Baljinder Kumar Bansal Dhiraj Kumar Gupta Shukriti Sharma Tawheed Ahmad Shafi Gursimran Filia Vol 9(9), 65-79 DOI- http://dx.doi.org/10.5455/ijlr.20180516043453
Emergence of antibiotic resistance in Staphylococcus aureus and in particular community associated methicillin-resistant S. aureus (MRSA) is one of the most serious problem. In the present study, 97 isolates of S. aureus from cases of clinical (n=49) and subclinical (n=48) mastitis were evaluated for phenotypic antibiotic resistance patterns. A total of 28 antibiotics belonging to 10 groups of antibiotics were tested. Isolates from clinical cases of mastitis had higher resistance than those from subclinical mastitis and resistance was observed in many newly developed antibiotics as well. Multidrug resistance (MDR) was observed in 83 of the studied isolates, and of these 18 exhibited extreme drug resistance (XDR) and one isolate demonstrated resistance to all the antibiotics (pan drug resistance, PDR). Though phenotypic methicillin resistance was observed in 25 isolates, mecA was present in only 3 isolates. The antibiotic resistance is mainly attributed to acquisition of resistance genes by genetic exchange. However, the present study revealed that there may be some other mechanisms associated with methicillin resistance in S. aureus.
Keywords : Mastitis mecA Methicillin Resistance Staphylococcus aureus
Mastitis, the inflammation of mammary gland, is one of the costliest diseases of dairy cattle resulting in the reduction of milk yield and quality. Annual economic losses due to subclinical and clinical mastitis in India have been estimated to be Rs. 4151.16 and Rs. 3014.35 crores, respectively with a total of Rs. 7165.51 crores (Bansal and Gupta, 2009). Mastitis is the single most common cause of antibiotic use in dairy farms. Mastitis therapy has a potential to develop antibiotic resistance, as antibiotics employed to treat mastitis often have a short duration of therapy. β-lactams, commonly used in therapy, have little or no activity against gram negative bacteria; infusion of dry cow therapy increases exposure time of intra-mammary bacteria; and efficacy of many therapeutics in lactating cows is quite low especially in chronic infections. Moreover, the use of antibiotics for treatment and control of mastitis often results in antibiotic residues in milk (Bansal et al., 2011) above the maximum residue limits (MRLs) resulting in build-up of antibiotic-resistant organisms in human food chain (Jones and Seymour 1988; Seymour et al., 1988).
Staphylococcus aureus is a major pathogen responsible for bovine mastitis (Lowy, 2003). S. aureus is a cause of skin infections and serious hospital infections including bacteremia and pneumonia in human beings. The emergence of antibacterial resistance in this pathogen is of growing concern as S. aureus associated bacteremia results in 20-40% mortality despite treatment (Mylotte et al., 1987). In addition, community acquired-methicillin resistant S. aureus strains (CA-MRSA) that have evolved independently of hospitals are becoming widespread. Cattle, pigs and poultry are colonized with MRSA and the zoonotic transmission of such MRSA to humans via direct animal contact or environmental contaminations are a matter of concern (Kock et al., 2013) indicating the need for surveillance and biosecurity measures in the animal health sector.
Phentoypic antibiotic susceptibility patterns have been studied previously in detail from clinician point of view in India (Mir et al., 2014; Bansal et al., 2015) but the development of antibiotic resistance in S. aureus has not been focused from community health aspects. Therefore, the present study has been undertaken to determine in vitro antibiotic resistance pattern of S. aureus in clinical and subclinical cases of mastitis and correlate the presence of mecA with the phenotypic methicillin resistance.
Materials and Methods
Confirmation of S. aureus Isolates
The study was carried out on 97 isolates of S. aureus isolated from cases of clinical (n=49) and subclinical (n=48) mastitis in dairy cows and buffaloes. All the isolates (n=20) from buffaloes belonged to subclinical mastitis. These strains were isolated from bovine milk samples in mastitis and milk quality laboratory of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana from July 2012 to June 2013. S. aureus organisms were identified on the basis of colony characteristics on blood agar, gram staining, clumping factor, growth characteristics on manitol salt agar, DNase agar, Baird parker agar and tube coagulase test (Quinn et al., 2000). Individual isolates were stored in trypticase soy broth containing 30% glycerol in deep freeze (−80°C).
