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Prevalence and antibiotic resistance of Staphylococcus aureus and Streptococcus agalactiae in family-owned dairy herds in the state of Minas Gerais, Brazil

Bovine mastitis is the most frequent disease in dairy herds worldwide, causing great economic losses to both producers and the industry. This disease has direct implications in public health due to the zoonotic potential of some agents involved in its etiology and the presence of antimicrobial residues in milk. Knowledge of the agents involved in its etiology and the susceptibility profiles to antimicrobials is of paramount


Introduction
The Brazilian food production is intrinsically related to small farms, especially family farming, which represents approximately 84% of all properties and yields US$ 55.2 billion in revenue annually. The Brazilian family farming segment ranks in the eighth position amongst the largest food producers in the world (MAPA, 2017).
Milk production is one of the main activities developed in family farming, accounting for 52% of the revenue of these properties; it represents an important complement to household income, playing an important role in The economic losses caused by mastitis are associated mainly with reduction in milk production, increased labor costs, including veterinary services, drug spending, and milk discard of affected animals, early death or animal disposal (OVIEDO-BOYSO et al. al., 2007;LOPES et al., 2012). In addition to direct losses to producers, this disease promotes changes in milk composition associated with increased bulk milk somatic cell count (BMSCC) and low milk quality (LANGONI et al., 2011 indicative of low quality, especially when it comes to milk quality indicators such as total bacterial count (TBC) and BMSCC (PETER et al., 2013).
Bovine mastitis is a disease that requires antibiotics, which is mostly administered to treat clinical cases, and dry cow therapy (ERSKINE et al., 2004). This, however, increases the risk of antimicrobial residues in milk and derivates. The continuous, reckless antibiotic treatment is a determining factor for selecting resistant strains, potentially harmful to public health (OIE, 2010). The dry cow therapy, associated with high BMSCC and a history of mastitis, has potential for selecting antibiotic resistant strains (RAJALA-SCHULTZ et al, 2009).
The high prevalence of mastitis in Brazilian herds and its economic and health impacts require periodic monitoring of both the pathogens involved in its etiology and their antibiotic susceptibility. In addition, it is important to study the resistance of the bacteria involved in intramammary infections (IMI) to antibiotics that are commonly used against mastitis. This allows for monitoring the resistance dynamics and adopting more effective treatment protocols to combat the disease.
This study evaluates the prevalence of S. aureus and S. agalactiae in bulk milk tanks of small dairy herds in the Campo das Vertentes region, in the south of Minas Gerais. It also analyzes their resistance to the antibiotics that have been commonly used to treat bovine mastitis.

Material and methods
This study analyzed the bulk milk tank samples from 200 dairy herds located in the Campo das Vertentes region (approximated of 12,564 km 2 ).
Since the properties were family farms, i.e., had less than 4 fiscal modules (less than 120 hectares), they produced less than 350 liters of milk/day and To detect S. agalactiae, an aliquot of 50µL of each milk sample was spread in modified Edwards Agar Selective Medium Petri dishes enriched with 5% defibrinated sheep blood. The plates were incubated for 24-48 hours at 37°C. Colonies that had a bluish color, bright appearance and convex shape, 1-5 mm in diameter, with or without an opaque zone on the periphery, were considered positive (SAWANT et al., 2002).
After evaluating the growth in the selective media, five suggestive colonies of each agent of interest were selected at random and submitted to confirmatory tests for precise species identification, including sugar fermentation tests and CAMP test for characterization of S. agalactiae, and acetoin production (VP test), hemolysis, coagulase and mannitol fermentation for identification of S. aureus (NMC, 2004). After characterization, the microorganisms were stored in BHI containing glycerol (15% v/v) at -20ºC until susceptibility tests were performed.
For antibiotic susceptibility testing, three strains of S. aureus randomly selected from each of the S. aureus positive properties were pooled and used to evaluate antimicrobial susceptibility. The same procedure was done to evaluate the susceptibility of S. agalactiae isolates. In total, 74 antibiograms were performed for S. aureus and 65 for S. agalactiae.
In-vitro antibiotic resistance tests were performed by the disk diffusion method (CLSI, 2013). To this end, the strains were thawed, and an aliquot of 10 µl was cultivated in tubes containing 2 ml of Mueller Hinton broth, incubated at 37ºC for 18-24 hours after evaluation of growth and purity, and then diluted in sterile saline until 0.5 turbidity equivalent to McFarland standard scale to produce the inoculum.
The standardized inoculums were spread on a culture media plate surface containing Mueller-Hinton Agar, which is supplemented with 5% sheep blood for S. agalactiae susceptibility tests. The antibiotics tested were:  (1983). All statistical analyses were descriptive.

