Molecular typing of Staphylococcus aureus based on coagulase gene

Aim: This study was conducted to study the coagulase gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle using restriction fragment length polymorphism (RFLP) and their sequence-based phylogenetic analysis. Materials and Methods: A total of 192 different samples from mastitic milk, nasal cavity, and pus from skin wounds of cattle from Military Dairy Farm, Jammu, India, were screened for the presence of S. aureus. The presumptive isolates were confirmed by nuc gene-based polymerase chain reaction (PCR). The confirmed S. aureus isolates were subjected to coagulase (coa) gene PCR. Different coa genotypes observed were subjected to RFLP using restriction enzymes Hae111 and Alu1, to obtain the different restriction patterns. One isolate from each restriction pattern was sequenced. These sequences were aligned for maximum homology using the Bioedit software and similarity in the sequences was inferred with the help of sequence identity matrix. Results: Of 192 different samples, 39 (20.31%) isolates of S. aureus were confirmed by targeting nuc gene using PCR. Of 39 S. aureus isolates, 25 (64.10%) isolates carried coa gene. Four different genotypes of coa gene, i.e., 514 bp, 595 bp, 757 bp, and 802 bp were obtained. Two coa genotypes, 595 bp (15 isolates) and 802 bp (4 isolates), were observed in mastitic milk. 514 bp (2 isolates) and 757 bp (4 isolates) coa genotypes were observed from nasal cavity and pus from skin wounds, respectively. On RFLP using both restriction enzymes, four different restriction patterns P1, P2, P3, and P4 were observed. On sequencing, four different sequences having unique restriction patterns were obtained. The most identical sequences with the value of 0.810 were found between isolate S. aureus 514 (nasal cavity) and S. aureus 595 (mastitic milk), and thus, they are most closely related. While as the most distant sequences with the value of 0.483 were found between S. aureus 514 and S. aureus 802 isolates. Conclusion: The study, being localized to only one farm, yielded different RFLP patterns as observed from different sampling sites, which indicates that different S. aureus coagulase types have a site-specific predilection. Two coa patterns were observed in mastitic milk indicating multiple origins of infection, with 595 bp coa genotype being predominant in mastitic milk. The coa genotypes and their restriction patterns observed in the present study are novel, not published earlier. 514 and 595 coa variants of S. aureus are genetically most related

Phenotypic and genotypic characterization of methicillin-resistant Staphylococcus aureus from bovine mastitis

Aim: This study was conducted to determine the occurrence of methicillin-sensitive and Staphylococcus aureus (MSSA) methicillin-resistant S. aureus (MRSA) from bovine mastitis and to characterize them with respect to antibiotic resistance gene mecA. Materials and Methods: A total of 160 mastitic milk samples were screened for the presence of S. aureus. The presumptive positive isolates were confirmed using nuc and 23S rRNA gene-based polymerase chain reaction. All the confirmed isolates were subjected to in vitro antibiogram using a number of antibiotics. Isolates which showed resistance against methicillin were characterized for the presence of mecA gene. Results: Out of the total 160 milk samples, 36 (22.5%) samples yielded S. aureus. The in vitro antibiogram revealed that 16.6% S. aureus isolates were resistant to all antibiotics screened for and 5.5% isolates were sensitive to all of them. Furthermore, the study found 94.4%, 83.3%, 77.7%, 66.6%, 50%, and 27.7% of S. aureus isolates resistant to penicillin, ampicillin, amoxicillin-sulbactam, enrofloxacin, ceftriaxone, and methicillin, respectively. Out of the 36 S. aureus isolates, only 6 (16.6%) isolates were confirmed as MRSA while rest were MSSA. Conclusion: The higher occurrence of S. aureus-mediated mastitis was concluded due to improper hygienic and poor farm management. The multiple drug resistance reveals the indiscriminate use of drugs and presence of methicillin resistance gene determinant is an alarming situation as such infections are difficult to treat

The study of aerobic bacterial flora of the upper respiratory tract of equines from Jammu and Kashmir region of India

Aim:To isolate aerobic bacterial micro flora residing in the upper respiratory tract of equines used by the pilgrims and tourists in Jammu & Kashmir. Materials and Methods:88 apparently healthy equines and 53 equines with respiratory tract diseases were used in this study. Swab samples were collected from the upper respiratory tract of equines. Isolation and identification of the bacteria was conducted under aerobic conditions. Each of the sample processed yielded at least one type of bacteria species. Results: A total of 321 bacterial isolates were recovered from both groups of equines. The majority of the isolates were Gram positive (84.11%) and the rest were Gram negative (15.88%). Bacterial isolates identified in order of the magnitude were Streptococcus equi subsp. zooepidemicus (17.44%), Micrococcus spp. (9.96%), Corynebacterium (9.65%), Staphylococcus intermedius (9.65%), Staphylococcus aureus (8.72%), Bacillus spp. (7.16%), Streptococcus pneumonia (5.60%), Staphylococcus chromogens (5.60%), Streptococcus equismilis (5.29%), Pseudomonas aeruginosa (5.29%), Rhodococcus equi (3.73%), Escherichia coli (3.73%), Klebsiella pneumoniae (3.42%), Proteus vulgaris (3.42%), and Streptococcus equisubsp. equi(1.24%). Conclusion: The present study reveals the predominance of Gram positive bacteria in both healthy and diseased animals. Bacteria were recovered at a higher rate from diseased equines than from apparently healthy animals. Streptococcus equi subsp. zooepidemicuswas mainly found to be associated with respiratory tract infections