Antibiotic resistance and molecular characterization of Staphylococcus species from mastitic milk

Species within the Staphylococcus genus are important mastitis pathogens. Studies to describe virulence and antibiotic resistance as well as rapid techniques that permit analyses strains are needed. The aims were to identify and characterize Staphylococcus spp. isolated from mastitic milk, and to optimize multiplex polymerase chain reactions (PCR). Staphylococci previously isolated from milk of dairy cows with subclinical mastitis were analyzed. PCR was completed to amplify nuc, sodA, spa, agr locus, virulence factors, and antibiotic resistance genes. DNA sequencing of sodA and spa genes was performed and antibiograms were carried out on all isolates. In a group of 49 staphylococci, S. aureus was the most prevalent, followed by S. hyicus, S. xylosus, S. chromogenes. Following optimization of multiplex PCR, virulence factor genes were identified in the majority of isolates. The enterotoxin genes, seh and selx were highlighted. All hemolysin genes were detected in 28.6% of isolates. Antibiotic resistance was evaluated and the majority of isolates (69.4%) were resistant to penicillin. Among the genes encoding antibiotic resistance, mecA was identified, while two methicillin-resistant S. aureus were typed as spa type 605, agr type II, and one identified as SCCmec type IVa. The types t605 and agr II were detected in the majority of S. aureus assessed. The findings emphasized the importance of preventing Staphylococcus infection in dairy cows. Effective dairy herd management and information on milk quality are essential to prevent mastitis pathogens.


INTRODUCTION
Bovine mastitis affects the dairy industry worldwide, and is associated with reduced milk quality and production and subclinical mastitis (Ote et al., 2011;Rajic-Savic et al., 2015), and the most important causative agent in this bacterial group is Staphylococcus aureus (Ote et al., 2011).In addition, the relevance of coagulase-negative staphylococci (CNS) as a cause of mastitis in dairy cows has also been shown (Silva et al., 2014).CNS research has predominantly focused on humans, and the enterotoxigenic potential of CNS has not been extensively explored, although it has been suggested that CNS from bovine intrammamary infection (IMI) could be a potential source of staphylococcal superantigens (SAgs) (Park et al., 2011).
SAgs, e.g.staphylococcal enterotoxin (SE) and toxic shock syndrome toxin-1 (TSST-1), were first identified in S. aureus (Park et al., 2011), and have been well characterized.The emetic activity of SEs has been demonstrated (Hu and Nakane, 2014); thus, there is a potential to cause foodborne disease (Jorgensen et al., 2005).Staphylococcus aureus may carry genes for production of other toxins such as Panton-Valentine leukocidin, toxic shock syndrome toxin and exfoliative toxins (Jarraud et al., 2002).
The importance of virulence factors in Staphylococcus genus and the highly clonal structure within the S. aureus population have been highlighted in medicine, and could potentially help in treatments (Ote et al., 2011).However, antibiotic resistance is a concern since studies have demonstrated the emergence of resistant isolates from bovine mastitis (Moon et al., 2007;Silva et al., 2014).Thus, the aims of the present study were to identify, and characterize Staphylococcus spp.isolated from mastitic milk, and to optimize several multiplex polymerase chain reaction (PCR) in order to simultaneously identify the presence of different virulence factor genes.

Origin and collection of isolates
The collection of bacterial isolates belonging to Hygiene and Dairy Laboratory, University of São Paulo, w as used.From this collection, isolates from mastitic milk previously identified as Staphylococcus spp.w ere selected.Forty-nine isolates w ere selected from three different dairy farms located in São Paulo State, region of Piracicaba city, Brazil.
The isolates w ere obtained in a previous study performed by Hygiene and Dairy Laboratory's group, in w hich dairy cow s w ere diagnosed w ith subclinical mastitis after screening using California Mastitis Test.Mastitic milk samples collected from September to October of 2013 w ere used.For the bacterial culturing, standard microbiological methods included colony morphology on Baird Parker Agar (BPA , Difco BD ® , Nova Jersey, EUA) w ith egg yolk tellurite supplement (Laborclin ® , Pinhais, Brazil), Gram staining, catalase, and coagulase test w ere completed to identify staphylococci, and all isolates w ere stored at -20°C.

