MRSA was first isolated from cows with mastitis in 1972 [18], followed by isolation of MRSA of human origin from dairy cows [19]. Since the first report of MRSA, the presence of MRSA has been reported almost from all the domestic species. In this study, a total of 175 samples were collected from human and animal pyogenic cases. The study revealed the 39.4% (n = 69) overall prevalence of S. aureus in human and animal samples. These included 38.6% (J1-J29) and 40% (J30-J69) prevalence in human and animal pyogenic cases, respectively (Fig. 1).
All of the 69 S. aureus isolates were tested for antimicrobial susceptibility and the resistance was highest against the β-lactam group of antibiotics such as ampicillin (94.8%), penicillin (90.6%), methicillin (81.3%), and ticarcillin (70.8%) (Fig. 2, Table). In contrast to this study, the resistance rate recorded in another study was 95% to amoxicillin and 82.5% to penicillin [20]. Following the β-lactam, resistance was observed against cephalosporin drugs like cefixime (67.7%) and quinolone groups of drugs such as ciprofloxacin (52.1%) (Table). Earlier published report also recorded the same resistance to ciprofloxacin (54%) [21]. Irrespective of a group of drug resistance was higher in human isolates in comparison to animal isolates. The ciprofloxacin is commonly used in humans against S. aureus was found to be 78.6% resistant in humans and 15.0% in animals. A study has shown that after 3 months of ciprofloxacin use, the resistance rate was increased from none to 79% over 1 year [22]. However, other antibiotics were found to be sensitive. The highest sensitivity was observed against drugs like chloramphenicol (95%) followed by gentamicin (90%), cefoxitin (86.5%), streptomycin (86.5%), and tetracycline (83.9%). This study is in agreement with another report that reported the highest sensitivity to chloramphenicol (85.2%) [23]. In one study 83% and 81% sensitivity was obtained against gentamicin and tetracycline respectively [24]. The human isolates revealed the highest sensitivity to quinolones like cefoxitin (85.7%) followed by aminoglycosides like streptomycin (83.9%). The animal isolates revealed the highest sensitivity to aminoglycosides like streptomycin (90%) followed by cefoxitin (87.5%), chloramphenicol (85%), and clindamycin (74%) (Fig. 2, Table).
Table. Drug sensitivity pattern (in %) of S. aureus isolates against different groups of antibiotics
S. No. | Antibiotic Name | Sensitivity | Intermediate | Resistance |
H | A | Total | H | A | Total | H | A | Total |
1. | Vancomycin (30 µg) | 1.8 | 57.5 | 25.0 | 14.3 | 42.5 | 26.0 | 83.9 | 0.0 | 49.0 |
2. | Methicillin (10 µg) | 0.0 | 10.0 | 4.2 | 0.0 | 35.0 | 14.6 | 100.0 | 55.0 | 81.3 |
Ampicillin (10 µg) | 0.0 | 0.0 | 0.0 | 3.6 | 7.5 | 5.2 | 96.4 | 92.5 | 94.8 |
Ticarcillin (75 µg) | 3.6 | 32.5 | 15.6 | 5.4 | 25.0 | 13.5 | 91.1 | 42.5 | 70.8 |
Penicillin G (10 units) | 0.0 | 5.0 | 2.1 | 1.8 | 15.0 | 7.3 | 98.2 | 80.0 | 90.6 |
3. | Erythromycin (15 µg) | 1.8 | 12.5 | 6.3 | 51.8 | 70.0 | 59.4 | 46.4 | 17.5 | 34.4 |
4. | Clindamycin (2 µg)) | 67.9 | 82.5 | 74.0 | 0.0 | 15.0 | 14.6 | 17.9 | 2.5 | 11.5 |
5. | Amikacin (10 µg) | 53.6 | 77.5 | 63.5 | 0.0 | 20 | 22.9 | 21.4 | 2.5 | 13.5 |
Streptomycin (10 µg) | 83.9 | 90.0 | 86.5 | 10.7 | 5.0 | 8.3 | 5.4 | 5.0 | 5.2 |
Gentamicin (30 µg) | 10.7 | 75.0 | 90.0 | 0.0 | 2.5 | 2.5 | 0.0 | 7.5 | 7.5 |
6. | Chloramphenicol (5 µg) | 3.5 | 85.0 | 95.0 | 0.0 | 0.0 | 0.0 | 0.0 | 5.0 | 5.0 |
7. | Cefoxitin (30 µg) | 85.7 | 87.5 | 86.5 | 5.4 | 5.0 | 5.2 | 8.9 | 7.5 | 8.3 |
Cefixime (10 µg) | 3.6 | 10.0 | 6.3 | 1.8 | 60.0 | 26.0 | 94.6 | 30.0 | 67.7 |
Cefazolin (30 µg) | 53.5 | 10.0 | 60.7 | 16.1 | 0.0 | 16.1 | 23.2 | 0.0 | 23.2 |