Antibiotic Sensitivity Testing
Susceptibility testing of bacterial isolates to antibiotics was performed on Mueller-Hinton agar (HiMedia) using the disc diffusion method (Quinn et al., 2000). Briefly, a fresh colony of an individual isolate was transferred to a tube containing 5 ml nutrient broth. The mixture was incubated at 37ºC until light visible turbidity appeared; this was compared with the McFarland 0.5 turbidity standard. The suspension of test organism was streaked over the surface of Muller Hinton agar plates using a sterile disposable cotton swab. Commercially available discs (Hi-Media) of 28 antibiotics belonging to 10 groups were evaluated in the study. The discs were firmly placed on agar by means of sterile forceps and plates were incubated for 24 h at 37°C. The diameters of growth-inhibition were measured in millimeters and reported as susceptible, intermediate or resistant as per Clinical and Laboratory Standards Institute (CLSI 2008) guidelines. The antibiotic discs along with their concentration and interpretation criteria have been given in the Table 1. Antibiotics, for which interpretive criteria was not available as per CSLI guidelines, breakpoints of antibiotic in similar group were used. Thus, the breakpoint of ampicillin has been used for amoxicillin and that of oxacillin was used for cloxacillin. For streptomycin and neomycin, the breakpoints suggested by Petrovski (2011) were used.
Table 1: Antibiotic sensitivity test of S. aureus against different antibiotics
Group | Antibiotic (concentration in mcg) | Interpretation | Break Point (mm) | Cow | Buffalo | Total | |||||
CM | SCM | ||||||||||
N= 46 | % | N= 31 | % | N= 20 | % | N | % | ||||
β-Lactams | Oxacillin | S | ≥13 | 28 | 60.9 | 23 | 74.2 | 19 | 95 | 70 | 72.2 |
(OX:1) | I | 11-12 | 2 | 4.3 | 0 | 0 | 0 | 0 | 2 | 2.1 | |
R | ≤10 | 16 | 34.8 | 8 | 25.8 | 1 | 5 | 25 | 25.8 | ||
Penicillin-G | S | ≥29 | 5 | 10.9 | 14 | 45.2 | 11 | 55 | 30 | 30.9 | |
(P:2 U) | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
R | ≤28 | 41 | 89.1 | 17 | 54.8 | 9 | 45 | 67 | 69.1 | ||
Amoxycillin + | S | ≥20 | 27 | 58.7 | 23 | 74.2 | 17 | 85 | 67 | 69.1 | |
Clavulanic acid | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
(AC:30) | R | ≤19 | 19 | 41.3 | 8 | 25.8 | 3 | 15 | 30 | 30.9 | |
Ampicillin | S | ≥29 | 8 | 17.4 | 17 | 54.8 | 14 | 70 | 39 | 40.2 | |
(A:10) | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
R | ≤28 | 38 | 82.6 | 14 | 45.2 | 6 | 30 | 58 | 59.8 | ||
Amoxycillin + | S | ≥15 | 24 | 52.2 | 23 | 74.2 | 17 | 85 | 64 | 66 | |
Sulbactum | I | 12-14 | 1 | 2.2 | 1 | 3.2 | 1 | 5 | 3 | 3.1 | |
