Molecular Characterization of Antibiotic Resistance Gene Pattern of Staphylococcus aureus and Escherichia coli in Mastitis Affected Dairy Cows

Study area
 
The present study was carried out in the department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, TANUVAS, Chennai-600007. The samples were collected from the Large Animal Medicine Unit, Department of Clinics, Madras Veterinary College Hospital, TANUVAS, Chennai-600 007. The study was conducted between January 2018 and March 2019.
 
Antimicrobial sensitivity testing
 
The milk samples were collected from mastitis affected cows following strict aseptic conditions. California Mastitis Test was used as a screening test for mastitis. The sensitivity of the isolates to various antibiotics was performed using disc diffusion method.  The disc diffusion method was performed using the procedure followed by Pu et al., (2014).
 
Isolation and identification of Staphylococcus aureus
 
Bacteriological examination of the milk sample was carried out within 24 hrs of collection of the milk sample. Nutrient broth was prepared and 10 ml was transferred into sterile test tubes, autoclaved, cooled and stored. Milk swab was dipped in 10 ml of nutrient broth containing test tube, plugged with cotton and incubated at 37°C for 18 hrs. The inoculum was streaked onto aseptically prepared Mannitol Salt Agar plates and incubated at 37°C for 16-20 hrs. Growth of yellow coloured colonies indicated the presence of Staphylococcus organisms. The test was performed following the procedure of Kampf et al., (1998) with certain modifications. The isolates were stored in 50% glycerol solution in eppendroff tubes for further use.
 
Isolation and identification of Escherichia coli
 
Nutrient broth was prepared and 10 ml of it transferred to sterile test tube, autoclaved, cooled and then stored. The milk swab was dipped into 10 ml nutrient broth containing test tube, plugged with cotton and incubated at 37°C for 18 hrs. The inoculum was streaked onto Eosin Methylene Blue agar plates. Distinctive metallic green sheen on EMB indicates the presence of E.coli. The test was performed following the procedure of Horvath and Ropp (1974).
 
Biochemical tests
 
The isolated organisms were identified according to routine microbiological diagnostics, including cultural properties, catalase (Igbinosa et al., 2016) and coagulase (Boerlin, 2003), utilization of mannitol and triple sugar iron agar and gram staining.
 
Antibiotic susceptibility
 
It was determined by the standardized agar diffusion test on Muller-Hinton agar (HiMedia) using the following disks (HiMedia): ampicillin (10mcg), amoxicillin + clavulanic acid (20/10mcg), azithromycin (15mcg), ciprofloxacin (5mcg), cefuroxime (30mcg), ceftriaxone (30mcg), gentamicin (10mcg), penicillin-G (10U), oxytetracycline (30mcg), tetracycline (30mcg), vancomycin (30mcg). Isolates were classified as susceptible and resistant based upon interpretative criteria developed by the Clinical and Laboratory Standards Institute (CLSI) (2005).
 
Minimum inhibitory concentration (MIC)
 
Microdilution method modified with resazurin dye as a redox indicator was utilized to determine the MIC value of resistant drugs. Inoculum was prepared by suspending overnight agar plate cultures of the isolates in sterile normal saline equivalent of 0.5 McFarland turbidity standards. The resazurin solution was prepared by dissolving 0.002g of resazurin powder (HiMedia) in 100mL of distilled water. The method was followed as reported by Sarkar et al., (2007) with slight modifications.
 
Plates were prepared under aseptic conditions. A sterile 96 well plate was labeled. A volume of 25µL of the broth medium was pipetted into columns 2 to 9, 10 and 12 of every row. To all other wells in the same row and to the second column, a volume of 25µL of the highest concentration of the drug was added. Serial dilutions were done from the second well to the ninth well by pipetting out 25µL from each well. The last 25µL from the ninth well was discarded. 100µL of the inoculum diluted to 0.5 McFarland standard was added to all the wells other than columns 10 and 11 which acted as the negative control for the media and the drug dilutions; 100µL of un-inoculated broth was added to the tenth and the eleventh wells instead. After incubating the well plate for 24 hours at 35±2°C, 5µL of 0.002% resazurin was added to all wells and was further incubated at 37°C for 3 hours.
 
DNA extraction for PCR analysis
 
DNA extraction was done by methods suggested by Fang and Goran (2003). A bacterial suspension prepared from a loopful of colonies from an overnight agar culture was prepared in 100µL of Nuclease free water. The suspension was boiled at 100 for 5 minutes and was chilled immediately at -20°C for 10 minutes. The clear supernatant obtained by centrifuging the chilled suspension at 10,000 rpm for 2 minutes served as the template for PCR reactions.
 
PCR analysis
 
Polymerase chain reaction analysis of the mecA, tetK, tetM, blaSHV and tetB genes were carried out using the primers and the respective amplifications program. Reaction was performed in a final volume of 25µL of mixture containing 12.5µL of Master Mix (AmpliconTaqPol), 1µL each of the forward and reverse primer, 3µL of the template DNA and 7.5µL of nuclease free water. The products were analyzed by electrophoresis through 1.2% agarose gel. The PCR was carried out using appropriate protocol (Garibyan and Avashia, 2014). The primer sequences for identification of Staphylococcus aureus and Escherichia coli and their resistant genes are depicted in Table 1, 3, 4. The cyclic conditions of Polymerase Chain Reaction for uspA and nuc gene is depicted in Table 2 and for amplification of resistance gene in Table 5.
 

