A total of 53 LAB isolates (out of an initial set of > 200 novel strains) from cattle feces and different food sources including meat, fruits, and vegetables that showed initial antagonistic activity against L. monocytogenes, Salmonella, and E. coli O157:H7 were obtained from the stock culture collection of the International Center of Food Industry Excellence at Texas Tech University (ICFIE: TTU). Isolates were streaked for isolation onto de Man Rogosa and Sharpe (MRS) agar plates (Merck, Darmstadt, Germany), and incubated aerobically at 37°C for 48 h to obtain well-isolated colonies. A single colony was selected and grown in 9-ml of fresh MRS broth (Criterion, Hardy Diagnostics, CA, United States) at 37°C for 12–18 h. Nine of the 53 LAB strains did not further grow in MRS broth and were removed from the study. Pure cultures of 44 remaining LAB strains were grown up in MRS broth as described above, preserved on cryobeads (Key Scientific, Stamford, TX, United States) and stored at -80°C until further use. Foodborne pathogen isolates including L. monocytogenes, Salmonella and E. coli O157:H7 were streaked onto Tryptic Soy Agar (TSA, Becton, Dickinson, Le Pont de Chaix, France) and incubated at 37°C for 18–24 h. A single colony of each pathogen was selected and grown individually in 9-ml of Brain Heart Infusion (BHI) broth (Merck, Darmstadt, Germany). Overnight cultures of LAB and pathogenic strains were used to perform agar-well diffusion and competitive exclusion assays as detailed below to determine the antagonistic activity of all 44 LAB strains against each pathogen.

This set of experiments was performed based on the method described previously by Vinderola et al. (2008) with slight modifications. Overnight cultures (incubated in nutrient rich media for 12–18 h at 37^oC) of foodborne pathogens were diluted to achieve a concentration of 10⁵ CFU/mL, swabbed onto BHI plates and incubated at 37^oC for 24 h to create a lawn. To assay the antagonistic activity of each LAB strain against each foodborne pathogen, five 6-mm wide wells cut into a BHI agar plate were filled with a 100 μl aliquot of the following: (i) 10⁸ CFU/mL of each LAB strain overnight culture in duplicate wells, (ii) cell-free filtrate (CFF) of LAB overnight cultures, passed through a sterile 0.45 μM filter (Sigma-Aldrich, St. Louis, MO, United States), in duplicate wells, and (iii) MRS broth (control) in a single well. Plates were first incubated at 4°C for 2 h to allow suspensions to diffuse in the agar followed by incubation for 24 h at 37°C (Zavisic et al., 2012). Antagonistic activity was determined by the development of clear zones of inhibition around each well and measured using a caliper. Overall scores of inhibition were calculated by summing up values observed for overnight cultures and CFF across all three pathogens, and potential LAB probiotic strains were ranked for antagonistic activity against all three foodborne pathogens.

Overnight cultures of each pathogen and LAB strains were prepared as described above for agar well diffusion assays and co-inoculated at 10⁵ and 10⁶ CFU/mL, respectively in Tryptic Soy Broth (TSB; Oxoid Ltd., Basingstoke, United Kingdom) supplemented with 1 g l^-1 Tween 80 (Acros, Organics, NJ, United States) and incubated at 37°C with slight agitation (130 rpm). A previous study demonstrated that addition of tween to TSB allows growth of both LAB and gram-negative pathogens, including E. coli O157:H7 and Salmonella (Cálix-Lara et al., 2012). Samples were diluted and plated onto Modified Oxford Agar (MOX; Becton, Dickinson and Company), Xylose Lysine Tergitol 4 Agar (XLT4; Becton, Dickinson and Company), MacConkey agar with sorbitol (SMAC; Criterion, Hardy Diagnostics, CA, United States), and MRS agar plates to enumerate L. monocytogenes, Salmonella, E. coli O157:H7, and LAB strains, respectively, at 0, 6, 12, and 24 h of co-inoculation. MOX, XLT4, and SMAC plates were incubated at 37°C for 24 h, and MRS plates were incubated at 37°C for 48 h. Antagonistic activity of each LAB strain was determined by pathogenic reduction with respect to control samples (pathogen cultures without LAB) at each time point. As for agar well diffusion assays, pathogenic reductions were summed up across all time points and across all three pathogens, and potential LAB probiotic strains were ranked based on their antagonistic effect. The top 20 ranking LAB strains from the agar well diffusion and competitive exclusion broth assays (n = 28 LAB strains in total) were selected for further phenotypic and genotypic evaluation by the assays below.

