Background
Staphylococcus aureus is a skin and mucosal commensal of humans and animals, and an important human pathogen involved in various infections, ranging from localized to life-threatening invasive diseases. Although human colonization varies with geographic location, seasonality, age and sex, ca. 30% individuals are nasal carriers of
S. aureus [
1].
S. aureus rapidly adapts to the selective pressure imposed by antimicrobial therapy, and methicillin-resistant
S. aureus (MRSA) has spread in both healthcare (hospital-associated MRSA, HA-MRSA) and community (community-associated MRSA, CA-MRSA) settings [
2]. Over the last decade, particular MRSA lineages also emerged in livestock animals (livestock-associated MRSA, LA-MRSA), with variable prevalence in different geographic regions. The predominant LA-MRSA clonal complex (CC) in Europe and North America is CC398, with the majority of the strains belonging to sequence type (ST) 398 (reviewed by ref. [
3]), whereas ST(CC)9 LA-MRSA predominates in Asia [
2].
LA-MRSA ST398 is recognized as an occupational hazard for people working in the intensive animal husbandry or living in high-density livestock production areas ([
4,
5] reviewed by ref. [
3]). Several studies have reported a positive correlation between human colonization by LA-MRSA and intensity of animal contact, especially in farmers, abattoir workers, and veterinarians [
6‐
10]. Colonization by MRSA is a prerequisite to human infection, and cases of severe infections caused by LA-MRSA ST398 have been reported, resembling those caused by CA-MRSA ([
11,
12] reviewed by ref. [
3]).
LA-MRSA usually do not possess the same repertoire of virulence factors [i.e. staphylococcal enterotoxins (SEs), Panton-Valentine leukocidin (PVL)] and pathogenic properties (i.e. adhesion, internalization and immune evasion abilities) as human-adapted lineages (HA- and CA-MRSA) [
3]. A phylogenomic study proposed that LA-MRSA ST398 evolved from a human methicillin-susceptible
S. aureus (MSSA) clone which acquired resistance to both methicillin and tetracycline and lost the human-specific immune evasion gene cluster (IEC) after the jump from humans to livestock, attenuating its potential to cause zoonotic infection [
13]. Based on evolutionary history and irrespective of methicillin-resistance, a formal definition of two major host-associated
S. aureus ST398 clades has been proposed: the livestock clade (tetracycline resistant and IEC-negative) and the ancestral human clade (tetracycline susceptible and IEC-positive) [
13,
14]. However, conventional definitions might be blurred in future due to the fast rate of MRSA evolution, i.e. changes in antibiotic susceptibility pattern and genetic signatures [
2].
A high prevalence of multi-drug resistant (MDR) ST398 LA-MRSA has recently been documented by our group in a large cohort of healthy pigs farmed in southern Italy [
15]. Given the potential exchange of MRSA between animals and humans, and the infection risk associated with human colonization by MDR LA-MRSA, the present study has been conducted to investigate: (
i) the prevalence, genetic characteristics and antimicrobial resistance profile of MRSA isolated from swine farm workers in southern Italy, and (
ii) the genome-based relatedness of human and animal MRSA isolates, for a better understanding of MRSA transmission to professionally-exposed farm workers.
Methods
Sampling
From January to March 2018, a cross-sectional prevalence study investigating
S. aureus and MRSA carriage among 475 swine in 32 farms (25 with an intensive type of breeding and 7 with a non-intensive type) in the Calabria Region in southern Italy was conducted [
15]. Farms were selected by both geographic distribution and convenience, mainly based on willingness to participate in the survey. Selected farms accounted for 8.99% of all swine farms from Calabria region (15,082 km
2), and were located in all the five provinces: Catanzaro (CZ; 11 farms), Reggio Calabria (RC; 10 farms), Cosenza (CS; 5 farms), Vibo Valentia (VV; 3 farms), Crotone (KR; 3 farms). Among the 32 selected farms, 25 practiced intensive breeding, in which animals were in crowded conditions (i.e. animals confined to indoor fences), and 7 adopted non-intensive breeding systems (i.e. animals living in free-range conditions).
A total of 88 workers belonging to the previously selected swine farms, were sampled. All farm workers over the age of 18 who were present at the time of the visit were sampled, and all participants signed an informed consent form. The study was approved by the Ethical Committee of Azienda Ospedaliera Universitaria Policlinico “G. Martino”, Messina, Italy (decrete no.1158/2018).
On February 2019, resampling of workers was conducted, and 7 out of 88 previously sampled workers agreed to participate. In both samplings, a saline pre-moistened nasal swab was collected from both nostrils of each participating worker, and immediately transferred into 5 ml of high-salt enrichment broth [Mueller Hinton Broth (MHB) (Becton Dickinson) supplemented with 6.5% (w/vol) sodium chloride]. Tubes were incubated for 24 h at 37 °C.
