolate that is resistant to at least one antibiotic in three or more drug classes) strains [71,72]. Notably, a high MAR index (Mean = 0.74) was observed in this study, and these findings were higher than that of the previous study [73]. This suggest that the isolates originate from a high-risk environment. Furthermore, the results obtained in this study revealed that L. monocytogenes harbored antimicrobial resistant genes ant(3")-la (92%), dfr1S (84%), sul1 (80%), and ermC (80%), while the lowest were tetA and tetB at 8%). Contradictory to our findings, Heidarzadeh et al. reported tetM was detected at high rates (70%) of tetracycline-resistant isolates [70]. Approximately, 27.3% isolates carried dfrD. The ermB gene was detected in 83.3% of the erythromycin-resistant isolates [70]. Although antibiotic resistance in L. monocytogenes was rare, over the last two decades, there has been an increasing trend in resistance to one or multiple common antibiotics found in both clinical and environmental sources. In general, resistance genes are handed along by transferable plasmids and conjugative transposons, and L. monocytogenes resistant to antibiotics is no exception. Despite the fact that the study was unable to prove a direct transmission link between resistant genes in L. monocytogenes, our findings revealed several resistance genes in water and food samples.
A further objective of the study was to determine the presence of virulence genes in L. monocytogenes. The detection of prfA (100%), hlyA (68%), and actA (56%) genes indicates the potential pathogenicity of the isolates and their potential to cause foodborne infections. In addition, plcB genes were detected in five isolates. In contrast to our findings, Du et al. [74] found plcB gene in all isolates, whereas the inlB, actA, plcA, and iap genes were found in 71.4-90.5% of the isolates. Several studies have shown that the prfA gene in L. monocytogenes encodes a protein that induces the transcription of the listeriolysin gene (lisA) [75][76][77][78]. This signifies the prfA gene in the pathogenesis of listeriosis. Furthermore, the prfA gene in L. monocytogenes possessed two important crucial functions: response to environmental stress conditions and capacity to infect humans [75].
Biofilm formation, as well as the adaptation to stress, induced by cold temperatures and sub-lethal concentrations of disinfectants, coupled with the potential to inhabit ecological niches in facilities and equipment that are difficult to clean, have been identified as key contributors to the persistence of L. monocytogenes in the food chain [79]. Biofilm formation or 'glycocalyx' facilitates the adherence of bacteria to surfaces and plays a significant role in the success of pathogenic bacteria; thus, the prevention of adhesion could be an effective way to combat infections and cross-contamination [80]. This study evaluated the biofilm formation potentials of 25 L. moncytogenes strains isolated from different sources. L. monocytogenes has the potential to multiply within a wide range of temperatures (2-45 • C); due to that, three various temperatures were evaluated, which comprised 37 • C (representing the optimum growth temperature range between 30 and 37 • C), 25 • C (representing the room temperature), and 4 • C (which stand for refrigeration temperature of foods in retail) [15,[81][82][83]. Our findings reveal that strong biofilm attachments were formed at 4 • C for 24, 48, and 72 h in all isolates (n = 25; 100%), followed by moderate biofilm formation for two (8%) isolates at 48 h and five (20%) L. monocytogenes isolates at 72 h. Similar results by Lado et al. [81] showed that L. monocytogenes isolates produced a strong biofilm community at 10 and 37 • C. All L. monocytogenes isolates utilized in this study were from food sources, as well as food processing equipment.
In addition, PCR-based biofilm formation genes showed that 18 out of 25 (72%) were amplified for flaA, followed by 14 (56%) for luxS. A total of nine (36%) isolates from food samples were found to carry the gene luxS, followed by five (20%) from water and food samples harboring the flaA gene. In a recent study, it was proven that L. monocytogenes biofilm was produced at optimum of neutral pH and 37 • C; supplementary data showed temperature as a key player in L. monocytogenes biofilm formation. It was concluded that the impacts of various factors on biofilm production are strain-dependent [74]. In Egypt, particularly Sohag city, 295 samples of ice cream, vaginal swabs, stool, and the urine of aborted women were collected and examined for the virulence ability of L. monocytogenes. Biofilm investigation was conducted by PCR detection of biofilm genes, such as luxS and flaA. The luxS gene was detected at 81.8%, followed by flaA at 72.7% in ice cream. In aborted women, the luxS gene amplified 81.8%, followed by flaA (63.6%), which contradicted our results [84]. In another study, Hassanien and Shaker assessed the biofilm potential of L. monocytogenes and showed that out of 38 isolates amplified, 23 (60.52%) and 1 (2.63%) were positive to luxS and flaA genes respectively [85].
Over the past two decades, researchers have shown the impact of biofilm in the food processing industries. These biofilms contribute to cross contamination by protecting and harboring harmful or spoilage microorganisms, including L. monocytogenes [86]. Antibiotic resistance is higher in the context of biofilm development than in planktonic cells [35,87]. Hence, this poses a problem in the control of L. monocytogenes from food sources, water, and food processing surfaces, as well as equipment.