The human epithelial intestinal cell line Caco-2 was used to evaluate the in vitro ability of the top ranking LAB strains to adhere to the GIT. Caco-2 cells (ATCC HTB-37^TM) were maintained in Eagle’s Minimum Essential Medium (EMEM, ATCC 30-2003^TM) supplemented with 20% fetal bovine serum (FBS, ATCC 30-2020^TM) and 1% of penicillin/streptomycin solution (10,000 U/mL of penicillin and 10,000 μg/mL streptomycin) (PenStrep) (Gibco, Thermo Fisher Scientific) at 37°C in a water-jacketed incubator with 5% CO₂. Three days before attachment assays were performed, Caco-2 cells were seeded into 24-well tissue culture plates at a target density of 5 × 10⁴ cells/well to achieve a confluent density of 1 × 10⁵ cells/well in EMEM supplemented with 20% FBS and 1% PenStrep. For each attachment assay, cell culture media (1 mL/well) was removed and replaced with antibiotic-free EMEM medium. Duplicate confluent Caco-2 cell monolayers were then inoculated with an average 1.6 × 10⁷ CFU/ ml (CI 95%: 1.1 × 10⁷–2.0 × 10⁷) of each LAB strain to be analyzed resulting in a multiplicity of infection (MOI) of 52.4, as determined by plating the LAB inoculum on MRS plates and counting the Caco-2 cells in a Neubauer chamber. Inoculated Caco-2 cell plates were returned to the water-jacketed incubator for 30 min to allow for LAB attachment. Caco-2 cell monolayers were washed once with 1 mL sterile phosphate-buffered saline (PBS, Thermo Scientific, Rockford, IL, United States) to remove non- or loosely adherent bacteria. Caco-2 cells were then lysed by addition of 1 mL of ice-cold water to release adherent LAB bacteria. Appropriate serial dilutions were plated onto MRS plates, incubated and LAB were enumerated as detailed above. The attachment efficiency of each LAB strain was assayed in two biologically independent experiments each with two technical replicates. Attachment efficiency was calculated by dividing the average number of adherent bacterial cells (across biological and technical replicates) by the number of Caco-2 cells in each well.

The in vitro cytotoxicity of the top ranking LAB strains against Caco-2 cells was evaluated by using a CytoTox 96 non-radioactive cytotoxicity kit (Promega, Madison, WI, United States), which measures release of lactate dehydrogenase (LDH) upon Caco-2 cell lysis, following manufacturer’s recommendations. Briefly, Caco-2 cells were seeded on a 24-well plate and grown to confluence as described above. Caco-2 monolayers were inoculated with each LAB strain at a level of 1.6 × 10⁷ CFU/ml (CI 95%: 1.1 × 10⁷–2.0 × 10⁷) and cytotoxicity was evaluated after 24 h by measuring absorbance (release of LDH) in a microplate reader (Biotek Instruments, Winooski, VT, United States) at 490 nm. The cytotoxicity of each LAB strain was evaluated in two biologically independent experiments each containing two technical replicates. In each independent experiment, two un-inoculated Caco-2 monolayers were included and used as maximum lysis controls (according to the kit instructions) and two un-inoculated Caco-2 monolayers were included to determine the background absorbance associated with the medium. Background absorbance was averaged and subtracted from all individual observed values for Caco-2 wells inoculated with a LAB strain within each independent experiment. Percent cytotoxicity was expressed as the adjusted average absorbance value (subtracting background absorbance) for each LAB strain divided by the absorbance for the maximum lysis control.