DNA extraction, polym erase chain reaction and m olecular typing
Each isolate w as inoculated into Brain Heart Infusion (BHI, Oxoid TM , Hampshire, UK) broth and incubated at 37°C for 24 h.Aliquots of each culture w ere centrifuged and the supernatant w as discarded.The pellet w as used to extract DNA using "AxyPrep TM Blood Genomic DNA Miniprep kit" (Axygen Scientific Inc., Union City, USA), according to manufacturer's instructions.Agarose gel electrophoresis w as completed to verify the extraction, and the genomic DNA w as stored at -20°C.
Coagulase-positive and coagulase-negative staphylococci previously identified by coagulase test w ere confirmed by detecting the coa gene through PCR.The PCR amplification of coa gene described by Aarestrup et al. (1995) w as modified by using 0.75 mM of MgCl2 in each reaction, and the PCR cycles used w ere as follow s: 95°C for 5 min; 30 cycles at 95°C for 30 s, 55°C for 2 min, and 72°C for 4 min; and finally at 72°C for 10 min.When confirmed as coagulase-positive, multiplex PCR w as performed to identify S. aureus, S. intermedius and S. hyicus according to Sasaki et al. (2010).Other strains w ere identified by amplifying the sodA gene, and through DNA sequencing using Sanger method (Silva et al., 2014).
Single PCR w as initially performed for genes and positive (extracted DNA from strains belonging to Hygiene and Dairy Laboratory collection, University of São Paulo) and negative controls w ere incorporate into each run.Next, primers w ere combined in the same reaction w hen possible depending on amplification characteristics e.g.annealing temperature, number of PCR cycles and concentration of MgCl2.The multiplex reactions w ere as follow s: 1X PCR Buffer, 1U GoTaq ® Hot Start Polymerase (Promega Corporation, Madison, USA), MgCl2 (Promega Corporation, Madison, USA) concentration w as variable, 10 ρmol of each primer (synthesis by Sigma-Aldrich ® , São Paulo, Brazil), 200 µM deoxynucleotides (Promega Corporation, Madison, USA), template DNA (approximately 40 ng) and ultrapure w ater to bring the final reaction volume to 25 µL.Genes that w ere not incorporate into multiplex PCR, w ere amplified by uniplex PCR using 2.5 mM of MgCl2 and thermally cycled at 94°C for 5 min, 30 cycles at 94°C for 2 min, 48°C for 1 min, and 72°C for 1 min, and then once at 72 °C for 10 min.
agr type w as developed according to Shopsin et al. (2003) and the amplification of spa region w as carried out follow ing the w ebsite http://w w w .ridom.com/, the repeats w ere identified for spa types detection after sequencing by Sanger method.

