First isolation of ST398 methicillin-resistant Staphylococcus aureus carrying staphylococcal cassette chromosome mec type IVd from pig ears in Japan.

The emergence and increasing prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) are a global concern. To investigate the prevalence and characteristics of sequence type 398 (ST398) MRSA in pig ears, 102 pig's ears were collected from 102 animals shipped from 51 farms at an abattoir. Eight ST398 MRSA isolates were isolated from the ears of eight pigs shipped from seven farms. Of the eight ST398 isolates, seven had the staphylococcal cassette chromosome mec (SCCmec) type IVd and these were obtained from seven pigs shipped from six farms. Single nucleotide polymorphisms ranging from 13 to 26 were observed in the core-genome regions in the seven SCCmec type IVd isolates. We believe that this is the first report on the isolation of ST398 MRSA SCCmec type IVd in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major drug-resistant pathogen responsible for a variety of infections worldwide, ranging from mild skin infections to life-threatening invasive diseases. In recent decades, the emergence and increasing prevalence of livestock-associated MRSA (LA-MRSA), particularly sequence type 398 (ST398) in pigs, have become a global concern [11, 25,26,27,28]. Since ST398 MRSA was isolated from pigs in Japan in 2012, it has been isolated from pigs in the Kanto and Tohoku regions of Japan as well [20, 21]. Typing of both staphylococcal cassette chromosome mec (SCCmec) and region X of the protein A gene (spa) revealed that divergent types of MRSA are distributed in Japan. Two SCCmec types, IVa and V, of ST398 MRSA are dominant with spa types t034, t011, t3934, and t16450 in Japan [20, 21]. Moreover, as ST398 MRSA was found in imported pigs at animal quarantine stations in Japan [12], the divergence of ST398 MRSA may increase.

The MRSA isolation rate is reportedly higher in swabs of the skin behind the ear than in nasal swabs [2]. Because pig ears are edible parts and shipped from abattoirs for consumption, data on the prevalence and characteristics of LA-MRSA, particularly ST398, in pig ears are required to estimate the risk of transmission from contaminated ears to humans. This study aimed to investigate the prevalence and characteristics of ST398 MRSA in the ears of pigs slaughtered at abattoirs.

Sampling was conducted at an abattoir in the Kanto region for 10 days in December 2018. We collected ears of 102 pigs (two per farm) shipped from 51 farms. Of the 51 farms, 10 were located in the region of Tohoku, 40 in Kanto, and one in Kyushu. An ear was removed from the heads of slaughtered pigs and placed in a plastic bag. The collected samples were stored at 4°C and delivered to the National Institute of Health Sciences.