Antimicrobial susceptibility profiling was performed following the National Antimicrobial Resistance Monitoring System (NARMS) protocol (FDA, 2016). LAB strains were streaked onto TSA plates and incubated at 37°C for 24 h. A single colony of each LAB strain was selected and sub-streaked onto TSA plates containing 5% defibrinated sheep blood (HemoStat Laboratories, Dixon, CA, United States) and incubated at 37°C for 24–48 h. Antibiotic susceptibility was evaluated using the Sensititre^TM Gram-positive MIC plate assayed on the Sensititre^TM automated antimicrobial susceptibility system (Trek Diagnostic Systems, Westlake, Ohio) following manufacturer’s instructions. Enterococcus faecalis ATCC 29212 was used as a quality control organism. Thirteen antimicrobial agents, included in the MIC plate assayed, were evaluated in this study including: ampicillin (AMP), clindamycin (CLI), daptomycin (DAP), erythromycin (ERY), gentamicin (GEN), levofloxacin (LVX), linezolid (LZD), penicillin (PEN), rifampicin (RIF), synercid (SYN), tetracyclin (TET), trimethoprim/sulfamethohazole (SXT), and vancomycin (VAN). The Minimum Inhibitory Concentration (MIC) breakpoints for the antimicrobials tested were interpreted based on the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2017).

A total of 28 novel LAB strains with antagonistic characteristics toward L. monocytogenes, E. coli O157:H7 and Salmonella were selected for further genotypic characterization. LAB strains were cultivated in MRS broth as detailed above, genomic DNA (gDNA) was isolated and purified using the Invitrogen Purelink DNA Extraction kit (ThermoFisher Scientific, Waltham, MA, United States). Pure gDNA was quantified using a Qubit^® 2.0 Fluorometer (Life Technologies, CA, United States), and used for library preparation with the Nextera XT v2.0 kit (San Diego, CA, United States) as per manufacturer’s recommendations. DNA libraries were subjected to paired-end sequenced using the 2 × 250 basepair (bp) V2 sequencing kit on an Illumina MiSeq platform (Illumina Inc., United States). Raw reads were preprocessed and filtered using Trimmomatic version 0.36 (Bolger et al., 2014), which was followed by de novo assembly using SPAdes version 11 (Bankevich et al., 2012). Resultant scaffolds were annotated using Prokka v1.13 (Seemann, 2014) and 31 conserved amino acid coding sequences were identified through the AMPHORA2 pipeline (Wu and Scott, 2012). The 31 conserved amino acid coding sequences were aligned and a concatenated alignment was created to compare our novel LAB strains to a large background of other strains representing genera in the LAB order. Taxonomic identification for each of our LAB strains was determined based on the highest confidence gene set.

Bacteriocins, virulence factors, and potential AMR genes were identified by comparing genome untranslated gene sequences identified during genome annotation to the BAGEL3, Virulence Finder, ResFinder and PlasmidFinder databases, respectively (Zankari et al., 2012; van Heel et al., 2013; Joensen et al., 2014). Un-gapped alignments with higher than 95% identity and 95% query coverage were identified as positive for the virulence factors and AMR genes were used to confirm the presence of these genes. A phylogenetic tree was generated using the concatenated alignment in RAxML (Randomized Axelerated Maximum Likelihood) (Stamatakis, 2014) on the CIPRES science gateway (Miller et al., 2010).

Antimicrobial resistance-encoding genes identified by using the ResFinder v3.0 (Zankari et al., 2012) and PlasmidFinder v2.0 (Carattoli et al., 2014) pipelines from the Center for Genomic Epidemiology website¹ were compared using BLASTn against the GenBank nucleotide database using default settings for sequence identification. A multiple genome alignment was created using Mauve software (v2.4.0) to compare plasmid sequences identified in strains L22, L24-A, and L25 against plasmid sequence data from Enterococcus faecium (accession number KJ645709).

Confidence intervals (95%) for attachment and cytotoxicity assay data were estimated using the mean and standard deviation of the ratio of bacterial cells to Caco-2 cells (attachment efficiency) and percent cytotoxicity (calculated by dividing adjusted average absorbance values by the maximum lysis control absorbance value in each experiment), respectively. Attachment efficiency and percent cytotoxicity values were analyzed using an ANOVA followed by the Bonferroni familywise error correction for multiple comparisons. Strain to strain comparisons were made to identify statistically significant differences using R coding language (R CoreTeam, 2017). The R packages: ggplot, phangorn, tidytree, ggtree, phylotools, and ape (Wickham, 2009; Schliep, 2011; Popescu et al., 2012; Revell, 2012; Yu et al., 2017) were used to describe the relationship between attachment efficiency, percent cytotoxicity and number of predicted bacteriocin and virulence genes.

