key: cord-1031010-muqytwcl
authors: Beier, Ross C.; Andrews, Kathleen; Hume, Michael E.; Sohail, Muhammad Umar; Harvey, Roger B.; Poole, Toni L.; Crippen, Tawni L.; Anderson, Robin C.
title: Disinfectant and Antimicrobial Susceptibility Studies of Staphylococcus aureus Strains and ST398-MRSA and ST5-MRSA Strains from Swine Mandibular Lymph Node Tissue, Commercial Pork Sausage Meat and Swine Feces
date: 2021-11-22
journal: Microorganisms
DOI: 10.3390/microorganisms9112401
sha: f6aa8a80d14b2ce100340459064fb388d43519fa
doc_id: 1031010
cord_uid: muqytwcl

Staphylococcus aureus (S. aureus) causes gastrointestinal illness worldwide. Disinfectants are used throughout the food chain for pathogenic bacteria control. We investigated S. aureus bioavailability in swine Mandibular lymph node tissue (MLT) and pork sausage meat (PSM), established susceptibility values for S. aureus to disinfectants, and determined the multilocus sequence type of MRSA strains. Antimicrobial and disinfectant susceptibility profiles were determined for 164 S. aureus strains isolated from swine feces (n = 63), MLT (n = 49) and PSM (n = 52). No antimicrobial resistance (AMR) was detected to daptomycin, nitrofurantoin, linezolid, and tigecycline, while high AMR prevalence was determined to erythromycin (50.6%), tylosin tartrate (42.7%), penicillin (72%), and tetracycline (68.9%). Methicillin-resistant S. aureus (MRSA) strains, ST398 (n = 6) and ST5 (n = 1), were found in the MLT and PSM, 4 MRSA in MLT and 3 MRSA strains in the PSM. About 17.5% of feces strains and 41.6% of MLT and PSM strains were resistant to chlorhexidine. All strains were susceptible to triclosan and benzalkonium chloride, with no cross-resistance between antimicrobials and disinfectants. Six MRSA strains had elevated susceptibilities to 18 disinfectants. The use of formaldehyde and tris(hydroxylmethyl)nitromethane in DC&R was not effective, which can add chemicals to the environment. Didecyldimethylammonium chloride and benzyldimethylhexadecylammonium chloride were equally effective disinfectants. ST398 and ST5 MRSA strains had elevated susceptibilities to 75% of the disinfectants tested. This study establishes susceptibility values for S. aureus strains from swine feces, mandibular lymph node tissue, and commercial pork sausage against 24 disinfectants. Since it was demonstrated that S. aureus and MRSA strains can be found deep within swine lymph node tissue, it may be beneficial for the consumer if raw swine lymph node tissue is not used in uncooked food products and pork sausage.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen important worldwide because of its human health effects [1] [2] [3] [4] [5] . Toxic shock syndrome is often caused by toxins produced by S. aureus bacteria [6, 7] , and S. aureus is a well-known cause of hospital and community acquired diseases involving the bloodstream, endocarditis, lungs, meningitis, sepsis, skin, and soft tissue infections [8] [9] [10] [11] [12] [13] [14] . The Centers for Disease Control (CDC) has selected S. aureus as one of the top five pathogens causing foodborne illness in cides used are lower than that required to kill the target bacteria, cross-resistance may be exacerbated [69, [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] , and surviving bacteria may develop biofilms resulting in biocide tolerance and increased AMR [70, [83] [84] [85] . The QAC didecyldimethylammonium chloride (DDAC) (referred to as C10AC in our laboratory to indicate the carbon chain length), was found to adsorb physicochemically onto the cell membrane where it could damage and disrupt the S. aureus membrane structure and function [86] . Further mode-of-action studies of C10AC and a mixture of N-alkylbenzyldimethylammonium chlorides (BACs) against S. aureus showed that C10AC formed a double monolayer, and the BACs formed a single monolayer that covered the bacterial cells, resulting in substantial depletion of the potassium pool [87] . A study of the QAC benzalkonium chloride (BKC) against meat-associated Staphylococcus spp. demonstrated an open reading frame (ORF) on the plasmid pST827 that was similar to the QAC resistance genes qacC, ebr, and smr [88] . Hydrogen peroxide and sodium hypochlorite [89, 90] were more effective against S. aureus biofilms than quaternary ammonium chloride (QAC) disinfectants [91] . However, these studies were very limited in the number of S. aureus bacteria studied and the number of disinfectants tested [89] [90] [91] . Previously, our laboratory has also investigated the effects of a wide array of disinfectants on the inhibition of foodborne pathogens, Escherichia coli O157:H7 [92] , Pseudomonas aeruginosa [93] , non-O157 Shiga toxin-producing E. coli strains (STECs) [94] , Salmonella spp. [95] , Campylobacter coli [96] , C. jejuni [70] , and vancomycin-resistant enterococci (VRE) [97] . In all studies C10AC resulted in the best bacterial inhibition by an ammonium chloride except against C. coli, in which both C10AC and the BACs appeared to perform equally well, and synergistically in the complex disinfectant P-128 [96] .