Results and discussion
The results showed a high prevalence of S. aureus (71.0%) and S.
agalactiae (68.0%) pathogens in the farms. In the herds, 49.5% had both agents, 91.0% had one single agent and only 9.0% of the studied properties were negative for both pathogens (see Table 1). The high prevalence found in the present study for S. aureus and S.
agalactiae (see Table 1) indicates that control measures for contagious mastitis have not been applied correctly. This points to a need for specific measures to control these contagious pathogens that impact the productivity of herds and the quality of milk, especially when it comes to the BMSCC (RODRIGUES et al., 2017).
The results of the antibiogram tests (see   Table 3 shows that 38 (51.35%) in the 74 S. aureus strains were resistant to more than five out of the 20 antibiotics tested in this study. The antibiotic susceptibility tests for S. agalactiae indicated that 64 stains (98.46%) were resistant to more than five antibiotics. These results point to the high frequency of multi-resistance strains for both pathogens.
The present in-vitro results point to higher resistance rates for S.
agalactiae as compared to S. aureus. Several studies have pointed to the greater difficulty in treating intramammary infections (IMI) caused by S.
aureus as compared to IMI caused by S. agalactiae. This can be explained due to both increased antibiotic resistance rates and the mechanisms of escape to  According to Myllys et al. (1994)

Conclusions
The findings point to a high prevalence of S. aureus and S. agalactiae pathogens in family-owned dairy herds in Campo das Vertentes, State of Minas Gerais, Brazil. High levels of resistance and multidrug resistance of S. aureus and S. agalactiae were also found. Multi-resistance was found for both agents, with higher levels for S. agalactiae. *** Prevalence and antibiotic resistance of Staphylococcus aureus and Streptococcus agalactiae in family-owned dairy herds in the state of Minas Gerais, Brazil ABSTRACT Bovine mastitis is the most frequent disease in dairy herds worldwide, causing great economic losses to both producers and the industry. This disease has direct implications in public health due to the zoonotic potential of some agents involved in its etiology and the presence of antimicrobial residues in milk. Knowledge of the agents involved in its etiology and the susceptibility profiles to antimicrobials is of paramount importance, give the multifactorial aspect of the disease and the importance of milk production in family farming. This study evaluated the prevalence of Staphylococcus aureus and Streptococcus agalactiae in family-owned herds in the Campo das Vertentes region, State of Minas Gerais, and the resistance of these pathogens to the most commonly used antibiotics in the treatment of mastitis in dairy herds. The study involved 200 properties, covering an area of approximately 12,564 km 2 . The prevalence of pathogens was carried out through microbiological analyses of milk samples from each farm's milk tank. Modified Baird-Parker Agar medium was used to detect S. aureus, and modified Edwards Agar medium, enriched with 5% defibrinated sheep blood, was used to detect S. agalactiae. Disc diffusion method was used to evaluate resistance to antibiotics. The results showed high prevalence of S. aureus pathogens (71.0%) and S. agalactiae (68.0%) as well as high levels of multidrug resistance. The results show that the control of contagious mastitis in the herds is flawed, which indicates the need for more effective control of these pathogens and more judicious use of antibiotics to minimize resistance.