Thirty-eight of the LAB strains showed an antagonistic effect against at least one of the pathogens tested (Salmonella, E. coli O157:H7 and L. monocytogenes), the remaining six did not inhibit or reduce the growth of the pathogens analyzed in this study. Thirty-seven of the LAB strains showed antimicrobial activity against L. monocytogenes with clear zones of inhibition ranging from 8.5 to 24 mm when overnight LAB cultures were used and from 6.5 to 21 mm when CFF was used. Twenty strains were antagonistic against E. coli O157:H7 with a maximum inhibition zone of 12 mm when L15 strain was used, and 18 were antagonistic against Salmonella, with a maximum zone of inhibition of 11.5 mm produced by strain L24-B. No zones of inhibition were observed for either E. coli O157:H7 or Salmonella when only CFF was used. Overall scores of inhibition summed across overnight culture and CFF for all three pathogens ranged from 33.5 to 45 mm for the top 20 LAB strains. Strain L20-B produced the highest ranking inhibition but was not inhibitory against Salmonella or E. coli O157:H7. Strain L28 was the highest ranking antagonistic strain that was effective against all three pathogens. Twelve of the top 20 LAB strains were originally isolated from a bovine source (i.e., cattle feces or raw meat), while the eight remaining isolates were isolated from fruits (n = 6) or vegetables (n = 2). Overall, increased antimicrobial activity was found when overnight cultures were used compared to CFF (Table 1).

The antimicrobial activity of all LAB strains evaluated in this study is shown in Supplementary Table 1. Pathogen reductions were analyzed at 6, 12, and 24 h after co-inoculation with respect to un-inoculated control cultures; for E. coli O157:H7 and Salmonella highest reductions were observed 6 h after co-inoculation (2.03 and 2.53 log₁₀ CFU/ml, respectively). At 12 h, highest reductions were 1.12 and 1.36 log₁₀ CFU/ml for E. coli O157:H7 and Salmonella, respectively. Twenty-four hours after co-inoculation, 43 of the strains reduced L. monocytogenes by 0.12–9.06 log₁₀ CFU/ml. Strains L28 and L20-B had the highest antimicrobial activity against L. monocytogenes with reductions of 9.06 and 6.96 log₁₀ CFU/ml, respectively. Forty-one of the strains reduced E. coli O157:H7 by 0.02 to 0.84 log₁₀ CFU/ml, where L4-B and L20-B achieved the highest reductions of 0.84 and 0.82 log₁₀ CFU/ml, respectively. Thirty-two of the LAB strains reduced Salmonella by 0.05–0.94 log₁₀ CFU/ml with L24-B and L20-B as the most antagonistic with reductions of 0.94 and 0.84 log₁₀ CFU/ml, respectively. Greater antimicrobial effects were observed against L. monocytogenes compared with the other pathogens evaluated, notably strain L28 completely eliminated L. monocytogenes after 24 h after co-inoculation (Supplementary Table 1).

Pathogen reductions were summed up across time points and all three pathogens, and novel LAB strains were ranked based on their increased antimicrobial effect; strain L28 had the highest reduction across all pathogens tested in this study (Supplementary Table 1). Overall scores for agar-well diffusion and competitive exclusion assays were summed up to determine the top 20 LAB strains (within each assay), L20-B, L28, J7 ranked as the LAB strains with the greatest antagonistic activity. The top 20 strains from each assay (n = 28 strains collectively) were further characterized by cell culture assays, antimicrobial susceptibility and whole genome sequencing.

The ability of LAB strains to adhere to Caco-2 cells after 30 min was evaluated in this study. LAB attachment ranged from 4 to 84 bacterial cells/Caco-2 cell (Table 2). According to Candela et al. (2005) classification of microorganisms based on bacterial adhesive properties, five including L4-B, L8-A, L3-A, L15, and L2A were classified as highly adhesive, with > 40 bacterial cells/Caco-2 cells; 21 of the strains were classified as adhesive with 5–40 bacterial cells/Caco-2 cells and two were classified as non-adhesive with < 5 adherent bacterial cells/Caco-2 cells. The cytotoxic activity of our LAB strain panel was determined based on the release of the stable cytosolic enzyme LDH in the culture medium 24 h after bacterial inoculation. Percent cytotoxicity for the panel ranged from -5 to 8%, with L6B being the least cytotoxic and J16 the most cytotoxic (Table 2 and Figure 1).