MRSA isolation and characterization
All samples were processed according to a previously described procedure for
S. aureus and MRSA detection [
15]. Briefly, aliquots of the enrichment broth (0.5 ml) were transferred to Phenol-Red Mannitol Broth (PRMB, 4.5 ml) (Becton Dickinson) and PRMB supplemented with 4 μg/ml of oxacillin (PRMB+OX, 4.5 ml). The two tubes were incubated for up to 48 h at 37 °C. If red-to-yellow colour change was observed in PRMB and PRMB+OX, 10-μl samples from PRMB+OX were plated on selective MRSA plates (Brilliance MRSA 2 agar, Oxoid). Suspected MRSA (blue) colonies were streaked on Muller Hinton Agar (MHA) (Becton Dickinson) supplemented with 4 μg/ml OX. If only the PRMB (without OX) turned yellow, presumptive
S. aureus identification was obtained by the Staphytect plus test (Oxoid) on the bacterial pellet. The tubes that did not change colour after 48-h incubation at 37 °C were considered negative for the presence of both
S. aureus and MRSA. All MRSA negative samples underwent a second screening procedure (look-back) to exclude the presence of MRSA in the first enrichment broth, as outlined previously [
15].
Genomic DNA of MRSA isolates was extracted by the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s recommendations, except for the addition of 50 μg/ml lysostaphin (Sigma Aldridch) to improve staphylococcal cell lysis. A multiplex PCR with primers annealing to the 16S rDNA,
nuc and
mecA genes [
16] was performed to confirm
S. aureus identification and methicillin resistance.
MRSA isolates were characterized by
spa, staphylococcal chromosomal cassette
mec (SCC
mec) and multi-locus sequence typing (MLST), as previously described [
17‐
20].
The presence of
pvl genes (
lukS-
lukF) coding for the PVL,
scn and
tet(M) was tested as previously described [
14,
21]. The qPCR detection of enterotoxin-producing MRSA was performed using primers
SA-U and
Sa3-r [
22]. The ST398-specific PCR was carried out with primer sets A07f/A07r [
23]. Analysis of the A07 fragment was performed by amplicon sequencing with primers A07f/A07r.
Rep-MP3 and RAPD (Random Amplification of Polymorphic DNA) PCR were carried out as previously described using primer RW3A [
24] and ERIC-2 [
25], respectively. Fingerprints were digitally compared using the BioNumerics software (Version 6.6; Applied Maths). Cluster analysis with Dice similarity index (S
D) based on the unweighted pair group method with arithmetic averages (UPGMA) was applied to generate dendrograms illustrating the relationships among fingerprints with the following comparison settings: optimization, 1.5%; minimum height, 0%; minimum surface, 0%; tolerance, 1%; tolerance change, 1%. An arbitrary cut-off value of 90% was chosen to assign rep-PCR clusters, named A to F.
S. aureus ATCC 43300 was included as a control strain for analysis.
Antimicrobial susceptibility testing was performed by Vitek2 system (bioMérieux), using the AST-P588 card. All human MRSA isolates were tested using the same antibiotic panel as previously reported for swine isolates [
15]. According to the CLSI interpretative criteria [
26,
27], MRSA isolates were classified as susceptible, intermediate, or resistant. Strains classified as resistant and intermediate were included in the same group (non-susceptible).
Whole genome sequencing (WGS), assembly and analysis
DNA libraries were prepared using Nextera XT v.3 (Illumina, San Diego, CA, USA) kit. WGS was performed using MiSeq (Illumina) platform with paired-end (2X 250-bp) operating mode. Fastq files of the paired-end reads were used as input for genome assemblies through the MEGAnnotator pipeline [
28]. Multiple whole-genome alignments were performed and visualized using the Mauve progressive algorithm with default parameters [
29].
Pairwise average nucleotide identity (ANI) was calculated with Jspecies v1.2.1 using the standard MUMmer algorithm [
30]. Genome-wide single nucleotide polymorphism (SNP) analysis was performed using the CSI Phylogeny 1.4 server [
31]. Sequences were aligned with the LA-ST398 MRSA reference strain S0385 genome (NC_017333, 2,872,582 nucleotides in size) for SNPs calls along 2,500,938 positions (87.1% of reference chromosome). A phylogenetic tree based by SNPs was visualized with MEGA (version X; ref. [
32]).
Data access
WGS data were submitted to the NCBI Sequence Read Archive (SRA) under BioProject PRJNA546229.
Statistical analysis
Data analyses were performed using Sigma Plot software version 12.0 (Systat Software). Categorical variables were compared with the χ2 test or Fisher’s exact test when appropriate. Significance was defined as P ≤ 0.05.
