Introduction
In the 2014 report of the World Health Organization, it was reported that when compared to methicillin susceptible Staphylococcus aureus (MSSA), infections caused by methicillin resistant Staphylococcus aureus (MRSA) were associated with a significantly higher risk of septic shock, all-cause mortality and bacterial-related mortality, and that they were associated with a longer hospital and intensive care unit stay (1). Considering these data, it is very important to detect the presence of MRSA quickly and accurately in patients.
In clinical practice, MRSA strains are considered resistant to all beta-lactam antibiotics (2). This is because MRSAs produce penicillin-binding proteins (PBP2a’) that bind with lower affinity to beta-lactams. PBP2a’s are encoded by the mecA gene located on the staphylococcal chromosome cassette (SCCmec) genetic region (3,4).
The presence of MRSA is detected by phenotypic and genotypic methods in clinical microbiology laboratories. The most common method is to look at the susceptibility of oxacillin/cefoxitin. With this method, the presence of MRSA can be demonstrated the next day. Genotypic methods are used in cases where phenotypic methods are not conclusive (eg, limit values in disc diffusion) or when rapid results are required (5). PCR tests to show the presence of the mecA gene in bacteria can give results in hours, and quick card tests can give results in minutes.
In 2011, isolation of S. aureus (LGA251) strains isolated from milk and different clinical specimens, which were phenotypically resistant to methicillin but could not be detected by tests for the mecA gene, and which were determined to have a different mecA gene, was reported for the first time (5). This different mecA gene (mecALGA251) was later named the mecC gene (6). These studies have shown that MRSA strains with the mecC gene can be overlooked in clinical practice. After this date, similar studies have been carried out from different countries and MRSA strains carrying mecC have been reported from many European countries (7).
Studies on the presence of mecC in our country have been relatively limited. It is clear that the presence and frequency of MRSA isolates carrying the mecC gene is also important in the development of public health policies, as it affects the treatment. Bezmialem Vakıf Hospital as a university hospital providing tertiary healthcare with a central location in Istanbul, accepts patients from all over Turkey. Therefore, it would not be wrong to assume that the data obtained from this hospital will also include some country-wide clues.
In this study, the presence of mecC was investigated in MRSA strains isolated and archived in the cultures of various clinical samples in the microbiology laboratory between November 2014 and March 2018 in order to identify MRSA isolates carrying the mecC gene in our hospital and to obtain information on the distribution of similar isolates throughout the country.
Methods
Bacterial Isolates and Antibiotic Susceptibility Tests
One hundred and twenty six consecutive MRSA strains isolated and stocked from various clinical samples of patients admitted to the outpatient clinics of Bezmialem Vakıf Hospital and hospitalized between November 2014 and March 2018 were included in the study. These strains were not previously passaged for any reason.
Antibiotic susceptibility of bacteria was determined with the Vitek-2 automated system (bioMerieux, France) in accordance with the manufacturer’s recommendations.
DNA Isolation
The MRSA strains in the stocks were passaged on sheep blood agar and incubated overnight. After the incubation, 4-5 colonies were taken from the cultures that grew and placed in 1 mL saline filled Eppendorf tubes and thoroughly mixed with vortex. Bacterial suspensions were incubated at 95 °C for 5 minutes in a heat block. Afterwards, the eppendorfs were centrifuged at 3,000 rpm for 1 minute, and the supernatants were separated to be used in the PCR process and stored at -20 °C.
Detection of mecC, mecA, spa and pvl Genes
According to the protocol published by the European Union Reference Laboratories National Food Institute in 2012, the primer mix used for the detection of mecC, mecA, spa and pvl genes was prepared and multiplex PCR was performed according to this protocol (8). PCR conditions were set at 94 °C for 5 minutes, 30 cycles (94 °C 30 seconds, 59 °C 1 minute, 72 °C 1 minute), and 72 °C for 10 minutes. S. aureus ATCC 43300 for mecA and S. aureus NCTC 13552 for mecC were used as positive controls.
The PCR for spa detection was performed according to the protocol published by Votintseva et al. (9) in 2014 for the strains of which spa gene could not be detected.
Results
According to the results of multiplex PCR of 126 MRSA strains studied, mecA gene was detected in 126 (100%), spa gene in 107 (85%) and pvl gene in 3 (2%), and mecC gene was not detected in any isolate. An example gel image is shown in Figure 1.
The PCR was applied to 19 strains in which the spa gene could not be detected, with a different method for spa detection, and spa gene could be detected in all 19 isolates (Figure 2) (9).
According to the historical VITEK 2 minimum inhibitory concentration results, all isolates were resistant to cefoxitin and oxacillin, and susceptible to vancomycin and linezolid. Inducible clindamycin resistance was reported in 24 isolates. The susceptibility results of some antibiotics are given in Table 1.
Discussion
The MRSA is one of the most important causes of community and hospital-acquired infections. MRSA outcomes reported from intensive care units are increasing day by day. MRSA is an important pathogen due to its multi-antibiotic resistance. In addition to being resistant to all beta lactam antibiotics, it also shows resistance to lincosamide, aminoglycoside and macrolides (10). It is very important to detect MRSA in the laboratory in order to select an appropriate and effective antibiotic.