All LAB isolates evaluated were susceptible to AMP, PEN, and LZD. Resistance to LVX was the most commonly found in our study (n = 17), followed by CLI (n = 9), VAN and SYN (each with n = 7), TET and SXT (each with n = 6), and RIF and GEN (each with n = 1). Intermediate resistance was mostly commonly observed for ERY, and DAP with 20 and 13 LAB isolates, respectively. Additionally, 7, 4, 3, and 2 LAB isolates exhibited intermediate resistance to GEN, TET, RIF, and SYN, respectively (Table 3). Three of the LAB isolates exhibited resistance to one antimicrobial agent (LVX); 16 were resistant to two antimicrobials, LVX-DAP was the most common AMR profile in this study with 9 of the isolates. Additionally, nine of all the LAB isolates in this study exhibited multidrug-resistance (MDR) with LVX-VAN-SXT as the most common AMR profile with 4 isolates, followed by DAP-TET-CLI-SYN with 3MDR isolates (Table 4).

Genotypic identification by WGS confirmed all as members of the LAB group; Enterococcus was the genus most frequently isolated accounting with 21 of the total LAB isolates. The seven remaining strains were identified as members of the Pediococcus and Lactobacillus genus with 4 and 3 isolates, respectively. E. faecium was the most common species identified in this study with 11 isolates, followed by E. hirae, and P. acidilactici with 5 and 4 LAB isolates, respectively (Table 5 and Figure 2).

In this study between one and six putative bacteriocins were identified in 24 of the top LAB strains analyzed by genome comparison against the BActeriocin GEnome mining tool Database. Putative bacteriocins identified included enterolysin A, enterocin, lactacin F, sactipeptides, pediocin, closticin, lasso peptide, lanthipeptide, salivaricin, colicin V, and carnocinCP52. Enterolysin A was the most common being found in 15 of the isolates, followed by enterocins, and lactacin F found in 9, and 7 of the LAB strains, respectively (Table 6). No virulence factors were identified for 5 of the isolates, and virulence factors associated with cell adhesion were identified for 13 of the LAB isolates (Table 5).

A total of 23 of the investigated strains carried from one to four acquired genes associated with antimicrobial resistance. The most common AMR-encoding gene identified among the LAB strain set was msr(C) gene (n = 11) encoding for an ABC transporter associated with resistance to erythromycin, other macrolides, or streptrogramin B antibiotics. The msr(C) gene was always found alone and was associated with the phenotypes showing resistant to less antimicrobials (LVX and LVX DAP profiles). A total of four strains exhibited MDR phenotypic profiles (DAP TET CLI SYN, and DAP TET CLI SYN GEN), three of the these strains shared the same genotype: erm(B), aac(6′)-Iid, ant(6)-Ia and tet(M), encoding for a rRNA adenine N-6-methyltransferase, an aminoglycoside 6′-N-acetyltransferase, an aminoglycoside nucleotidyltransferase and a tetracycline resistance protein, TetM, involved in antibiotic target modification, respectively. Additionally, six different plasmid incompatibility types (rep1, rep2, repUS15, rep6, rep9, and repUS1) were characterized to determine if AMR-encoding genes were carried on the chromosome or plasmid. Seventeen strains harbored from one to three plasmids supporting the majority of AMR-genes were likely carried on plasmids. Further investigation elucidated that only plasmids belonging to the incompatibility type repUS1 were carrying antimicrobial genes (Figure 3). LAB strains, L22, L24-A, and L25, carried such plasmid harboring the erm(B) and ant(6)-Ia genes, involved in resistance to macrolides and aminoglycosides, respectively. BLASTn comparison showed that the repUS1 plasmids had 99% similarity out of 47, 48, and 68 of the total plasmid sequence registered under the accession number KJ645709, for the L22, L24-A, and L25 LAB strains, respectively.