(AS:30/15) | R | ≤11 | 21 | 45.7 | 7 | 22.6 | 2 | 10 | 30 | 30.9 | |
Amoxycillin | S | ≥29 | 5 | 10.9 | 14 | 45.2 | 11 | 55 | 30 | 30.9 | |
(AMX:10) | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
R | ≤28 | 41 | 89.1 | 17 | 54.8 | 9 | 45 | 67 | 69.1 | ||
Cloxacillin | S | ≥13 | 37 | 80.4 | 29 | 93.5 | 19 | 95 | 85 | 87.6 | |
(COX:10) | I | 11-12 | 2 | 4.3 | 1 | 3.2 | 0 | 0 | 3 | 3.1 | |
R | ≤10 | 7 | 15.2 | 1 | 3.2 | 1 | 5 | 9 | 9.3 | ||
Ceftrioxone + | S | ≥21 | 17 | 37 | 22 | 71 | 16 | 80 | 55 | 56.7 | |
Sulbactum | I | 14-20 | 23 | 50 | 4 | 12.9 | 2 | 10 | 29 | 29.9 | |
(CIS:30/15) | R | ≤13 | 6 | 13 | 5 | 16.1 | 2 | 10 | 13 | 13.4 | |
Ceftrioxone + | S | ≥21 | 29 | 63 | 21 | 67.7 | 16 | 80 | 66 | 68 | |
Tazobactum | I | 14-20 | 13 | 28.3 | 5 | 16.1 | 3 | 15 | 21 | 21.7 | |
(CIT:30/10) | R | ≤13 | 4 | 8.7 | 5 | 16.1 | 1 | 5 | 10 | 10.3 | |
Ceftrioxone | S | ≥21 | 17 | 37 | 22 | 71 | 15 | 75 | 54 | 55.7 | |
(CI:10) | I | 14-20 | 21 | 45.7 | 4 | 12.9 | 3 | 15 | 28 | 28.9 | |
R | ≤13 | 8 | 17.4 | 5 | 16.1 | 2 | 10 | 15 | 15.5 | ||
Ceftazidime | S | ≥18 | 11 | 23.9 | 19 | 61.3 | 11 | 55 | 41 | 42.3 | |
(CA:30) | I | 15-17 | 11 | 23.9 | 5 | 16.1 | 5 | 25 | 21 | 21.7 | |
R | ≤14 | 24 | 52.2 | 7 | 22.6 | 4 | 20 | 35 | 36.1 | ||
Cefoperazone | S | ≥24 | 7 | 15.2 | 16 | 51.6 | 13 | 65 | 36 | 37.1 | |
(CS:75) | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
R | ≤23 | 39 | 84.8 | 15 | 48.4 | 7 | 35 | 61 | 62.9 | ||
Ceftixozime | S | ≥20 | 26 | 56.5 | 22 | 71 | 17 | 85 | 65 | 67 | |
(CK:30) | I | 15-19 | 3 | 6.5 | 2 | 6.5 | 2 | 10 | 7 | 7.2 | |
R | ≤14 | 17 | 37 | 7 | 22.6 | 1 | 5 | 25 | 25.8 | ||
Cefuroxime | S | ≥18 | 28 | 60.9 | 24 | 77.4 | 17 | 85 | 69 | 71.1 | |
(CU:30) | I | 15-17 | 2 | 4.3 | 1 | 3.2 | 1 | 5 | 4 | 4.1 | |
R | ≤14 | 16 | 34.8 | 6 | 19.4 | 2 | 10 | 24 | 24.7 | ||
Aminogylcosides | Gentamicin | S | ≥15 | 34 | 73.9 | 21 | 67.7 | 9 | 45 | 64 | 66 |
(G:10) | I | 12-14 | 7 | 15.2 | 9 | 29 | 10 | 50 | 26 | 26.8 | |
R | ≤11 | 5 | 10.9 | 1 | 3.2 | 1 | 5 | 7 | 7.2 | ||
Neomycin | S | ≥15 | 27 | 58.7 | 19 | 61.3 | 11 | 55 | 57 | 58.8 | |
(N:30) | I | 13-14 | 9 | 19.6 | 7 | 22.6 | 5 | 25 | 21 | 21.7 | |
R | ≤12 | 10 | 21.7 | 5 | 16.1 | 4 | 20 | 19 | 19.6 | ||
Streptomycin | S | ≥14 | 37 | 80.4 | 27 | 87.1 | 18 | 90 | 82 | 84.5 | |
(S:25) | I | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
R | ≤13 | 9 | 19.6 | 4 | 12.9 | 2 | 10 | 15 | 15.5 | ||
Amikacin | S | ≥17 | 26 | 56.5 | 24 | 77.4 | 12 | 60 | 62 | 63.9 | |
(AK:30) | I | 15-16 | 11 | 23.9 | 2 | 6.5 | 6 | 30 | 19 | 19.6 | |
R | ≤14 | 9 | 19.6 | 5 | 16.1 | 2 | 10 | 16 | 16.5 | ||
Floroquinolones | Enrofloxacin | S | ≥22 | 23 | 50 | 27 | 87.1 | 17 | 85 | 67 | 69.1 |
