Profiles of Non-aureus Staphylococci in Retail Pork and Slaughterhouse Carcasses: Prevalence, Antimicrobial Resistance, and Genetic Determinant of Fusidic Acid Resistance

Yu Jin Yang1, Gi Yong Lee2, Sun Do Kim2, Ji Heon Park2, Soo In Lee2, Geun-Bae Kim2, Soo-Jin Yang1,*
Author Information & Copyright
1Department of Veterinary Microbiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea
2Department of Animal Science and Technology, School of Bioresources and Bioscience, Chung-Ang University, Anseong 17546, Korea
*Corresponding author : Soo-Jin Yang, Department of Veterinary Microbiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea, Tel: +82-2-880-1185, Fax: +82-2-885-0263, E-mail:

© Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Sep 22, 2021 ; Revised: Dec 10, 2021 ; Accepted: Dec 20, 2021

Published Online: Mar 01, 2022


As commensal colonizers in livestock, there has been little attention on staphylococci, especially non-aureus staphylococci (NAS), contaminating meat production chain. To assess prevalence of staphylococci in retail pork and slaughterhouse carcass samples in Korea, we collected 578 samples from Korean slaughterhouses (n=311) and retail markets (n=267) for isolation of staphylococci and determined antimicrobial resistance phenotypes in all the isolates. The presence of and prevalence of fusB-family genes (fusB, fusC, fusD, and fusF) and mutations in fusA genes were examined in fusidic acid resistant isolates. A total of 47 staphylococcal isolates of 4 different species (Staphylococcus aureus, n=4; S. hyicus, n=1; S. epidermidis, n=10; Mammaliicoccus sciuri, n=32) were isolated. Fusidic acid resistance were confirmed in 9/10 S. epidermidis and all of the 32 M. sciuri (previously S. sciuri) isolates. Acquired fusidic acid resistance genes were detected in all the resistant strains; fusB and fusC in S. epidermidis and fusB/C in M. sciuri. Multi-locus sequence type analysis revealed that ST63 (n=10, 31%) and ST30 (n=8, 25%) genotypes were most prevalent among fusidic acid resistant M. sciuri isolates. In conclusion, the high prevalence of fusB-family genes in S. epidermidis and M. sciuri strains isolated from pork indicated that NAS might act as a reservoir for fusidic acid resistance gene transmissions in pork production chains.

Keywords: non-aureus staphylococci; antimicrobial resistance; retail pork; slaughterhouse carcass


Staphylococci are frequent inhabitants of the normal microbiota of skin and mucous surfaces in humans and animals (Davis, 1996). Although less frequent than the coagulase-positive staphylococci such as S. aureus, coagulase-negative staphylococci (CoNS) can cause many nosocomial and community-associated infections including skin and soft tissue infections, urinary tract infections, endocarditis, and blood stream infections (Piette and Verschraegen, 2009). Moreover, several recent studies have reported the potential role of non-aureus staphylococci (NAS) in transmission of antimicrobial resistance by acting as a reservoir for antimicrobial resistance genes (Archer and Niemeyer, 1994; Nemeghaire et al., 2014a).

Fusidic acid (FA) is a bacteriostatic steroid antibiotic originated from Fusidium coccineum, previously used to treat staphylococcal skin infections since the early 1960s (Godtfredsen et al., 1962). It targets elongation factor G (EF-G) that functions as ribosomal translocase and interact with ribosomal recycling factor to release ribosomal complexes (Fernandes, 2016). However, FA resistance can occur due to spontaneous mutations in fusA, which encodes EF-G (Turnidge and Collignon, 1999). Point mutations in fusE, encoding ribosomal protein L6, are also associated with FA resistance in staphylococci (Norström et al., 2007). In addition, FusB-family proteins bind to EF-G and protect them from FA binding (O’Neill and Chopra, 2006). The FusB-family proteins are produced by the fusB, fusC, fusD, and fusF genes and frequently mediate low-level resistance to FA (Fernandes, 2016). These fusB-family genes have been reported in S. aureus and NAS isolates, either carried on a plasmid, phage-associated resistance islands, or staphylococcal cassette chromosome (SCC) (Chen et al., 2015; O’Neill and Chopra, 2006; O’Neill et al., 2007). It has been well known that S. saprophyticus and S. cohnii subsp. urealyticus possess fusD and fusF genes for their intrinsic FA resistance (Chen et al., 2015; O’Neill et al., 2007).

The significance of food-producing animals as carriers of foodborne zoonotic pathogens and antimicrobial resistance genes has been demonstrated in many countries including Korea (Hung et al., 2015; Nam et al., 2011; Nemeghaire et al., 2014a). In contrast to coagulase-positive S. aureus, well recognized as a major causative pathogen of staphylococcal food poisoning and antimicrobial resistance, occurrence of NAS in foods of animal origin, their antimicrobial resistance phenotypes, and the genetic factors associated with the resistant phenotypes have not been well investigated. Therefore, we aimed to investigate i) the profiles of NAS in pork and carcass samples collected from retail markets and slaughterhouses in Korea, ii) the antimicrobial resistance phenotypes of the staphylococcal isolates, and iii) the occurrence and distribution of fusB-family genes (fusB, fusC, fusD, and fusF), and iv) the point mutations in fusA genes by sequencing analyses.

Materials and Methods

Sample collection and isolation of staphylococci

We obtained a total of 578 swab or pork samples from seven slaughterhouses (311 carcass samples) and 35 retail markets (267 pork samples) across eight Korean provinces in 2018. Slaughterhouse carcass samples were obtained from a single visit at seven different slaughterhouses: Gyeonggi (46 swabs), Gangwon (46 swabs), Chuncheong (46 swabs), Jeolla (two slaughterhouses, 40 and 42 swabs), and Gyeongsang (two slaughterhouses, 45 and 46 swabs). Each carcass swab was prepared on an area of (10×10 cm) per site on the back and chest of pig carcasses within 8 h of slaughter. Fresh pork samples were collected from four to five retail markets in each province. All samples were kept at 4°C and processed for isolation of staphylococci within 24 h of collection.