The current study evaluated the susceptibility of 164 S. aureus strains isolated from swine feces, swine mandibular lymph node tissue (MLT), and commercial pork sausage meat (PSM) against 16 antimicrobials, 17 disinfectants, and 7 disinfectant components. Multilocus sequence typing was conducted on the seven MRSA strains found in the MLT and PSM. The disinfectant component susceptibilities in some complex disinfectants were calculated and their individual potencies discussed. The potency of various individual disinfectant component ammonium chlorides with respect to the alkyl carbon chain length of the ammonium chlorides are discussed.

The 164 S. aureus strains were previously isolated from swine feces (n = 63), MLT (n = 49), and PSM (n = 52) [98] . The isolated bacteria were held at −72 • C until used. Prior to experimentation, each S. aureus strain was grown for 24 h at 37 • C on plates containing Trypticase™ Soy Agar with 5% sheep blood (TSA II™) (BD BBL™ Stacker™ Plate, Becton, Dickinson and Company, Sparks, MD, USA).

Antimicrobial susceptibility testing (AST) and disinfectant susceptibility testing (DST) were performed on the 164 S. aureus strains using standard broth microdilution methods according to the Clinical and Laboratory Standards Institute [99, 100] . Mueller-Hinton broth had previously been shown to not influence the results of suspension tests with disinfectants and E. coli DSM 682 or S. aureus ATCC 6538 [101] . The lowest concentration of the antimicrobial that had no visible S. aureus growth was determined to be the minimum inhibitory concentration (MIC) [102] .

AST was used to determine the S. aureus MICs against 16 antimicrobials using the National Antimicrobial Resistance Monitoring System (NARMS) Gram-positive plate (CMV3AGPF). The strains were adjusted to the proper concentration using a 0.5 McFarland standard and dilution tubes containing demineralized water (5 mL) obtained from Remel Inc. (Lenexa, KS, USA). The inoculated demineralized water (30 µL) was added to tubes containing 11 mL of cation-adjusted Mueller-Hinton broth (MHB) with TES (Tris, EDTA, and NaCl, pH 8) and dose heads (#E3010) obtained from Remel Inc. were used to inoculate the antimicrobial-containing plates. MICs of the 164 S. aureus strains were obtained for 16 antimicrobials (Aminoglycosides: gentamicin (GEN), kanamycin, streptomycin (STR); Amphenicols: chloramphenicol (CHL); Cyclic lipopeptides: daptomycin; Fluoroquinolones: ciprofloxacin (CIP); Glycopeptides: vancomycin; Lincosamides: lincomycin; Macrolides: erythromycin (ERY), tylosin tartrate (TYLT); Nitrofurans: nitrofurantoin; Oxazolidinones: linezolid; Penicillins: penicillin (PEN); Streptogramins: quinupristin/dalfopristin (SYN); and Tetracyclines: tetracycline (TET), tigecycline) by following the manufacturer's instructions for the Sensititre ® susceptibility system (Trek Diagnostic Systems Inc., Independence, OH, USA). Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used as control strains for AST.

MRSA strains were determined by traditional cefoxitin and oxacillin susceptibility tests of all isolated S. aureus strains followed by confirmation by polymerase chain reaction (PCR) methods [103] . A QIAmp ® DNA Mini Kit (51306, Qiagen, Valencia, CA, USA) was used to isolate and purify genomic DNA from colonies of pure culture of S. aureus isolates. The colonies were grown on TSA II™ plates for 24 h at 37 • C. A loop-full (10-µL loop) of colonies were collected from the plate. Qiagen Protocol D under Protocols for Bacteria was followed for DNA isolation. For protocol D, 200 µg/mL of lysostaphin (L7386-1MG, Sigma-Aldrich, St. Louis, MO, USA) was used to pre-incubate the Gram-positive Staphylococcus cells. A NanoDrop™ One (13400518PR2, Thermo Fisher Scientific, Madison, WI, USA) spectrophotometer was used to obtain DNA concentrations and sample purity. Two-microliters of DNA-containing solution was used for each measurement.

