Antibiogram and Antibiotic Resistance Genes among Coagulase-Negative Staphylococci recovered from Bovine Mastitis

| Coagulase-negative Staphylococci (CoNS) have emerged as an important Staphylococci species implicated in bovine mastitis in dairy herds. This study was conducted to determine the prevalence, antibiogram, and antibiotic resistance genes among coagulase-negative Staphylococci recovered from cattle suffer from subclinical mastitis in Egypt. A total of 110 (36.7%) milk samples collected from 300 lactating cows were positive for the California mastitis test. On the Mannitol Salt Agar medium, out of 110 subclinical mastitis samples 62 (56.36%) were identified as CoNS isolates. Antibiotic sensitivity test conducted against nine types of antibiotics for CoNS strains that exhibited high susceptibility to most of the tested antibiotics, with particularly resistance pattern to oxicillin (41.9 %), and 11 (17.7%) CoNS isolate exhibited multidrug resistance (MDR). A total of 15 randomly selected isolates were subjected for detection of antibiotic-resistant genes among CoNS. The results indicated that mec A (73.3%) was the most identified gene, followed by tet K (60%) and erm B (13.3%) genes, whereas no detection for bla Z and van A genes. In conclusion, our results indicate the importance of the regular surveillance of phenotypic and genotypic profiles of CoNS, isolates to ensure effective control measures and minimize the evolution of MDR strains and recommended the applying of antibiotic sensitivity test before treatment or random selection of antibiotics in field cases to avoid the emerging of resistance phenomena.

generally associated with less severe signs (Taponen et al., 2006;Becker et al., 2014;Srednik et al., 2015). In most countries, Coagulase negative Staphylococci (CoNS) are progressively recognized as etiological agents associated with intramammary infections (Unal et al., 2012). Some studies presumed CoNS as emerged causative mastitis pathogens with major virulence determinants (Zhang and Maddox, 2000), with a high degree of resistance to antimicrobials (Rajala-Schultz et al., 2009), and the capability to cause chronic infectious diseases . Several strains of CoNS species have been implicated in intramammary infections, although the most frequently isolated CoNS from bovine mastitis are S. chromogenes, S. simulans, S. xylosus, S. epidermidis, S. hyicus, and S. haemolyticus (Thorberg et al., 2009;Park et al., 2011). Due to the increasing importance of CoNS in bovine mastitis, the identification of CoNS species is necessary to design effective control approaches for CoNS mastitis (Sawant et al., 2009).
Treatment with antimicrobial agents is the most widely used protocol for the treatment and control of mastitis (Gomes and Henriques, 2016). However, the excessive and abuse of antibiotics in humans and animals' practices is strongly linked to the evolution of antibiotic resistance against several antimicrobial groups particularly in veterinary medicine (Schwarz et al., 2018). Also, CoNS may have a role in the transmission of resistance genes (Becker et al., 2014), and emerging of new multidrug resistant strains (Otto, 2012;Vitali et al., 2014). In recent study Qu et al. (2019) reported that CoNS harbored various antimicrobial resistance genes such as mecA, tetK, tetL, tetM, dfrG, and ermB which are responsible for methicillin, tetracycline, trimethoprim and erythromycin respectively.The constant surveillance and monitoring of the antimicrobial resistance profiling of the CoNS isolates will provide valuable database about the efficacy of the antibiotics in control of mastitis and manipulative an effective treatment and control measures for bovine mastitis (Veras et al., 2008). As result of the limited available information about the phenotypic and genotypic resistance profile of CoNS strains isolated from subclinical mastitis in Egypt as recent reported by (Nayel at el., 2020) who revealed the emerging of CoNS in clinical mastitis and subclinical mastitis with prevalence 60% and 67.27% respectively in Egyptian cows with high resistance against penicillin and oxacillin in Menofiuya Governorate. Therefore, this study was planned to investigate the phenotypic and molecular detection of antibiotics resistance genes in CoNS strains from bovine mastitis, Egypt.

saMplE collEction and study arEa
A total of 300 mixed breed dairy cows aged from 3-7 years old from individual cases from Sadat City, Menoufiya Governorate, Egypt, were examined during the period from December 2019 to March 2020. One hundred and ten cows showed positive reaction with California mastitis test (CMT), which indicated subclinical mastitis. CMT was performed according to the methodology described by (Schalm and Noorlander, 1957). The CMT-positive milk samples were collected aseptically and transferred under cold conditions (4°C) at the earliest possible to the laboratory for bacterial isolation and identification.

bactErioloGical isolation and idEntification
For the isolation of Staphylococci, the milk samples were centrifuged and the sediment was then cultured in Mannitol Salt Agar (Oxoid Ltd. UK) and incubated for 1-2 days at 37°C. Confirmatory identification of CoNS was implemented by Gram staining then subjected to catalase and finally by tube coagulase test (Tortora et al., 2013). Pink color colonies were considered as CoNS which were confirmed by standard biochemical activities according to (Murray et al., 2003). Evaluation of hemolytic activity on sheep blood agar (5-7%) was performed as described by (Quiblier et al., 2011). Biofilm activity on Congo red agar medium (Arciola et al., 2015). The positive Congo red activities produced black colonies while non-producer gave red colonies.

antibioGraM profilE of cons isolatEs rEcovErEd froM bovinE Mastitis
The identified CoNS isolates were subjected for in vitro using the disk diffusion method as described by (CLSI, 2017) against different antibiotic groups to determine the susceptibility and resistance pattern to nine antimicrobial disks (Oxoid Ltd.). The used antibiotics: penicillin: P

MolEcular charactErization of antibiotic rEsistancE GEnEs in cons isolatEs
Freshly grown typical CoNS colonies were harvested, and DNA extraction was performed according to the manufacturer's guidelines using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).