Swab samples from slaughterhouses were inoculated into 6 mL of tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) containing 10% sodium chloride (NaCl) for enrichment at 37°C. Each pork sample (25 g) was homogenized in 225 mL of 10% NaCl-TSB. After overnight incubation, 15 μL aliquots of the pre-enriched NaCl-TSB cultures were streaked onto Baired-Parker agar (BPA; Difco Laboratories) supplemented with potassium tellurite and egg yolk, and then grown at 37°C for up to 48 h. Next, up to two presumptive staphylococcal colonies from each plate were re-streaked on BPA plate for identification. For genomic DNA isolation, individual isolates were inoculated into fresh TSB, cultured at 37°C for 18–24 h, and bacterial cell pellets were subjected to the Genmed DNA extraction kit (Genmed, Seoul, Korea) according to the manufacturer’s protocols. Identification of staphylococcal species was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Bruker Daltonics GmbH, Bremen, Germany) and 16S rRNA sequencing. For bacterial identification using MALDI-Biotyper Realtime Classification system, presumptive staphylococci were placed on a target plate coated with specific energy-absorbent agent, the matrix. The sample within the matrix was then ionized in an automated mode with a laser beam as recommended by the manufacturer. Next, peptides in bacterial sample converted into protonated ions and the peptide mass fingerprints were used to identify bacterial species based on the spectral database (MALDI Biotyper 3.1, Bruker Daltonics GmbH). Score values ≥2.0 were used for an identification of staphylococcal species. For 16S rRNA sequencing analyses, 16S rRNA genes were PCR-amplified using a universal primer set (16S_27F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 16S_1492R: 5’-TAC GGY TAC CTT GTT ACG ACT T-3’) (Mendoza et al., 1998). PCR amplifications were performed as follows: Denaturation at 94°C for 30 s followed by 28 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min. After a final 10 min extension at 72°C, the samples were purified using PCR purification kit (Bionics, Seoul, Korea), and then sequenced at Bionics.

Antimicrobial susceptibility assays

Standard disc diffusion methods were used to determine the antimicrobial susceptibility of each isolate according to Clinical and Laboratory Standards Institute’s (CLSI) recommendations for the following antimicrobial agents: penicillin (PEN, 10 μg), ampicillin (AMP, 10 μg), cefoxitin (FOX, 30 μg), chloramphenicol (CHL, 30 μg), clindamycin (CLI, 2 μg), erythromycin (ERY, 15 μg), FA (10 μg), ciprofloxacin (CIP, 5 μg), mupirocin (MUP, 200 μg), trimethoprim/sulfamethoxazole (SXT, 1.25/23.75 μg), tetracycline (TET, 30 μg), rifampicin (RIF, 5 μg), gentamycin (GEN, 10 μg), quinupristin/dalfopristin (SYN, 15 μg). The minimum inhibitory concentrations (MICs) to FA (Liofilchem, Roseto degli Abruzzi, Italy) and oxacillin (OXA; bioMérieux, Marcy l’Etoile, France) were determined by standard E-test. OXA E-test was performed on MH BBL II agar (Becton Dickinson, Sparks, MD, USA) supplemented with 2% NaCl. The breakpoint for FA resistance (>1 μg/mL) was based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2021) guidelines. The S. aureus ATCC 25923 strain was included as a reference for the antimicrobial susceptibility analyses.

mecA detection and staphylococcal cassette chromosome (SCC)mec type determination

All the strains showing resistance to OXA or FOX were examined for the presence of mecA gene using the PCR method as described previously (Geha et al., 1994). The mecA positive staphylococcal strains were subjected to SCCmec typing as previously described (Kondo et al., 2007). A series of multiplex PCR reactions were employed to amplify mec regulatory elements (mec) and chromosomal cassette recombinase (ccr) genes. The combinations mec complexes and ccr types were used to determine the SCCmec types of the staphylococcal strains. The PCR was preceded as previously described (Kondo et al., 2007).

Detection of fusidic acid (FA) resistance determinants

The carriage of acquired FA resistance genes fusB,fusC, fusD, and fusF were detected by PCR methods as described before (Chen et al., 2010; Chen et al., 2015; McLaws et al., 2008). The primers used to amplify fusB-family genes are listed in Table 1. For detection of fusB-family gene homologues in M. sciuri (previously S. sciuri) strains, a specific primer set was designed based on the published sequences of 7 M. sciuri strains ( carrying the fusB/C-family genes (Table 1). The PCR conditions for detecting fusB/C in M. sciuri were as follows: Denaturation at 95°C for 2 min, followed by 28 cycles of denaturation at 94°C for 35 s, annealing at 53°C for 40 s, extension at 72°C for 45 s, and final extension at 72°C for 10 min.

Table 1. Primers used to detect fusidic acid resistance determinants in this study
Primer name Sequence (5’→3’) Product size (bp) Target gene References
BF CTA TAA TGA TAT TAA TGA GAT TTT TGG 431 fusB (McLaws et al., 2008)
CF TTA AAG AAA AAG ATA TTG ATA TCT CGG 332 fusC (McLaws et al., 2008)
B/C-F CTT AAA AGC TAC GTC GTC CCA 299 fusB/C This study
A-S1 TTA ATT GAA GCT GTT GCT GA Sequencing only
DF AAT TCG GTC AAC GAT CCC 465 fusD (Chen et al., 2010)
FF CTA AAA TAG ACA TTT ATC AGC AG 427 fusF (Chen et al., 2015)
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To detect point mutations within fusA genes in FA resistant isolates, DNA sequencing analyses were performed. Using the specific primer set (AF and AR) as shown in Table 1, fusA gene was amplified from genomic DNA samples purified from S. epidermidis and M. sciuri strains. The PCR amplicons were sequenced with AF and two additional primers A-S1 and A-S2 at Bionics (Seoul, Korea). The fusA sequence data were then compared with the published sequences of S. epidermidis (GenBank: NZ_CP035288.1) and M. sciuri (GenBank: CP071138.1).

