Molecular epidemiology of methicillin-resistant Staphylococcus aureus from dairy farms in North-eastern Italy

Highlights

• S. aureus was frequently isolated from milk, though MRSA was rarely detected.

• Most of MRSA isolates from milk belonged to ST398 and were multidrug-resistant.

• Epidemiological link between MRSA isolates from workers and milk was proven.

• Phylogenetic analysis supported two lineages of Italian ST398 t899 isolates.

• Enterotoxin and PVL genes were detected in a minority of MRSA isolates.

Abstract

Transmission of Staphylococcus aureus along the dairy production chain is an emerging public health problem with human, veterinary, and food safety issues. The prevalence of multidrug-resistant, particularly methicillin-resistant S. aureus (MRSA), has steadily increased in several European countries. In this study, the prevalence of S. aureus in raw cow milk and farm workers was investigated, and the trajectories of MRSA transmission at the primary stage of the dairy chain were assessed. To this purpose, a longitudinal survey was conducted in 618 dairy farms in two contiguous regions with high livestock density in North-eastern Italy. S. aureus contamination of bulk tank milk (BTM) was observed in more than 80% of farms, while MRSA prevalence was 3.6% and 15.9% in BTM and farm workers, respectively. The majority of MRSA isolates from both BTM and farm workers were assigned to ST398, and showed a worrisome multidrug-resistant phenotype. Enterotoxin and Panton-Valentine leukocidin genes were detected in 11.5% and 4.9% of MRSA isolates from both sources. Nearly all MRSA isolates from workers belonged to the same epidemiological type as BTM isolates from the corresponding farm, denoting a bidirectional MRSA transmission pattern. A focus on the ST398 spa type t899 MRSA lineage in the Italian livestock system highlighted the presence of two major clusters whose dissemination was likely facilitated by the selective pressure imposed by antimicrobial use in animal farming. Our findings emphasize the need for continuous monitoring of MRSA along the dairy production chain, not only to avoid transmission between animals and exposed workers, but also to contain the risk of raw milk and dairy product contamination by multidrug resistant and toxigenic strain.

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.