(EX:10) | I | 18-21 | 11 | 23.9 | 3 | 9.7 | 2 | 10 | 16 | 16.5 | |
R | ≤17 | 12 | 26.1 | 1 | 3.2 | 1 | 5 | 14 | 14.4 | ||
Ciprofloxacin | S | ≥21 | 13 | 28.3 | 22 | 71 | 15 | 75 | 50 | 51.6 | |
(CF:5) | I | 16-20 | 17 | 37 | 6 | 19.4 | 4 | 20 | 27 | 27.8 | |
R | ≤15 | 16 | 34.8 | 3 | 9.7 | 1 | 5 | 20 | 20.6 | ||
Moxifloxacin | S | ≥24 | 5 | 10.9 | 19 | 61.3 | 12 | 60 | 36 | 37.1 | |
(MO:5) | I | 21-23 | 7 | 15.2 | 5 | 16.1 | 3 | 15 | 15 | 15.5 | |
R | ≤20 | 34 | 73.9 | 7 | 22.6 | 5 | 25 | 46 | 47.4 | ||
Sulphonamide | Co-Trimoxazole | S | ≥16 | 20 | 43.5 | 23 | 74.2 | 18 | 90 | 61 | 62.9 |
(CO:25) | I | 11-15 | 7 | 15.2 | 4 | 12.9 | 0 | 0 | 11 | 11.3 | |
R | ≤10 | 19 | 41.3 | 4 | 12.9 | 2 | 10 | 25 | 25.8 | ||
Macrolide | Erythromycin | S | ≥23 | 6 | 13 | 9 | 29 | 7 | 35 | 22 | 22.7 |
(E:10) | I | 14-22 | 38 | 82.6 | 21 | 67.7 | 13 | 65 | 72 | 74.2 | |
R | ≤13 | 2 | 4.3 | 1 | 3.2 | 0 | 0 | 3 | 3.1 | ||
Tetracycline | Tetracycline | S | ≥19 | 29 | 63 | 26 | 83.9 | 17 | 85 | 72 | 74.2 |
(T:10) | I | 15-18 | 4 | 8.7 | 1 | 3.2 | 1 | 5 | 6 | 6.2 | |
R | ≤14 | 13 | 28.3 | 4 | 12.9 | 2 | 10 | 19 | 19.6 | ||
Lincosamide | Clindamycin | S | ≥21 | 15 | 32.6 | 14 | 45.2 | 8 | 40 | 37 | 38.1 |
(CD:2) | I | 15-20 | 25 | 54.3 | 13 | 41.9 | 11 | 55 | 49 | 50.5 | |
R | ≤14 | 6 | 13 | 4 | 12.9 | 1 | 5 | 11 | 11.3 | ||
Oxazolidinone | Linezolid | S | ≥21 | 31 | 67.4 | 25 | 80.6 | 11 | 55 | 67 | 69.1 |
(LZ:30) | I | NA | 0 | 0 | NA | NA | NA | NA | NA | NA | |
R | ≤20 | 15 | 32.6 | 6 | 19.4 | 9 | 45 | 30 | 30.9 | ||
Chloramphenicol | Chloramphenicol | S | ≥18 | 42 | 91.3 | 28 | 90.3 | 18 | 90 | 88 | 90.7 |
(C:30) | I | 13-17 | 4 | 8.7 | 3 | 9.7 | 2 | 10 | 9 | 9.3 | |
R | ≤12 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Glycopeptide | Teicoplanin | S | ≥14 | 17 | 37 | 21 | 67.7 | 10 | 50 | 48 | 49.5 |
(TE:30) | I | Nov-13 | 22 | 47.8 | 8 | 25.8 | 9 | 45 | 39 | 40.2 | |
R | ≤10 | 7 | 15.2 | 2 | 6.5 | 1 | 5 | 10 | 10.3 |
S: Sensitive, I: Intermediate, R: Resistant, NA: Not applicable, N: Number of isolates, CM: Clinical mastitis, SCM: Subclinical mastitis, Break Point: Zone of inhibition in mm
Criteria for Phenotypic Antibiotic Resistance
Phenotypic antibiotic resistance pattern was interpreted as per guidelines of Magiorakos et al. (2012). Isolates resistant to three or more antibiotics belonging to different groups were classified as multidrug resistant (MDR). Among MDR isolates, isolates susceptible to only two antibiotics belonging to two different groups were considered extreme drug resistant (XDR), while resistance to all the antibiotics was considered as pan-drug resistant (PDR).