Genomic DNA was sent to a commercial laboratory for Multilocus Sequence Typing (MLST) (Molecular Research Laboratory, Shallowater, TX, USA). S. aureus ATCC ® 43300 and S. aureus ATCC ® 29213 were used as controls for MLST testing. Molecular typing of the S. aureus isolates was performed by MLST software version 2.19.0 using Galaxy tools [104] . Alleles of each locus were compared, and sequence types were assigned based on the S. aureus MLST database [67] .

In this work, 17 disinfectants and 7 disinfectant components, a total of 24 chemicals were evaluated by DST for inhibition of 164 S. aureus isolates [98] by methods similar to those previously described [97] . The sources and recommended uses for 21 of 24 chemicals tested were previously reported [95] . Briefly, a list of the 21 chemical disinfectants and disinfectant components with their abbreviations and with the added exponent "CP" to indicate a commercial product are listed as follows (name, abbreviation): benzalkonium chloride, BKC; Betadine CP (10% povidone-iodine), P-I; cetylpyridinium bromide hydrate, CPB; DC&R CP , DC&R CP ; ethylhexadecyldimethylammonium bromide, CDEAB; Food Service Sanitizer CP , FSS; F-25 Sanitizer CP , F25; Final Step 512 Sanitizer CP , FS512; cetylpyridinium chloride hydrate, CPC; cetyltrimethylammonium bromide, CTAB; Novasan Solution CP (chlorhexidine diacetate), chlorhexidine; Odoban CP , Odoban CP ; P-128 CP , P-128 CP ; Tek-Trol CP , Tek-Trol CP ; triclosan (ergasan), triclosan; didecyldimethylammonium chloride, C10AC; benzyldimethyldodecylammonium chloride, C12BAC; benzyldimethyltetradecylammonium chloride, C14BAC; benzyldimethylhexadecylammonium chloride, C16BAC; J.T. Baker 37% formaldehyde, formaldehyde; and tris(hydroxylmethyl)nitromethane, THN. The additional three chemicals tested are the following: CaviCide CP is a multi-purpose disinfectant that can be used on the hands as well as to decontaminate non-porous surfaces. CaviCide CP kills bacteria and viruses including but not limited to TB, Norovirus, HIV-1, HBV and HCV [105] . Triclocarban, or 3,4,4 -trichlorocarbanilide (TCC), is used worldwide as an antimicrobial both in personal care products (such as, bar soap, cleansing lotions, and deodorants) [106] , and in pharmaceuticals [107] . Both CaviCide CP and TCC were obtained from Sigma-Aldrich (Milwaukee, WI, USA). TCC has been shown to be an endocrine disruptor [108, 109] . The chemical, dioctyldimethylammonium chloride (C8AC), is a disinfectant component used in complex disinfectants and was obtained from Lonza Inc. (Fairlawn, NJ, USA). To aid the solubility of some chemicals, dimethyl sulfoxide (DMSO) (MilliporeSigma, St. Louis, MO, USA) was added to purified reverse-osmosis water ( RO H 2 O) obtained using a water purification system from MilliporeSigma (Bedford, MA, USA).

Many disinfectants have multiple active components, and the percentages of active components in the complex disinfectants used in this study were previously provided [95] . The following disinfectants are mixtures of active components: F25, FS512, FSS, DC&R CP , P-128 CP , and Tek-Trol CP . S. aureus MICs for the disinfectants containing multiple active components have been determined using the authentic complex disinfectants. Triclosan resistance was determined by using the published susceptible/resistant criterion [110] ; S. aureus bacteria were considered susceptible at MICs < 0.5 µg/mL, were intermediate with MICs from 0.5 to 2 µg/mL and were considered resistant at MICs > 2 µg/mL triclosan. The breakpoint used for chlorhexidine against S. aureus was the same as previously used [111] for staphylococci bacteria; MICs ≥ 1 µg/mL were resistant. The susceptible/resistant criterion used for BKC was previously defined [112] ; S. aureus at MICs < 30 µg/mL were considered susceptible, MICs from 30 to 50 µg/mL were assigned low-level resistance, and S. aureus at MICs > 50 µg/mL were considered resistant to BKC.