DISCUSSION
In this study, in a limited group of staphylococci were identified and S. aureus was the dominant species; however, CNS was also present.In addition, several virulence factor genes were identified in the majority of isolates by multiplex PCR as well as antibiotic resistance to one or more antibiotics tested by diffusion method.Regarding SEs, the importance of seh, and selx genes corresponding to SEH and SElX is emphasized due their high incidence, while low frequency or absence of classical SEs were observed.The mecA positive isolates detected were spa type 605, and agr type II, which were also identified in the majority isolates.
Herein, high frequency of S. aureus was detected, this species has been identified as the primary pathogen associated with mastitis (Ote et al., 2011;Silva et al., 2014), and previous studies have identified a high  5) t267( 4) t9129 ( 1) I ( 5) II ( 5) II ( 4) frequency of this pathogen in Brazil (Silva et al., 2013;Lange et al., 2015).Giannechini et al. (2002) also detected high frequency of S. aureus, and low frequency of S. hyicus coagulase-positive among isolates from subclinical mastitis cases.The coa gene amplification also showed that the minority of the isolates belonged to CNS; which are capable of causing opportunistic mastitis (Moon et al., 2007).Lange et al. (2015) reported S. chromogenes at a frequency of 38.5%, which highlights the importance of coagulase-negative strains; however, in this study, the detection of CNS was low.S. xylosus, coagulase-negative, were also detected, within this species there are strains that can potentially be hazardous, and they are related to animal opportunistic infections (Dordet-Frisoni et al., 2007).The low frequency of classical SEs is in agreement with a previous study in which S. aureus associated with bovine mastitis were analyzed (Ote et al., 2011).In this study, classical SEs were not identified in S. chromogenes, S. xylosus and S. hyicus; however, classical SEs have been reported in these species (Park et al., 2011).Among the other SEs, the frequency of seh was highly detected.SEH has emetic activity and staphylococcal food poisoning associated with S. aureus carrying the seh gene has been reported (Jorgensen et al., 2005;Argudín et al., 2010).Considering the potential of SEH to cause foodborne disease, strains from our collection that carry the seh gene should be tested forenterotoxin protein expression in further investigations.
The staphylococcal enterotoxin-like toxin X (SElX) also demonstrated a high frequency.The selx gene is encoded in the core genome of S. aureus, which explains the frequency of selx.However, its emetic activity has not yet been tested (Hu and Nakane, 2014).In addition, it is suggested in this case to further study allelic diversification.Other genes (seb, seg, sej and ser) that encode for SEs with emetic activity were detected; it shows that milk quality control needs to be strict in order to avoid the pathogen or significant count of it, and consequently the possibility of milk contamination with SEs.sek , sel, sem, sen, seo, sep, seq and seu were detected; however, these have not exhibited emetic activity in primate models or emetic activity has not been tested for some genes.Several SEs profiles were identified, this finding demonstrates the high distribution of SEs genes in the species studied; for example, 32 superantigenic toxin genotypes were observed across 166 isolates (69 food poisoning isolates, and 97 healthy human nasal swab isolates) in the study performed by Omoe et al. (2005).All hemolysin genes were identified, hla, hlb, hld and hlgAC were also detected by Ote et al. (2011), and they identified frequencies between 78.6 and 100% in strains.In this study, hemolysin gene frequencies were between 32.7 and 55.1%.The most prevalent was hlb, which is in agreement with other study that assessed isolates from raw milk products (Morandi et al., 2009).Genes encoding exfoliative toxins, pvl and tst were not identified; previously Ote et al. (2011) identified eta and tst genes in isolates associated with bovine mastitis.
Regarding antibiotic resistance, penicillin resistance is commonly detected in Staphylococcus spp.(Moon et al., 2007;Gómez-Sanz et al., 2010), and this was demonstrated in the present study.Silva et al. (2013) did not detect resistance to erythromycin in their isolates, although they detected one strain of S. aureus with resistance to chloramphenicol.Erythromycin and tetracycline resistance genes were observed (Table 2); these genes have been detected in Staphylococcus sp.(Silva et al., 2014;Gómez-Sanz et al., 2010).It is important to highlight that all isolates were tested for the presence of mecA and mecC genes as well as other resistance genes.These results on mecA, and SCCmec type are in line with Silva et al. (2014), where they assessed methicillin-resistant coagulase-negative staphylococci in milk from cows with mastitis in Brazil.Herein, SCCmec type I to V was investigated due to the availability of positive controls, further studies to assess types I to XI are necessary due to their importance in methicillin resistance.Meanwhile, the absence of a novel mecA homologue could be expected because it is of rare occurrence (Cuny et al., 2011).The absence of mec genes in cefoxitin and oxacillin resistant strains can indicate the potential presence of modified S. aureus (MODSA); MODSA possesses a modification of its penicillin-binding proteins (PBPs), which is different of classical mechanism of MRSA (Bhutia et al., 2012).
Four spa types were detected, and on the farm A only one spa type (t605) was observed.This suggests that the spa type t605 is common and it can be endemic in the region causing subclinical bovine mastitis.The t605 type was initially detected in Austria, France, Germany, Netherlands, Norway, Spain, Sweden and United Kingdom, and represents 0.1% of relative global frequency of spa type occurrences in accordance with the website, http://www.ridom.com(http://spaserver.ridom.de-data collected on June 2015).Other studies in Brazil also detected this spa type in strains isolated from milk from bovine and others animals (Aires- de-Sousa et al., 2007;Silva et al., 2013).On the other hand, the spa type t127 were the most detected by Silva et al. (2013).The agr types detected were I and II, which were also detected in a previous study with isolates from bovine mastitis (Silva et al., 2013).

Conclusion
The majority of isolates were identified as S. aureus.Other isolates also identified were S. hyicus, S. xylosus and S. chromogenes.The majority virulence factor genes identified using multiplex PCR, in total eleven different multiplex reactions were successfully optimized and applied in this study.The most isolates carried virulence factor genes, seh, and selx were the most detected among SEs.Hemolysins genes were widely identified, presenting several profiles as well as SEs.Antibiotic resistance was widely detected for penicillin; in addition, MRSA strains were observed which presents a concern to public health.The most prevalent spa type was t605, which suggests that this could be an endemic spa type in the herds sampled.In summary, data regarding molecular variability, and antibiotic resistance for a small group of staphylococci isolated from mastitic milk was shown, which confirms that more studies should be completed to identify and understand strains/clones in specifics regions, and thus to help prevent Staphylococcus infection in dairy cows.

Table 1 .
Primers sequences used to amplify virulence factors.

Table 2 .
Species, spa typing, virulence factor genes and antibiotic resistance genes detected by farm.

Table 3 .
Conditions of the multiplex PCR optimized in this study.