Prevalence of mecA- and mecC-Associated Methicillin-Resistant Staphylococcus aureus in Clinical Specimens, Punjab, Pakistan

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically prevalent bacterium and is resistant to many drugs. Genetic factors such as mec genes are considered to be responsible for this resistance. Recently, Staphylococcal Cassette Chromosome mec (SCCmec) element mutations produced mecC, a new genetic variant that encodes a transpeptidase enzyme (63% similarity with mecA-encoded PBP2a). This cross-sectional study was conducted to establish the prevalence of the mecA and mecC genes among phenotypically identified MRSA and their effectiveness against different antibiotics in clinical specimens. The prevalence of Staphylococcus aureus was 10.2% (n = 102) in the total number of clinical specimens collected (n = 1000). However, the prevalence of MRSA was 6.3% (n = 63) of the total samples collected, while it was 61.8% among total Staphylococcus aureus isolates. mec genes were confirmed in 96.8% (n = 61) isolates of MRSA, while 3.2% (n = 2) were found to be negative for mec genes. The combination of mecA and mecC was detected in 57.1% (n = 36) of the MRSA isolates. The prevalence of lone mecA was 31.8% (n = 20) and that of lone mecC was 7.9% (n = 5) among all the MRSA samples. Penicillin and amoxicillin/clavulanic acid were the most resistant antibiotics followed by norfloxacin (91.2%), levofloxacin (87.1%), ciprofloxacin (83.9%), azithromycin (78.6%), erythromycin (77.4%), moxifloxacin (69.8%), and sulfamethoxazole/trimethoprim (54.9%). On the other hand, vancomycin and teicoplanin (98.4%) were more effective drugs against MRSA followed by linezolid (96.7%), clindamycin (84.6%), chloramphenicol (83.7%), fusidic acid (70.6%), gentamicin (67.7%), and tetracycline (56.8%). In conclusion, a significant prevalence of mecA and mecC has been found among MRSA isolated from clinical specimens, which is likely responsible for antibiotic resistance in MRSA in our clinical settings. However, vancomycin, teicoplanin, and linezolid were found the top three most effective drugs against MRSA in our clinical settings. Thus, MRSA endemics in local areas require routine molecular and epidemiological investigation.


Introduction
Staphylococcus aureus (S. aureus) is a clinically important Gram-positive bacteria found in the normal flora of the skin and nasal cavity. It can cause endocarditis, osteoarthritis, dermal and soft tissue infections, pulmonary and aerobic vaginitis, and even death [1]. for analysis as soon as possible after collection and transportation to the Microbiology Department. After processing, urine samples were preserved at 2-8 • C in the refrigerator and the blood culture vials were kept at room temperature for one week. The preserved samples were used for troubleshooting or confirmation of the results if any doubt was found in the findings [15,16].
The Institutional Review Board (IRB) of the Institute of Molecular Biology and Biotechnology (Reference No. 334/A) granted ethical approval for this cross-sectional study, which was designed and conducted at Bahauddin Zakariya University, Multan, from June 2021 to December 2021.

Isolation and Confirmation of Staphylococcus aureus
To isolate Staphylococcus species, urine samples and routine samples were cultured on nutrient media and differential media as blood agar (MSA, Oxoid Ltd., Basingstoke Hampshire, UK) and mannitol salt agar (MSA, Oxoid Ltd., Basingstoke Hampshire, UK), respectively, by using a sterile swab. After inoculation, plates were placed at 37 • C in the incubator. The next day bacterial growth was evaluated. If growth was found, then Gram staining and other biochemical tests were performed using a pure and well-isolated colony. If growth was not observed on the first day, the plates were reincubated for overnight incubation. On the second day, these samples were reported as "No Growth" if growth was not seen. Similarly, for blood samples, samples from BD vials were inoculated onto blood and mannitol salt agar plates 3 days after sample collection and incubation of vials at room temperature, following the same steps as in urine and routine samples. According to the morphology of S. aureus, the golden yellow colonies from plates were selected for further study [17]. Initial identification of Staphylococcus aureus was performed by Gram staining and biochemical testing including catalase and coagulase tests [18]. S. aureus was positive for the catalase and coagulase test. Additionally, the molecular confirmation of S. aureus was performed by using specific primers of the nuc gene for polymerase chain reaction (PCR) (Table 1, Figure 3). After confirmation, pure colonies of S. aureus were taken from the mannitol salt agar plates and preserved using the glycerol stock method for further analysis of genetic variants corresponding to MRSA [19].