DNA Extraction
After overnight inoculation of an individual bacterial colony in brain heart infusion (BHI, HiMedia) broth, 1 ml culture was pelleted at 7500 rpm for 5 min in refrigerated centrifuge (Thermo Scientific). The pellet was suspended in 180 µI lysozyme enzyme solution and incubated at 37ºC for 30 min. Bacterial DNA was extracted using QIAamp DNA mini kit (Qiagen) as per manufacturer guidelines. Eluted genomic DNA was stored at −20°C until use.
Amplification of mecA and Agarose Gel Electrophoresis
The PCR assays used 100 pg of DNA template in a 25 µl reaction mixture with 13 µl of Taq DNA Master Mix (Qiagen) and 250 nM of each oligonucleotide primer (Genaxy) for amplification of mecA (Murakami et al., 1991). PCR products were analyzed using conventional agarose gel electrophoresis in 1% w/v agarose (Genaxy). The amplified products were run in agarose gel in 1x TBE buffer (Genaxy) containing ethidium bromide at 0.1 mg/ml. Quantitative DNA Markers (Genaxy) were used as molecular size markers. The DNA bands were visualized and imaged using the Molecular Imager® ChemiDocTM XRS+ imaging system (Bio-Rad).
Results
Clinical Interpretation of Antibiotic Sensitivity Testing
A total of 97 S. aureus isolates consisting of 77 isolates from cows (subclinical=31, clinical=46) and 20 isolates from buffaloes (subclinical=17, clinical=3) were evaluated for antibiotic sensitivity testing. Susceptibility of individual S. aureus isolates to antibiotics is presented in Table 1. It is clear from the table that isolates from clinical cases of cows had higher resistance than isolates from subclinical mastitis and resistance was also observed against linezolid (30.9%), an antibiotic that has been developed recently. To simplify the results of antibiotic sensitivity test from clinician point of view (shown in Table 1), susceptibility profile of S. aureus isolates to different antibiotics has been presented in Table 2. Clinical isolates demonstrated resistance to most of the antibiotics used (ampicillin, amoxycillin, ceftazidime, clindamycin, ciprofloxacin, ceftrioxone-sulbactum, co-trimoxazole, cefoperazone, ceftrioxone, erythromycin, moxifloxacin, penicillin, teicoplanin), while in comparison, subclinical isolates demonstrated resistance to a fewer antibiotics (amoxycillin, clindamycin, erythromycin, penicillin, gentamicin). Overall, chloramphenicol, co-trimoxazole, cloxacillin, oxacillin and streptomycin showed substantial in vitro susceptibility to S. aureus.