Multi-locus sequence type (MLST) analysis

Except one S. hyicus strain, whose MLST scheme has not yet been developed, MLST was performed on all S. aureus, S. epidermidis, and M. sciuri isolates as described previously (Enright et al., 2000; Schauer et al., 2021; Thomas et al., 2007). For MLST analyses, internal fragments of seven housekeeping genes from each strain were PCR amplified and sequenced. The seven genes amplified from each species of staphylococci are: arcC, aroE, gmk, glpF, pta, tpi, and yqiL for S. aureus (Enright et al., 2000); arcC, aroE, gtr, pyrR, mutS, yqiL, and tpiA for S. epidermidis (Thomas et al., 2007); ack, aroE, glpK, ftsZ, gmk, pta1, and tpiA for M. sciuri (Schauer et al., 2021). The alleles and sequence types (STs) of each staphylococcal species were assigned according to the MLST databases (


Profiles of staphylococci isolated from pork meat and carcass samples

As presented in Table 2, 44 staphylococci (44/311, 14.1%) of four different species were isolated from slaughterhouse carcass samples, and only three strains of M. sciuri (3/267, 1.1%) were isolated from retail pork samples obtained over the 10-month study period. The most frequent staphylococci identified in the carcasses were M. sciuri (previously S. sciuri), comprising ~66% (29/44) of the staphylococcal isolates from slaughterhouse samples. While four different species (S. aureus, S. hyicus, S. epidermidis, and M. sciuri) were identified in the slaughterhouse samples, only three strains of M. sciuri were cultured from retail pork meat samples. All of the 47 isolates used in this investigation were obtained from different carcass or meat samples.

Table 2. Profiles of staphylococci isolated from pork meat and carcass samples
Bacterial species No. of methicillin-resistant strains SCCmec type (No. of strains)
Staphylococci from slaughterhouses (44/311 samples)
Staphylococcus aureus (n=4/311, 1.29%) 2 SCCmec IV (1), SCCmec V (1)
Staphylococcus hyicus (n=1/311, 0.32%) 0 -
Staphylococcus epidermidis (n=10/311, 3.22%) 5 SCCmec IV (4), SCCmec V (1)
Mammaliicoccus sciuri (n=29/311, 9.32%) - -
Staphylococci from retail markets (3/267 samples) 0 -
Mammaliicoccus sciuri (n=3/267, 1.12%) 0 -
Total 47 strains 7

SCC, staphylococcal cassette chromosome.

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MLST analyses of the S. aureus and S. epidermidis isolates revealed four and six different STs, respectively, with four non-typeable S. epidermidis strains (Fig. 1). The 32 strains of M. sciuri were assigned to three different STs: ST63 (n=10, 31.3%), ST30 (n=8, 3%), and ST96 (n=1, 3.1%) with 13 non-typeable strains under the current M. sciuri MLST scheme.

Fig. 1. MLST profiles of staphylococci isolated from retail pork and carcass samples in Korea. SA, Staphylococcus aureus; SE, Staphylococcus epidermidis; MS, Mammaliicoccus sciuri; MLST, multi-locus sequence type.
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Methicillin-resistant staphylococci in retail pork and pig carcass

Seven methicillin-resistant staphylococci, two methicillin-resistant S. aureus (MRSA) and five methicillin-resistant S. epidermidis (MRSE) strains, were identified among the 47 staphylococcal isolates, indicating 14.9% methicillin resistance prevalence (Table 2). All seven methicillin-resistant staphylococcal strains were mecA positive and exhibited resistant phenotype to OXA, OXA MIC ≥4 μg/mL (S. aureus), OXA MIC ≥0.5 μg/mL (S. epidermidis) (Table 3). Each of the five MRSE strains were assigned to five different STs of ST172, ST130, ST80, ST173, and ST768. The two MRSA strains were ST398 with SCCmec V and ST2084 with SCCmec IV, respectively (Table 2). Although all 32 M. sciuri strains showed OXA MICs of ≥0.5 μg/mL, none of the strains were FOX-resistant (Table 3). Furthermore, all the M. sciuri strains were negative for the mecA gene.