Dilutions of disinfectants and disinfectant components were made using RO H 2 O followed by filter sterilization using 0.2 µm × 25 mm syringe filters (#431224, Corning Inc., Corning, NY, USA). Some chemicals were not sufficiently soluble with pure RO H 2 O and required an addition of DMSO to achieve solubility. The chemicals that required added DMSO are listed using the following format: Chemical (% DMSO added, % DMSO in final solution). The chemicals requiring DMSO for solubility were the following: CPB (100%, 5%); CPC (30%, 4.5%); CTAB (100%, 4.5%); C14BAC (20%, 1%); C16BAC (60%, 1.5%); TCC (80%, 2.5%); Tek-Trol CP (93%, 4%); THN (60%, 2.5%); and triclosan (100%, 4%). The final working solutions of chemicals did not contain more than 5% DMSO. The methods used to perform DST of S. aureus strains were similar to the DST conducted on Salmonella spp. [95] and Campylobacter coli [96] . The following concentration gradients were tested against the 164 S. aureus strains. BKC, 0.25-256 µg/mL; CaviCide CP , 1-1024 µg/mL; chlorhexidine, 0.008-8 µg/mL; CDEAB, 0.016-16 µg/mL; CPB, 0.016-16 µg/mL; CPC, 0.016-16 µg/mL; CTB, 0.016-16 µg/mL; C8AC, 0.062-64 µg/mL; C10AC, 0.062-64 µg/mL; C12BAC, 0.25-256 µg/mL; C14BAC, 0.062-64 µg/mL; C16BAC, 0.062-64 µg/mL; DC&R CP , 1-1024 µg/mL; formaldehyde, 2-2048 µg/mL; FSS, 0.062-64 µg/mL; FS512, 0.062-64 µg/mL; F25, 0.062-64 µg/mL; OdoBan CP , 0.062-64 µg/mL; P-I, 32-32,768 µg/mL; P-128 CP , 0.016-16 µg/mL; Tek-Trol CP , 0.25-256 µg/mL; triclosan, 0.002-2 µg/mL; THN, 2-2048 µg/mL; and TCC, 0.016-16 µg/mL. Staphylococcus aureus ATCC 29213 was used as the control strain for DST.

The following calculations were used to obtain the theoretical MICs ( theo MICs) for the active components in complex disinfectants. The theo MICs estimate the concentration levels of individual active ingredients in a disinfectant mixture. The active ingredients in P-128 CP consists of a mixture of the BACs (C12BAC-40%, C14BAC-50%, and C16BAC-10%) at 3.38% and C10AC at 5.07%. The theo MICs for the active components of P-128 CP can be calculated similarly to the DC&R CP components above. Briefly, the theo MICs P-128 of the two active components in P-128 CP , theo MICs BACs P-128 and theo MICs C10AC P-128 can be obtained by multiplying each determined P-128 CP MIC by the percentage of each component, 3.38 and 5.07, respectively, and then dividing by the sum of the component percentages in P-128 CP , which is 8.45. Table 1 shows the AMR profiles among the 164 S. aureus strains isolated from swine feces, MLT, and PSM. Table 1 provides the MIC 50 , MIC 90 , MIC range of recorded values, the number of resistant strains and breakpoint used for each of the 16 antimicrobials tested against the S. aureus strains. No resistant strains were found against four different antimicrobials: daptomycin, nitrofurantoin, linezolid, and tigecycline. A low level of resistant strains was observed for the five antimicrobials: gentamicin (0.6%), streptomycin (2.4%), chloramphenicol (3.8%), vancomycin (0.6%), and quinupristin/dalfopristin (3.7%). There were 21/164 (12.8%) resistant S. aureus strains against ciprofloxacin. However, there was an observed high level of resistant strains against the four antimicrobials, erythromycin, tylosin tartrate, penicillin, and tetracycline at percentage levels of 50.6, 42.7, 72, and 68.9%, respectively. When a bacterial strain exhibits a MIC [102] less than the breakpoint value for an antimicrobial it is considered susceptible to the antimicrobial. When the MIC is higher than the breakpoint it is considered resistant to the antimicrobial. The susceptible/resistant determination for S. aureus cannot be determined for kanamycin and lincomycin using the CMV3AGPF susceptibility plates since the lowest value for kanamycin on the susceptibility plate is 128 µg/mL but the kanamycin breakpoint is only ≥64 µg/mL. The highest value of lincomycin on the susceptibility plate is 8 µg/mL and is far lower than the lincomycin breakpoint for S. aureus (≥32 µg/mL). The individual S. aureus AMR profiles for strains from swine feces is presented in Supplementary Table S1, the AMR profiles for the strains from the MLT are in Supplementary Table S2 , and the AMR profiles for the strains isolated from the PSM are in Supplementary Table S3 .

