Introduction

Salmonella Typhimurium is one of the major zoonotic bacteria that can cause serious gastroenteritis, typhoid fever, diarrhea, and sepsis (Taylor and Winter, 2020). This bacterium is usually transmitted to humans via the consumption of poultry products, or sometimes by contaminated surfaces (Shen et al., 2022). Although Chinese official data for Salmonella contamination in foods is not available, many studies have indicated that the prevalence of Salmonella was found to be 10.7–71.8%, with S. Typhimurium being one of the most predominant serovars (Zhou et al., 2018). In addition, studies have declared that the major concerns of S. Typhimurium in the food industry are the ability to acquire antimicrobial resistance and form biofilms on surfaces (Merino et al., 2019; Wang et al., 2022).

Antibiotics are used to both control the animal infections caused by Salmonella, and alleviate the widespread of salmonellosis among humans. Unfortunately, with the overuse of antibiotics in agriculture, S. Typhimurium is becoming highly resistant to a variety of antibiotics including lincomycin, tetracycline, quinolones, sulfamethoxazole, and oxytetracycline (Ingle et al., 2021; Tian et al., 2021). The occurrence of multidrug-resistant S. Typhimurium could cause treatment failure in animal husbandry and constitute huge threats to public health.

Biofilm formation has been proved to be a key factor that contributes to the ineffectiveness of antibiotics, which allows for the persistence of S. Typhimurium in the food industry (Yuan et al., 2021). Defined as the assemblage of microbes within the self-produced extracellular polymeric substances (EPS), biofilms are a regular mode for bacterial growth in nature, which account for around 80% of bacterial infections globally (Yuan et al., 2020). Many studies have indicated the occurrence of S. Typhimurium on meat products, glasses, plastics, stainless steel, and rubber in poultry processing environments, and the -presence in the above environs is strongly linked to its biofilm-forming capacity (Lee et al., 2020). Once formed, S. Typhimurium biofilms are believed to show much higher resistance to antibiotics compared to their free-living counterparts, leading to the continuous transmission of foodborne diseases in the food industry (Yuan et al., 2020).

In this study, the biofilm-forming capacity and antibiotic resistance profiles of S. Typhimurium isolates from a swine slaughterhouse were evaluated. In addition, genome sequencing was performed to determine the genes of a unique S. Typhimurium isolate with multi-characteristics of high biofilm-forming ability and multidrug resistance. The results could provide new insights into the persistence of S. Typhimurium in the food industry, which would be valuable to effectively control Salmonella contamination in the food industry.

Materials and Methods

Bacterial strain and culture conditions

In this study, 39 S. Typhimurium isolates were isolated from a swine slaughterhouse in Jiangsu, China, from October 2016 to April 2017 (Zhou et al., 2018). The cultures of S. Typhimurium were stored at -80°C in tryptic soy broth (TSB, Difco, USA), which contained 20% (v/v) glycerol.

Biofilm formation by S. Typhimurium

Biofilm formation by S. Typhimurium was determined according to the crystal violet staining assay described by Yuan et al. (2018). In brief, aliquots of 200 μL bacterial culture at a concentration of 10⁴ CFU/mL in TSB were added into six wells of a 96-well plate (Costar, Corning, USA). Six negative controls with only TSB were also included. After incubation for 24 h at 37°C, the medium was gently poured, and each well was washed with sterile phosphate buffer saline (PBS), followed by fixing with methanol. Each well was stained by 200 μL of 0.05% (w/v) crystal violet solution (Sigma, USA) for 10 min, and washed with PBS. Adhered crystal violet in each well was then solubilized in 200 μL of 33% (v/v) acetic acid. The microplate reader (Thermo Fisher, USA) was used to measure optical density (OD) of the solution in wells at 594 nm. Biofilm-forming ability of each S. Typhimurium isolate was expressed by cut-off OD (ODc), which was defined as the mean OD of negative control plus three standard deviations (SD). Biofilm-forming capacity of each S. Typhimurium isolate was classified as the following groups: strong biofilm former (OD > 4ODc), moderate biofilm former (4ODc ≥ OD > 2ODc), weak biofilm former (2ODc ≥ OD > ODc), and no biofilm former (ODc ≥ OD) (Diaz et al., 2016). All results are expressed as mean ± SD obtained from assays with three replicates.

