ABSTRACT
Livestock-associated methicillin-resistant Staphylococcus aureus sequence type (ST) 398 (LA-MRSA ST398) is a genetic lineage for which pigs are regarded as the main reservoir. An increasing prevalence of LA-MRSA ST398 has been reported in areas with high livestock density throughout Europe. In this study, we investigated the drivers contributing to the introduction and spread of LA-MRSA ST398 through the pig farming system in southern Italy. Whole-genome sequencing (WGS) of LA-MRSA ST398 isolates collected in 2018 from pigs (n = 53) and employees (n = 14) from 10 farms in the Calabria region of Italy were comparatively analyzed with previously published WGS data from Italian ST398 isolates (n = 45), an international ST398 reference collection (n = 89), and isolates from Danish pig farms (n = 283), which are the main suppliers of pigs imported to Italy. Single-nucleotide polymorphisms (SNP) were used to infer isolate relatedness, and these data were used together with data from animal trading to identify factors contributing to LA-MRSA ST398 dissemination. The analyses support the existence of two concurrent pathways for the spread of LA-MRSA ST398 in southern Italy: (i) multiple introductions of LA-MRSA ST398 through the import of colonized pigs from other European countries, including Denmark and France, and (ii) the spread of distinct clones dependent on local trading of pigs between farms. Phylogenetically related Italian and Danish LA-MRSA ST398 isolates shared extensive similarities, including carriage of antimicrobial resistance genes. Our findings highlight the potential risk of transboundary transmission of antimicrobial-resistant bacterial clones with a high zoonotic potential during import of pigs from countries with high LA-MRSA prevalence.
IMPORTANCE Over the past decade, livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 (LA-MRSA ST398) has spread among pig holdings throughout Europe, in parallel with the increased incidence of infections among humans, especially in intensive pig farming regions. Despite the growing prevalence of LA-MRSA ST398 in Italian pig farms, the transmission dynamics of this clone in Italy remains unclear. This work provides genome-based evidence to suggest transboundary LA-MRSA ST398 transmission through trading of colonized pigs between European countries and Italy, as well as between farms in the same Italian region. Our findings show that both international trading and local trading of colonized pigs are important factors contributing to the global spread of LA-MRSA ST398 and underscore the need for control measures on and off the farm to reduce the dissemination of this zoonotic pathogen.
INTRODUCTION
Staphylococcus aureus is an opportunistic human pathogen that can cause a variety of diseases, ranging from skin and soft tissue infections to life-threatening invasive infections. Some of these infections are caused by drug-resistant strains, primarily methicillin-resistant S. aureus (MRSA). Since the mid-2000s, MRSA clones colonizing livestock animals, called livestock-associated MRSA (LA-MRSA) (1), have emerged. The most common LA-MRSA lineage in the European Union (EU) is sequence type 398 (ST398).
Since the first EU baseline survey in 2008 (2), an increase in the prevalence of LA-MRSA ST398 has been documented in several EU countries (3–6). Worryingly, this lineage has spread beyond the farm setting, showing increasing prevalence among humans living in high-density livestock production areas (7, 8).
The application of high-throughput whole-genome sequencing (WGS) has unveiled potential drivers for LA-MRSA ST398 dissemination, and trading of colonized pigs, contaminated transport vehicles, and human carriers have been suggested as potential vectors for both local and transboundary transmission of LA-MRSA (4, 5, 9).
Italy is the sixth largest pork producer in the EU (10), and in 2008, two nationwide surveys estimated the prevalence of LA-MRSA ST398 among pig farms in Italy to be 14% to 28% (2, 11). Since then, the prevalence of LA-MRSA ST398 in the Italian pig farming system has steadily increased, especially in southern Italy, where the percentage of positive farms has been estimated to be ca. 60% (6, 12). However, the transmission routes that have caused such a major increase of LA-MRSA ST398 prevalence have not been investigated so far.
In this study, we integrated WGS data for LA-MRSA ST398 isolated from pigs farmed in southern Italy (6, 13) with genome data available in international sequence repositories in order to trace the local and transboundary dissemination dynamics of LA-MRSA ST398.