Resistance profiles for the 164 S. aureus strains are provided in Table 2 according to sample type, the number of multidrug-resistant (MDR) stains within each sample type, the number of antimicrobial-resistant strains, and the resistance profiles for each resistant strain. MDR strains are resistant to 3 or more classes of antimicrobials [113] . Out of 63 S. aureus strains isolated from swine feces, 32 (50.8%) were shown to be MDR with the overall major resistance profile of ERY-TET-PEN-TYLT. There were 49 S. aureus strains isolated from the MLT, and 16 strains (32.7%) were determined to be MDR with 4 (25.0%) of these strains also determined to be MRSA [98] . There was no major resistance pattern in this group, except that MDR strains had seven unique resistance profiles. The two most prevalent resistance profiles found in the MLT MRSA strains were TET-PEN and TET-CIP-PEN. Out of 52 S. aureus strains, isolated from the PSM, only 15 strains (28.8%) were MDR, including 3 (20.0%) MRSAs [98] . Here too there was no single major resistance phenotype among the PSM MDR strains. However, there were 11 different resistance profiles among the PSM MDR strains, and the two resistance profiles found for the MRSA strains were TET-CIP-PEN and ERY-TET-CIP-PEN-TYLT. 

The multilocus sequence typing data are presented in Table 3 , which provides the sample number, bacteria type and MLST designation with the determined allelic profile. Six of the seven isolated MRSA strains from the MLT and PSM were determined to be strain ST398 and one strain was ST5.

The MICs for the 63 S. aureus strains, isolated from swine feces determined against 24 disinfectants and disinfectant components, are shown in Table 4 . The data for the swine feces set of strains are shown in a table by themselves because these strains differed in their interactions with disinfectants compared to strains isolated from the MLT and PSM, which are shown in Table 5 . The S. aureus MICs for strains from the MLT and PSM were determined to be higher than the MICs for strains from swine feces against many of the disinfectants, such as DC&R CP , CaviCide CP , P-128 CP , P-I, FSS, F25, FS512, OdoBan CP , CPB, CPC, CDEAB, CTAB, C8AC, C10AC, C12BAC, C14BAC, and THN. Swine feces strains were 17.5% resistant to chlorhexidine, whereas the group of strains from the MLT and PSM were determined to be 41.6% resistant to chlorhexidine. All strains studied showed no resistance to triclosan. Practically no difference in MICs between the two groups of strains was observed for BKC, C16BAC, formaldehyde, TCC, and Tek-Trol CP . The elevated numbers highlighted in yellow in Table 5 Table 6 shows the correlation between S. aureus strains having increased disinfectant susceptibility with S. aureus MDR and MRSA strains. There were no strains from swine feces with elevated susceptibility to disinfectants. However, six (12.3%) of the strains from the MLT and five (9.6%) of the strains from the PSM had increased disinfectant susceptibility levels. There were four MRSA strains among the MLT strains and 3 MRSA strains among the PSM strains. One of the four MRSA strains from the MLT did not have elevated disinfectant susceptibility levels. Therefore, six of the seven MRSA strains had elevated disinfectant susceptibilities. All three MRSA strains isolated from PSM had elevated disinfectant susceptibility levels. Table 4 for the BACs, (C12BAC, C14BAC, and C16BAC), Form (formaldehyde), THN (tris(hydroxylmethyl)nitromethane), and C10AC (didecyldimethylammonium chloride) against S. aureus strains from feces. † Component MICs obtained from Table 5 for the BACs, (C12BAC, C14BAC, and C16BAC), formaldehyde, THN (tris(hydroxylmethyl)nitromethane), and C10AC (didecyldimethylammonium chloride) against S. aureus strains from the MLT and PSM. ‡ Percentage of strains at the indicated MIC. Figure 1 depicts the curves generated for the inhibition of 63 S. aureus strains from swine feces by the ammonium chloride disinfectant components C8AC, C10AC, C12BAC, C14BAC, and C16BAC in µmol/L (µM). Figure 2 shows the curves generated for the inhibition of 101 S. aureus strains from the MLT and PSM by the ammonium chloride disinfectant components C8AC, C10AC, C12BAC, C14BAC, and C16BAC in µM. C10AC and C16BAC were equally the most effective disinfectant components against S. aureus. While C8AC, C10AC, and C12BAC required progressively higher levels to inhibit some of the MLT and PSM strains than for the swine feces S. aureus strains.

1.0 (1.6%) 1.2 (12.9%) 2.0 (7.9%) * Component MICs obtained from Table 4 for the BACs, (C12BAC, C14BAC, and C16BAC), Form (formaldehyde), THN (tris(hydroxylmethyl)nitromethane), and C10AC (didecyldimethylammonium chloride) against S. aureus strains from feces. † Component MICs obtained from Table 5 for the BACs, (C12BAC, C14BAC, and C16BAC), formaldehyde, THN (tris(hydroxylmethyl)nitromethane), and C10AC (didecyldimethylammonium chloride) against S. aureus strains from the MLT and PSM. ‡ Percentage of strains at the indicated MIC. Figure 1 depicts the curves generated for the inhibition of 63 S. aureus strains from swine feces by the ammonium chloride disinfectant components C8AC, C10AC, C12BAC, C14BAC, and C16BAC in µmol/L (µM). Figure 2 shows the curves generated for the inhibition of 101 S. aureus strains from the MLT and PSM by the ammonium chloride disinfectant components C8AC, C10AC, C12BAC, C14BAC, and C16BAC in µM. C10AC and C16BAC were equally the most effective disinfectant components against S. aureus. While C8AC, C10AC, and C12BAC required progressively higher levels to inhibit some of the MLT and PSM strains than for the swine feces S. aureus strains. 

