Genetic diversity and antimicrobial resistance in Staphylococcus aureus and coagulase‐negative Staphylococcus isolates from bovine mastitis in Minas Gerais, Brazil

Abstract The aims of this study were to determine the antimicrobial susceptibility profile and genetic diversity of Staphylococcus spp. isolated from dairy cows in Minas Gerais, Brazil, and to assess the relationship among the isolates’ susceptibility profiles and pulsed‐field gel electrophoresis (PFGE) genotypes. Seventy‐nine isolates were used, including S. aureus (n = 71) and coagulase‐negative staphylococci (CoNS) (n = 8). Susceptibility to 12 antimicrobial agents was performed. All Staphylococcus spp. were subjected to PFGE. Staphylococcus aureus and CoNS isolates exhibited full susceptibility only to cephalothin. The greatest percentages of resistance among Staphylococcus spp. were observed to penicillins, folate pathway inhibitors, and tetracyclines. Twelve S. aureus and four CoNS were classified as multidrug resistance strains. Percentage of MRSA was also higher among CoNS (75%), compared to S. aureus isolates (2.81%). Adopting 100% of similarity, 34 different genotypes were identified. Association of minimum‐spanning tree (MST) analysis with data from municipalities, herds, methicillin‐resistant S. aureus (MRSA), and resistance patterns for all isolates did not show any clustering. However, a clustering pattern of bacterial species was observed. Results from this study indicate a high frequency of antimicrobial resistance, especially among CoNS, and a high genetic diversity among Staphylococcus spp. isolated from dairy cows with mastitis in Minas Gerais, Brazil.

symptoms. From the epidemiological point of view, it is critical to define the microorganisms involved in the etiology of the bovine mastitis, to determine the reservoirs and the sources of infection, and to adopt appropriate hygienic measures and a strict monitoring program.
Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are the major agents of contagious bovine mastitis in several countries and are recognized worldwide as a major public health issue, since they cause serious diseases in humans and animals and have great ability to become resistant to antibiotics (Aires-de-Sousa, 2017;Botrel et al., 2010;Santos et al., 2016). Methicillin-resistant S. aureus (MRSA), which is resistant to all beta-lactams antibiotics, are of particular concern, as recently, these strains have also emerged as a widespread cause of infections in the community and in animals, besides hospitals (Chambers & DeLeo, 2009). Moreover, albeit majority of bovine mastitis pathogens are susceptible to the antimicrobials used for treatment, some studies have indicated an increasing resistance among S. aureus and especially CoNS to β-lactam antibiotics, including previous study carried out in Staphylococcus strains from Minas Gerais, Brazil (Botrel et al., 2010;Kalmus, Aasmae, Karssin, Orro, & Kask, 2011;Santos et al., 2016).Surveillance of resistant staphylococci and the accurate diagnosis of these bacteria are critical for appropriate mastitis treatment and for the identification of risks to the general population, as strains generated in the livestock environment can spread to the community by contact with infected animals or contaminated animal products.
However, besides the identification of the pathogens involved in mastitis, it is also important to use highly discriminatory methods for the characterization of these isolates, which will allow to trace back the outbreaks, to understand the transmission routes, and to control the disease spread (Boffetta, 2000). The pulsed-field gel electrophoresis (PFGE) of bacterial isolates from mastitis has shown a high discriminating power, especially for S. aureus, being considered the standard method for epidemiological studies of this species (Goering, 2010). This technique has been extensively used for the comprehension of the epidemiology of both endemic and epidemic MRSA strains from different origin (livestock, community, and hospital), contributing for the elucidation of the epidemiology of staphylococcal infection in all regions of the world. Thus, the aims of present study were (a) to determine the antimicrobial susceptibility profile and (b) genetic diversity, using PFGE, of Staphylococcus spp. strains isolated from dairy cows with mastitis in Minas Gerais, Brazil, and (c) to assess the relationship among the susceptibility profiles of the isolates, epidemiological data, and PFGE genotypes.

| Bacterial isolates and culture conditions
Seventy-nine isolates of Staphylococcus spp. (S. aureus n = 71 and CoNS n = 8) previously isolated from milk samples of dairy cows with mastitis and S. aureus ATCC 25923 T were used in the present study (Santos et al., 2016).