At the laboratory, MRSA isolation was conducted within 48 hr of sampling. One whole ear per pig was inoculated in 100 mL of Mueller Hinton (MH) broth (Kanto Chemical, Tokyo, Japan) containing 6.5% NaCl and incubated for 18–24 hr at 37°C for enrichment. After incubation, a loopful of the MH culture was plated onto CHROMagarTM MRSA medium (CHROMagar, Paris, France) and Pourmedia® MRSA II medium (Eiken Chemical, Tokyo, Japan) and incubated for 48 hr at 37°C. When suspected MRSA colonies were observed on these media, up to two colonies per sample were selected and subsequently identified as Staphylococcus aureus using commercial identification kits (CycleavePCRTMStaphylococcus aureus [DnaJ gene] Detection Kit; TaKaRa Bio, Kusatsu, Japan). The isolates were tested for the presence of the pvl and hlg genes encoding Panton–Valentine leukocidin and γ-hemolysin, respectively, using polymerase chain reaction (PCR), as previously described [17]. The S. aureus ATCC strain BAA-1556 (USA 300 clone) was used as a control in the identification procedures. One MRSA isolate per sample was subjected to molecular typing and antimicrobial susceptibility testing. MRSA isolates were characterized via multilocus sequence typing [10] and spa typing [13]. SCCmec typing was also performed using two multiplex PCR assays [15, 29]. The isolates were tested for the presence of czrC, the gene encoding a protein that confers cadmium and zinc resistance, using PCR as previously described [5]. MRSA isolates were also tested for susceptibility to ampicillin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, trimethoprim, gentamicin, teicoplanin, tetracycline, oxacillin, and vancomycin. Oxacillin was used to confirm the identity of the isolate as MRSA. The minimal inhibitory concentrations (MICs) of these antimicrobials were determined using the broth microdilution method on dried plates (Eiken Chemical, Tokyo, Japan) according to the guidelines of the Clinical and Laboratory Standards Institute [6, 7]. S. aureus ATCC 29213 was used as the quality control strain. The MIC of zinc chloride was determined using a previously reported agar dilution assay for zinc chloride (0.25 to 16 mM) [1]. An MIC value >2 mM was used as the cut-off value to designate resistance in accordance with a previous report [1]. In addition, the presence of antimicrobial resistance genes and phylogenetic relationships in eight ST398 MRSA isolates was confirmed using draft whole-genome sequencing (WGS). Briefly, a DNA library was prepared using a QIAGEN QIAseq FX DNA library kit (QIAGEN, Hilden, Germany) and subjected to WGS using an Illumina iSeq sequencer (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The obtained data of sequence reads were submitted to ResFinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/). Core-genome phylogenetic analysis was performed according to our previous report [4] and S. aureus 08BA02176 genome (accession number: CP003808.1) was used as a reference. The draft-WGS read data of the eight ST398 MRSA isolated in this study were deposited in the DNA Data Bank of Japan Sequence Read Archive on accession numbers from DRR381280 to DRR381287.

MRSA was isolated from the ears of 16 (15.7%) pigs shipped from 13 (25.5%) farms (Table 1). On three farms (farm codes a, h, and k), MRSA was isolated from both tested pigs. All the isolates possessed γ-hemolysin genes but not pvl. The 16 isolates were subjected to molecular typing. Eight isolates were classified as ST398 and all of them were classified as spa type t011. ST398 MRSA was isolated on five out of ten sampling days. The SCCmec type of seven ST398 isolates from pig ears from six farms (farms codes a to f) were IVd. The SCCmec type of one isolate from one of the farms (farms code g) was V. ST398 MRSA SCCmec type IVd isolates were obtained from the Kanto (prefecture codes A to D) and Tohoku (prefecture E) regions. All the ST398 isolates were resistant to ampicillin, tetracycline, and erythromycin. In addition, all the ST398 isolates possessed the czrC gene and were resistant to zinc chloride. Furthermore, six isolates were classified as ST5 and among them, four and two were classified as spa types t1560 and t002, respectively. ST5 MRSA was obtained from the Kanto region. The SCCmec type of ST5/t1560 was V. ST5/t002 isolates had the class A mec gene complex but were negative for ccr and were classified as atypical. In contrast, ST5/t1560 was resistant to ampicillin, ciprofloxacin, and erythromycin, whereas ST5/t002 was susceptible to both ciprofloxacin and erythromycin. Two isolates were classified as ST8 and spa types t1767 or t18785. The SCCmec type of ST8 was IVa. ST8 MRSA was resistant to ampicillin, gentamicin, and kanamycin. None of the ST5 and ST8 isolates possessed czrC and all were susceptible to zinc chloride.

Table 1.

