ISOLATION AND MOLECULAR IDENTIFICATION OF STAPHYLOCOCCUS SPP. FROM BOVINE MASTITIS MILK SAMPLES

Khushal Patel 1 , Chaitanya Joshi 2 and * Anju Kunjadiya 3 . 1. Ashok & Rita Patel Institute of Integrated Study & Research in Biotechnology and Allied Sciences, New Vallabh Vidyanagar-388121, Gujarat, India. 2. Department of Animal Biotechnology, College of Veterinary Science and A. H., Anand Agricultural University, Anand-388001, Gujarat, India. 3. Center for Interdisciplinary Studies in Science and Technology, Sardar Patel University, Vallabh Vidyanagar388121, Gujarat, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Mastitis is an inflammation of mammary gland caused by major etiological agent Staphylococcus sp. mainly antibiotic resistant strains. In this study, antibiotic resistant Staphylococcus spp. were isolated, characterized and confirmed by molecular techniques from a total of 120 lactating cows including 30 buffaloes (480 quarter milk samples). They were screened for mastitis using Somatic Cell Count (SCC) and California Mastitis Test (CMT). Isolation and identification of Staphylococcus spp. was done using standard microbiological methods by species specific media as well as various biochemical tests. Antibiotic susceptibility tests (ABST) were performed to screen resistant isolates. Genotypic identification was confirmed using 23S rRNA amplification and 16S rDNA sequencing. A total of 193 nonduplicated Staphylococcus spp. were isolated having higher than 500 somatic cells/μl and CMT score was 1 or 2. Methicillin resistance in 31 and other multidrug resistivity in 17 Staphylococcus spp. were observed. Multidrug resistant strains were identified using Sanger sequencing and assigned accession number KX821621 to KX821638 for 11 Staphylococcus aureus and 7 coagulase negative Staphylococcus spp. These findings suggested that milk represents a potential infection risk threat of multidrug-resistant S. aureus in India due to neglected hygienic practices during lactation and maintenance of herd.

ISSN: 2320-5407
Int. J. Adv. Res. 5(7), 1080-1088 1081 Natural immunity to fight against environment of S. aureus has been detected in clinical and subclinical bovine mastitis cases (Davis MP and Gamier P. 2013). Staphylococcus aureus infection causes main concern regarding treatment using experimental antimicrobials and leads challengeable tasks for veterinarians. Intramammary infections in animal caused by methicillin-resistant staphylococci (MRS) are indeed challenging task which include health risk of animal as well as milkers (Gómez et al. 2015).
Coagulase negative Staphylococcus spp. (CNS), which are in acquisition of interest for researchers because of its augmented exposure as an infection causing agents including human (Embden and Barkema 2009). At present, CNS are generally deliberated to be opportunistic pathogens in the research field. Epidemiology of CNS is not clear, which is sole reason for not having treatment against this pathogen. CNS group consists of about 40 different Staphylococcus species (Shittu and Lin 2006).
Majority of mastitis causing pathogens are normally found intermediate resistant to the antimicrobials used for treatment, but in past two decades few studies have signposted an augmented resistance of S. aureus and CNS to βlactam antibiotics (Trojan et al. 2016). In a view of this, the emergence of methicillin resistance among Staphylococcus spp. isolated from bovine milk has been reported in different countries and continents (Bergonier et al. 2014).
The surveillance of methicillin-resistant Staphylococci should be prioritized because the accurate diagnosis of these bacteria is paramount for appropriate mastitis treatment. The purpose of this study was to identify and characterize multidrug resistant including methicillin in Staphylococcal isolates from bovine mastitis milk samples.

Methodology:-
Sample collection and processing:-A total of 480 quarters milk samples from 120 lactating cows and buffaloes were collected from the different regions of Anand and Kheda district in Gujarat. Sample collection was done by maintaining proper hygiene conditions as described (Patel et al. 2017), At beginning, few drops of milk were discarded and samples were collected in autoclaved 50mL falcon tube (Tarson). California Mastitis Test was executed to check infections level simultaneously (Bhutto et al. 2012). Samples were brought to the laboratory in desired cooling condition and subjected to further microbiology evaluation. Somatic cells present in milk sample were analyzed on Fossomatic TM Cell Minor (Foss, Denmark) (Bhutto et al. 2012).

Isolation of Staphylococci and culture Conditions:-
For isolation of Staphylococci, milk samples were diluted and streaked on Baird-Parker agar base (Himedia, Catalog No. M043, India) plate with added 3.5 % potassium tellurite and incubated at 37°C for 24 to 36 h. S. aureus colonies were identified on the basis of colony morphology as mentioned in Himedia reference (Oliveira et al. 2011). Such colonies were further transferred to mannitol salt agar (Himedia, Catalog No. MH118, India) for differentiation and selection of pathogenic Staphylococcus aureus and Staphylococci sp. (Kateete et al. 2010). Confirmed isolates were cultured in Luria Broth (Himedia M575, India) at 37°C with aeration and 25% glycerol stocks were prepared for storage purpose. Isolates were further confirmed as S. aureus by a coagulase test using coagulase rabbit plasma (HiMedia, FD248) and species specific biochemical tests. Gram's morphology was also observed by preparing smear from the isolated culture on clean grease free microscopic glass slide and stained with gram's method of staining (Carles et al. 2004).