 

 

 

 

Isolation, identification and molecular characterization
 
The phenotypic and biochemical analysis shows 100% and 94% prevalence respectively for S. aureus and the genotypic prevalence of S. aureus was found to be 12.9%. The prevalence of E. coli was found to be 12.9% based on the phenotypic test and 100% on biochemical test. The genotypic analysis reports 50% prevalence for E. coli. The results were found to be similar to earlier studies conducted by Chandrasekaran et al., (2014). The results accumulated from the tests are depicted in Table 6.
 

 
Antibiotic sensitivity testing
 
The antimicrobial susceptibility pattern of S. aureus and E. coli isolates from mastitis specimens against antimicrobial agents of different classes were found to be highly variable. Antibiotic Sensitivity Pattern showed that the isolates were resistant to Ampicillin, Amoxicillin and Penicillin-G, Tetracycline and Oxytetracycline, Vancomycin, intermediately sensitive to Azithromycin and sensitive to Gentamicin, Enrofloxacin and Ciprofloxacin. There is abundant evidence to corroborate the emergence of resistance of penicillin and tetracycline for both Staphylococcus aureus and Escherichia coli (Foster, 2017; Ragbelti et al., 2016; Arabzadeh et al., 2018). The emergence of resistance can be ascribed to indiscriminate antibiotic usage and non-compliance to the recommended dose regimens. The antibiotic sensitivity pattern is depicted in Fig 1 and 2.
 

 

 
Minimum inhibitory concentration
 
The MIC values of Penicillin against S. aureus and E. coli were found to be 26.90µg/ml and 13.54µg/ml respectively and the MIC values of Tetracycline against S. aureus and E. coli were 243.75µg/ml and 960.93µg/ml respectively. The data generated has been depicted in Table 7.
 
Polymerase chain reaction
 
The genotypic analysis of the antibiotic resistance in isolated microorganisms has brought forward the prevalence pattern of resistance genes in them. The prevalence resistance genes in S. aureus was 51.67% tet (K), 67.7%, tet (M) for tetracycline and 87.09% mec (A) for penicillin. In E. coli isolates the prevalence of resistance genes were recoded to be 50% for bla_SHV and 75% for tet (B). Tetracyclines exert their antimicrobial effect by binding to the 30S ribosome and thereby preventing protein sysnthesis. Bacterial resistance to tetracycline is most commonly mediated by energy-dependent pumping of tetracycline out of the bacterial cell (Karami et al., 2006). The most common resistance mechanism of Escherichia coli to tetracycline is through energy dependent efflux pump which is encoded by tetA, tetB, tetC, tetD and tetG, with tetA and tetBgenes (Olowe et al., 2013). The resistance of Escherichia coli to tetracycline may be attributed to the presence of tetB gene which was detected in the current study with the help of PCR with a prevalence rate of 75%. Staphylococcus aureus acquires resistance to tetracycline by active efflux, resulting from the acquisition of the plasmid-located tetK and tetM genes (Emaneini et al., 2013).. In the current study, presence of tetK and tetM gene were detected by using PCR with a prevalence rate of 51.61% and 67.7% respectively. The resistance of Staphylococcus aureus to tetracycline can be ascribed to the efflux pump encoded by tetK and tetM genes. In Staphylococcus aureus, biosynthesis of the peptidoglycan is accomplished by the membrane-bound enzymes known as penicillin-binding proteins (PBPs) (Sauvage et al., 2008). Staphylococcal resistance to penicillin is usually conferred by the acquisition of a non-native gene encoding a penicillin-binding protein (PBP2a), with significantly lower affinity for β-lactams. (Peacock et al., 2015)  High-level resistance to methicillin is caused by the mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a (Weilders et al., 2002). Detection of mecA gene in the Staphylococcus aureus isolates leads us to presume that some of the resistance of Staphylococcus aureus to Penicillin G may be owing to the alternative penicillin binding protein, PBP 2a synthesis. Each bla gene of extended-spectrum β-lactamase-producing E. coli was further subtyped to be bla_CTX-M-15, bla_CTX-M-104, bla_TEM-1 and bla_SHV-12 which are responsible for conferring resistance to Beta lactam antibiotics (Ombarak et al., 2018).  In the present study, bla_SHV gene was detected in Escherichia coli with a prevalence rate of 50% using PCR technique. The resistance of Escherichia coli to penicillin may be attributed to the presence of blaSHV gene which confers resistance by production of beta lactamase enzyme.The results of the PCR are depicted in Table 8 and 9, Fig 3, 4, 5 and 6.
 

 

 

 

From the current study, it can be concluded that mastitis in dairy cows are highly resistant to various antimicrobial drugs.  Present study further indicates Staphylococcus aureus and Escherichia coli are both resistant to Penicillin and Tetracycline with very high MIC. There is a need to carry out further studies in this direction to better understand the resistance pattern of these antibiotics and also arrive at a dosage regimen.

Authors are thankful to the Professor and Head, Department of Clinics, Madras Veterinary College Teaching Hospital, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tami Nadu, India for providing the facilities required to conduct this study.