Table 3. Genotypes, antimicrobial resistance phenotypes, and fusidic acid resistance determinants of staphylococci isolated from pork meat and carcass samples
ID Species Resistance profiles MICs to mecA SCCmec type FA resistance genes fusA mutation MLST Score value1)
OXA (μg/mL) TET (μg/mL) FA (μg/mL)
SSA1 MRSA OXA-AMP-FOX-PEN-CHL-CIP-OXA-TET 6 64 0.25 + V - NA ST398 2.304
SSA2 MRSA OXA-AMP-FOX-PEN 192 0.5 0.125 + IV - NA ST2084 2.144
SSA3 MSSA AMP-PEN 0.5 1 0.19 - NA - NA ST5 2.204
SSA4 MSSA AMP-PEN-CHL-MUP 0.5 4 0.125 - NA - NA ST9 2.279
SSE1 S epidermidis OXA-AMP-FOX-PEN-ERY-FA 64 1 24 + IV fusB - ST172 2.362
SSE2 S. epidermidis OXA-AMP-PEN-FA 64 4 24 + IV fusB - ST130 2.32
SSE3 S. epidermidis OXA-AMP-FOX-PEN-FA 32 0.5 8 + IV fusB, fusC - ST80 2.293
SSE4 S. epidermidis OXA-AMP-PEN-CHL-ERY-FA-MUP-SXT 8 4 32 + V fusB - ST173 2.436
SSE5 S. epidermidis AMP-PEN-FA 0.25 2 8 - NA fusB - NT 2.375
SSE6 S. epidermidis AMP-PEN-FA 0.25 4 8 - NA fusB - NT 2.249
SSE7 S. epidermidis OXA-FA-MUP-TET 16 32 24 + IV fusB - ST768 2.293
SSE8 S. epidermidis AMP-PEN-FA 0.25 4 12 - NA fusB - NT 2.376
SSE9 S. epidermidis AMP-PEN-FA 0.25 2 24 - NA fusB V599I (GTT→ATT) NT 2.264
SSE10 S. epidermidis AMP-PEN 0.25 0.5 0.25 - NA fusB - ST1017 2.247
SSH1 S. hyicus AMP-PEN-CLI-SXT-TET 0.25 16 0.19 - NA - NA NA 2.138
RMS1 M. sciuri OXA-CLI-FA 2 0.5 12 - NA fusB/C - ST63 2.273
RMS2 M. sciuri OXA-FA 0.5 0.25 16 - NA fusB/C - NT 2.377
RMS3 M. sciuri OXA-FA 1 0.25 16 - NA fusB/C - ST30 2.274
SMS4 M. sciuri OXA-FA-TET 1 16 8 - NA fusB/C - ST63 2.227
SMS5 M. sciuri OXA-FA 1 1 16 - NA fusB/C - NT 2.433
SMS6 M. sciuri OXA-FA 1 0.5 12 - NA fusB/C - NT 2.324
SMS7 M. sciuri OXA-FA 1 0.5 16 - NA fusB/C - NT 2.212
SMS8 M. sciuri OXA-FA 1 0.5 24 - NA fusB/C - ST63 2.012
SMS9 M. sciuri OXA-CLI-FA 2 0.5 16 - NA fusB/C - ST63 2.01
SMS10 M. sciuri OXA-FA 0.5 0.25 12 - NA fusB/C - NT 2.343
SMS11 M. sciuri OXA-FA 2 1 8 - NA fusB/C - NT 2.257
SMS12 M. sciuri OXA-FA 1 1 12 - NA fusB/C - NT 2.13
SMS13 M. sciuri OXA-CLI-FA 0.5 0.25 16 - NA fusB/C - ST63 2.248
SMS14 M. sciuri OXA-FA 0.5 0.25 8 - NA fusB/C - NT 2.17
SMS15 M. sciuri OXA-FA 0.5 0.25 12 - NA fusB/C - NT 2.028
SMS16 M. sciuri OXA-CLI-FA 0.5 0.25 6 - NA fusB/C - NT 2.077
SMS17 M. sciuri OXA-FA 1 0.25 8 - NA fusB/C - ST63 2.214
SMS18 M. sciuri OXA-PEN-FA 2 0.25 8 - NA fusB/C - NT 2.056
SMS19 M. sciuri OXA-FA 0.5 0.25 12 - NA fusB/C - NT 2.203
SMS20 M. sciuri OXA-FA 1 8 12 - NA fusB/C - ST63 2.29
SMS21 M. sciuri OXA-FA-TET 1 16 8 - NA fusB/C - ST63 2.185
SMS22 M. sciuri OXA-FA 1 0.5 12 - NA fusB/C - ST63 2.203
SMS23 M. sciuri OXA-FA 1 0.25 8 - NA fusB/C - NT 2.336
SMS24 M. sciuri OXA-FA-TET 1 16 8 - NA fusB/C - ST30 2.218
SMS25 M. sciuri OXA-FA-TET 1 16 12 - NA fusB/C - ST30 2.277
SMS26 M. sciuri OXA-FA-TET 1 16 8 - NA fusB/C - ST30 2.272
SMS27 M. sciuri OXA-FA-TET 1 16 12 - NA fusB/C - ST30 2.085
SMS28 M. sciuri OXA-FA-TET 1 16 12 - NA fusB/C - ST30 2.081
SMS29 M. sciuri OXA-FA 1 0.25 12 - NA fusB/C - ST30 2.398
SMS30 M. sciuri OXA-FA 1 0.25 8 - NA fusB/C - ST30 2.325
SMS31 M. sciuri OXA-FA 1 0.5 12 - NA fusB/C - ST63 2.295
SMS32 M. sciuri OXA-PEN-FA 1 8 8 - NA fusB/C - ST96 2.282

1) Strains with score value of ≥2.000 were used in this study.

MICs, minimum inhibitory concentrations; OXA, oxacillin; TET, tetracycline; FA, fusidic acid; SCC, staphylococcal cassette chromosome; MLST, multi-locus sequence type; MRSA, methicillin-resistant S. aureus; AMP, ampicillin; FOX, cefoxitin; PEN, penicillin; CHL, chloramphenicol; CIP, ciprofloxacin; TET, tetracycline; NA, not applicable; MSSA, methicillin-susceptible S. aureus; MUP, mupirocin; ERY, erythromycin; SXT, trimethoprim/sulfamethoxazole; NT, not-typable stain; CLI, clindamycin; S. epidermidis, Staphylococcus epidermidis; M. sciuri, Mammaliicoccus sciuri.