Biochemical examination:-
Various biochemical tests specific for confirmatory Staphylococcus sp. identification named methyl red test, Voges-Proskauer test, hemolytic test, catalase test, urea hydrolysis, indole and ammonia production were performed for characterization of isolates (Makki 2011).
Extraction of genomic DNA:-DNA was extracted from 24 hr grown Staphylococcal cultures using Proteinase-K-SDS method mentioned with slight modification (Prosperi et al. 2013). Concisely, 400 μl of culture was taken, centrifuged at 8000 rpm, supernatant was removed and dissolved in 500 μl TE buffer (10 mm of Tris HCl, 1 mM of EDTA, pH 8.0). Treatment of lysozyme (Sigma-Aldrich), 10% w/v freshly prepared SDS and 10 μl proteinase-k (10 mg/ml) (Sigma-Aldrich) was given t culture and incubated at 37 o C for 1 hr. Subsequently, addition of 10% w/v CTAB and 200 μl NaCl (5M) in the suspension and was re-incubated for 10 min at 56 o C in boiling water bath. The undesirable contaminants that are chiefly made of proteins were removed by Phenol: Chloroform: isoamyl alcohol (25:24:1) and Chloroform: isoamyl alcohol (24:1). 100 µl of 5M sodium acetate is added to the contents and is mixed gently. The salts and alcohol remnants are removed by washing with 80% alcohol. After removal of alcohol by centrifugation, desiccated the tubes and elution was done in 50 μl warm TE buffer. Quantity of extracted DNA was checked using Nanodrop 1000 (ThermoScientific, USA) and quality on 0. Nucleotide sequencing:-Purified 16S products of isolates which includes, multidrug resistivity against conventional antibiotics (ABST assay) and positive 23S rRNA gene amplification were sent for Sanger deodoxy sequencing (SciGenome Pvt. Ltd., Kerala). Obtained Sequences were blasted with the GenBank NCBI nr database (http://www.ncbi.nlm.nih.gov/). The highest identity was selected as the identified and according to the results, sequences were submitted for species assignment (Hijazin et al. 2011).

Results:-
Out of total 120 animals harboring 480 quarter milk samples, 128 (26.66%) and 52 (10.83%) were positive for CMT score 1 and 2 respectively. SCC score more than 500 cells/μl was found in 140 (29.17) quarters (Table 1). Milk samples from these contagious quarters were proceeded further for isolation of Staphylococcus spp. Identification of isolates using 16S rDNA sequencing:-Output sequences of sanger dideoxy sequencing were submitted to GenBank under the accession number KX821628 to KX821638 for Staphylococcus aureus (11 strains) and KX821621 to KX821627 for coagulase negative Staphylococcus spp (07 strains).

Discussion:-
In this study, we have isolated multidrug resistant Staphylococcus spp. from bovine mastitis milk. Staphylococcus aureus remains one of the most significant micro-organisms associated with bovine mastitis worldwide. The isolation of MRSA from animals was first reported in 1972 (Devriese et al. 1972). For identification of bacteria, majority of the researchers tend to use isolation on selective media and molecular identification. In this study, biochemical characterization of each isolate was used to eliminate sequencing cost for molecular identification at later stage.
Since the use of antibiotic has been boundless in recent years, presence of MRSA in dairy farm has been recognized (Haran et al. 2012a). Many studies on prevalence of MRSA investigation have been reported in cattles in India; however, limited information with respect to the characterization of MRSA in cattle milk is available (Sumathi et al. 2008).
For veterinarians, one of the paramount microbial threats of the twenty-first century is developing antimicrobial resistance in pathogens. One of the pathogen, S. aureus has always been a stumbling block for antimicrobial chemotherapy and the introduction of new classes of antimicrobial agents is usually followed by the emergence of resistant forms of this pathogen (Shittu and Lin 2006;Harrison et al. 2013). Therefore, surveillance on the antimicrobial susceptibility patterns of S. aureus mainly methicillin resistant is of utmost importance in understanding new and emerging resistance trends and in the management. The CoNS species in our data, S. hominis and S. pasteuri has been reported most frequently in numerous studies on bovine intramammary infection (IMI) or mastitis. S. chromogenes has been isolated most commonly in almost all studies but we couldn't isolate it in our study (Lange et al. 2015).
The presence of MRSA isolates in milk may surge a potential public health risk, and MRSA may spread between animals and humans due to many reasons like milk practice and improper hygiene maintenance. Spread of such microbes may be depending upon environmental condition in the herd. Virulence factors associated to this pathogen helps to protest against antimicrobial agents. The indiscriminate use of antibiotics/antimicrobials agents for therapeutic purpose could be the reasons for increased antimicrobial resistance of S. aureus. Different strains other than Staphylococcus spp. are also resistant to the antibiotics commonly used to treat all the microbes in therapeutic protocols of many human and animal infections. Further study on virulence factors and antimicrobial resistance genes may highlight the strain's response and strength in diverse condition during treatment with antimicrobials.

Conclusion:-
In this study eleven Coagulase positive methicillin resistant Staphylococcus aureus, six coagulase negative Staphylococcus spp. (two S. hominis, four S. pasteuri) and one Staphylococcus sp. having multidrug resistivity including methicillin were found from mastitis cases. Stimulatingly, obtained coagulase negative Staphylococcus sp. in this study are usually found in human origins, which leads us to understand that the mobile pathway of such pathogen into milk udders may be because of human activities during milking process. Increase in population of MRSA is affected by persistent use of antibiotics for treatment. Further study on virulence factors might help to understand the pathogenicity and treatment. Careful monitoring of the resistance status of S. aureus in dairy environments is needed, as Staphylococcus spp. transmission is dynamic and involves human approach, animal health, and likely the farm production environment.