Table 2: Susceptibility profile of S. aureus isolates to different antibiotics
Species | Mastitis Status | Susceptibility of S. aureus isolates | |||
High (≥90%) | Moderate (75-89.9%) | Low (50-74.9%) | Resistant (<50%) | ||
Cow | Clinical (n=46) | C | COX, S | AK, AC, AS, CIT, CU, CK, EX, G, LZ, N, OX, T | A, AMX, CA, CD, CF, CIS, CO, CS, CI, E, MO, P, TE |
Subclinical (n=31) | C, COX | AK, CU, EX, LZ, S, T | A, AC, AS, CA, CF, CIS, CIT, CO, CS, CI, CK, G, N, MO, OX, TE | AMX, CD, E, P | |
Buffalo (n=20)
|
17 subclinical + 3 clinical | C, CO, COX, OX, S | AC, AS, CF, CIS, CIT, CI, CU, CK, EX, T | AK, A, AMX, CA, CS, LZ, MO, N, P, TE | CD, G, E |
OX: oxacillin P: penicillin AC: amoxycillin + clavulanic acid A: Ampicillin AS: amoxycillin + sulbactum AMX: amoxycillin COX: cloxacilliin CIS: ceftrioxone + sulbactum CIT: ceftrioxone + tazobactum CI: ceftrioxone CA: ceftazidime CS: cefoperazone CK: ceftixozime CU: cefuroxime G: gentamicin N: neomycin S: streptomycin AK: amikacin EX: enrofloxacin CF: ciprofloxacin MO: moxifloxacin CO: co-trimoxazole E: erythromycin T: tetracycline CD: clindamycin LZ: linezolid C: chloramphenicol TE: teicoplanin
Phenotypic Antibiotic Resistance Patterns
Eighty three out of 97 isolates were found to be MDR. Of these, 18 isolates were XDR, and one isolate was resistant to all the antibiotics tested (PDR). The antibiotic resistance status of an individual isolate against all the antibiotics within 10 groups has been presented in Fig. 1 and their relationship in terms of MDR, XDR and PDR is shown in the diagram (Fig. 2). Interestingly, only 14 isolates (14.4%) were identified as non-MDR indicating high level of resistance in S. aureus strains isolated from cases of bovine mastitis.
Isolate | Antibiotics (1-28) within Antibiotic groups (A-J) | Resistance Status | |||||||||||||||||||||||||||
No* | A | B | C | D | E | F | G | H | I | J | |||||||||||||||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | ||
1. | Non-MDR | ||||||||||||||||||||||||||||
2. | MDR | ||||||||||||||||||||||||||||
3. | MDR | ||||||||||||||||||||||||||||
4. | MDR | ||||||||||||||||||||||||||||
5. | XDR | ||||||||||||||||||||||||||||
6. | Non-MDR | ||||||||||||||||||||||||||||
7. | MDR | ||||||||||||||||||||||||||||
8. | MDR | ||||||||||||||||||||||||||||
9. | Non-MDR | ||||||||||||||||||||||||||||
10. | Non-MDR | ||||||||||||||||||||||||||||
11. | Non-MDR | ||||||||||||||||||||||||||||
12. | Non-MDR | ||||||||||||||||||||||||||||
13. | MDR | ||||||||||||||||||||||||||||
14. | MDR | ||||||||||||||||||||||||||||
15. | Non-MDR | ||||||||||||||||||||||||||||
16. | MDR | ||||||||||||||||||||||||||||
17. | MDR | ||||||||||||||||||||||||||||
18. | MDR | ||||||||||||||||||||||||||||
19. | MDR | ||||||||||||||||||||||||||||
20. | XDR | ||||||||||||||||||||||||||||
21. | MDR | ||||||||||||||||||||||||||||
22. | MDR | ||||||||||||||||||||||||||||
23. | MDR | ||||||||||||||||||||||||||||
24. | MDR | ||||||||||||||||||||||||||||
25. | MDR | ||||||||||||||||||||||||||||
26. | MDR | ||||||||||||||||||||||||||||
27. | MDR | ||||||||||||||||||||||||||||
28. | MDR | ||||||||||||||||||||||||||||
29. | MDR | ||||||||||||||||||||||||||||
30. | MDR | ||||||||||||||||||||||||||||
31. | MDR | ||||||||||||||||||||||||||||
32. | Non-MDR | ||||||||||||||||||||||||||||
33. | Non-MDR | ||||||||||||||||||||||||||||
34. | Non-MDR | ||||||||||||||||||||||||||||
35. | MDR | ||||||||||||||||||||||||||||
36. | Non-MDR | ||||||||||||||||||||||||||||
37. | MDR | ||||||||||||||||||||||||||||
38. | MDR | ||||||||||||||||||||||||||||
39. | XDR | ||||||||||||||||||||||||||||
40. | XDR | ||||||||||||||||||||||||||||
41. | MDR | ||||||||||||||||||||||||||||