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Antimicrobial resistance profiles

All 47 isolates were susceptible to rifampin, gentamicin and SYN (Table 4). Multidrug resistance was observed in 17 staphylococcal isolates (36.2%), which showed resistance to ≥3 different antimicrobials agent classes. As shown in Table 4, 41/47 (87.2%) isolates were resistant to FA, displaying the highest frequency of resistance for FA. Among the 47 isolates, 9/10 (90%) S. epidermidis strains and all 32 M. sciuri strains were FA-resistant. However, all the FA resistant S. epidermidis and M. sciuri strains displayed FA MICs ranging from 6–24 μg/mL, indicating low-level resistance (Fernandes, 2016).

Table 4. Prevalence of antimicrobial resistance in staphylococci isolated from pork meat and carcass samples
Frequency of resistance Total (n=47, %)
MRSA (n=2) MSSA (n=2) Staphylococcus hyicus (n=1) Staphylococcus epidermidis (n=10) Mammaliicoccus sciuri (n=32)
OXA 2 - - 5 32 39 (83.0)
AMP 2 2 1 9 - 14 (29.8)
FOX 2 - - 2 - 4 (8.5)
PEN 2 2 1 9 2 16 (34.0)
CHL 1 1 - 1 - 3 (6.4)
CIP 1 - - - - 1 (2.1)
CLI - - 1 1 4 6 (12.8)
ERY - - - 2 - 2 (4.3)
FA - - - 9 32 41 (87.2)
MUP - 1 - 2 - 3 (6.4)
SXT - - 1 1 - 2 (4.3)
TET 1 - 1 1 7 10 (21.3)
RIF - - - - - -
GEN - - - - - -
SYN - - - - - -

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; OXA, oxacillin; AMP, ampicillin; FOX, cefoxitin; PEN, penicillin; CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; FA, fusidic acid; MUP, mupirocin; SXT, trimethoprim/ sulfamethoxazole; TET, tetracycline; RIF, rifampicin; GEN, gentamycin; SYN, quinupristin/dalfopristin.

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Genetic determinants of fusidic acid (FA) resistance

All S. epidermidis and M. sciuri isolates showing resistance phenotype to FA were examined for the presence of fusB, fusC, fusD, and fusF. All nine FA-resistant S. epidermidis strains were fusB positive, with one carrying the fusC gene (Table 3). Similarly, all 32 M. sciuri isolates were positive for a fusB-family homolog, fusB/C. None of the FA resistant isolates were carrying fusD nor fusF.

Besides the fusB-family genes, point mutations within the fusA open reading frame encoding EF-G have been associated with FA resistance (Turnidge and Collignon, 1999). As shown in Table 3, a V599I mutation was confirmed in the fusA in only one of the S. epidermidis (SSE9) isolates. No mutation was identified in the fusA gene of M. sciuri isolates from the sequencing analyses.


High prevalence of antimicrobial resistance is a significant threat to public health as it undermines treatment options for bacterial infections (Sugden et al., 2016). Since staphylococci are frequently associated with commensal microbiota of skin and mucous surface of various food-producing animals, the development and spread of antimicrobial resistance among staphylococci in the food chain is considered an important threat to food safety (Founou et al., 2016). While coagulase-positive S. aureus has been well investigated for its ability to develop antimicrobial resistance and zoonotic potentials to infect human and animal hosts, relatively few studies have focused on the role of NAS, such as CoNS, in antimicrobial resistance development and transmission. Indeed, several recent studies demonstrated that the antimicrobial resistance in CoNS has been increasing over the past decades (Piette and Verschraegen, 2009), and they act as reservoir for resistance genes that can be transferred to other bacteria (von Wintersdorff et al., 2016).

In this study, we assessed prevalence of staphylococci in retail pork meat and slaughterhouse carcass samples collected from eight provinces of Korea. Overall, the prevalence of staphylococci in the retail pork and slaughterhouse carcasses was 1% and 16.5%, respectively. As shown in Table 2, only 4/44 (9.1%) staphylococci from slaughterhouse carcass samples were S. aureus, and this high proportion of NAS over S. aureus was similar to previous report (Fijałkowski et al., 2016). Among the three different species of NAS from slaughterhouses, M. sciuri displayed the highest prevalence (65.9%) followed by S. epidermidis (22.7%). While previous studies reported much higher levels of S. aureus prevalence in retail pork meat samples in China (18.6%; Wu et al., 2018), Denmark (60%; Tang et al., 2017), and USA (16%–66%; Hanson et al., 2011; O’Brien et al., 2012), no S. aureus was detected from retail pork meat samples in this study. At least several factors such as sample treatment, enrichment/isolation method, and geographical location may have affected the differences in prevalence of S. aureus and other staphylococci. It should also be noted that the use of different sampling methodology (swab samples on ~100 cm2 surface versus 25 g of pork meats) to isolate staphylococci from slaughterhouse carcass samples and retail pork meat samples would have affected the overall prevalence and proportion of each species presented in this investigation.

The occurrence and prevalence of antimicrobial-resistant NAS in retail pork and slaughterhouse carcass samples have not been well investigated in Korea. Recent reports of methicillin-resistant CoNS from food-producing animals have raised concerns regarding transmission of these antimicrobial-resistant staphylococci through the meat production chain (Huber et al., 2011; Nemeghaire et al., 2014b). In this study, 7/44 (15.9%) staphylococci from slaughterhouse carcass samples were methicillin-resistant staphylococci (two MRSA and five MRSE strains) (Table 2). Out of the two MRSA strains, only one strain of S. aureus (SSA1) was ST398 carrying SCCmec V, which has been frequently reported S. aureus genotype in pigs and pork meat worldwide (Chuang and Huang, 2015; Golding et al., 2010; Lozano et al., 2009). Consistent with previous reports (Garza-González et al., 2010; Ruppé et al., 2009), 4/5 MRSE carried SCCmec IV for methicillin resistance. As presented in Table 3, all the M. sciuri isolates displayed a low-level of the OXA resistance phenotype (0.5–2 μg/mL). However, all of these OXA-resistant isolates were susceptible to FOX (Tables 3 and 4), and none of them were positive for the mecA gene. Previously, it has been reported that CoNS isolates other than S. epidermidis strains that displays OXA MICs of 0.5–2 μg/mL may lack mecA (Feßler et al., 2010), and have been defined as methicillin-susceptible strains (CLSI, 2015). It has been suggested that the mecA–negative OXA-resistant CoNS may overexpress penicillinase (Kolbert et al., 1995).