Characteristics of methicillin-resistant Staphylococcus aureus isolated in this study

Prefecture code	Region	Farm code	Isolate	MLST		spa	SCCmec	Antimicrobial resistance profile	Zinc resistance		Other antimicrobial resistant genes
				CC	ST				czrC	MIC (mM)
A	Kanto	a	a-1	398	398	t011	IVd	ABPC, TC, EM	+	4	blaI, blaR1, blaZ, erm(C), mecA, tet(38), tet(M)
			a-2	398	398	t011	IVd	ABPC, TC, CP, EM	+	4	blaI, blaR1, blaZ, erm(C), fexA, mecA, str, tet(38), tet(M)
A	Kanto	b	b-1	398	398	t011	IVd	ABPC, TC, CP, EM	+	4	blaI, blaR1, blaZ, erm(C), fexA, mecA, qacG, str, tet(38), tet(M)
B	Kanto	c	c-1	398	398	t011	IVd	ABPC, TC, CP, EM	+	4	blaI, blaR1, blaZ, erm(C), fexA, mecA, qacG, tet(38), tet(M)
C	Kanto	d	d-1	398	398	t011	IVd	ABPC, TC, EM	+	4	blaI, blaR1, blaZ, erm(C), mecA, qacG, tet(38), tet(M)
D	Kanto	e	e-1	398	398	t011	IVd	ABPC, TC, EM	+	4	blaI, blaR1, blaZ, erm(C), mecA, str, tet(38), tet(M)
E	Tohoku	f	f-1	398	398	t011	IVd	ABPC, TC, EM	+	4	blaI, blaR1, blaZ, erm(C), mecA, qacG, tet(38), tet(M)
A	Kanto	g	g-1	398	398	t011	V	ABPC, TC, CP, EM, TMP	+	4	ant (6)-Ia, blaI, blaR1, blaZ, dfrG, erm(C), fexA, lnu(B), lsa(E), mecA, spw, tet(38), tet(K), tet(M)
A	Kanto	h	h-1	5	5	t1560	V	ABPC, CPFX, EM	-	1	Not tested
			h-2	5	5	t1560	V	ABPC, CPFX, EM	-	1	Not tested
B	Kanto	i	i-1	5	5	t1560	V	ABPC, CPFX, EM	-	1	Not tested
F	Kanto	j	j-1	5	5	t1560	V	ABPC, CPFX, EM	-	1	Not tested
B	Kanto	k	k-1	5	5	t002	Atypical	ABPC	-	1	Not tested
			k-2	5	5	t002	Atypical	ABPC, CP	-	1	Not tested
E	Tohoku	l	l-1	8	8	t1767	IVa	ABPC, GM, KM,	-	1	Not tested
D	Kanto	m	m-1	8	8	t18785	IVa	ABPC, GM, KM, TC, CPFX, EM	-	1	Not tested

MLST: multlocus sequence typing, CC: clonal complex, ST: sequence type, SCCmec: staphylococcal cassette chromosome mec, ABPC: ampicillin, TC: tetracycline, TMP: trimethoprim, GM: gentamicin, EM: erythromycin, CP: chloramphenicol, MIC: minimal inhibitory concentration.

In Europe and North America, ST398 of SCCmec type V accounts for the majority of ST398 MRSA isolated from pigs [8, 14, 18, 19]. In contrast, seven ST398 MRSA isolates in the present study possessed SCCmec type IVd and the remaining one ST398 MRSA possessed SCCmec V. To the best of our knowledge, the isolation of ST398 MRSA carrying SCCmec type IVd has not been reported in Japan or elsewhere. We speculate that we have isolated ST398 MRSA carrying SCCmec type IVd for the first time, at least in Japan. Recent studies indicate that the use of zinc compounds, such as zinc sulfate and zinc oxide, leads to the selection and persistence of MRSA SCCmec type V in pig farms [3, 24]. This is because zinc resistance is encoded by czrC, which is expressed by MRSA SCCmec type V [5]. The ST398 MRSA of SCCmec type IVd obtained in the present study expressed czrC and can be selected by using zinc compounds similar to the approach used for MRSA of SCCmec type V.