Sample Area and Sample Collection
A total of 207 samples were collected randomly from different sources (water, meat, vegetable, milk, and cattle feces) around the North West province in South Africa, between June 2018 and March 2020. The samples comprised 59 water (42 from boreholes and 17 from rivers) and 47 meat (12 wors (a South African traditional home-made sausage that is commercially available), 10 Russian (a South African sausage-shaped packed meat with a 'strong meaty flavour and an intense smoky finish), 10 mince, and 15 red meat) samples. In addition, 23 vegetable samples (comprising 6 carrots, 5 cucumber, 4 lettuce, 4 cabbage, and 4 spinach), 34 milk (22 pasteurized and 12 unpasteurized), and 44 cattle feces samples were collected. The water samples were collected from eight sites, including Mafikeng, Potchefstroom, Klerksdorp, Zeerust, Vryburg, Marikana, Lichtenburg, and Groot Marco (Figure 2). For the collection of cattle feces, aseptic procedures were followed, and samples were obtained directly from the rectum of the animals, using sterile arm-length gloves, which were changed for each sample collected. The food samples were collected from the shelves of different shops and displayed for consumption by the consumers. Non-pasteurized milk was collected from dairy farms during the milking of cattle and distributed into sterile 250 mL bottles. All samples were transported on ice to the laboratory and processed within 24 h of collection. Figure 2 shows the different areas from which samples were collected.

Bacterial Strains
The reference strains L. monocytogenes (ATCC 19115) and L. ivanovii (ATCC 19119) were used as positive controls, while Escherichia coli (ATCC 25922) was used as negative control in the study. These American-type culture collection strains were purchased from Davies Diagnostics, Randburg, South Africa.

Isolation of Listeria spp.
To process the fecal, meat, and vegetable samples, 25 g of each sample was homogenized in 225 mL of half-Fraser broth (HFB, Acumedia, Lansing, MI, USA), while, for milk samples, 25 mL of each sample was transferred into HFB. Aliquots of 100 mL of water samples were filtered through a 0.45-μm Whatman filter paper (Whatman ® glass microfiber GS filter paper, Separation Scientific SA (Pty) Ltd., Johannesburg, South Africa) using a vacuum pump machine (model N035AN.18, Freiburg, Germany). The membrane filters were inoculated in 225 mL of HFB and incubated aerobically at 30 °C for 24 h. Following incubation, aliquots of 100 µ L from each sample were transferred into 10 mL of Fraser broth (Acumedia) and incubated at 30 °C for 24 h. Tubes with black cultures, resulting from the hydrolysis of esculin, were considered to be presumptively positive for Listeria species, and 100 µ L aliquots were spread-plated on Listeria isolation medium (Oxford formulation, Acumedia) [88]. The plates were incubated at 30 °C for 24-48 h. Pure isolates with greenish-grey morphologies and a concave center surrounded by a black halo (due

Bacterial Strains
The reference strains L. monocytogenes (ATCC 19115) and L. ivanovii (ATCC 19119) were used as positive controls, while Escherichia coli (ATCC 25922) was used as negative control in the study. These American-type culture collection strains were purchased from Davies Diagnostics, Randburg, South Africa.