RESULTS
Selection of LA-MRSA ST398 isolates for WGS.WGS was performed on 67 recently isolated LA-MRSA ST398 strains representative of the major clones, as defined by spa typing, circulating among pigs and farm workers in a large area of southern Italy (Calabria region; 15,222 km2). Fifty-three strains originated from pigs and 14 from farm workers. All of them were isolated in intensive farms during a survey conducted in 2018 (6, 13). Antibiotic resistance profiles and spa and SCCmec types were previously determined (see Table S1 in the supplemental material). Isolates were selected according to the criteria described in Materials and Methods.
Phylogeographic context and comparative genomics of LA-MRSA ST398.Animal movement is considered a driver for LA-MRSA ST398 spread among pig farms (5, 9, 14). To trace the source of recently identified Italian LA-MRSA ST398 isolates to a potential country of origin, the genomes of the 67 LA-MRSA ST398 isolates from southern Italy were compared with 45 genomes from previously sequenced Italian spa type t899-related ST398 isolates (15) and an international reference collection of 89 genomes of methicillin-resistant and -susceptible S. aureus ST398 (16) strains, including the S. aureus ST398 reference strain S0385. In total, 201 genomes were investigated to reconstruct phylogenetic relationships based on single-nucleotide polymorphisms (SNPs).
After removal of 327 sites in recombinant regions, 6,400 core genome SNPs in the 201 isolates were used to construct a rooted maximum-likelihood tree (Fig. 1; also, see Fig. S1 in the supplemental material). The analysis revealed a nonuniform distribution of the isolates from southern Italy, which appeared intermingled throughout the phylogeny and did not cluster according to geographic origin (Fig. 1). Seven groups (designated A to G) (Fig. 1) comprising the recent isolates from southern Italy with their closest neighbors from the international reference collection (16) were arbitrarily defined. All groups were supported with bootstrap values of >90%. Groups A, B, C, and F were composed solely of isolates originating from single farms (designated 01CZ, 05CS, 07KR, and 29RC, respectively). Six isolates from farm 07KR clustered with isolates from farm 18CS (group D). Groups E and G were composed of isolates originating from three farms (03CZ, 19RC, and 32RC) and two farms (02CZ and 11RC), respectively.
Rooted maximum-likelihood phylogeny of 67 recent LA-MRSA ST398 isolates from southern Italy, 89 S. aureus ST398 isolates from the international reference collection (16), and 45 Italian LA-MRSA ST398 t899-related isolates (15). The tree was rooted according to the outgroup used in reference 16. Bootstrap values above 90% are illustrated by filled circles at the ends of branches. Colored filled circles correspond to farms yielding isolates. The scale bar represents the number of nucleotide substitutions per variable site. A to G represent groups of the recent Italian isolates and their closest neighbors from the international reference collection.
The relatedness of isolates from southern Italy and other countries was examined using the phylogeny (Fig. 1). Groups A, B, and G did not exhibit any close relationship with isolates from countries other than Italy. Conversely, the closest neighbors of Italian isolates clustering in groups C, D, E, and F originated from different EU countries (16). Group C was related to isolates from Austria and Slovenia, whereas group D was related to one French isolate. Both groups E and F were closely related to isolates from Denmark.
Evidence of transboundary and local transmission of LA-MRSA ST398.To investigate any possible transboundary and/or local dissemination of LA-MRSA ST398 via pig movement, farms from which the 67 isolates originated from were queried about the source of their pigs (Table 1). Three farms (03CZ, 19RC, and 29RC) reported that they had purchased animals from Denmark (Table 1), which in 2015 was the country with the highest number of pigs imported into Italy (17) (Fig. 2) and which has experienced a remarkable increase in LA-MRSA ST398 prevalence in pig farming (18). Since the structure of the LA-MRSA ST398 population in Danish pigs was recently characterized by WGS analysis (5), Denmark was selected as the study case to uncover potential LA-MRSA ST398 transmission through pig trading to southern Italy. To this purpose, additional genome data for isolates from Danish pig farms (n = 283) (5) were incorporated in the analyses.