The core-genome phylogenetic analysis showed relatively small numbers of single nucleotide polymorphisms (SNPs) (range, 13–26) in the seven ST398 MRSA isolates (a-1, a-2, b-1, c-1, d-1, e-1, and f-1) carrying SCCmec type IVd (Fig. 1). Conversely, the core-genome phylogenetic analysis showed relatively large numbers of SNPs (range, 478–486) between the ST398 MRSA isolate (g-1) carrying SCCmec type IVd isolate and the seven ST398 MRSA isolates carrying SCCmec type V. Duchêne et al. [9] reported that the mean substitution rate in the clonal complex (CC) 398 S. aureus (genome size, 2.8 megabase pairs) was 2.43 × 10−6 substitutions per site per year. The mean substitution rate can be interpreted as an average of 6.8 SNPs occurring in the CC398 genome per year. Therefore, the small numbers of SNPs among the seven ST398 MRSA of SCCmec type IVd indicate that they originated from a common ancestor a few years ago. In Japan, pigs are annually imported for breeding purposes. Furuno et al. [12] reported that 12 of 125 pigs imported from Europe and North America tested positive for MRSA ST398 during the quarantine period. A herd of pigs infected with ST398 MRSA of SCCmec type IVd may have been imported a few years ago, and the infected animals delivered to pig farms in the Tohoku and Kanto regions after the quarantine period.

Fig. 1.

Phylogenetic tree of ST398 methicillin-resistant Staphylococcus aureus isolates constructed with maximum-likelihood phylogenetic analysis based on single nucleotide polymorphisms (SNPs) in the core-genome and excluding homologous recombination sequences. The core-genome region was 92.5% (2,574,746/ 2,782,313 bp) of the genome of the reference strain, S. aureus 08BA02176 (accession number: CP003808.1). The scale distance corresponds to the number of SNPs per site.

Ears contaminated with ST398 MRSA originated from pigs in the Tohoku and Kanto regions. In contrast to the characteristics of ST398 MRSA reported in the two regions [20,21,22], the characteristics of the isolates in the present study were different. Fifty-eight ST398 MRSA isolates were obtained in the Kanto region between 2012 and 2014, and the most frequent spa type was t16450 (33 isolates), followed by t034 (14 isolates), and t3934 (11 isolates) [20]. The SCCmec types of t16450 and t034 were atypical and V, respectively. In the Tohoku region, 13 ST398 MRSA isolates were obtained in 2017, and the most frequent spa type was t034 (11 isolates) followed by t011 (two isolates) [21]. The SCCmec types of t034 were IVa (three isolates) and V (eight isolates).

The eight ST398 isolates were all positive for blaI, blaZ, ermC, tet(38), and tet(M). Of the seven SCCmec type IVd isolates, three had the chloramphenicol/florfenicol resistance gene (fexA) and four had the disinfectant resistance gene (qacG). One SCCmec type V isolate was multidrug-resistant and had ant(6)-Ia (aminoglycoside resistance), dfrG (trimethoprim resistance), fexA, lnu(B) and lsa(E) (both lincomycin resistance), and tet(K) (Table 1). A similar profile of resistance genes in ST398 MRSA SCCmec type V was reported in Korea [16]. However, the existence of the florfenicol resistance gene (fexA) and lincomycin resistance genes lnu(B) and lsa(E), which had not been reported in Japan, was verified. In addition, the resistance gene profiles of SCCmec type IVd isolates were obtained in this study.

In the present study, the prevalence of ST398 MRSA in pig ears was 7.8% (8/102, 95% confidence interval 3.8–14.9). The prevalence of MRSA in 40 domestic pork products was investigated in 2017 [23] but no ST398 MRSA was isolated from any of the pork products tested. However, other STs (ST97 and ST8) were isolated from two domestic pork products. Moreover, the prevalence of MRSA in swabs of the skin behind the ear and surface of the carcass collected from 276 pigs was investigated in 2019 and the prevalence of ST398 MRSA was statistically higher in skin swabs taken from behind the ear (14.5%, 40/276) than those from the carcasses (0.4%, 1/276) [22]. The risk of ST398 MRSA transmission to humans could be higher from ears than from pork.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to disclose.