The 164 S. aureus strains isolated from swine feces, MLT, and PSM demonstrated no AMR to daptomycin, nitrofurantoin, linezolid, and tigecycline. These same strains showed very low AMR prevalence to gentamicin, streptomycin, chloramphenicol, vanco- 

The 164 S. aureus strains isolated from swine feces, MLT, and PSM demonstrated no AMR to daptomycin, nitrofurantoin, linezolid, and tigecycline. These same strains showed very low AMR prevalence to gentamicin, streptomycin, chloramphenicol, vancomycin, and quinupristin/dalfopristin antimicrobials. Some strains showed AMR to ciprofloxacin; however, high AMR was demonstrated among the strains against erythromycin, tylosin tartrate (macrolides), penicillin, and tetracycline. A high level of S. aureus macrolide resistance has been observed and was suggested to be due to excessive use of the macrolide antibiotics [114] . As many as 60 resistance genes have been identified in S. aureus that can confer resistance to different classes of antimicrobials, such as β-lactam antibiotics, tetracyclines, and the macrolides [115] . The resistance profiles of the S. aureus strains studied here show swine feces strains had a limited number of resistance profiles compared to either the MLT or PSM strains. The MDR strains from both the MLT and PSM contained all the MRSA strains detected, and pork sausage has been shown to be an excellent growth medium for S. aureus [32, 37] . Of the S. aureus strains isolated from the MLT, 32.7% were MDR and 25.0% of the MDR strains were MRSA. Of the S. aureus strains isolated from PSM, 28.8% were MDR and 20.0% of those were MRSA strains. Even though the number of MDR MLT strains were greater than the number of MDR PSM strains, the number of different resistance profiles were greater for the PSM MDR strains (13 profiles) than for the MLT MDR strains (nine profiles). Seven MRSA strains were isolated from the MLT and PSM, but no MRSA strains were observed among the strains isolated from swine feces. It was previously demonstrated that intracellular contamination of the MLT and PSM by MRSA was 8.2% and 5.8%, respectively [98] ; suggesting that the contamination levels of MRSA in swine tissues cannot be eliminated.

Multilocus sequence typing determined six of the seven MRSA strains isolated from the MLT and PSM belonged to strain ST398 while one belonged to strain ST5. A new clone of MRSA with the sequence type ST398 was first described in 2005 [45] , and the first study showing a direct association between food animal and human carriage of ST398 was in 2010 [116] . The MRSA strain ST398 was demonstrated to be present in pigs [117] [118] [119] and in pig farmers [48, 120] . MRSA strain ST398 was observed in humans and food animals in Central Europe [121] , in humans in Northern Austria [122] , Canada [123] , the Dominican Republic and New York City [124] , and in Midwestern U.S. swine and swine workers [48] . MRSA strain ST398 was observed in fresh pork meat in Germany [125] and in general can be found in final meat products if the pigs were colonized with ST398 [4] . Severe endocarditis, pneumonia, blood steam, and other infections can be caused by CA-MRSA ST398 [59, [126] [127] [128] [129] [130] . Resistance gene analysis of LA-MRSA CC398 has demonstrated commonly found genes in S. aureus and other staphylococci but also novel resistance genes have been described [42] . It was observed at a hospital in South Korea that children were only infected at 6.8% with strain ST5, while adults were infected at a rate of 58% with strain ST5 [131] .