42. | MDR | ||||||||||||||||||||||||||||
43. | XDR | ||||||||||||||||||||||||||||
44. | XDR | ||||||||||||||||||||||||||||
45. | MDR | ||||||||||||||||||||||||||||
46. | Non-MDR | ||||||||||||||||||||||||||||
47. | MDR | ||||||||||||||||||||||||||||
48. | MDR | ||||||||||||||||||||||||||||
49. | Non-MDR | ||||||||||||||||||||||||||||
50. | MDR | ||||||||||||||||||||||||||||
51. | MDR | ||||||||||||||||||||||||||||
52. | MDR | ||||||||||||||||||||||||||||
53. | MDR | ||||||||||||||||||||||||||||
54. | MDR | ||||||||||||||||||||||||||||
55. | XDR | ||||||||||||||||||||||||||||
56. | XDR | ||||||||||||||||||||||||||||
57. | PDR | ||||||||||||||||||||||||||||
58. | MDR | ||||||||||||||||||||||||||||
59. | MDR | ||||||||||||||||||||||||||||
60. | MDR | ||||||||||||||||||||||||||||
61. | MDR | ||||||||||||||||||||||||||||
62. | MDR | ||||||||||||||||||||||||||||
63. | XDR | ||||||||||||||||||||||||||||
64. | MDR | ||||||||||||||||||||||||||||
65. | MDR | ||||||||||||||||||||||||||||
66. | XDR | ||||||||||||||||||||||||||||
67. | MDR | ||||||||||||||||||||||||||||
68. | MDR | ||||||||||||||||||||||||||||
69. | MDR | ||||||||||||||||||||||||||||
70. | MDR | ||||||||||||||||||||||||||||
71. | Non-MDR | ||||||||||||||||||||||||||||
72. | MDR | ||||||||||||||||||||||||||||
73. | MDR | ||||||||||||||||||||||||||||
74. | XDR | ||||||||||||||||||||||||||||
75. | MDR | ||||||||||||||||||||||||||||
76. | MDR | ||||||||||||||||||||||||||||
77. | MDR | ||||||||||||||||||||||||||||
78. | MDR | ||||||||||||||||||||||||||||
79. | XDR | ||||||||||||||||||||||||||||
80. | MDR | ||||||||||||||||||||||||||||
81. | XDR | ||||||||||||||||||||||||||||
82. | MDR | ||||||||||||||||||||||||||||
83. | MDR | ||||||||||||||||||||||||||||
84. | MDR | ||||||||||||||||||||||||||||
85. | MDR | ||||||||||||||||||||||||||||
86. | XDR | ||||||||||||||||||||||||||||
87. | MDR | ||||||||||||||||||||||||||||
88. | MDR | ||||||||||||||||||||||||||||
89. | MDR | ||||||||||||||||||||||||||||
90. | MDR | ||||||||||||||||||||||||||||
91. | MDR | ||||||||||||||||||||||||||||
92. | XDR | ||||||||||||||||||||||||||||
93. | MDR | ||||||||||||||||||||||||||||
94. | XDR | ||||||||||||||||||||||||||||
95. | XDR | ||||||||||||||||||||||||||||
96. | MDR | ||||||||||||||||||||||||||||
97. | XDR |
A: β-Lactams B: Aminoglycosides C: Floroquinolones D: Sulphonamide E: Macrolide F: Tetracycline G: Lincosamide H: Oxazolidinone I: Chloramphenicol J: Glycopeptide; 1: oxacillin 2: penicillin 3: amoxicillin + clavulanate 4: ampicillin 5: amoxicillin + sulbactum 6: amoxycillin 7: cloxacilliin 8: ceftrioxone + sulbactum 9: ceftrioxone + tazobactum 10: ceftrioxone 11: ceftazidine 12: cefaperazone 13: ceftixozime 14: ceforoxime 15: gentamicin 16: neomycin 17: streptomycin 18: amikacin 19: enrofloxacin 20: ciprofloxacin 21: moxifloxacin 22: cotrimazole 23: erythromycin 24: tetracycline 25: clindamycin 26: linezolid 27: chloramphenicol 28: teicoplanin; *1-48 isolates are from cases of subclinical mastitis, and 49-97 are from clinical mastitis
Fig. 1: Antibiotic resistance status of individual S. aureus isolates
Phenotypic Antibiotic Resistance Patterns
Eighty three out of 97 isolates were found to be MDR. Of these, 18 isolates were XDR, and one isolate was resistant to all the antibiotics tested (PDR). The antibiotic resistance status of an individual isolate against all the antibiotics within 10 groups has been presented in Fig. 1 and their relationship in terms of MDR, XDR and PDR is shown in the diagram (Fig. 2). Interestingly, only 14 isolates (14.4%) were identified as non-MDR indicating high level of resistance in S. aureus strains isolated from cases of bovine mastitis.