For the last ten years, over a two-fold increase (from 15% to 34%) in FA resistance in clinical isolates of S. aureus has been reported in Korea (Hong et al., 2016). More recently, it has been reported that ~27% of S. pseudintermedius isolates from canine pyoderma and otitis were resistant to FA (Lim et al., 2020). In the current study, as shown in Table 4, 9/10 S. epidermidis strains and the 32 M. sciuri strains displayed FA resistance. Similarly, the high rates of resistance to FA in S. saprophyticus, S. xylosus, and M. sciuri isolates collected from ready-to-eat foods have been reported in Taiwan (Wang et al., 2019). CoNS isolated from meat products also displayed FA resistance rates of 79.2% and 43% in Nigeria (Okoli et al., 2018) and Poland (Fijałkowski et al., 2016) respectively. Recent studies from Taiwan and the UK reported 14% and 46% prevalence, respectively, of FA resistance in clinical isolates of S. epidermidis (Chen et al., 2011; McLaws et al., 2008). The widespread occurrence of FA resistance in non-aureus staphylococcal isolates indicate that NAS such as S. epidermidis and M. sciuri could become a significant public health concern, serving as a reservoir of antimicrobial resistance through food chains. In line with previous reports (Chen et al., 2011; Lee et al., 2018; McLaws et al., 2008), FA resistance in S. epidermidis isolates from slaughterhouse carcasses in this study was mediated by the fusB gene (Table 3). Although one strain of S. epidermidis (SSE3) was double positive for fusB and fusC genes, this strain showed a FA MIC of 8 μg/mL, indicating that the presence of the two fusB-family genes does not confer a high-level FA resistance phenotype (≥ MIC of 128 μg/mL). None of the S. epidermidis isolates displaying FA resistance were positive for fusD nor fusF (Table 3). Sequencing analyses of fusA in S. epidermidis revealed that the SSE9 strain had V599I mutation within the linker site between domain IV and V of EF-G. Amino acid sequence substitutions in EF-G have frequently been associated with high-levels of FA resistance (Fernandes, 2016). However, the location of V599I mutation within EF-G and the relatively low-level FA resistance (MIC of 24 μg/mL) indicate that the point mutation in V599I is not causing FA resistance in the S. epidermidis strain.

The high prevalence of FA resistance in M. sciuri isolated from ready-to-eat-foods (99%) (Wang et al., 2019), healthy chickens (100%) (Nemeghaire et al., 2014b), and livestock (100%) (Bagcigil et al., 2007) has recently been reported. Similar to these reports, all of the M. sciuri isolates from retail pork (n=29) and slaughterhouse carcasses (n=3) exhibited FA resistance (Tables 3 and 4). To determine genetic factors involved in the FA resistance in M. sciuri isolates, a specific primer set detecting homologues of fusB-family genes (fusB/C) was designed in the current study using the published sequences of seven M. sciuri strains in NCBI databases. All the M. sciuri isolates carried the fusB/C gene for the FA resistance phenotype (Table 3), and the sequencing analyses of the amplified PCR products confirmed the sequences of fusB/C genes (data not shown). As shown in Table 5 and Fig. 2, FusB/C protein from M. sciuri displayed 42%–47% of similarity to amino acid sequences of previously characterized FusB-family proteins. None of the 32 M. sciuri strains had point mutation in the fusA gene, correlating with the low-level FA resistance phenotypes observed in the M. sciuri strains. These results combined with the MLST analyses suggests that various genetic lineages of S. epidermidis and M. sciuri strains contribute to the high prevalence of FA resistance in NAS isolated from retail pork and slaughterhouse carcass samples in Korea.

Table 5. Similarities of FusB-family protein amino acid sequences among staphylococci and mammaliicocci
FusB-family proteins Species Accession number Amino acid size Amino acid sequence similarity
FusB (%) FusC (%) FusD (%) FusF (%) MS-FusB/C (%)
FusB Staphylococcus aureus CAL23838.1 213 - 45 47 53 44
FusC Staphylococcus aureus WP_001033157.1 212 45 - 41 42 47
FusD Staphylococcus saprophyticus BAE19310.1 213 47 41 - 68 42
FusF Staphylococcus cohnii AVL76727.1 214 53 42 68 - 46
MS-FusB/C Mammaliicoccus sciuri QYG31551.1 214 44 47 42 46 -
MF-FusB/C Mammaliicoccus fleurettii WP_078357269.1 214 44 46 43 47 92
MV-FusB/C Mammaliicoccus vitulinus QQT15456.1 214 44 44 43 48 87
ML-FusB/C Mammaliicoccus lentus QMU10254.1 214 41 43 41 46 81
Download Excel Table
Fig. 2. Amino acid sequence alignment of FusB/C from mammaliicocci and FusB-family proteins. GenBank accession numbers are FusB/C, (in Mammaliicoccus sciuri, QYG31551.1; Mammaliicoccus fleurettii, WP_078357269.1; Mammaliicoccus vitulinus, QQT15456.1; Mammaliicoccus lentus, QMU10254.1); FusB, CAL23838.1; FusC, WP_001033157.1; and FusD, BAE19310.1. Amino acids highlighted are conserved sequences. (The key residues associated with the interaction with EF-G are underlined.) Sequences were pairwised with dots for identities.
Download Original Figure

In summary, our results suggest that i) a relatively higher level of NAS than S. aureus are present in pork production chains, particularly in slaughterhouse carcass samples, ii) there is a high prevalence of FA resistance in NAS isolates, especially in S. epidermidis and M. sciuri isolates, and iii) fusB-family genes, rather than fusA mutations, caused the high occurrence of FA-resistant S. epidermidis and M. sciuri. Our results demonstrate a high prevalence of FA-resistant NAS in pork meat production chains, which may act as reservoirs for FA resistance. To the best of our knowledge, this is the first study to report the genetic determinants and prevalence of FA resistance in NAS collected from pork meat production chains in Korea.

