Molecular epidemiology of methicillin-resistant Staphylococcus aureus from dairy farms in North-eastern Italy
Introduction
Staphylococcus aureus is a human and animal pathogen involved in a wide range of diseases, ranging from mild to life-threatening infections. In veterinary medicine, S. aureus is among the most frequent etiological agents of intramammary infections in dairy ruminants, causing both clinical and subclinical mastitis, which result in a major economic burden on the dairy industry (Fluit, 2012).
Besides the ability to colonize and/or infect both humans and animals, some S. aureus strains have the potential to cause foodborne intoxications from contaminated foods, including milk and dairy products (Kümmel et al., 2016). Two major factors contribute to the importance of S. aureus in the dairy chain: i) the clinical and subclinical infections of cows, which act as the first contamination source of milk and other dairy products (Akineden et al., 2001; Heidinger et al., 2009), and ii) production of enterotoxins (ETs), which is variable among strains and can eventually cause food poisoning (Papadopoulos et al., 2019).
S. aureus enters the dairy production chain through various routes. At the farm level, S. aureus can a priori be shed in raw milk by infected cows or introduced during milking (Kümmel et al., 2016; Papadopoulos et al., 2019). Contamination of dairy products can also occur during food processing and handling, via either human carriers or contaminated fomites (Asao et al., 2003; Johler et al., 2018).
The overuse of antibiotics in medicine and animal husbandry has contributed to the emergence of drug-resistant S. aureus strains, among which methicillin-resistant S. aureus (MRSA) are the cause of major public health concern. MRSA are conventionally named according to their epidemiological origin, including healthcare-associated (HA-MRSA), community-associated (CA-MRSA) and livestock-associated (LA-MRSA) lineages (Pantosti and Venditti, 2009). In the One Health context, LA-MRSA is considered a serious problem for the risks of zoonotic transmission not only to people with occupational livestock exposure, e.g. dairy farm workers (Alba et al., 2015; Becker et al., 2017; Feltrin et al., 2016), but also to the community through the food chain (Kluytmans, 2010). Worryingly, the spread of LA-MRSA in animal husbandry has been associated with an increased incidence of LA-MRSA infections among humans, especially in areas of high livestock density (Monaco et al., 2013; van de Sande-Bruinsma et al., 2015).
The MRSA clonal complex (CC) 398, including sequence type (ST) 398 and related STs, is currently the most prevalent lineage among LA-MRSA (Ballhausen et al., 2017). However, other S. aureus lineages colonizing animals have acquired methicillin resistance (Butaye et al., 2016), and these include ST(CC)9 (Chuang and Huang, 2015), the mastitis-associated ST(CC)97 (Feltrin et al., 2016; Wang et al., 2015) and the ST(CC)1, which has increasingly been reported among pigs farmed throughout Europe since 2010 (Alba et al., 2015; Battisti et al., 2010; Elstrøm et al., 2019).
While swine are generally considered the main reservoir of LA-MRSA, recent reports provided evidence that LA-MRSA circulates also among cattle in several countries (Kadlec et al., 2019; Papadopoulos et al., 2019; Unnerstad et al., 2018), especially in Italy (Alba et al., 2015; Basanisi et al., 2017; Cortimiglia et al., 2016; Feltrin et al., 2016; Luini et al., 2015; Parisi et al., 2016), which is the sixth largest bovine milk producer in the European Union (EU) (EUROSTAT, 2019).
In this study, the circulation of S. aureus at the beginning of the dairy production chain, and its transmission between animals and farm workers was investigated. To this aim, the prevalence of methicillin-sensitive S. aureus (MSSA) and MRSA in bulk tank milk (BTM) from dairy farms and in farm workers was estimated in two regions of North-eastern Italy, both characterized by high livestock density. In addition, the genetic relatedness and antibiotic-resistance traits of MRSA isolates from BTM and workers were examined to trace the patterns of MRSA transmission in dairy farming.
Section snippets
Sample collection
Between March 2013 and April 2014, a longitudinal study was carried out in 618 dairy farms located in two contiguous regions of North-eastern Italy, spanning an area of 26,269 km2, namely Veneto (408 farms; Fig. S1) and Friuli-Venezia Giulia (210 farms; Fig. S2). Farms accounted for 10% and 19% of all dairy herds of Veneto and Friuli-Venezia Giulia regions, respectively, and were selected by both geographic distribution and convenience, mainly based on willingness to participate in the survey.
Prevalence of S. aureus in dairy farms and farm workers
The geographical distribution of farms in Veneto and Friuli-Venezia Giulia regions is illustrated in Figs. S1 and S2, respectively. Among the 618 surveyed farms, MSSA and MRSA were detected in at least one BTM sample from 491 [79.4%; 95% confidence interval (CI): 79.2–79.7%] and 22 (3.56%; 95% CI: 3.55–3.57%) farms, respectively (Table 1). Considering all three samplings, the MSSA and MRSA prevalence in BTM were 56.6% (95% CI: 56.5–56.8%) and 1.94% (95% CI: 1.93–1.95%), respectively (Table 1).
Discussion
We conducted an extensive longitudinal survey in a high-density dairy farming area of North-eastern Italy in order to evaluate the prevalence of S. aureus in milk and farm workers, and to gain insights into the possible routes of MRSA transmission within farms. Our findings reveal a high proportion of BTM contamination by MSSA, occurring in almost 80% of the farms and in multiple BTM samples for more than half of farms. Nevertheless, we report a relatively low prevalence of MRSA in both BTM and
Conclusion
Consumption of raw-milk and unprocessed dairy products has shown an increasing trend in Europe, posing the risk of transmission of milk-associated pathogens to consumers (EFSA, 2015). This study highlights the high prevalence of S. aureus, and to a lesser extent of MDR MRSA strains, at the beginning of the dairy production chain in North-eastern Italy. Transmission of toxigenic MRSA clones between milk and workers has been documented, and it is predicted to enhance the risk of both human
Funding
This work was supported by a grant from the Italian Ministry of Health (National Centre for Disease Prevention and Control), Programme CCM 2012 “Surveillance and prevention of methicillin-resistant Staphylococcus aureus (MRSA) in north-eastern Italy dairy farming and in exposed workers” (No. E85C12000470005) to A.B., A.P., B.C., F.A., M.Pa., N.V. and, P.V., and by the Excellence Departments grant from the Italian Ministry of Education, University and Research (MIUR-Italy) (Art. 1, commi 314-337
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
Authors wish to thank the contribution of the staff from Istituto Zooprofilattico Sperimentale delle Venezie, particularly Dr. L. Bonfanti for having inspired and planned this study, and Dr. L. Gagliazzo for epidemiological data management. The outstanding technical assistance in MRSA genotyping and antimicrobial susceptibility testing of P. Alba, G. Cordaro, and F. Feltrin is also acknowledged.
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Contributed equally to this work.