Association between source of pigs and distribution of LA-MRSA ST398 isolates across the phylogeny
Data on import of pigs to Italy by country of origin and year. White dots denote the total import value of pigs per year, expressed in millions of U.S. dollars, with the solid black line showing the yearly trend. Data were retrieved from the Observatory of Economic Complexity (17).
A total of 484 isolates were included in this analysis, and they differed in 6,059 core genome SNPs, after the removal of 591 sites falling into recombination regions. The rooted SNP-based maximum-likelihood tree is shown in Fig. 3 (see Fig. S2 for additional details). The isolates from the three Italian farms importing animals from Denmark (farms 03CZ, 19RC, and 29RC) (Table 1) clustered with the prevalent L1 and L3 Danish lineages (5) (Fig. 3). Two farms (07KR and 32RC) reported importing pigs from France, despite the fact that their isolates exhibited a close relatedness with the Danish L2 and L3 lineages.
Rooted maximum-likelihood phylogeny of 67 recent LA-MRSA ST398 isolates from southern Italy, 283 LA-MRSA ST398 Danish isolates (5), 89 S. aureus ST398 isolates from the international reference collection (16), and 45 Italian LA-MRSA ST398 t899-related isolates (15) (see Fig. S2 in the supplemental material for further annotations, including bootstrap values). The tree was rooted according to the outgroup used in reference 16. Colored filled circles correspond to farms yielding isolates. The scale bar represents the number of nucleotide substitutions per variable site. IT, Italian cluster; L1, Danish lineage 1; L2, Danish lineage 2; L3, Danish lineage 3; t899, Italian t899 lineage.
Three unique Italian clusters are defined in Fig. 3; two of them are composed of isolates from farms 01CZ and 05CS (corresponding to groups A and B in Fig. 1, respectively). Farm 01CZ imported animals from Spain, while farm 05CS had an autonomous breeding system (Table 1). The third Italian cluster (corresponding to group D in Fig. 1) was composed of isolates from farms reporting interfarm trading of pigs (farm 07KR sold pigs to farm 18CS [6]) (Table 1). Interestingly, farm 07KR imported animals from France (Table 1), and the isolate from the international reference collection most closely related to this cluster also originated from France (Fig. 3). Lastly, all the t899-related isolates clustered together (t899 cluster) (Fig. 3), supporting the notion that spa type t899 represents a monophyletic entity (15, 16). Within this cluster, all isolates from southern Italy appeared to be closely related (group G in Fig. 1), originating from two farms importing animals from northern Italy (farms 02CZ and 11RC) (Table 1).
Distribution of antimicrobial resistance genes.The distribution of antimicrobial resistance genes among the 67 LA-MRSA ST398 isolates from southern Italy is reported in Table 2 and Fig. S3. The previously documented presence of mecA and tet(M) (6, 13) was confirmed in all isolates. Aminoglycoside resistance genes were present in >80% of the isolates, with ant(9)-Ia (synonymous with spc) being the most prevalent, followed by aadD and aac(6′)-aph(2′′) (Table 2). Interestingly, the zinc-cadmium resistance gene czrC was exclusively detected in isolates carrying SCCmec type V (85% of the isolates) (Fig. S3). The erm(B) and erm(C) genes, conferring resistance to macrolide-lincosamide-streptogramin B (MLSB), were present in 18% and 19% of the isolates, respectively. Lincosamide resistance was encoded by lnu(B) and lnu(A) in 55.2% and 7.5% of the isolates, respectively. Similarly, trimethoprim resistance was encoded by dfrG and dfrK in 34.3% and 7.5% of the isolates, respectively.
Antimicrobial resistance genes in Italian and Danish isolates belonging to lineages L1, L2, and L3a
The expansion of the L1, L2, and L3 lineages among Danish LA-MRSA ST398 isolates was suggested to be driven by the use of antimicrobials (5). To investigate the transmission of antimicrobial resistance genes along with pig movement, the antibiotic resistomes in Italian and Danish isolates of lineages L1, L2, and L3 were compared (Table 2). Italian isolates clustering within L1, L2, and L3 lineages showed resistance gene patterns very similar to those of the Danish isolates from the same lineages. When data for all three lineages were combined, significant differences between Italian and Danish isolates were observed for only three genes; specifically, ant(9)-Ia (spectinomycin resistance) was significantly more frequent among Italian isolates, whereas aph(6)-Ic (synonymous with str; streptomycin resistance) and erm(B) (macrolide resistance) were more frequent among Danish isolates (Table 2).