Biofilm formation of S. Typhimurium isolates on stainless steel surface was measured by following the assay as described by Yuan et al. (2018). Stainless steel coupons (with a square size of 1 × 1 cm) were immersed in acetone, washed with 75% (v/v) ethanol, rinsed with water, and then autoclaved before use. The overnight culture of each S. Typhimurium isolate was inoculated in 100 mL TSB in a flask containing coupons to obtain the final bacterial concentration of 10⁴ CFU/mL. After the incubation at 37°C for 24 h, stainless steel coupons were removed, washed with sterile PBS (pH 7), and transferred to a tube containing 10 g of glass beads and 10 mL of sterile PBS (pH 7). Adhered cells were detached from stainless steel surface by vortex mixing for 120 s. Each bacterial suspension was decimally diluted and plated onto TSA plates, followed by the incubation for 48 h at 28°C. All results are expressed as mean ± SD obtained from assays with three replicates.

Antimicrobial resistance of the high-biofilm former S. Typhimurium M3

Antibiotic resistance of the high-biofilm former S. Typhimurium M3 was assessed based on the diffusion method of Clinical Laboratory Standards Institute (CLSI) guidelines (2016). Commercial antibiotic discs (HiMedia, India) contain 22 antibiotics (Table 1). In brief, 100 μL of overnight S. Typhimurium M3 culture was spread onto TSA plates. Then, antibiotic discs were placed on plates, followed by the incubation for 20 h at 37°C. Inhibition zones (mm) were measured and categorized as susceptible (S), intermediate (IR), and resistant (R) according to each zone diameter of ≥20, 15–19, and ≤14 mm, respectively.

Defined as the lowest concentration of antibiotics that can inhibit bacterial growth, the MIC of antibiotics in S. Typhimurium M3 was assessed by the broth microdilution method (Dawan and Ahn, 2020). In brief, different dilutions of antibiotics and S. Typhimurium M3 were inoculated in 96-well plates at 37°C for 24 h.

Genome sequencing of S. Typhimurium M3

Genomic DNA of S. Typhimurium M3 was extracted by E.Z.N.A.^® Bacterial DNA Kit (Omega, USA) according to the instructions. DNA quality was assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Preparation for library was performed based on Illumina’s TruSeq Nano DNA Sample Prep Kit. Whole genome of S. Typhimurium M3 was sequenced by Illumina novaseq 6000 (Illumina Inc, San Diego, USA). Raw paired-end reads were trimmed and filtered using Trimmomatic, and sequences were assembled by ABySS. Genes in the genome of S. Typhimurium M3 were predicted with GeneMarkS. All gene models were blastp against nonredundant (NR) database, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Cluster of Orthologous Group (COG) for functional annotation. A circular map of S. Typhimurium M3 genome was drawn using Circos. Antibiotic resistance genes in the S. Typhimurium M3 genome were predicted by Resistance Gene Identifier against the Comprehensive Antibiotic Resistance Database (CARD).

