One of the remaining effective treatments for MRSA infections is Vancomycin (Moise-Broder et al., 2004). Nasal colonization in humans can begin during the first few days of life (Maayan-Metzger et al., 2017). 7/8 of the mobile strains were found to be genetically identical to nares isolates, according to genotyping (Chang et al., 2017).
Stubbs et al., 1994) conducted an interesting study in which they investigated the nasal transport of S. MRSA carriers are uncommon (0.2%) in persons who have never had any interaction with the health care system (Salgado et al., 2003). The bacterium displayed the high level vancomycin resistance gene VanA and was methicillin resistant (Sievert et al., 2002).
Recently, prevalence rates of CA-MRSA have been reported through a meta-analysis of studies (Salgado et al., 2003). Catalase test: This test is performed for the evaluation of gas bubbles of hydrogen peroxide (Rusenova et al., 2017). The toxins present in the samples clump the latex particles together and determine the test result (Berube et al., 2013).
Cefoxitin activates the MRSA-mecA gene and the results resemble PCR (Broekema et al., 2009).
CHAPTER – III Materials and Methods
The tube coagulase test was performed by adding 0.2 mL of the overnight culture grown in brain heart infusion fluid to 0.5 mL of horse plasma in a glass tube. The presence of coagulates was considered when large organized coagulation of the entire contents of the tube occurred, which did not dissociate when inverted (Brasil, 2003). All suspected staphylococcal isolates were confirmed by PCR using the primers described by Shome et al.
Blood agar was used to pick a loopful of fresh colonies (approximately 3-4), which were then transferred to a 1.5 mL Eppendorf tube containing 200 µL of ultrapure water. A vent hole is drilled in the top of each tube to allow extra fumes to escape while the tubes are boiling. After freezing, the tubes were again immersed in 99°C hot water for 10 minutes, and the boiled tubes were immersed in -20°C for five minutes.
Finally, the melted agarose was added to the gel tray and left for approx. 20 minutes to allow the gel to solidify. The gel was placed in an electrophoresis tank which had 50 ml of 1X TAE buffer previously added. To compare the amplicon size of the gene product, a hole was filled with a DNA marker (Thermo scientific O'Gene ruler 100 kb).
The antimicrobial susceptibility testing of the obtained isolates was performed according to CLSI guidelines (CLSI, 2020) with a panel of 11 antimicrobial agents, including Ampicillin, Cefoxitin, Ceftriaxone, Ciprofloxacin, Erythromycin, Gentamicin, Meropenem, Oxacillin, Teprimacyclin, Penicillin, Penicillin. Bauer-Kirby disk diffusion procedure (Bauer et al., 1966) was used to perform the antimicrobial susceptibility test. Staphylococcal isolates showing resistance to at least three groups of antimicrobial agents (≥3) were defined as multi-drug resistant (MDR) isolates ( Li et al., 2014 ).
All oxacillin- and cefoxitin-resistant isolates were considered for predicting mecA-mediated resistance in staphylococci (CLSI, 2020). The phenotypically resistant isolates were further examined for the presence of the mecA gene by PCR (Larsen et al., 2008). DNA extraction of the isolates was performed by the boiling method as described in the previous section. To perform PCR assays, concentrations of 20 pmol/μL of each primer were used.
CHAPTER – IV Results
Like medical students, all staphylococcal isolates from veterinary students were resistant to Ampicillin and Penicillin. Individual antibiogram profiles of all isolates from medical and veterinary students are illustrated in Figure 9 and Figure 10, respectively. A total of 7 and 22 resistance patterns with different combinations of antimicrobial agents were observed in coagulase-positive S.
32 | P a g e Figure 9: Heat map showing distribution of antimicrobial resistance phenotype of methicillin-resistant Staphylococcus aureus and methicillin-resistant CoNS isolates obtained from medical students. 33 | P a g e Figure 10: Heat map showing distribution of antimicrobial resistance phenotype of methicillin-resistant Staphylococcus aureus and methicillin-resistant CoNS isolates obtained from veterinary students. A total of 5 and 14 resistance patterns with different combinations of antimicrobial agents were observed in coagulase-positive S .
About 50% of the total coagulase-positive isolates showed multidrug resistance (ie, resistance to ≥3 antimicrobial classes) with a range of 3 to 5 different antimicrobials (Table 15), while about 81% of the total CoNS isolates showed multidrug resistance . Among the 39 isolates obtained from medical students were positive for the mecA gene, and 6 (22.2%) out of the 27 isolates from veterinary students carried the mecA gene. Notably, all mecA genes were carried by both CoPS and CoNS isolates and eventually classified as methicillin-resistant isolates (Figure 11).
However, none of the variables or factors were suitable for multivariable logistic regression analysis.
CHAPTER – V Discussion
MRSA codes for the mecA gene, which allows the bacteria to produce penicillin-binding proteins that are difficult for bacteria to bind to medicines. Unfortunately, the risk factors include abuse or overuse of antibiotics such as cephalosporins, fluoroquinolones, long-term intensive care facilities, colonization, contact, very poor hand washing, living in crowded or unsanitary conditions, or the use of immunosuppressive medications such as corticosteroids (Ralston et al ., 2018). To prevent MRSA colonization among medical and veterinary students, preventive measures should be implemented, such as maintaining high hygiene standards, washing and drying hands thoroughly before and after caring for a patient, and not touching potentially contaminated equipment or bandages.
When combating AMR, it is critical to reduce the spread and transmission of resistant pathogens, both within and between animal and human populations. It is challenging to determine the exact origin of resistant bacterial strains due to the ability of bacteria to spread from one environment to another, sometimes over large distances and between different populations. Therefore, more research into the sources and routes of transmission of antibiotic-resistant microorganisms is warranted, ideally from a One-Health perspective.
It is critical that we improve our understanding of how interactions and animal trade (direct transmission), farm management and the wider farm environment (indirect transmission) contribute to the spread of antimicrobial resistance and pinpoint effective countermeasures to this phenomenon.
CHAPTER-VI Conclusions
Questionnaire
- Particulars of the patient
