Characterization of Coa Gene and Antimicrobial Profiles of Staphylococcus Aureus Isolated from Bovine Clinical and Subclinical mastitis

| Staphylococcus aureus (S. aureus) is one of the major pathogens involved in bovine mastitis. Monitoring of antibiotic use could assess the hazard of S. aureus in cow’s milk. The objective of this study was to determine the prevalence of S. aureus in mastitic dairy cows in some localities in Dakahlia and Damietta Governorates, Egypt and to phenotypically characterize the antimicrobial susceptibility of the obtained isolates. In that context, a total of406 milk samples were collected from dairy cows suffering from clinical (n=100) and subclinical (n=306) mastitis from four different dairy farms. Milk samples were subjected to conventional bacteriological isolation techniques, and confirmed as S.aureus by using PCR assay targeting nuc (S. aureus-specific thermonuclease) and coa (coagulase) genes. Interestingly, 28 S. aureus strains tested negative to coagulase enzyme phenotypically were positive for Cao gene. The prevalence rate of S. aureus in clinical and subclinical mastitic milk samples was 66% (66/100) and 30.72% (94/306) respectively with an overall prevalence of 39.40% (160/406). All S.aureus isolates were tested against 10 different antimicrobials belonged to 7 antimicrobial classes. S.aureus strains exhibited a high rate of resistance to vancomycin (93.75%), penicillin (86.25%), trimethoprim (60%), and oxacillin (58.75%). A medium rate of resistance was observed to clindamycin (41.25%), ciprofloxacin (41.25%) and erythromycin (37.5%).On the other hand; S.aureus isolates displayed a low frequency of resistance to gentamicin (21.25%), ampicillin/sulbactam (20%) and chloramphenicol (18.75%). The multidrug-resistance to three or more classes of antimicrobials was detected in 108isolates (67.5 %). In conclusion, antimicrobial resistance of S.aureus was prevalent in dairy herds in the study area which represents a public health hazard. Hence, control and prevention policies should be implemented to minimize the dissemination of resistance trend.


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other staphylococcus species by possessing coagulase enzyme and called coagulase positive staphylococcus (CPS) (Cunha, 2009).S.aureus in milk can affect the consumer in case of ineffective pasteurization or also in dairy products with raw milk; this represents an important public health hazard (Silva et al., 2013).It can also cause outbreaks of variety of diseases such as food poisoning (nausea, violent vomiting, and abdominal cramping with or without diarrhea) (Le Loir et al., 2003), osteomyelitis, septic arthritis or septicemia (Peles et al., 2007).
Antimicrobial therapy is an important tool in mastitis therapeutic programs.In the last decades, failure of antimicrobial therapy against Staphylococcus spp.has been recorded (Vintov et al., 2003).This therapeutic failure is associated with antimicrobial resistance of S. aureus, intracellular location of S. aureus or microabscess formation deep inside the udder tissues (Fox et al., 2001;National Mastitis Council, 2004).S. aureus strains have been observed for resistance against different antimicrobial agents including beta-lactams, aminoglycosides, fluoroquinolones, lincosamides, macrolides, andstreptogramins that are commonly used by veterinarians worldwide to treat mastitis (Hendriksen et al., 2008;Wang et al., 2008).
There is a great concern that the antibacterial-resistant agents in food animals can be transmitted to humans by dairy food chain (Irlinger, 2008).Therefore, this study was designed to determine the prevalence of S. aureus in mastitic dairy cows in Dakahlia and Damietta Governorates, Egypt and to genetically characterize the isolated strains using nuc (S. aureus-specific thermonuclease) and coa (coagulase) genes.In addition, the antimicrobial susceptibility patterns of isolated strains against common antimicrobial agents were also determined.

samples ColleCtion
A total of 406 quarter milk samples were collected from 285 animals affected with mastitis.Samples were collected from four different dairy farms located at Dakahlia and Damietta Governorates, Egypt during the period between June 2016 and August 2017.Forms of mastitis were determined based on physical examination of the udder and California Mastitis Test (CMT) findings.Clinical mastitis was diagnosed based on the presence of abnormal changes in milk, mammary gland, teat, and systemic reaction (Quinn et al., 2004).Milk samples were collected aseptically.Quarters were washed with clean warm water and dried and the teat ends were disinfected with 75% ethyl alcohol then 8 to 10 ml milk samples were collected in sterile tubes after discarding the first three streams of milk.Milk samples were held in an ice box and transported im-mediately to laboratory for bacteriological analysis.