| DNA extraction and pulse field gel electrophoresis (PFGE)
Preparation of DNA of S. aureus and CoNS strains was performed as described by Mulvey et al. (2001). All Staphylococcus spp. isolates were subjected to restriction-endonuclease digestion and PFGE strictly following the procedures as described by Mork, Tollersrud, Kvitle, Jorgensen, and Waage (2005).
The number and size of the DNA fragments obtained in PFGE were assessed using the software BioNumerics 7.5 (Applied Maths, Belgium). Clustering analysis was performed based on the Dice coefficient and the unweighted pair group method with arithmetic mean (UPGMA) using the same software. The Hunter and Gaston Diversity Index (HGDI) was calculated (Hunter & Gaston, 1988) (http://insilico.ehu.eus/mini_tools/discriminatory_power/index.php). The minimum-spanning tree (MST) was generated to assess the association of clustering patterns of the isolates and antimicrobial susceptibility profiles, herds, MRSA, or municipalities. MST presented is the one with the highest overall reliability score and was performed using the UPGMA to calculate the distance matrix Prim's algorithm associated with the priority rule and the permutation resampling (Feil, Li, Aanensen, Hanage, & Spratt, 2004).

| RE SULTS AND D ISCUSS I ON
Staphylococcus spp., particularly S. aureus, stands out as one of the most common agents involved in the etiology of bovine mastitis.
TA B L E 1 Susceptibility of Staphylococcus spp. isolated from milk samples of dairy cows with mastitis from municipalities of the Zona da Mata Region, Minas Gerais, Brazil to 12 antimicrobial agents, 2010-2011 Antimicrobial Zone diameter cutoff (mm)

S I R S (%) I (%) R (%) S (%) I (%) R (%)
Ampicillin  In addition to veterinary importance, there are also implications in public health due to the potential for zoonotic spread of these microorganisms, besides the emergence of resistant and multidrug-resistant pathogens among animal isolates (Chambers & DeLeo, 2009 to seven of the twelve antimicrobial tested was observed, which is of special concern, since this agent is ubiquitously distributed in nature among several species inhabiting different ecosystems and have be- Similarly to what was observed in other studies, in addition to oxacillin, all our MRSA isolates were also resistant to penicillin and tetracycline (Bardiau et al., 2013;Fessler et al., 2010). The low frequency of MRSA strains among S. aureus isolates from bovine mastitis observed in the present study has also been commonly reported elsewhere (Bardiau et al., 2013;Fessler et al., 2010;Moon et al., 2007). However, even at low frequency, it is worth noting that livestock-associated MRSA (LA-MRSA) can also contribute to human MRSA infections, which will vary in importance depending on other epidemiological variables present, as consumption of raw milk and milk products. In addition to resistance to all beta-lactams, isolates resistant to three or more antimicrobial drugs were also considerably observed, representing 11 of the 22 different antimicrobial susceptibility profiles depicted ( Figure 2). As the antimicrobial used in animal production is the same used to treat several zoonotic infections in humans, these finding strengthen the argument in favor of the prudent use of antibiotics by farmers and veterinarians.
F I G U R E 2 Dendrogram (a) and minimum-spanning tree (MST) (b) of the 79 isolates of Staphylococcus spp. isolated from cows with mastitis in dairy herds in Minas Gerais state using PFGE data. The numbers in parentheses correspond to the number of isolates within each PFGE profile (P). The MST presented is the one with the highest overall reliability score and was calculated using UPGMA (unweighted pair group method using arithmetic averages) associated with the priority rule and the permutation resampling Adopting 100% of similarity, 34 different genotypes were identified among the 79 Staphylococcus spp. isolates evaluated by PFGE. Figure 2 shows the cluster analysis (dendrogram and MST)

ACK N OWLED G M ENTS
This work was supported through funding from São Paulo Research

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S CO NTR I B UTI O N
ASG, MAVPB, and MBH conceived the study. EMSD, ASG, APL, and MBH participated in design of the study and wrote the paper.
MDAMF, JAPA, CRP, and HBM participated in data acquisition and analysis. All authors read and approved the final manuscript.

E TH I C S S TATEM ENT
This research did not involve any human or animal subjects and therefore did not require any ethics oversight or approval in these respects.

DATA ACCE SS I B I LIT Y
All data are provided in full in the results section of this paper.