Discussion
LA-MRSA ST398 is an occupational hazard for people in direct contact with livestock animals. Indeed, swine are a major reservoir of this staphylococcal lineage in western countries, including Italy, where a high prevalence of ST398 has been documented in swine farms over the past decade [
9,
15,
34]. Worryingly, an increasing number of LA-MRSA ST398 infections has been reported in professionally exposed workers or in people living nearby high-density swine farming areas [
3‐
5]. The clinical spectrum of LA-MRSA ST398 infection varies from SSTIs to invasive infections, including bloodstream infections, pneumonia and bone and joint infections (reviewed by ref. [
3]). Of note, LA-MRSA ST398 has previously been reported to cause pelvic multiloculated abscess and severe necrotizing fasciitis in two Italian farm workers [
11,
12].
To gain insight of the risk associated with professional exposure to MRSA-colonized pigs and trace the epidemiological trajectories of MRSA in pig farming, we investigated the MRSA prevalence in workers of swine farms which had concomitantly been screened for MRSA colonization of farmed pigs [
15].
A high rate (55.7%) of
S. aureus nasal carriage was observed in farm workers, with an MRSA prevalence of 21.6%. Although high MRSA colonization rates have been documented for swine farmers in different European countries [
7,
8,
10], here we report higher rates of MRSA nasal colonization in swine farm workers from southern Italy, compared with previous surveys from the same region (7.3–8%; refs [
9,
35]).
MRSA colonization was significantly higher in workers reporting direct contact with swine (farm workers and veterinarians), compared with other farm employees, indicating that the carrier status is associated with direct animal contact. In agreement with a previous report, MRSA colonization was age-related [
8], and workers aged <50 years had a higher chance of being MRSA nasal carriers, compared with their older peers. Remarkably, farms which had adopted a non-intensive breeding system showed lower colonization rates with
S. aureus and never yielded MRSA, in line with previous studies [
7,
36]. Although limited in sample size, follow-up screening provided evidence of LA-MRSA persistence or re-colonization in some workers, given that carriage of the same MRSA strain was demonstrated 1 year after primary sampling. This strengthens the notion that frequent animal contact, especially in intensive swine breeding, is a major risk factors for persistent colonization with LA-MRSA in farm workers [
36,
37]. In our previous survey, a trade of pigs between farm ID 07KR (seller) and farm ID 18CS (purchaser) has been documented [
15], and MRSA isolates with an identical epidemiological type (t011, V, C) were detected in the majority of animals from these two farms. Intriguingly, workers of these farms were colonized by the same MRSA strain detected in animals (see Table
2, and Additional file
3: Figure S1), and two of them were found to be (re)colonized by the same strain (ID 07KR005U and 18CS002U) upon follow-up screening (Additional file
4: Figure S2). This observation suggests that inter-farm pig movements drive the spread of MRSA ST398 clones, leading to an increased risk of MRSA transmission to workers [
38]. Therefore, periodical screening of LA-MRSA carriage in farm workers and animals should be implemented to reduce the inter-farm spreading of LA-MRSA.
Irrespective of the source (either workers or swine), all ST398 MRSA isolates analysed in this survey belong to the livestock clade, being
tet(M)-positive and
scn-negative, and displayed epidemiological profiles (i.e.
spa and SCC
mec type) mirroring the diversity of LA-MRSA ST398 so far reported in Europe [
7,
10]. Moreover, both human and swine MRSA isolates were MDR, and showed similar resistance profiles, especially to drugs commonly used in pig husbandry (tetracyclines, lincosamides, macrolides and fluoroquinolones). Nonetheless, all isolates were susceptible to vancomycin and all but one to rifampicin, and were negative for PVL and ETs production, consistent with the attenuated virulence potential reported for LA-MRSA ST398 [
3]. However, the possibility that LA-MRSA can evolve towards a more virulent pathotype should not be disregarded, as many staphylococcal virulence genes reside on mobile genetic elements [
39] and it has recently been demonstrated that lysogenization of LA-MRSA CC398 strains by virulence-associated phages leads to the production of new virulence factors [
40].
By combining epidemiological typing (
spa, SCC
mec, MLST) with DNA fingerprinting (Rep- and RAPD PCR) and whole genome analysis results, identity or close relatedness between human and swine MRSA isolates from the same farm was demonstrated. Our study highlights the power of WGS in epidemiological investigations, since apparent difference in DNA fingerprints could be explained by insertion of the
Staphylococcus phage Sebago within identical genome scaffolds. Thus, typing data suggest unidirectional transmission of LA-MRSA from pigs to workers, either by direct animal contact or indirectly, through the farm environment, given that LA-MRSA can survive in dust for weeks [
41].
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