BaCterioloGiCal examination
Milk samples were subjected to S.aureus isolation procedures based on the standard technique previously established by Wang et al. (2012).Briefly, milk samples were vortexed, and 10 µL of milk was plated on Baird-Parker agar plates (BPM, Oxoid, Basingstoke, UK) with 5% egg yolk and 1% potassium tellurite and incubated at 37°C for 24 h.For purification, one or two presumptive colonies per plate (black colonies surrounded by hallow zone) were transferred to trypticase soy agar (TSA; Oxoid, UK) plates.S.aureus isolates were subjected to Gram staining, coagulase test and the standard biochemical tests (Boerlin, et al 2003;De Freitas Guimarães et al., 2013).All isolates were stored in 20% glycerol solution at −80°C for further investigation.
Pcr assay: PCR assay was performed for the detection of the nuc (encoding for the S. aureus-specific thermonuclease) gene and coa gene encoding for coagulase as previously described by Sallam et al. (2015) and Himabindu et al. (2009), respectively.The primers sequences (Hokkaido System Science Co.Ltd., Hokkaido, Sapporo, Japan and Metabion, Germany) and its amplicons size are listed in Table 1.Each PCR reaction was performed in a total vol

statistiCal analYsis
Differences between different antimicrobial agent in resistant, intermediate and susceptible Staphylococcus aureus strains were tested using the Chi square (χ2) test.χ2 was calculated 830.95 and the degree of freedom was 18. Differences among means with P < 0.000001 were considered as statistically highly significant.

S.aureuS prevalenCe
The overall prevalence of S. aureus in this study was 39.4% (160/406) out of 406 milk samples from both clinical and subclinical mastitis.According to the type of mastitis, S. aureus was recovered at a prevalence rate of 66% (66/100) and 30.72% (94/306) from clinical and subclinical mastitis, respectively.
S. aureus isolates were identified with the nuc gene using PCR assay in all the biochemically identified isolates (Figure 1).Coagulase gene encoded by coa was also used for the confirmation of the isolated S. aureus, all the recovered S. aureus harbored the specific amplified products of coa gene at the expected size 500 bp except in ten isolates, in which two variants of coa gene were detected, one with the size of 500bp and the second variant was 600bp (two isolates), 700bp (four isolates), 800bp (two isolates) and 900bp (one isolate) (Figure 2).Amplification of coa gene showed that 28 isolates, which had been identified as coagulase negative staph aureus, contained coa gene (Table 2).3).The multidrug-resistance (resistance to three or more classes of antimicrobials) was detected in 108 isolates (67.5 %) (Table 4).In the present study, the overall prevalence of S. aureus was 39.4%, being higher in cows with clinical mastitis (66%) than those having subclinical mastitis (30.72% S. aureus isolates in this study were subjected to phenotypic and molecular characterization using the coa gene.Coagulase is a genetic marker for S. aureus strains responsible for coagulation of plasma and is considered as a significant virulence determinant of S. aureus (Watanabe et al., 2009).Among all S. aureus isolates in this study, 28 strains harbored coa gene, but tested negative with coagulase test.This suggests that coagulase gene is not functional in these 28 isolates.A similar finding of mutated coagulase has been reported previously (Phonimdaeng et al., 1990 al., 2011).Furthermore, confirmation of S. aureus was performed by PCR assay targeting nuc gene encodes the S. aureus-specific region of the thermonuclease which can degrade both DNA and RNA, and its enzymatic activity can resist 100°C for at least 1 h.PCR amplification for nuc gene is considered as an accurate, rapid, and safe screening method for S. aureus detection (Shrestha et al, 2002).Moreover, phenotypic methods are considered time consuming, expensive, and less accurate method so molecular screening of S.aureus is reported to be more efficient than other phenotypic methods (Aras et al., 2012).
In this study, S. aureus isolates exhibited a high resistance to vancomycin (93.75%).But vanomycin resistance was not recorded against S. aureus isolates from mastitic cow's milk (Kumar et al., 2010;Wang et al., 2014).In addition, the high resistance of S. aureus to oxacillin (58.25%) contradicted the results obtained by many other authors (De Oliveira et al., 2000;Gentilini et al., 2000;Guler et al., 2005;Kumar et al., 2010) who reported susceptibility of S.aureus to oxacillin.
Concerning penicillin resistance, a higher rate of resistance was revealed in this study (86.25%).This finding went parallel with those reported in many studies (Guler et al., 2005;Turtoglu, et al., 2006;Li et al., 2009;Shi et al., 2010;Wang et al., 2014;Akindolire et al. 2015).On the other side, much lower resistance was previously recorded by others (Kumar et al., 2010;Haran et al., 2012).The variety in penicillin resistance between investigations could be attributed to many reasons including the differences in animal production systems and the use of antimicrobial drugs in each country (Guler et al., 2005).
Comparing to the results of the current study, Kumar et al.
For clindamycin and ciprofloxacin resistance, a high rate of resistance against these antimicrobial agents was also observed by Wang et al. (2008).
Taken together, it can be inferred that the isolated S. aureus had a high resistance to the antibiotics which are frequently used in the country.Besides, bovine multidrug resistant S. aureus strains could be zoonotic pathogens (Soares et al., 2012).Given that Vancomycin and oxacillin are important antibiotics for human, isolates.Our results indicated high resistance rate for oxacillin which is responsible for methicillin resistance.This may be an indication for contamination or even infection by human isolates (Spanu et al., 2012).Moreover, the difference of animal welfare system and the medication by the antimicrobial drugs caused this high prevalence of antimicrobial resistance (Guler et al., 2005).The antimicrobial agents being used for treatment of mastitic cases were unexpected to possess this increased resistance (Goni et al., 2004).This may be attributed to modification of the antibiotics by modifying enzymes (Goni et al., 2004) or the influence of excessive use (Turutoglu et al., 2006).

