Genetic characterization of livestock-associated methicillin-resistant Staphylococcus aureus isolated in Greece

Abstract

The interest in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains is increasing due to their wide distribution and transmission even in persons without previous contact with livestock, and these strains pose a public health threat. The aim of the study was the genetic characterization of the whole genome of two epidemiologically unrelated t034 LA-MRSA strains previously isolated from the nasal cavities of a goat and a farmer in Greece. Both strains were assigned to the ST398-Vc-t034 type and they were carrying a single transposon identical to Tn6133. They harbored genes conferring resistance to several antibiotics (aminoglycosides, β-lactams, macrolides, streptogramin B, tetracycline, and trimethoprim), and genes associated with virulence (enterotoxins, γ-hemolysins, and aureolysin). The present study can serve as baseline for further LA-MRSA epidemiological and evolutionary studies in Greece, while awareness and increased surveillance are needed to avoid their spread.

Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a severe threat to public health as it causes hospital-acquired (ΗΑ) and community-acquired (CA) infections [1], while a third group, livestock-associated MRSA (LA-MRSA), was identified in the early 2000s when strain exchange was observed between pigs and farmers in France and the Netherlands [2, 3]. Since then, LA-MRSA is often being detected even in patients who had no previous contact with livestock, while nosocomial transmissions of LA-MRSA have been also confirmed [4–6]. The initially detected predominant sequence types (STs) of LA-MRSA were ST9, ST398, and ST433 [2, 3]. Among them, LA-MRSA ST398 has become a rapidly emerging cause of human infections [7].

In Greece, LA-MRSA was detected in patients with or without prior contact with animals [8–11]. In a study conducted during November 2016–June 2017 in 40 dairy (cattle, sheep, and goat) farms in northern Greece, S. aureus and MRSA were detected in 47.8% and 4.1% of 387 samples from raw milk, farmers, and livestock, respectively [12]. Two of the MRSA strains originating from that study, isolated in 2017 from the nasal cavity of a 4-year-old goat (G-MRSA) and an asymptomatic cattle farmer (H-MRSA) working in a distantly located cattle farm, were assigned to spa type t034 [12]. Since t034 spa type is strongly associated with LA-MRSA clonal complex (CC) 398 [13], the aim of the present study was to genetically analyze the whole genome sequence of these two isolates focusing on the analysis of antimicrobial resistance genes, virulence factors, and mobile genetic elements. Both isolates were resistant to penicillin, oxacillin, trimethoprim/sulfamethoxazole, trimethoprim, erythromycin, tetracycline, and amoxicillin/clavulanic acid, and they were biofilm producers and non-typeable by SmaI-PFGE. Both carried the mecA gene and the enterotoxin sec gene, while H-MRSA carried also the sed gene [12].

DNA was extracted from each isolate using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The concentration of double-strand (ds) DNA was measured using Qubit ds DNA HS assay kit (Q32851, Life Technologies). NGS was performed using the Ion Torrent S5 PGM Platform (Life Technologies Corporation, Grand Island, NY, USA). Shearing, purification, ligation, barcoding, size selection, library amplification and quantitation, emulsion PCR, and enrichment were performed according to the manufacturer’s instructions and the products were loaded on a 316 chip. The Ion PGMHi-Q (200) chemistry (Ion PGM Hi-Q Sequencing kit, A25592) was applied. De novo assembly was performed with SPADES version 5. Strain RIVM1295 (accession number CP013616) was used as a reference sequence, while the Tn6133 sequence (accession number FR772051) was used as a reference sequence for the transposon. Identification of MLST, antimicrobial resistance and virulence genes, SCCmec elements, and plasmids was performed applying the tools using MLST 2.0, Resfinder 4.1, KmerResistance 2.2, Comprehensive Antibiotic Resistance Database (CARD), VirulenceFinder 2.0, SCCmecFinder 1.2, and PlasmidFinder 2.1 [14–19]. Detection and identification of insertion sequences (ISs) were performed using the ISFinder software [20] and the database URL (http://www-is.biotoul.fr). Genome annotation of virulence and pathogenesis genes was performed using the Rapid Annotation using Subsystem Technology (RAST) v.2.0 server [21].