Phenotypic Identification of MRSA and Antimicrobial Susceptibility Testing
The Clinical Laboratory and Standards Institute (CLSI) 2020 guidelines were followed for both phenotypic identification of MRSA and antimicrobial susceptibility testing (AST) using the Kirby Baur disk diffusion method [23]. In a test tube, one colony from the MSA plate was suspended in 200 µL of 0.9% germ-free normal saline solution. Using a sterile swab, the inoculum was streaked homogeneously on Muller Hinton Agar (MHA, Oxoid, Hampshire, United Kingdom) plates. According to CLSI, MRSA has resistance to cefoxitin (FOX) antibiotic. Thus, phenotypical identification of MRSA was performed by dispensing cefoxitin (FOX) on an MHA plate by using sterile forceps after preparing the lawn of the inoculum. The same procedure was followed for AST against antibiotics, i.e., penicillin (P), amoxicillin/clavulanic acid (AMC), norfloxacin (NOR), levofloxacin (LFX), ciprofloxacin (CIP), azithromycin (AZM), erythromycin (E), moxifloxacin (MXF), sulfamethoxazole/trimethoprim (SXT), tetracycline (TE), gentamycin (G), fusidic acid (FD), chloramphenicol (C), clindamycin (DA), linezolid (LZD), vancomycin (VA), teicoplanin (TEC), and identification of MRSA for each sample. Plates were incubated at 37 • C for 24 h. After 24 h, the zone of inhibition (ZOI) was measured, and the results were interpreted according to the CLSI guidelines. The isolates showing ZOI ≤ 19 mm were characterized as MRSA and with ≥22 as MSSA [24].

DNA Extraction for PCR
The modified CTAB (Cetyltrimethylammonium bromide) method was used for DNA extraction from bacteria [25]. The DNA was extracted from purified (on Tryptic Soya Broth (TSB) for 24 h at 37 • C) and biochemically confirmed Staphylococcus aureus. Briefly, 10% Sodium Dodecyl Sulfate (SDS), Proteinase K (Thermo Scientific™ Proteinase K), 10% CTAB/Sodium chloride (NaCl), and 5M NaCl were used for cell lysis. Extracted DNA was suspended in 100 µL of Tris-Ethylenediaminetetraacetic acid buffer (TE Buffer) and stored at −20 • C. Qualitative measurement of DNA was performed using a 0.5% agarose gel. The Gel Doc system (BIO-RAD Gel Doc TM XR + with Image Lab tm Software, BioRad, Hercules, CA, USA) was used for gel visualization.

Molecular Detection of mecA and mecC Genes in MRSA
Antibiotic resistance genes (mecA and mecC) were amplified by using a basic Thermal Cycler PCR (BIO-RAD T100 TM Thermal Cycler, Massachusetts, USA) in which specific primers were used for both mecA and mecC genes ( Table 1). A total volume of the PCR reaction (15 µL) contained 2 µL (10 ng/µL) of DNA, 7.5 µL of 2X Taq master mix (Vazyme Biotech Co., Nanjing, China), 1 µL (10 µM) of forward primer, 1 µL (10 µM) of reverse primer, and 3.5 µL of deionized water. PCR for both genes (mecA and mecC genes) was performed by applying the following conditions: first denaturation at 95 • C for 5 min followed by 35 cycles of 95 • C for 1 min, 55 • C for 30 s, 72 • C for 1 min, and 72 • C for 5 min [21,22]. Molecular detection of mecA and mecC was performed by loading 6-7 µL of 100 bp DNA ladder into the first well and the remaining wells were loaded by PCR products using 1.5% agarose gel containing 0.5 mg/mL of ethidium bromide in Tris-borate-EDTA (10 mM+1mM EDTA; pH 8.0) buffer used by applying 120 V for an hour. DNA bands were observed under ultraviolet light (UV-light) using the Gel Doc system (BIO-RAD Gel Doc TM XR + with Image Lab tm Software, BioRad, Hercules, CA, USA).

Quality Control
Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) strains from the American Type Culture Collection (ATCC) were used as a control to evaluate the growthsupporting ability of mannitol agar, blood agar, CLED, and MHA agar to maintain the quality of growth throughout the investigation. Gram staining was performed on samples, along with quality control strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) as a Gram-positive cocci and a Gram-negative rod, respectively. Using ATCC strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli, the accuracy and reproducibility of biochemical test results were maintained.

Statistical Analysis
In this study, statistical analysis was performed on the data using the chi-square test. GraphPad Prism 9 was used for statistical analysis (GraphPad Software, San Diego, CA, USA) and a p-value ≤ 0.05 was used to illustrate the statistically significant differences.
Sixty-one isolates (n = 61/63; 96.8%) were positive for mec genes, while two isolates (n  A total of fifty-six (n = 56) isolates were confirmed for mecA with PCR from total isolates of MRSA (n = 63). The prevalence of the mecA gene was 88.8% (n = 56/63), but the mecA gene was not detected in seven isolates (n = 7/63; 11.1%). Remarkably, these isolates showed complete resistance to cefoxitin and penicillin in the antibiotic sensitivity test. The mecC gene was identified in forty-one isolates (n = 41/63; 65.0%) and the remaining twenty-two showed no amplification (n = 22/63; 34.9%).