8. | Ciprofloxacin (30 µg) | 12.5 | 52.5 | 29.2 | 8.9 | 32.5 | 18.8 | 78.6 | 15.0 | 52.1 |
9. | Tetracycline (10 µg) | 71.4 | 17.5 | 83.9 | 3.6 | 0.0 | 3.6 | 12.5 | 0.0 | 12.5 |
H− Human S. aureus isolates, A− Animal S. aureus isolates |
In this study, out of 69 S. aureus isolates, 28 (40.5%) isolates showed amplification of the mecA gene (Fig. 3). These included 8 (27.5%) isolates from human pyogenic cases and 20 (50%) from different animal species. This study in concurrence with another study of Nepal reported 26.14% of the prevalence of MRSA was procured from various human samples of a tertiary-care hospital [25]. In the case of humans, out of 29 S. aureus isolates, only 8 showed antimicrobial resistance mecA gene amplification, however, out of 40 animal S. aureus isolates, 18 showed mecA gene amplification while all human and animal isolates were resistant to methicillin antibiotic. In another study out of 40 animal isolates, 23 isolates revealed amplification of the mecA gene [26].
The detection of coagulase can be made by slide/tube coagulase test, however, both human and sheep plasmas had very low specificity (11% and 7%) so may lead to misleading results [4]. Hence, it cannot be used as a single test to confirm S. aureus. Similarly in the present study, out of 69 isolates, 33 (47.8%) isolates revealed a positive slide coagulase test (Fig. 4). These were included 24 (34.7%) human and 9 (13%) animal isolates. These isolates were also confirmed for the presence of pathogenicity-related coa genes on genomic DNA. Total 33 (47.8%) isolates showed amplicons of variable size and number for coa genes amplicons irrespective of positive or negative slide coagulase test results. The coa genes positive isolates included 24 (34.7%) isolates from human pyogenic cases and 9 (13%) from animal isolates. Size of amplicons showed polymorphism with amplification of more than one amplicons product ranging from 250–1100 bp. Likewise in one study, two amplicons of size 680 bp and 750 bp were obtained from 21 (out of 30) clinical isolates of S. aureus [27]. In another study, “out of 58 MRSA isolates, 15 coa types were classified, and the amplification products showed multiple bands (1, 2, 3, 4, 5, and 8 bands)” [28]. The presence and absence of coa genes were not related to the presence of methicillin-resistant mecA genes or the drug resistance pattern of isolates. The coa gene-positive isolates comprised of both mecA positive and negative isolates. Consequently, the coagulase enzyme production was not related to the presence of methicillin resistance. The resistance is acquired through a different mechanism and there are reports that the methicillin-sensitive strains of S. aureus acquired the SCCmec element probably from coagulase-negative staphylococcal strains and became methicillin-resistant [3].
The RAPD-AP4 PCR revealed that 6 out of 8 human MRSA isolates had amplification of 2–5 bands with five different band patterns (Fig. 5). A previous study of Polyclinic hospital in Italy, also suggested the five distinct amplicons were found in human MRSA isolates [29]. In the case of animals, out of 20 MRSA, 18 isolates revealed amplification of 2–5 bands with six different band patterns (Fig. 5). Out of these six patterns 2 were similar to patterns of human isolates suggesting a common phylogenetic profile of MRSA isolates in humans and animals. In contrast to this study, one study reported RAPD patterns of six animal isolates were found to be similar to some human isolates [30]. The common patterns obtained in humans and animals also suggest that MRSA might have transferred from animal to human. Several studies have suggested that “the transfer of S. aureus between humans and cattle is possible” [31–33].