Results and Discussion

Biofilm formation by S. Typhimurium isolates

Biofilm formation by S. Typhimurium in food industry may cause huge economic losses and serious safety issues globally. In this study, the biofilm--forming capacity of each S. Typhimurium isolate was quantified by the crystal violet staining assay. The OD₅₉₄ values of S. Typhimurium isolates ranged from 0.017 to 1.098, and the highest OD₅₉₄ value was obtained from S. Typhimurium M3. Biofilm-forming ability was proved to be in a strain--dependent way, as 9, 12, and 18 isolates were classified as weak, moderate, and strong biofilm formers, respectively (Figure 1A). High biofilm-forming ability of S. Typhimurium was also observed in previous studies, providing them survival strategies against antibiotics (Lee et al., 2020). Characteristics of different food-contact surfaces could influence bacterial attachment and biofilm formation of microorganisms. In this work, isolates belonging to S. Typhimurium were also measured for their biofilm--forming capacity on stainless steel surface to simulate the real situation in the food industry. Cell counts recovered from biofilms of all S. Typhimurium isolates were in a range from 2.96 to 8.06 log CFU/cm² (Figure 1B). High biofilm-forming capacity (7.95 log CFU/cm²) of S. Typhimurium M3 on stainless steel coupon was also found. A previous study proved that Salmonella could be transferred from biofilms formed on stainless steel coupons to poultry products, posing safety issues to consumers (Wang et al., 2015). Therefore, the high biofilm-forming ability of S. Typhimurium observed in this study indicates the need to optimize the cleaning and disinfection procedures to avoid Salmonella biofilm contamination.

Figure 1. Biofilm formation of 39 Salmonella Typhimurium isolates on both polystyrene (A) and stainless steel coupons (B).

Antibiotic resistance of S. Typhimurium M3

The wide spread of multidrug-resistant Salmonella has made it a threat to public health. The high biofilm--forming isolate S. Typhimurium M3 was selected to test its susceptibility to 22 antibiotics by the diffusion method (Table 1). S. Typhimurium M3 was resistant to oxacillin, lincomycin, rifampicin, tetracycline, and clindamycin, and intermediate resistant to gentamicin, streptomycin, polymyxin B, novobiocin, doxycycline, azithromycin, and erythromycin. Furthermore, the MICs of oxacillin, lincomycin, rifampicin, tetracycline, and clindamycin were 512, 32, 16, 64, and 64 ug/mL, respectively. Occurrence of resistance to these antibiotics of S. Typhimurium have already been proved in different countries (Dawan and Ahn, 2020; Harb et al., 2018). It is speculated that the resistance to multiple antibiotics may be caused by several mechanisms, including altering the cell membrane permeability, modifying the site of drug action, interfering with DNA synthesis, and affecting the structure of cell wall (Enrique et al., 2020).

Genome properties of S. Typhimurium M3

This work also aims to determine the genome properties of S. Typhimurium M3 with unique multi--characteristics of high biofilm-forming and multidrug resistance. The draft genome sequence of S. Typhimurium M3 had a length of 4881503 bp, a GC content of 52.17%, which contained 4579 coding sequences, 81 tRNAs, 8 rRNAs (6 of 5S rRNA, 1 of 16S rRNA, and 1 of 23S rRNA) (Table 2). The circular representation of S. Typhimurium M3 draft genome is shown in Figure 2.

Figure 2. Genomic circle map of Salmonella Typhimurium M3.

The proteins with functional assignments of S. Typhimurium M3 included 2276 with GO assignments, 3882 with eggNOG, 138 with CARD, and 3171 with KEGG pathway mapping. The COG protein database was generated by the comparison of predicted and known proteins in completely sequenced microbial genomes to infer sets of orthologs (Figure 3). In this study, the COG annotation showed that transcription, carbohydrate transport and metabolism, energy production and conversion, and amino acid transport and metabolism were the most abundant categories. Furthermore, KEGG annotation showed that 2003, 485, 263, and 245 genes were participated in overview maps, carbohydrate metabolism, amino acid metabolism, and membrane transport, respectively (Figure 4).

Figure 3. Cluster of orthologous group annotation of genes in Salmonella Typhimurium M3.

Figure 4. Kyoto encyclopedia of genes and genomes pathway enrichment results of genes in Salmonella Typhimurium M3.