DISCUSSION
WGS of recent LA-MRSA ST398 isolates from southern Italy was performed to gain insights into the transmission dynamics within and between countries. Our findings strongly suggest the existence of two concurrent modes for LA-MRSA ST398 introduction and spread in southern Italy. The first is multiple introductions of LA-MRSA ST398 strains by trading of piglets with other EU countries, including France and Denmark. The second is the expansion of independent ST398 clones, especially in farms trading animals with other Italian farms.
With 13 million exported animals per year, Denmark is the leader in pig exports to other EU countries (19, 20), and our analysis revealed that Denmark has been the main provider of pigs to Italy since 2015. Denmark has experienced a dramatic increase in the prevalence of LA-MRSA ST398 in pig farms, and in 2018, >80% (83% to 89%) of the conventional pig farms were found to be positive for MRSA (18). This increase has been linked to the clonal expansion of three dominant lineages (L1, L2, and L3) (5), which have spread beyond the farm level and have been detected in the Danish food production chain and health care facilities (8, 21, 22). Since Denmark is the primary country of origin of pigs imported to Italy, genomic comparison of our isolates to Danish isolates revealed that isolates from Italian farms that reported pig imports from Denmark clustered within the dominant Danish lineages L1 and L3. This supports transmission of Danish LA-MRSA ST398 with pigs traded to Italy. Interestingly, farms purchasing animals from France also appeared to be colonized by strains belonging to the Danish lineages L2 and L3. It could be hypothesized that Danish lineages L2 and L3 previously spread to France and from there they were imported to Italy. However, only a few French strains were available in the ST398 reference data set (16), and they were scattered across the phylogeny. Thus, a larger and more recent collection of French isolates, as well as other national collections of LA-MRSA ST398 isolates from Europe, should be inspected to confirm our hypothesis.
Interestingly, Italian and Danish isolates clustering within the predominant Danish lineages (L1, L2, and L3) also shared a high similarity in carriage of antibiotic resistance genes which confer resistance to antimicrobials commonly used in the Danish pig farming system (18). The different frequency of ant(9)-Ia (spectinomycin) and aph(6)-Ic (streptomycin) gene carriage between Italian and Danish isolates could reflect the different use of aminoglycosides in the two countries. Moreover, recent isolates from southern Italy showed higher frequency of the zinc/cadmium resistance gene czrC (85%) than previously reported (56%) (23). This is consistent with the predominant SCCmec type V found among the Italian isolates and the extensive use of zinc oxide to prevent postweaning diarrhea in pigs (5, 24), which is still common practice in Italy.
This study has some limitations. First, despite the evident genetic relatedness between Danish and Italian MRSA isolates, the skewness of the Danish data set may have caused a bias toward the suggested geographical origin of MRSA in Italian pigs. Second, unique isolates from only 10 farms in a large region with high density of pig farming were analyzed, and the number of isolates per farm was not uniform (2 to 17 isolates). Third, ST398 has been shown to spread through the environment (e.g., via water outflow and manure or dust effusion), via contaminated fomites, and from other hubs of dissemination which we cannot exclude. For example, farm 19RC not only imported colonized pigs from Denmark but also employed a colonized farmer (19RC002U) who regularly visited pig farms in Denmark. In this case, the possibility of travel-associated human colonization and/or human-to-pig transmission cannot be ruled out. However, no such links were documented in the other farms. Thus, our observations strongly suggest that local and transboundary transmission of multidrug-resistant ST398 occurred via trading of colonized animals, as already reported in Denmark, Norway, and New Zealand (4, 5, 9).
In summary, this study sets the groundwork for future WGS-based epidemiological investigations of LA-MRSA ST398 in Italy. The dissemination of this lineage is known to be facilitated by animal movements, and trading of ST398-positive pigs between Italian pig farms and holdings from other EU countries may have contributed to the spread of this lineage. Our findings underscore the need for control measures on and off the farm to reduce the dissemination of this zoonotic pathogen.