Isolation of Listeria spp.
To process the fecal, meat, and vegetable samples, 25 g of each sample was homogenized in 225 mL of half-Fraser broth (HFB, Acumedia, Lansing, MI, USA), while, for milk samples, 25 mL of each sample was transferred into HFB. Aliquots of 100 mL of water samples were filtered through a 0.45-µm Whatman filter paper (Whatman ® glass microfiber GS filter paper, Separation Scientific SA (Pty) Ltd., Johannesburg, South Africa) using a vacuum pump machine (model N035AN.18, Freiburg, Germany). The membrane filters were inoculated in 225 mL of HFB and incubated aerobically at 30 • C for 24 h. Following incubation, aliquots of 100 µL from each sample were transferred into 10 mL of Fraser broth (Acumedia) and incubated at 30 • C for 24 h. Tubes with black cultures, resulting from the hydrolysis of esculin, were considered to be presumptively positive for Listeria species, and 100 µL aliquots were spread-plated on Listeria isolation medium (Oxford formulation, Acumedia) [88]. The plates were incubated at 30 • C for 24-48 h. Pure isolates with greenish-grey morphologies and a concave center surrounded by a black halo (due to hydrolysis of esculin) were selected for further identification tests. Two to three colonies were picked from each plate and subjected to Gram staining and biochemical testing, such as the fermentation of carbohydrates (mannitol, rhamnose, dextrose, maltose, and xylose), motility, catalase activity, and β-hemolysis [89]. Samples with positive β-hemolysis and catalase tests fermented most sugars, including rhamnose, dextrose, and maltose, but not mannitol or xylose, were identified as Listeria spp. and sent to molecular identification for confirmation.

Identification of Listeria spp., Virulence Genes, Antibiotic Resistance Genes and Biofilm Genes Using PCR (Polymerase Chain Reaction) Technique
Except otherwise mentioned, all PCR reagents used in this study were from Fermentas (Waltham, MA, USA), and all primers were synthesized by Inqaba Biotechnical (Pty) Ltd. (Pretoria, South Africa). DNA was subjected to bacterial 16S rRNA gene amplification, and the amplicons were sent to Inqaba Biotechnical (Pty) Ltd. (Pretoria, South Africa) for the 16S rRNA sequencing identification. The prs gene was also used for the detection of Listeria spp., and the primers (jograyi, lin0464, liv22228, lmo1030, iseelin and lwe1801) were used for the detection of Listeria specific species (Table S1). DNA from L. monocytogenes ATCC 19115 and L. ivanovii ATCC 19119 were used as positive controls, while nuclease-free water was used as a negative control. The C1000 Touch TM DNA thermal cycler (Bio-Rad, Singapore) was used to perform the amplifications. PCR primer sequences, target genes, amplicon sizes, and cycling conditions are shown in Table S1.
All PCR amplicons were separated by electrophoresis on a 2% (w/v) agarose gel comprising 0.001 µg/mL ethidium bromide. A 100 bp DNA molecular weight marker (Fermentas) was incorporated in each gel run. Electrophoresis was conducted at 60 Volt for 120 min and the images captured using a ChemiDoc Imaging System (Bio-Rad, UK).

Antimicrobial Susceptibility Test
The Kirby-Bauer disc diffusion technique was used to determine the antimicrobial susceptibility profiles of the isolates [93]. A total of 12 antibiotics (Mast Diagnostics, UK) were used, including ampicillin (AMP), 10 µg; erythromycin (E), 15 µg; kanamycin (K), 30 µg; norfloxacin (NOR), 10 µg; novobiocin (NB), 30 µg; tetracycline (TET), 30 µg; oxacillin (OX), 5 µg; clindamycin (DM), 2 µg; nalidixic acid (NA), 30 µg; gentamicin (GM), 15 µg; meropenem (MEM), 10 µg; and trimethoprim and sulfamethoxazole (SXT), 1.25 + 23.75 µg. The selection of these antibiotics was based on the treatment regimen for listeriosis or their extensive use as prophylactics in beef and dairy cattle farming in South Africa. Overnight pure cultures were prepared in 15mL TSB (tryptic soy broth) and incubated at 37 • C for 24 h. The bacterial suspension was adjusted by performing serial dilution until reaching 10 6 cells/mL on the McFarland standards scale of 0.5. After 24 h, the bacterial cell suspension were spread on Mueller Hinton agar (Lasec SA (Pty) Ltd, Johannesburg, South Africa) plates using a sterile swab, the plates were incubated aerobically for 24 h at 37 • C, and the diameter of the zones of inhibition around the discs were measured [94]. The results were interpreted based on the antibiotic growth inhibition zone diameter critical values, as recommended by the Clinical Laboratory Standards Institute (CLSI) guidelines [95], in order to classify the isolates as sensitive, intermediate, or resistant to a particular antibiotic. For quality control, L. monocytogenes ATCC 19115 and L. ivanovii ATCC 19119, were used as reference.