The genome analysis of S. Typhimurium M3 showed several genes related to antibiotic resistance by antibiotic efflux, antibiotic target alteration, and antibiotic inactivation (Table 3). Antimicrobial resistance genes were predicted based on the CARD database, and S. Typhimurium M3 contained 18 genes connected with resistance to tetracycline (tetA), fluoroquinolone (gyrB, parC, parE), triclosan (gyrA), sulfonamide (folP), nitrofuran (nfsA), elfamycin (tuf), aminoglycoside (AAC6), rifamycin (rpoB), fosfomycin (uhpA, uhpT, ptsI, murA, cyaA, glpT), cephalosporin (ampH), and isoniazid (fabI). Remarkably, in this study, 36 genes belonged to several gene families, including ATP-binding cassette (ABC) antibiotic efflux pumps (soxS, soxR, phoP, msbA, tolC, yojI), resistance-nodulation–cell division (RND) antibiotic efflux pumps (acrA, acrE, acrR, baeR, baeS, cpxA, golS, marR, mdsA, mdsB, mdsC, sdiA, crp, marA, acrB, acrD, acrF, mdtA, mdtB, mdtC, ramA), major facilitator superfamily (emrA, emrB, emrR, hns, mdtG, mdtH, mdtM), multidrug and toxic compound extrusion (MATE) transporters (mdtK), and KdpDE (kdpE) identified in the genome of S. Typhimurium M3. The presence of efflux pumps makes S. Typhimurium isolates insensitive to tetracycline, oxacillin, lincomycin, rifampicin, streptomycin, clindamycin, kanamycin, ciprofloxacin, norfloxacin (Ge et al., 2022; Zwama and Nishino, 2021).

In this study, key genes encoding for chemotaxis, motility, surface attachment, quorum sensing (QS), matrix protein-encoding genes, biofilm transcriptional regulators, and matrix polysaccharide synthesis genes were also identified (Table 4). Some of the adhesion factors, such as flagella and pili, are vital for biofilm formation (Yuan et al., 2020). Genes related to flagellar (flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, flgL, flgM, flgN, flgO, flgP) identified in this study are essential for attachment but can serve different roles during Salmonella biofilm formation. Gene coding for -chemotaxis (cheV) in this study is involved in the transmission of sensory signals from the chemoreceptors to the flagellar motors. QS is the bacterial cell-to-cell communication signaling mechanism, which can regulate bacterial motility and biofilm formation (Yuan et al., 2018). The luxS gene detected in this study is responsible for the production of QS signaling molecule by converting S-ribosyl-L-homocysteine into homocysteine and DPD. EPS of S. Typhimurium biofilms is composed of cellulose, curli, and biofilm--associated protein, and can support the adherence and surface attachment of S. Typhimurium onto different surfaces (Maruzani et al., 2019). In this study, the genes for cellulose biosynthesis (bcsA, bcsB, bcsC, bcsE, bcsF, bcsG, bcsQ), curli production (csgA, csgB, csgE, csgF, csgG), fimbriae biogenesis (fimA, fimC, fimF, fimH, fimW, fimY, lpfB, yhcD, yhcA, mrkB) were identified. Biofilm formation by Salmonella has proved to be regulated by a complex genetic network involving interactions between regulators, and the major regulators including csgD, adrA, and flk were identified in this study. CsgD encoded by csgD is the biofilm control center to regulate expressions of major Salmonella biofilm constituents, control the transition between planktonic and biofilm cells, and even maintain the 3-D structure of biofilms (Chen et al., 2021). AdrA regulates genes encoding cellulose biosynthesis of Salmonella by changing cellular levels of c-di-GMP during biofilm formation (Chen et al., 2021). The large protein BapA (encoded by bapA) of S. Typhimurium was shown to be necessary for bacterial aggregation and pellicle formation by interconnecting individual cells.

Conclusion

In summary, the high biofilm-forming capacity and multi-drug resistance of S. Typhimurium from swine slaughterhouse evidenced by in vitro and genomic sequencing tests confirmed their persistence in the food industry. The results have significant implications for the food safety and public health globally, highlighting the importance of good hygienic practices and appropriate use of antibiotics in the industry. Furthermore, the development of more appropriate and effective approaches for control of Salmonella contamination control in the food industry are also critical.