Mastitis casEs
Of the total 300 dairy cows examined in this study, 110 (36.7%) were diagnosed with subclinical mastitis depending on the CMT result. Among the CMT-positive samples, 62 were found to be positive on the Mannitol Salt Agar medium (56.36%) CoNS isolates as showed in Table  (

MolEcular dEtEction of cons antiMicrobial rEsistancE
Regarding to the prevalence of antibiotic-resistant, mecA was the most detected gene in 11 (73.3%) isolates of CoNS (Fig.1A). Furthermore, the blaZ not detected at any of the tested CoNS isolates. Regarding the tetK gene, it was detected in 9 isolates with a prevalence of 60% in CoNS (Fig.1B). The ermB gene was identified in 2 isolates with a prevalence of 13.3% in CoNS (Fig. 1C).

dIScuSSIon
Bovine mastitis is the most economic and productive disease affecting the livestock industry which can be graded as clinical or sub-clinical mastitis (Krishnamoorthy et al., 2017). A large drop in milk production due to subclinical mastitis is a major financial loss (Romero et al., 2018).
The present study revealed that CoNS species was isolated in 62 (56.63%) among the CMT-positive samples and S. epidermidis, S. saprophyticus, S. heamolyticus were the detected CoNS species with 30 (48.4%), 20 (32.3%), 12(19.4%) respectively. Our results agree with those of several scientific reports that described CoNS as a major bacterial etiology connected in bovine mastitis. For example, (Dieser et al., 2013;Zigo et al., 2017) reported that CoNS was the most isolated bacterial group from milk samples of dairy cows with subclinical mastitis. Furthermore, (Zigo et al., 2019) reported that CoNS accounted for 43.4% and 50.0% in two cattle herd examined for mastitis. In contrast to our study, (Mahmoud et al., 2015) recorded a lower percentage (8.9%) of CoNS isolates from subclinical mastitis samples in Egypt. In a recent comparative study, (Sukur and Esendal, 2020) reported that S. chromogenes, S. capitis and S. simulans were the most prevalent CoNS species in subclinical mastitis with (41.2%), (14.7%) and (11.8%) respectively in North Cyprus. This was also supported by (Vanderhaeghen et al., 2015) who demonstrated that S. chromogenes, S. haemolyticus, S. epidermidis, S. simulans and S. xylosus are common isolated CoNS species isolated from bovine mastitis. Likewise, (Nayel et al., 2020) isolated S. epidermidis and S. saprophyticus from bovine clinical mastitis in Egypt.
In the current study, the antibiotic resistance profile of the 62 CoNS species that extremely showed susceptibility to gentamycin, ciprofloxacin, amoxicillin/clavulanic acid, chloramphenicol, tetracycline, penicillin, vancomycin, and erythromycin. Meanwhile, the resistance pattern of CoNS species against the oxacillin 41.94% was observe. This is not consistent with the result reported by (Phophi et al., 2019) who recorded that 90% of CoNS were resistant to at least one antimicrobial and 51% showed MDR, and the highest resistance against ampicillin (90%) and penicillin (89%). A low resistance (19.35%) against penicillin among CoNS species was observed in our study compared to that reported in mastitis of dairy cattle in Estonia (38.5%) (Pitkälä et al., 2007), Finland (32%) (Simojoki et al., 2012), and South Africa (63%) (Phophi et al., 2019).
Vancomycin resistance is very important due to its use in the treatment of MRSA cases, and in this study, the uncommon resistance of vancomycin among CoNS strains was (3.23%), which is in parallel to the study of (Phophi et

Advances in Animal and Veterinary Sciences
August 2021 | Volume 9 | Issue 8 | Page 1272 al., 2019), who observed no vancomycin resistance among CoNS strains recovered from mastitis in dairy cattle as well as was report of (Bengtsson et al., 2009) in Sweden revealed the resistance of CoNS to β-lactams antibiotics. The low resistance of CoNS strains to vancomycin may be attributed to the uncommon use of antibiotics such as vancomycin in systemic bovine mastitis as injectable drug but only used as local intrammamary infusion.
With reference to CoNS, 11/62 (17.74%) exhibited MDR to three to four groups of antibiotics. Previously similar findings have been described in several countries, including the USA, Switzerland, and the Netherlands (Sawant et al., 2009;Moser et al., 2013;Sampimon et al., 2011). Moreover, (Huber et al., 2011) found that 33%-49% of all 414 CoNS strains exhibited MDR to several antimicrobial groups. Moreover, (Van Duijkeren et al., 2004) reported MDR in CoNS strains isolated from animals with clinical diseases.
Concerning MRCoNS, 53.33% of the strains exhibited MDR in the phenotypic testing and possessed (two to three) antibiotic resistance genes. Correspondingly, (Frey et al., 2013) reported that CoNS isolated from bovine mastitis samples exhibited MDR profiles. Likewise, (Araujo et al., 2006) reported that biofilm-producing methicillin-resistant S. epidermidis isolates exhibited MDR compared with non-producer isolates.

concluSIon
In conclusion, this study concluded that CoNS were considered an emerging cause of subclinical intramammary infection in individual bovine mastitis cases. Moreover, the higher predominance of phenotypic and genotypic resistance of CoNS species in this study indicates the potential economic loss. Therefore, it can recommend that the hygiene regimen and regular investigation for MDRCoNS screening and recommended the significance for antibiotic sensitivity test to minimize the emerging of resistance CoNS strains in subclinical mastitis in cows in the studied area.