Fifty-three of the 164 (32.3%) S. aureus strains were resistant to chlorhexidine. Chlorhexidine resistance was observed in 17.5% of swine feces strains and 41.6% of the combined MLT and PSM strains, suggesting both swine MLT and PSM strains had a greater chance of becoming chlorhexidine resistant. In previous experiments, E. coli O157:H7 strains from cattle demonstrated only 11% resistance to chlorhexidine [92] and 32% of C. jejuni strains were resistant to chlorhexidine [70] , but 76% of VRE strains were resistant to chlorhexidine [97] . While strains from non-O157 STECs [94] , cattle Salmonella strains [95] and swine C. coli strains [96] were~90% resistant to chlorhexidine, and Ps. aeruginosa [93] and turkey Salmonella strains [132] were 100% resistant to chlorhexidine. All 164 S. aureus strains were susceptible to triclosan, which is similar as observed for Salmonella [95, 132] , E. coli O157:H7 [92] , and the non-O157 STEC strains [94] . Our laboratory regularly refers to triclosan as a pseudo-antibiotic since it is synthetic and not a natural product but functions similarly as an antibiotic [96] . Triclosan is described in the literature as a biocide, but functions like an antimicrobial since it has a specific bacterial cellular target [133] . Tri-closan inhibits the final enzyme in the fatty-acid biosynthesis elongation cycle, the highly conserved enzyme enoyl-[acyl-carrier-protein] reductase (NADH) [110] . Triclosan is well known to affect efflux pumps and membrane permeability by causing genetic mutations in at least five genes in E. coli resulting in MDR [134] , and Ps. aeruginosa [93] , VRE [97] , C. coli [96] , and C. jejuni strains [70] are also highly resistant to triclosan.

All 164 S. aureus strains were susceptible to BKC. BKC is a biocide commonly used to preserve human ocular medications, and to clean animal wounds and prevent skin infections. BKC can be used for sanitization in the dairy industry, in fisheries, and on poultry farms [135] , and can be used as a Covid-19 hand sanitizer [136] ; however, any disinfectant that will disrupt a lipid bilayer will cause virus inactivation. In previous studies it was shown that C. jejuni [70] , C. coli [96] and VRE [97] were susceptible to BKC. Some strains of E. coli O157:H7 [92] , non-O157 STECs [94] and Salmonella from cattle [95] , and most Salmonella strains from turkeys [132] have shown intermediate resistance to BKC, whereas 97.1% of 175 Ps. aeruginosa strains were resistant to BKC [93] .

The 164 S. aureus swine strains had similar susceptibilities for the disinfectants TCC, P-I, THN, and formaldehyde. The MLT and PSM strains had elevated susceptibilities for DC&R CP , Tek-Trol CP , CaviCide CP , P-128 CP , BKC, FSS, F25, FS512, OdoBan CP , CPB, CPC, CDEAB, CTAB, C8AC, C10AC, C12BAC, C14BAC, and C16BAC compared to the swine feces strains. The MLT and PSM MRSA strains showed elevated susceptibilities to all these disinfectants and disinfectant components, except for Tek-Trol CP when compared to the swine feces strains. The MLT and PSM strains have similar disinfectant susceptibilities as was observed in C. jejuni [70] , C. coli [96] , and VRE strains [97] . The disinfectant susceptibilities for previously tested Salmonella [95, 132] , E. coli O157:H7 [92] , and non-O157 STEC strains [94] were two-to four-fold higher than seen here for the MLT and PSM strains.

Whereas the susceptibilities for previously tested Ps. aeruginosa strains [93] were 32-to 64-fold higher than obtained here for the MLT and PSM strains. The highest susceptibility values observed were for S. aureus against P-I. These levels are similar to those observed previously for C. jejuni and are in excess of 49-to 98-fold less than the manufacturer suggested application rate of 100,000 µg/mL of a P-I solution directly applied to wound surfaces [70] . We were unable to observe cross-resistance between the antimicrobials tested and the disinfectants.

The number of MRSA strains correlated well with the strains that had increased disinfectant susceptibility. Out of the 164 S. aureus strains tested only 17 strains (10.4%) had elevated disinfectant susceptibility levels, and they were found among the MLT and PSM strains. Six of the seventeen strains with elevated disinfectant susceptibility levels were determined to be MRSA strains and were determined to have increased S. aureus susceptibility to 18 of 24 (75%) disinfectants tested. There was one MRSA strain among the MLT strains that did not have elevated disinfectant levels. There were two strains among the MLT strains and five strains among the PSM strains that had elevated disinfectant susceptibility but were not MDR. No strains were found with elevated disinfectant susceptibility levels or tested positive for MRSA among the swine feces strains.

When [93] , non-O157 STEC [94] , Salmonella [95] , and VRE [97] strains. However, the BACs component of P-128 CP was the most active against C. coli [96] , and the BACs component and C10AC appeared to act equally and synergistically against C. jejuni [70] .