MATERIALS AND METHODS
Selection criteria of LA-MRSA ST398 isolates from southern Italy.A total of 67 different isolates from a previous cross-sectional study conducted in the Calabria region of Italy during 2018 were selected for de novo WGS (6, 13). Isolates were selected according to the following criteria: (i) belonging to the predominant spa types circulating in EU countries, namely, t011 (n = 45), t034 (n = 12), and t899 (n = 10) (3, 15, 16, 25, 26); (ii) originating from farms (n = 10) in which LA-MRSA ST398 was isolated from both pigs and farm workers (13); and (iii) showing distinct genotypic (i.e., spa type or SCCmec type) and phenotypic (antimicrobial susceptibility pattern) traits compared with other isolates from the same farm (6, 13) (Table S1). Therefore, for each farm, isolates displaying identical epidemiological types and antimicrobial susceptibility profiles were considered duplicates, and only one representative isolate (from either pig or worker) was selected for WGS (Table S1).
DNA extraction and WGS.Genomic DNA was extracted by using the QIAamp DNA minikit (Qiagen) according to the manufacturer’s instructions, except for the addition of 50 μg/ml lysostaphin (Sigma-Aldrich) during the lysis step. DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA), and WGS was performed using a MiSeq (Illumina) platform with paired-end operating mode (2 × 250 bp).
Prediction of antimicrobial resistance genes.The ResFinder v.3.2 web-based pipeline at the Centre for Genomic Epidemiology (http://www.genomicepidemiology.org) was used to search for the presence of known antibiotic resistance genes using default settings (identity threshold, ≥90%; minimum length, ≥60%) (27). Genomes were screened for the czrC gene, encoding resistance to cadmium and zinc, by aligning sequence reads against the reference sequence (GenBank accession no. KF593809).
SNP calling and phylogenetic analysis.For comparative purposes, 89 S. aureus ST398 from a worldwide collection (16), plus 45 Italian t899 and t899-related ST398 isolates (15), and 283 Danish ST398 isolates (5) were included in the phylogenetic analysis. Metadata for all isolates is provided in Data Set S1 in the supplemental material.
Identification of SNPs was performed with NASP version v.1.0.0 (28) using the GATK Unified Genotyper with a filtering set to remove SNPs with less than 10-fold sequencing depth and 90% unambiguous variant calls after duplicated regions of the LA-MRSA ST398 reference chromosome S0385 (GenBank accession no. AM990992) (29) were excluded using NUCmer. SNPs caused by recombination events were identified and removed using Gubbins v.2.3.4 (30) prior to phylogenetic reconstruction using IQ-TREE version v.1.5.5 (31) with the best model found by the implemented ModelFinder and bootstrap analysis using 100 replicates. The tree was rooted according to the outgroup used in reference 16.
Statistical analysis.Data analyses were performed using GraphPad Prism v.6.1. Categorical data were compared with a two-sided Fisher’s exact test. Significance was defined as a P value of ≤0.05.
Data availability.WGS data for 10 isolates from farms 01CZ and 32RC were previously submitted to the NCBI Sequence Read Archive (SRA; available at https://www.ncbi.nlm.nih.gov/sra) under BioProject PRJNA546229. WGS data for the remaining 57 isolates have been submitted to the NCBI SRA under BioProject PRJNA607440.
ACKNOWLEDGMENTS
This work was supported by the BRIC ID 18/2016 grant from the Italian Workers’ Compensation Authority (INAIL) to F.C., G.S., and P.V., an Excellence Departments grant from the Italian Ministry of Education, University and Research (MIUR, Italy) (Art. 1, commi 314-337 Legge 232/2016) to the Department of Science, Roma Tre University, and the PRIN 2017 grant prot. 20177J5Y3P from MIUR to P.V. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We report no potential conflicts of interest.
FOOTNOTES
- Received 21 February 2020.
- Accepted 22 April 2020.
- Accepted manuscript posted online 1 May 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