concluSIonS
The widespread use of antibiotics on dairy farms and other food-producing animals could lead to emergence of antibiotic-resistant bacterial strains which represents a serious public health problem because of the possibility of dissemination of the antimicrobial-resistant bacteria to humans via food.Therefore; all possible control and prevention policies should be implemented to minimize the dissemination of resistance trend.

AcKnowlEdgEMEntS
We gratefully acknowledge Dr. Eman Abo El-fadl at the Department of Animal Husbandry and Development of Animal Wealth, Faculty of Veterinary Medicine, Mansoura University for her technical help in this study.

Figure 1 :
Figure 1: PCR products for nuc gene of Staphylococcus aureus strains showing amplified genes at the expected molecular size 660 bp from lane (1-12) and the last two for positive and negative controls, respectively.

Figure 2 :
Figure 2: PCR products for coa gene of Staphylococcus aureus strains showing amplified genes at the expected molecular size 500-1000 bp from lane (1-12) and the last two for positive and negative controls, respectively.

table 1 :
Primer and cyclic PCR conditions for PCR amplification of various genes specific for molecular identification of Staphylococcus aureus ume of 25 µL, consisting of the following components: 12.5 µl master mix (Thermo scientific, USA); 1 µl of each primer; 2 µl DNA template and volume of the reaction mixture was completed to 25 µl using DNase/RNase-free water.PCR reactions were performed using 96 well Applied Biosystem,2720thermal cycler and cyclic PCR conditions described in Table2according to the referenced authors.Eight µl of each PCR product was separated by agarose gel electrophoresis, 1.2 % agarose prepared in TBE.Gels were stained with ethidium bromide and then visualized by Gel Doc (cleaver scientific ltd UV gel documentation system, USA).

table 2 :
Difference between phenotypic and molecular typing of S. aureus Antimicrobial resistance was investigated against 10 anti-Advances in Animal and Veterinary Sciences April 2018 | Volume 6 | Issue 4 | Page 164

table 4 :
Antimicrobial resistance patterns ; Sunagar et Advances in Animal and Veterinary Sciences April 2018 | Volume 6 | Issue 4 | Page 165

Advances in Animal and Veterinary Sciences April 2018 | Volume 6 | Issue 4 | Page 166 al
. 2013).Hence, proper identification of coagulase positive and coagulase negative species can't be performed by phenotypic methods only but also, requires a combination of phenotypic and molecular assays (Akineden et