Non-MDR
(n=14) |
MDR
(n=83) |
XDR
(n=18) |
PDR
(n=1) |
Fig. 2: Diagram showing the extent of phenotypic antibiotic resistance in S. aureus
Phenotypic Methicillin Resistance and Presence of mecA
A high level of phenotypic methicillin resistance (25.8%) was observed in S. aureus (Table 1). To determine, whether this resistance is actually associated with the mecA presence, the genomic DNA was extracted and PCR amplification targeting mecA was performed on all S. aureus isolates. Surprisingly, mecA was present (Fig. 3) in only 3 isolates of cows with clinical mastitis.
533bp |
bp M N 1 2 3 |
50 |
200 |
100 |
150 |
250 |
1000 |
500 |
600 |
300 |
400 |
Fig. 3: Agarose gel electrophoresis of amplified DNA targeting mecA in S. aureus. Lanes 1-3: positive isolates revealing 533 bp product, M: Fermentas GeneRuler 50 bp DNA ladder, N: mecA negative isolate.
Discussion
MRSA strains were first reported from cases of bovine mastitis in 1972 (Devriese et al., 1972). Since then, MRSA strains have become a cause of major public health concerns due to their possible transmission between livestock species and human beings. Studies conducted on human S. aureus isolates in India have reported a high prevalence of MRSA ranging between 12 and 40% (Verma et al., 2000; Kali et al., 2013). In present study as well, 25 out of 97 (25.8%) S. aureus isolates from bovine milk exhibited phenotypic methicillin resistance. MRSA have been found to be resistant to most of the commonly used antibiotics including the aminoglycosides, chloramphenicol, flouroquinolones, lincosamides, macrolides, sulphonamides, tetracycline and trimethoprim-sulfamethaxazole (Mandell et al., 1995; Feng et al., 2008). Only a few antibiotics including linezolid and vancomycin are effective against MRSA and recent studies have indicated the emergence of resistance against these antibiotics as well (Thati et al., 2011; Gu et al., 2013).
To determine the reasons behind high prevalence of MRSA in Indian strains of S. aureus isolated from bovine mastitis, it was planned to compare the presence of methicillin resistance (n=25) with the presence of mecA. DNA was extracted from 97 individual S. aureus isolates and PCR amplification targeting mecA was carried out. The gene was present only in 3 of the 97 S. aureus isolates. Similar to our findings, mecA could be found in 3 of the 18 phenotypic methicillin resistant S. aureus isolates from bovine mastitis cases in Turkey (Turutoglu et al., 2009). Most of the previous studies have demonstrated lower (7 to 11.6%) mecA presence (McKay 2008; Jamali et al., 2014 and Reshma et al, 2017), however Pu et al (2014) has reported prevalence to be quite high (47.6%). The antibiotic resistance is mainly attributed to acquisition of resistance genes by genetic exchange. However, the present study confirmed the results of earlier workers who indicated that there might be some other mechanisms associated with phenotypic methicillin resistance (Vesterholm-Nielsen 1999; Olsen, 2006).
Conclusion
In conclusion, this study has shown the emergence of MDR, XDR and PDR in S. aureus of bovine mastitis origin. The presence of MRSA strains in bovine milk has public health implications, and needs elaborative research work.
Acknowledgements
The authors sincerely acknowledge the Director of Research, Guru Angad Dev Veterinary and Animal Sciences University and Dean, College of Veterinary Science for providing necessary facilities to carry out the research. The help and cooperation of the farmers are also duly acknowledged. Funds for this research work were provided from the non-plan project on mastitis being funded by Punjab State Government.
References