Discussion
Infections with MRSA are prevalent in both healthcare facilities and the general pop ulation. mecC is an emerging gene responsible for Staphylococcal methicillin resistance The antimicrobial susceptibility pattern of MRSA isolates having mecA and mecC gene differs from non-MRSA isolates, which frequently exhibit resistance to both penicillin an cefoxitin antibiotics. In contrast, the majority of genetic factors (mecA and mecC gene) con taining MRSA are resistant to penicillin and cefoxitin and are subsequently reported a MRSA, and may be susceptible to remaining antibiotics [26].
In this study, we reported the prevalence of mecA and mecC genes in MRSA isolate from our local population of southern Punjab, Pakistan. In addition, we also conducted study on the antibiotic resistance profile of MRSA against antibiotics other than penicilli and cefoxitin. Several studies reported antibiotic resistance of MRSA in this region; how ever, the genetic factor responsible for resistance was not broadly studied in our clinica settings. In this study, S. aureus was isolated from routine clinical samples includin blood, urine, sputum, pus, and body fluids collected from different hospital wards. A hig incidence (10.2%) of S. aureus in routine clinical samples indicates that S. aureus infection are a major contributor to infections in our clinical settings as compared to other Gram positive and Gram-negative bacteria [27]. Furthermore, the prevalence of MRSA wa 61.8% in total S. aureus isolates, while methicillin-susceptible S. aureus (MSSA) was con firmed in 38.2%. Similar to the previous study, all MRSA isolates were also found to b resistant to the β-lactam antibiotics including cefoxitin, amoxicillin, and penicillin [28]. I contrast to previous studies in Pakistan [29,30], MRSA isolates were found to be mor susceptible to non-β-lactam antibiotics, i.e., fusidic acid, gentamicin, tetracyclin clindamycin, and chloramphenicol in this study. Previously, MRSA isolates were show to be more resistant to non-β-lactam antibiotics, i.e., fusidic acid, tetracycline, clindamy 31.8% 7.9% 57.1%