It was shown that both MRSA isolates belonged to ST398, and the predicted SCCmec type was Vc (5C2&5). Genes conferring resistance to aminoglycosides (ant(9)-Ia), β-lactams (mecA and blaZ), macrolides and streptogramin B (vgaE and ermA), tetracycline (tetK and tetM), and trimethoprim (dfrG) were detected in both isolates (Table 1). The major detected virulence factors were related to adherence, exoenzymes, immune evasion, iron uptake, and toxins and are seen in Table 1, while the characteristics of the intact phages are seen in Table 2. The detected ISs, together with the respective score and e-values, are shown in Table 3. None of the isolates was found to carry PVL gene or any of the human-related immune evasion cluster (IEC) genes (Sa3int, sak, scn, sea, and sep). The two MRSA isolates contained a transposon of 11,475 bp, identical to the transposon Tn6133 identified in the IMD49-10 strain isolated from a pig in Switzerland (accession number FR772051). Both MRSA harbored one plasmid of the rep_7a family. The rep gene was 955-nt long and encoded the protein repC (413 aa). The subsystem categories of the virulence and pathogenesis genes of the two strains generated through RAST software are shown in Fig. 1. The whole genome sequences of G-MRSA and H-MRSA were submitted to European Nucleotide Archive (ENA) under the study PRJEB39748 and received the accession numbers ERS4944046 and ERS4944047, respectively.

Table 1.

Genetic characteristics of the two MRSA t034 isolates of the study

	G-MRSA	H-MRSA
Size (bp)	2,121,406	2,817,670
GC content (%)	32.9	32.8
Number of contigs (with PEGs)	1402	328
MLST	ST398	ST398
Antimicrobial resistance genes	mecA, blaZ, ant(9)-Ia, vgaE, ermA, tetK, tetM, dfrG, czrC	mecA, blaZ, ant(9)-Ia vgaE, ermA, tetK, tetM, dfrG, czrC
Efflux pump genes	mgrA, mepR, LmrS	mgrA, arlR, tet(K), mepR, norA, LmrS
Plasmid	rep7a	rep7a
Virulence factor (gene)
Adherence
Collagen binding protein (cna)	+	+
Clumping factor (clfA, clfB)	+	+
Elastin-binding protein (ebp)	+	+
Extracellular adherence protein/MHC analogous protein (map)	+	+
Fibronectin binding proteins (fnbA, fnbB)	+	+
Intercellular adhesion proteins (icaA, icaB, icaC, icaD, icaR)	+	+
Ser-Asp rich proteins (sdrC, sdrD, sdrE)	+	+
Exoenzyme
Aureolysin (aur)	+	+
Staphylocoagulase (coa)	+	+
V8 protease (sspA)	+	+
von Willebrand factor-binding protein (vWbp)	+	+
Immune evasion
Protein A (spa)	+	+
Iron uptake
Isd, srt	isdA, isdC, isdD, isdE, isdF, isdH, srtB	isdA, isdB, isdC, isdD, isdE, isdF, isdH, srtB
Toxins
γ-Hemolysin (hlgA, hlgB, hlgC)	+	+
Staphylococcal enterotoxins (sea, seb, sec, sec, sed, see, she, selk, selq)	sec	sec, sed

Table 2.

Intact prophage regions in the two MRSA isolates of the study

Strain	Intact phages	Length (kb)	GC (%)	CDS
H-MRSA	PHAGE_Staphy_YMC/09/04/R1988_NC_022758(19)	30.2	33.71	37
	PHAGE_Staphy_55_NC_007060(13)	24	36.60	31
G-MRSA	PHAGE_Staphy_phi2958PVL_NC_011344(15)	17.7	33.11	33

Table 3.

Insertion sequences in G-MRSA and H-MRSA isolates of the study

Sequences producing significant alignments	IS family	Group	Origin	Score (bits)	E-value
ISSau3	IS1182		S. aureus	3130	0.0
ISSau8	ISL3		S. aureus	2615	0.0
ISSau2	IS3	IS150	S. aureus	2016	0.0
ISSau1	IS30		S. aureus	1853	0.0
IS431mec	IS6		S. aureus	1542	0.0
IS431R	IS6		S. aureus	1530	0.0
IS257R2	IS6		S. aureus	1524	0.0
IS257R1	IS6		S. aureus	1511	0.0
IS431L	IS6		S. aureus	1497	0.0
IS257-3	IS6		S. aureus	1366	0.0
IS257-1	IS6		S. aureus	1346	0.0
IS257-2	IS6		S. aureus	791	0.0
ISSep1	IS1182		S. epidermidis	722	0.0

Fig. 1.