Biofilm Formation Phenotypic Detection
Biofilm formation by L. monocytogenes was determined using the microtiter plate assay described by Bertrand et al. [96] and Mahm [97] with slight modifications. L. monocytogenes strains were sub-cultured in tryptic soy broth (TSB) supplemented with 0.6% yeast extract (TSBYE) and incubated at 37 • C overnight. A 10 µL aliquot of each overnight culture at 10 5 CFU was used to inoculate 96-well plates containing 190µL of brain-heart infusion (BHI) broth (Lasec SA (Pty) Ltd, Johannesburg, South Africa) per well. Each isolate was loaded in triplicates. Two positive controls, Pseudomonas aeruginosa (strong biofilm producer) and L. monocytogenes ATCC 19115 were included, while a negative control well contained 200 µL of BHI only. For all samples, plates were incubated at three different temperatures (4, 25, and 37 • C) for 24, 48, and 72 h. After incubation, the plates were rinsed three times with PBS (300 µL, pH 7.4) to remove unattached microbial cells, and excess PBS was removed by blotting the plate using a clean paper towel and allowed to dry. The dye of crystal violet (0.1%, 200 µL) was added to each well to stain the biofilms formed by the cells on well bottom and incubated at room temperature for one hour. The wells were rinsed three times with 300 µL of PBS, and aliquots of 95% ethanol (200 µL) were added to each well for stain fixation, with incubation at room temperature for an hour. The microplate reader (Shenzhen Heales Technology Development Co., Ltd., Shenzhen, China) was used to read the optical density at 630 nm. The cut-off value of the negative control (ODc) was determined as three standard deviations (SD) above the mean OD of the negative control. As stipulated by Stepanovi'c et al. (2000), biofilm formation was categorized into four groups, according to the ODs obtained: OD ≤ ODc, non-adherent; ODc < OD ≤ 2 x ODc, weak biofilm formation; 2 x ODc < OD ≤ 4 x ODc, moderate biofilm formation; and 4 x ODc < OD, strong biofilm formation.

Conclusions
The current study revealed the occurrence of Listeria spp. from various sample types. Our finding showed that L. monocytogenes carried virulence genes and was resistant to several antibiotics. In addition, further investigation indicated strong biofilm formation by L. monocytogenes strains. This clearly suggests that L. monocytogenes have all the features that contribute to pathogenicity, while being difficult to control, due to the resistance. Hence, effective alternative measures (such as greater attention to good manufacturing and hygiene practices throughout the food production, distribution, and storage chains, including in the home environment) are needed to control and prevent resistant L. monocytogenes and reduce the risk of listeriosis.  Table S1: List of oligonucleotide primer sequences and PCR conditions used for the identification of Listeria spp.; Table S2: List of oligonucleotide primer sequences and PCR conditions used for detection of virulence genes in L. monocytogenes; Table S3. List of oligonucleotide primer sequences and PCR conditions used for detection of antibiotic resistance genes in L. monocytogenes; Table S4. List of oligonucleotide primer sequences and PCR conditions used for detection of genes associated with biofilm formation in L. monocytogenes; Refs [74,[96][97][98][99][100][101][102][103][104][105][106][107][108][109] are cited in the supplementary materials.  Table S1: List of oligonucleotide primer sequences and PCR conditions used for the identification of Listeria spp.; Table S2: List of oligonucleotide primer sequences and PCR conditions used for detection of virulence genes in L. monocytogenes; Table S3. List of oligonucleotide primer sequences and PCR conditions used for detection of antibiotic resistance genes in L. monocytogenes; Table S4. List of oligonucleotide primer sequences and PCR conditions used for detection of genes associated with biofilm formation in L. monocytogenes; Refs [74,[96][97][98][99][100][101][102][103][104][105][106][107][108][109] are cited in the supplementary materials.