The PCR to detect the mecA gene in bacteria and rapid agglutination tests for the presence of PBP2a may miss the presence of mecC (5). Although mecC has not yet been detected in human samples in our country, it has been detected in many European countries. Since the number of studies conducted in our country is very small, it can be said that isolates carrying mecC have not yet been detected in human samples in our country. In this case, it may be recommended to carry out studies looking for MRSA isolates with the mecC gene at regular intervals, to continue the scans, and to carry out studies with a large number of samples and necessary arrangements in order not to miss the possible mecC gene presence in the laboratory diagnosis of MRSA (7). Our study exemplifies such a survey.
In a study published in 2013, 896 S. aureus strains were studied with the VITEK 2 susceptibility system and it was aimed to detect MRSA carrying mecC. Of the 455 MRSA mecA positive strains, 98% were resistant to oxacillin and cefoxitin, 0.9% were susceptible to cefoxitin, while 1.1% were resistant to oxacillin and susceptible to cefoxitin, and no mecA MRSA strains were found to be susceptible to cefoxitin and oxacillin. Of the 62 mecC positive MRSA strains, 88.7% were susceptible to oxacillin, resistant to cefoxitin, and 11.3% were found to be resistant to both antibiotics, and similarly, no mecC MRSA strains were found to be susceptible to cefoxitin and oxacillin. Of 379 methicillin-susceptible strains (mecA and mecC negative), 98.8% were found to be sensitive to both antibiotics, while 1.1% were resistant to oxacillin, but sensitive to cefoxitin (11). All 126 MRSA strains in our study were found to have mecA gene and according to VITEK 2 results, none of the strains were sensitive to oxacillin or cefoxitin. VITEK 2 antibiotic susceptibility system is a frequently used method in our country. Work needs to be done with other commercial systems as well.
Panton-Valentine leukocidin, a cytotoxin known to be secreted from community-acquired S. aureus, which causes skin and soft tissue infections and pneumonia, is an important virulence factor (12). Detection of pvl gene in a MRSA strain suggests that that strain may be a community-acquired MRSA (13). Data on the origin of the MRSA strains examined in this study were not collected. Community-acquired MRSA strains are often susceptible to clindamycin, tetracycline, trimethoprim-sulfamethoxazole gentamicin, fluoroquinolones, and chloramphenicol, unlike hospital-acquired strains (10). The two MRSA strains we found to have pvl gene were also susceptible to these antibiotics. The result of antibiotic susceptibility and pvl positivity suggest that these strains may be of community origin.
One of the many virulence factors of S. aureus is staphylococcal protein A (spa). This protein binds to immunoglobulin (Ig)G and protects the bacteria from phagocytosis. Changes in the IgG binding region of the spa gene may cause the spa gene not to be detected by classical methods (9). In the multiplex PCR we performed in our study, we could not detect the spa gene, although it was repeated twice in 19 of 126 samples. Thereupon, we performed PCR again for the spa gene on these 19 isolates according to the protocol published by Votintseva et al. (9) in 2014, and we were able to detect the spa gene in all strains (Figure 2).
In many studies conducted in different countries around the world, the presence of mecC gene was investigated in S. aureus growing from human and animal samples, while mecC was not found in some of them (5,7,14). In our country, studies have begun to detect the presence of mecC gene in MRSA strains. In the study of Kılıç et al. (15) in 2015, the mecC gene was investigated in 1,177 MSSA and 523 MRSA strains isolated from humans, and no mecC gene was found in any of the strains. In the study conducted by Cikman et al. (16) in 2018, the presences of mecA and mecC genes were investigated in 494 MRSA strains isolated from humans, and mecA was detected in 315 of them by using PCR, but the presence of mecC was not found. In the study conducted by Sayın et al. (17) on cows with mastitis, the presences of mecA and mecC genes were investigated, and the mecA gene was found in 21 of 150 strains and mecC gene was found in 7 of 150 strains. In this study, in addition to the previous studies, we discussed MRSA strains from a different region. While we detected mecA gene in 126 of 126 MRSA strains isolated from humans in our study, we did not find mecC gene.
Studies show that especially molecular methods that look for mecA gene in MRSA detection and rapid tests for PBP2a’ may miss MRSA due to mecC gene, and these methods should be revised to catch mecC gene (5). Therefore, laboratories should review the methods by which they detect MRSA to make sure that they do not miss a possible mecC positive MRSA with quality control strains. Currently, the number of studies conducted in our country to detect the presence of mecC gene in humans and animals is limited. With these data, it would not be correct to say that mecC gene was not shown in human samples. In order to show the mecC gene status in our country, it is necessary to conduct studies with larger sample numbers from different locations of the country.
Study Limitations
In our study, since the hospital data of the MRSA strains in the stock could not be reached, from which patient samples it was reproduced or outpatient/inpatient interpretation could not be made.
Conclusion
While the mecA gene was detected in all of the 126 MRSA strains studied, no mecC gene was found in any of the strains. These data support the conclusion that MRSA isolates carrying the mecC gene have not yet emerged as an important public health problem in our country.
Ethics
Ethics Committee Approval: Approval was obtained from the Bezmialem Vakıf University Non-Invasive Clinical Research Ethics Committee (02.02.2021/2011-KAEK-42).
Peer-review: Externally peer reviewed.
Authorship Contributions
Surgical and Medical Practices: A.N.C., B.S., M.Z.D., Concept: A.N.C., B.S., M.Z.D., Design: A.N.C., B.S., M.Z.D., Data Collection or Processing: A.N.C., B.S., M.Z.D., Analysis or Interpretation: A.N.C., B.S., M.Z.D., Literature Search: A.N.C., B.S., M.Z.D., Writing: A.N.C., B.S., M.Z.D.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.