Subsystem categories of the virulence and pathogenesis genes of A H-MRSA and B G-MRSA generated through RAST software

Although the isolates were derived from different farms, with no evidence of epidemiological link, they were similar, differing only in the phage composition and few virulence factors. They belonged to ST398, a fact that explained their non-typeability by SmaI-PFGE due to DNA methylation which occurs in ST398 isolates including those of type t034 [22]. ST398 is considered the most prevalent livestock-associated ST in Europe [23]. There are reports of CC398 MRSA strains in Greece from patients [11, 24], but the present study reports the first WGS of LA-MRSA CC398 in livestock (goat) in Greece and a farmer working in close contact with cattle. Both G- and H-MRSA isolates harbored the SCCmec type Vc (5C2&5) element. The vast majority of LA-MRSA strains (including CC398 LA-MRSA) carry this specific type, which contains genes related to the detoxification of heavy metals, such as cadmium (cadmium zinc resistance C, czrC) and copper (copA) [25, 26]. It has to be mentioned that copper is routinely used as a growth enhancer in farming [27, 28], while the presence of copper resistance genes contributes to bacterial survival [29].

The two MRSA isolates carried a large variety of antimicrobial resistance genes which have been previously detected in ST398 LA-MRSA strains [26, 30–32]. It has been shown that isolates carrying the Vc(5C2&5) element co-harbor tet(K) in a higher proportion than isolates with other SCCmec elements [33].

Βoth G- and H-MRSA isolates carried the enterotoxin sec gene, and H-MRSA carried also the sed gene; however, they lacked scn, chp, and sak, as is the case for LA-MRSA [34], while aur gene has been reported in LA-methicillin-susceptible S. aureus ST398 strains [35]. The detection of hlgA, hlgB, and hlgC genes has been associated with human and livestock carriage [36, 37]. PVL and human-related IEC genes were not detected in the two isolates of the study, which is in accordance with other studies that reported that the majority of ST398 LA-MRSA strains lack major virulence factors, such as PVL, exfoliative toxins, and TSST-1 [38]. Subsequently, both isolates lacked the bacteriophage ΦSa3 containing the IEC genes, as previously described in ST398 LA-MRSA strains [39].

Several ISs were detected in the two isolates, most of them belonging to the IS6 family, while ISSau2 of the IS3 family was identified in LA-MRSA ST398 [40]. Furthermore, IS257 and IS431 elements detected in both strains have been previously reported in LA-MRSA strains [41, 42].

Both MRSA isolates carried a Tn6133 transposon as previously described in a human MRSA ST398 strain in Greece [43]. Similarly, LA-MRSA ST398-t034 strains carrying the Tn6133 transposon have been isolated from cattle and poultry in Germany [44].

LA-MRSA is prevalent in multiple animal species, with pigs being the predominant host worldwide [45]. Recently, it was shown that farmed mink in Denmark constitute a reservoir of MRSA CC398 posing a potential exposure risk to numerous mink farm workers, and it was demonstrated that pigs were the source of LA-MRSA CC398 in mink feed, mink, and mink farmers [46].

There is an ongoing evolution of LA-MRSA strains, as they can acquire antimicrobial resistance genes and virulence factors from other bacteria [47]. The only way to gain a better insight into their genetic characterization is through WGS. The present study provides a first insight into the genetic characterization of two LA-MRSA isolated in Greece and can serve as basis for future studies. Since LA-MRSA carriage represents mainly an occupational risk, awareness, infection control measures, and implementation of high hygiene standards in farms are critical to eliminating the spread of these strains in the community and the nosocomial settings. Further studies are needed to identify the prevalence of LA-MRSA in humans and livestock in Greece.

Author contribution

All the authors have contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript.

Funding

The current work was supported by the European Union’s Horizon 2020 grant VEO (grant number 874735).

Availability of data and material

The whole genome sequences of G-MRSA and H-MRSA were submitted to European Nucleotide Archive (ENA) under the study PRJEB39748 and received the accession numbers ERS4944046 and ERS4944047, respectively.

Declarations

Ethics approval and consent to participate

Ethical approval was not required as both isolates were obtained from previously published studies.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.