Discussion
Infections with MRSA are prevalent in both healthcare facilities and the general population. mecC is an emerging gene responsible for Staphylococcal methicillin resistance. The antimicrobial susceptibility pattern of MRSA isolates having mecA and mecC genes differs from non-MRSA isolates, which frequently exhibit resistance to both penicillin and cefoxitin antibiotics. In contrast, the majority of genetic factors (mecA and mecC gene) containing MRSA are resistant to penicillin and cefoxitin and are subsequently reported as MRSA, and may be susceptible to remaining antibiotics [26].
In this study, we reported the prevalence of mecA and mecC genes in MRSA isolated from our local population of southern Punjab, Pakistan. In addition, we also conducted a study on the antibiotic resistance profile of MRSA against antibiotics other than penicillin and cefoxitin. Several studies reported antibiotic resistance of MRSA in this region; however, the genetic factor responsible for resistance was not broadly studied in our clinical settings. In this study, S. aureus was isolated from routine clinical samples including blood, urine, sputum, pus, and body fluids collected from different hospital wards. A high incidence (10.2%) of S. aureus in routine clinical samples indicates that S. aureus infections are a major contributor to infections in our clinical settings as compared to other Gram-positive and Gram-negative bacteria [27]. Furthermore, the prevalence of MRSA was 61.8% in total S. aureus isolates, while methicillin-susceptible S. aureus (MSSA) was confirmed in 38.2%. Similar to the previous study, all MRSA isolates were also found to be resistant to the β-lactam antibiotics including cefoxitin, amoxicillin, and penicillin [28]. In contrast to previous studies in Pakistan [29,30], MRSA isolates were found to be more susceptible to non-β-lactam antibiotics, i.e., fusidic acid, gentamicin, tetracycline, clindamycin, and chloramphenicol in this study. Previously, MRSA isolates were shown to be more resistant to non-β-lactam antibiotics, i.e., fusidic acid, tetracycline, clindamycin, and chloramphenicol. This is likely due to less use of these antibiotics in past in our clinical patients, which may allow MRSA to restore sensitivity against these drugs.
In this study, MRSA isolates were highly resistant to erythromycin, norfloxacin, ciprofloxacin, levofloxacin, and azithromycin, which is in line with previously conducted studies in this region [31,32]. This indicates overuse of antibiotics in this area, or an evolutionary adaptation of bacteria [33]. Vancomycin and teicoplanin have already exhibited some (1.6%) resistance in our clinical settings. This is an alarming situation, as intermediate or resistant strains are already emerging in this area; vancomycin is considered to be the only effective drug against MRSA infections. A recently launched antibiotic called linezolid is being used to treat MRSA infections as well. It can bind to the 23S ribosomal RNA of the larger subunit of the bacterial ribosome and inhibits protein synthesis [34]. It is also a remarkable finding in this study that 3% of MRSA isolates are already showing resistance against linezolid. This obviates the importance of continuous monitoring of drug resistance in all clinical settings where the use of antibiotics is a common clinical practice.
Generally, the mecA gene has been considered the most prevalent in MRSA compared to mecC. For example, the prevalence of mecA has been reported at around 31.9% in MRSA [35,36]. Similarly, in this study, the prevalence of mecA alone was 31.8%, and the prevalence of mecC alone was 7.9% in MRSA. Remarkably, the prevalence of the mecA and mecC combination was the highest at 57.1% in MRSA. This indicates an accumulative effect of both genetic factors in the incidence of MRSA, which is an alarming situation for clinical settings. A similar incidence of mecA (100%; n = 50) and a combination of both mecA and mecC (6%; n = 3/50) has recently been reported for MRSA in Egypt in 2020 [37]. A decade before this, a low incidence (0.45%) of the mecC gene in MRSA was reported in other countries [38]. The mecC gene in MRSA was first reported in our hospital settings in 2020 [39]. Remarkably, we are reporting a distressing increase in the prevalence of mecC in MRSA in our clinical settings within recent years [39]. A previous study from Pakistan reflects a similar situation, with MRSA containing both mecA and mecC genes [39]; however, the prevalence of this combination reported in this study was lower. Further, the mecA and mecC genes were not detected in two clinical isolates in this study, although these were phenotypically and biochemically characterized as MRSA. This may indicate the need for other molecular confirmations along with conventional methods to characterize MRSA, as previously described [39,40]. Furthermore, it is also possible that another mutant may be circulating in our hospital-acquired MRSA. Further studies are still required to characterize these isolates.
The origin of MRSA-carrying mecC is not yet clear, but this novel gene has also been reported in other Staphylococcus species. [41]. This suggests that coagulase-negative Staphylococcus spp. may be the source of mecC in MRSA, as previously reported for mecA as well [42]. Thus, the genetic transformation from Staphylococci to S. aureus may play a pivotal role in increasing the incidence and prevalence of mecC in S. aureus in the last couple of years in our clinical settings. Therefore, clinical microbiologists should be aware of the risk of mecC transformation from other methicillin-resistant Staphylococcal pathogenic species [43].
However, mecC remained more prevalent in Denmark than in other countries of Europe and the subcontinent. Many cases of mecC-mediated MRSA were reported in Spain, especially in skin infections that supposedly emerge from livestock [28]. Now, countries in Asia have also reported cases of MRSA with a prevalence of the mecC gene [44]. The MRSA cases in this study are clinical isolates and also appeared to be multi-drug-resistant. These results indicate that these MRSA cases may carry an SCC mec element of type III [45]. SCC mec element IV is present in community-associated MRSA and they do not resist multiple antibiotics. The incidence of mecC in MRSA may have an impact on the multidrug resistance quality and make them difficult to treat with new antimicrobial agents. mecC-carrying MRSA harbors other antibiotic-resistant genes and their regulator genes for expression. We showed the resistance profile of clinical MRSA isolates for many antibiotics. The antibiotic resistance rate for non-β lactams antibiotics was low. While it may be possible that there was an absence or no expression of the genetic factors responsible for those antibiotics, non-β-lactam antibiotics may be used for MRSA infections in this area.

Conclusions
We concluded that the mecA gene was more prevalent compared to the mecC gene in MRSA isolated from our clinical settings. However, the prevalence of the mecC gene is increasing gradually. Vancomycin, linezolid, teicoplanin, chloramphenicol, and clindamycin can be used against infections caused by MRSA due to their lower resistance rate. The molecular investigation of the mecA and mecC genes in MRSA should be a routine practice in our clinical settings. The routine molecular epidemiology of mecC should be conducted using other clinical isolates so we can find ways to avoid the genetic transformation of mecC into mecC-negative bacterial species. The changing molecular basis of MRSA drug resistance affects not only new treatment strategies for MRSA but also impedes the control of MRSA infections. There is a need for more molecular and epidemiological investigations to prevent the spread of S. aureus in local areas and to understand the rise of multi-drug resistance in MRSA.