The potency of the five-ammonium chloride disinfectant components, C8AC, C10AC, C12BAC, C14BAC, and C16BAC was tested against the S. aureus strains and determined that C10AC and C16BAC were equally the most effective against S. aureus. C14AC, C12BAC, and C8AC, respectively, required progressively higher concentrations to inhibit the S. aureus strains. However, some of the MLT and PSM strains required higher levels of components C8AC and C12BAC for inhibition of S. aureus than the swine feces strains did. The length of the carbon chain attached to the ammonium chloride for each disinfectant component is incorporated into the abbreviated names of the components, C8, C10, C12, C14, and C16. In previous potency studies C10AC, C12BAC, and C14BAC were clearly the most effective against C. jejuni [70] and C. coli [96] , while C16BAC was the least effective against these two bacteria. Potency studies of disinfectant components against E. coli O157:H7 [92] , Ps. aeruginosa [93] , non-O157 STECs [94] , Salmonella [95] , and VRE [97] determined that C10AC was the most effective ammonium chloride disinfectant component against these bacteria.

A high prevalence of AMR was demonstrated by the 164 S. aureus strains to four antimicrobials, erythromycin (50.6%), tylosin tartrate (42.7%), penicillin (72%), and tetracycline (68.9%), and no AMR was detected to daptomycin, nitrofurantoin, linezolid, and tigecycline. The MLT and PSM strains demonstrated a wide array of resistance profiles. MRSA strains were found only in the MDR strains from the MLT (25.0%) and PSM (20.0%), but not among the swine feces strains. Multilocus sequence typing determined six of the seven MRSA strains isolated from the MLT and PSM were strain ST398 while one strain was ST5. About 17.5% of the swine feces strains and 41.6% of the combined MLT and PSM strains were resistant to chlorhexidine. All 164 strains were susceptible to the pseudoantibiotic triclosan and to BKC. The MLT and PSM strains had elevated susceptibilities for the disinfectants, DC&R CP , Tek-Trol CP , CaviCide CP , P-128 CP , BKC, FSS, F25, FS512, OdoBan CP , CPB, CPC, CDEAB, CTAB, C8AC, C10AC, C12BAC, C14BAC, and C16BAC compared to the swine feces strains. Six of the seven MRSA strains demonstrated increased MICs to 18 of 24 (75%) disinfectants evaluated compared to non-MRSA strains, and they correlated well with increased disinfectant susceptibility. No strains were found with elevated disinfectant susceptibility levels among the swine feces strains. It was determined that the BAC components of DC&R CP were responsible for the inhibition of S. aureus strains. The C10AC component in P-128 CP was responsible for S. aureus inhibition. The C10AC and C16BAC were equally effective against S. aureus. Some of the MLT and PSM S. aureus strains required higher levels of the components C8AC and C12BAC for inhibition compared to the swine feces strains. Since the S. aureus and MRSA strains were found deep within the MLT, this tissue may be a candidate for specialized treatments or even removal from the human consumption market. The use of formaldehyde and THN in the complex disinfectant DC&R CP is questionable since they are not effective against S. aureus at the concentrations present in DC&R CP , and the inclusion of formaldehyde and THN may result in additional unnecessary chemicals in the environment. This study establishes susceptibility values for S. aureus strains from swine feces, mandibular lymph node tissue, and commercial pork sausage against 24 disinfectants. Since it was demonstrated that S. aureus and MRSA strains can be found deep within swine lymph node tissue, it may be beneficial for the consumer if raw swine lymph node tissue was not used in food products and pork sausage.

The following are available online at https://www.mdpi.com/article/ 10.3390/microorganisms9112401/s1, Table S1 : Antimicrobial resistance profiles among 63 Staphylococcus aureus strains isolated from swine feces, Table S2 : Antimicrobial resistance profiles among 49 Staphylococcus aureus strains isolated from swine mandibular lymph node tissue, Table S3 : Antimicrobial resistance profiles among 52 Staphylococcus aureus strains isolated from commercial pork sausage meat, Table S4 : Distribution of disinfectant and disinfectant components susceptibility profiles for the 7 MRSA strains isolated from swine mandibular lymph node tissue and pork sausage meat, Table S5 : Distribution of disinfectant and disinfectant component susceptibility profiles for 49 Staphylococcus aureus strains isolated from swine mandibular lymph node tissue, Table S6 : Distribution of disinfectant and disinfectant component susceptibility profiles for 52 Staphylococcus aureus strains isolated from commercial pork sausage meat. 

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We thank Taryn Scholz for reviewing this and many other manuscripts. This work was funded by the USDA, Agricultural Research Service. Mention of trade names, proprietary products or specific equipment is solely for the purpose of providing specific information and does not constitute a guarantee, warranty or endorsement by the U.S. Department of Agriculture does not imply its approval to the exclusion of other products that may be suitable. Additionally, the views expressed in this article are those of the authors and do not necessarily reflect the official policy of the U.S. Department of Agriculture, or the U.S. Government.

The authors declare no conflict of interest.