Conflicts of Interest

The authors declare no potential conflicts of interest.


This work was supported by the New Faculty Startup Fund from Seoul National University and Research of Korea Centers for Disease Control and Prevention (Project No. 2020ER540500).

Author Contributions

Conceptualization: Yang SJ. Data curation: Yang YJ, Yang SJ. Formal analysis: Yang SJ. Methodology: Lee GY, Kim SD, Park JH, Lee SI. Investigation: Yang YJ, Lee GY, Yang SJ. Writing - original draft: Yang YJ, Yang SJ. Writing - review & editing: Yang YJ, Lee GY, Kim SD, Park JH, Lee SI, Kim GB, Yang SJ.

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.



Archer GL, Niemeyer DM. 1994; Origin and evolution of DNA associated with resistance to methicillin in staphylococci. Trends Microbiol. 2:343-347


Bagcigil FA, Moodley A, Baptiste KE, Jensen VF, Guardabassi L. 2007; Occurrence, species distribution, antimicrobial resistance and clonality of methicillin- and erythromycin-resistant staphylococci in the nasal cavity of domestic animals. Vet Microbiol. 121:307-315


Chen HJ, Hung WC, Lin YT, Tsai JC, Chiu HC, Hsueh PR, Teng LJ. 2015; A novel fusidic acid resistance determinant, fusF, in Staphylococcus cohnii. J Antimicrob Chemother. 70:416-419


Chen HJ, Hung WC, Tseng SP, Tsai JC, Hsueh PR, Teng LJ. 2010; Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother. 54:4985-4991


Chen HJ, Tsai JC, Hung WC, Tseng SP, Hsueh PR, Teng LJ. 2011; Identification of fusB-mediated fusidic acid resistance islands in Staphylococcus epidermidis isolates. Antimicrob Agents Chemother. 55:5842-5849


Chuang YY, Huang YC. 2015; Livestock-associated meticillin-resistant Staphylococcus aureus in Asia: An emerging issue?. Int J Antimicrob Agents. 45:334-340


Clinical and Laboratory Standards Institute [CLSI]. 2015 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 3rd ed.Clinical and Laboratory Standards Institute. Wayne, PA, USA: .


Davis CP. 1996 Chapter 6 Normal flora. In Medical microbiology. 4th ed. In: Baron S, editor.University of Texas Medical Branch at Galveston. Galveston, TX, USA: .


Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. 2000; Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 38:1008-1015


EUCAST. 2021 European committee on antimicrobial susceptibility testing. Available from [Accessed at Aug 2, 2021]. .


Feßler AT, Billerbeck C, Kadlec K, Schwarz S. 2010; Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother. 65:1576-1582


Fernandes P. 2016; Fusidic acid: A bacterial elongation factor inhibitor for the oral treatment of acute and chronic staphylococcal infections. Cold Spring Harb Perspect Med. 6:a025437


Fijałkowski K, Peitler D, Karakulska J. 2016; Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile. Int J Food Microbiol. 238:113-120


Founou LL, Founou RC, Essack SY. 2016; Antibiotic resistance in the food chain: A developing country-perspective. Front Microbiol. 7:1881


Garza-González E, López D, Pezina C, Muruet W, Bocanegra-García V, Muñoz I, Ramírez C, LLaca-Díaz JM. 2010; Diversity of staphylococcal cassette chromosome mec structures in coagulase-negative staphylococci and relationship to drug resistance. J Med Microbiol. 59:323-329


Geha DJ, Uhl JR, Gustaferro CA, Persing DH. 1994; Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J Clin Microbiol. 32:1768-1772


Godtfredsen WO, Jahnsen S, Lorck H, Roholt K, Tybring L. 1962; Fusidic acid: A new antibiotic. Nature. 193:987


Golding GR, Bryden L, Levett PN, McDonald RR, Wong A, Wylie J, Graham MR, Tyler S, van Domselaar G, Simor AE, Gravel D, Mulvey MR. 2010; Livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 in humans, Canada. Emerg Infect Dis. 16:587-594


Hanson BM, Dressler AE, Harper AL, Scheibel RP, Wardyn SE, Roberts LK, Kroeger JS, Smith TC. 2011; Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) on retail meat in Iowa. J Infect Public Health. 4:169-174


Hong SN, Kim J, Sung HH. 2016; A study on changes in antimicrobial resistant Staphylococcus aureus from wound isolates in a South Korean University Hospital for the past 10 years (2006, 2016). Korean J Clin Lab Sci. 48:335-342


Huber H, Ziegler D, Pflüger V, Vogel G, Zweifel C, Stephan R. 2011; Prevalence and characteristics of methicillin-resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons. BMC Vet Res. 7:6


Hung WC, Chen HJ, Lin YT, Tsai JC, Chen CW, Lu HH, Tseng SP, Jheng YY, Leong KH, Teng LJ. 2015; Skin commensal staphylococci may act as reservoir for fusidic acid resistance genes. PLOS ONE. 10e0143106


Kolbert CP, Connolly JE, Lee MJ, Persing DH. 1995; Detection of the staphylococcal mecA gene by chemiluminescent DNA hybridization. J Clin Microbiol. 33:2179-2182


Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu K. 2007; Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother. 51:264-274


Lee JYH, Monk IR, Gonçalves da Silva A, Seemann T, Chua KYL, Kearns A, Hill R, Woodford N, Bartels MD, Strommenger B, Laurent F, Dodémont M, Deplano A, Patel R, Larsen AR, Korman TM, Stinear TP, Howden BP. 2018; Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis. Nat Microbiol. 3:1175-1185


Lim YJ, Hyun JE, Hwang CY. 2020; Identification of fusidic acid resistance in clinical isolates of Staphylococcus pseudintermedius from dogs in Korea. Vet Dermatol. 31:267-e62


Lozano C, López M, Gómez-Sanz E, Ruiz-Larrea F, Torres C, Zarazaga M. 2009; Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain. J Antimicrob Chemother. 64:1325-1326


McLaws F, Chopra I, O’Neill AJ. 2008; High prevalence of resistance to fusidic acid in clinical isolates of Staphylococcus epidermidis. J Antimicrob Chemother. 61:1040-1043


Mendoza M, Meugnier H, Bes M, Etienne J, Freney J. 1998; Identification of Staphylococcus species by 16S-23S rDNA intergenic spacer PCR analysis. Int J Syst Bacteriol. 48:1049-1055


Nam HM, Lee AL, Jung SC, Kim MN, Jang GC, Wee SH, Lim SK. 2011; Antimicrobial susceptibility of Staphylococcus aureus and characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Korea. Foodborne Pathog Dis. 8:231-238


Nemeghaire S, Argudín MA, Feßler AT, Hauschild T, Schwarz S, Butaye P. 2014a; The ecological importance of the Staphylococcus sciuri species group as a reservoir for resistance and virulence genes. Vet Microbiol. 171:342-356


Nemeghaire S, Argudín MA, Haesebrouck F, Butaye P. 2014b; Molecular epidemiology of methicillin-resistant Staphylococcus sciuri in healthy chickens. Vet Microbiol. 171:357-363


Norström T, Lannergård J, Hughes D. 2007; Genetic and phenotypic identification of fusidic acid-resistant mutants with the small-colony-variant phenotype in Staphylococcus aureus. Antimicrob Agents Chemother. 51:4438-4446


O’Brien AM, Hanson BM, Farina SA, Wu JY, Simmering JE, Wardyn SE, Forshey BM, Kulick ME, Wallinga DB, Smith TC. 2012; MRSA in conventional and alternative retail pork products. PLOS ONE. 7e30092


Okoli CE, Njoga EO, Enem SI, Godwin EE, Nwanta JA, Chah KF. 2018; Prevalence, toxigenic potential and antimicrobial susceptibility profile of Staphylococcus isolated from ready-to-eat meats. Vet World. 11:1214-1221


O’Neill AJ, Chopra I. 2006; Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus. Mol Microbiol. 59:664-676


O’Neill AJ, McLaws F, Kahlmeter G, Henriksen AS, Chopra I. 2007; Genetic basis of resistance to fusidic acid in staphylococci. Antimicrob Agents Chemother. 51:1737-1740


Piette A, Verschraegen G. 2009; Role of coagulase-negative staphylococci in human disease. Vet Microbiol. 134:45-54


Ruppé E, Barbier F, Mesli Y, Maiga A, Cojocaru R, Benkhalfat M, Benchouk S, Hassaine H, Maiga I, Diallo A, Koumaré AK, Ouattara K, Soumaré S, Dufourcq JB, Nareth C, Sarthou JL, Andremont A, Ruimy R. 2009; Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries. Antimicrob Agents Chemother. 53:442-449


Schauer B, Szostak MP, Ehricht R, Monecke S, Feßler AT, Schwarz S, Spergser J, Krametter-Frötscher R, Loncaric I. 2021; Diversity of methicillin-resistant coagulase-negative Staphylococcus spp. and methicillin-resistant Mammaliicoccus spp. isolated from ruminants and New World camelids. Vet Microbiol. 254:109005


Sugden R, Kelly R, Davies S. 2016; Combatting antimicrobial resistance globally. Nat Microbiol. 1:16187


Tang Y, Larsen J, Kjeldgaard J, Andersen PS, Skov R, Ingmer H. 2017; Methicillin-resistant and -susceptible Staphylococcus aureus from retail meat in Denmark. Int J Food Microbiol. 249:72-76


Thomas JC, Vargas MR, Miragaia M, Peacock SJ, Archer GL, Enright MC. 2007; Improved multilocus sequence typing scheme for Staphylococcus epidermidis. J Clin Microbiol. 45:616-619


Turnidge J, Collignon P. 1999; Resistance to fusidic acid. Int J Antimicrob Agents. 12(Suppl 2):S35-S44


von Wintersdorff CJH, Penders J, van Niekerk JM, Mills ND, Majumder S, van Alphen LB, Savelkoul PHM, Wolffs PFG. 2016; Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Front Microbiol. 7:173


Wang YT, Lin YT, Wan TW, Wang DY, Lin HY, Lin CY, Chen YC, Teng LJ. 2019; Distribution of antibiotic resistance genes among Staphylococcus species isolated from ready-to-eat foods. J Food Drug Anal. 27:841-848


Wu S, Huang J, Wu Q, Zhang J, Zhang F, Yang X, Wu H, Zeng H, Chen M, Ding Y, Wang J, Lei T, Zhang S, Xue L. 2018; Staphylococcus aureus isolated from retail meat and meat products in China: Incidence, antibiotic resistance and genetic diversity. Front Microbiol. 9:1-14