Prevalence of MecC Gene Among Methicillin Resistant Staphylococcus aureus isolated from Patients in Ain- shams University hospital

Methicillin-Resistant Staphylococcus aureus (MRsA) is an important cause of healthcare associated infections globally. New mecA homologue (mecC), was first reported in the UK and Denmark. The mecC mediated MRSA is resistant only to Β-lactams antibiotics and is sensitive to other antibiotics. Detecting the prevalence of mecC MRSA provides more options in treatment of MRSA infections. The aim of this study was to prevalence of mecC gene in clinical isolates of MRSA in Ain-Shams university hospitals & to correlate Minimal Inhibitory concentration (MIC) of Oxacillin with the mecC gene expression in MRSA isolates. Fifty MRSA isolates were collected from different intensive care units (ICUs) of AinShams university hospital from April-December 2018. Methicillin resistance was detected by Cefoxitin disc, and antimicrobial susceptibility testing was done for all isolates and its results were interpreted according to Clinical & Laboratory Standards Institute (CLSI) guidelines 2018. Minimal Inhibitory Concentration of Oxacillin was detected using Oxacillin E-test and the results were interpreted according to the manufacturer’s instructions, then Polymerase Chain Reaction was done to detect mecA and mecC genes among MRSA isolates. Fifty isolates were identified as MRSA by Cefoxitin disc out of 163 samples. Twelve isolates were sensitive to Oxacillin while 38 isolates were resistant to Oxacillin. All isolates were positive to mecA gene while only 3 isolates were positive to both mecA and mecC genes. MecC is a new emerging gene responsible for methicillin resistance in staphylococci and was detected in 6 % of the isolates in this study.


INTRODUCTION
Methicillin-Resistant Staphylococcus aureus (MRSA) is considered as one of the most common causes of community and hospitalacquired infections, leading to high morbidity and mortality. MRSA poses a serious problem in hospitals and its detection is crucial for infection prevention and control 1 .
Resistance to beta-lactams antibiotics is conferred by the presence of mecA gene, which encodes a penicillin-binding protein (PBP2'). mecA is part of a mobile genetic element called the "Staphylococcal cassette chromosome (SCC) mec." 2 The report of MRSA carrying a new variant of the mecA gene in 2011 in humans was highly significant. This new variant was named mecC gene. The presence of this gene poses diagnostic problems due to the probability of misdiagnosis of isolates as methicillin-sensitive S. aureus which may affect the results of MRSA surveillance 3 .
The mecC gene shared only 70% DNA identity with the mecA gene. MRSA isolates carrying mecC gene have been reported in different European countries and from several host species 4 .
This mecC MRSA shows a characteristic antimicrobial susceptibility pattern compared to mecA MRSA. Where mecA MRSA displays resistance to both Oxacillin and Cefoxitin while most of mecC MRSA shows resistance to Cefoxitin and susceptibility to Oxacillin 5 .
There is limited available data about the prevalence of MRSA that carry mecC gene in Egypt, hence, this study was performed.

Collection of clinical samples
Different clinical samples were obtained from patients admitted to different ICUs of Ain-Shams university hospitals according to the regulations of scientific research Ethical Committee Faculty of Medicine-Ain Shams University during the period from April 2018 to December 2018. The collected specimens include blood, pus, sputum and swabs from burn and surgical wound.

Identification and antimicrobial susceptibility testing of the isolated organism
Sample collection and identification of the isolated organism were performed by conventional bacteriological methods according to Colle et al, 1996 6 and Cheesbrough, 2009 7 .
For identification of Staphylococcus aureus isolates, the samples were inoculated on Blood agar and Mannitol salt agar, the colonies were identified based on morphology of the isolated colonies, microscopic examination of Gram-stained films, the β-hemolytic effect on blood agar and yellow colonies on mannitol Salt agar.
Then detection of MRSA strains was done by Cefoxitin discs 30µg (FOX) supplied by (Oxoid, Basingstoke, UK) using Kirbey-Bauer disc diffusion method and the results were interpreted according to CLSI guidelines 2018 8 .

Detection of oxacillin minimal inhibitory concentration (MIC)
MIC was done for the Fifty MRSA isolates by using Oxacillin E-test supplied by LIOFILCHEM company, and the interpretation was done according to CLSI guidelines,2018.

Detection of mecA gene and mecC gene by Polymerase chain reaction (PCR)
All MRSA strains were subjected to PCR for detection of mecA and mecC gene, first DNA extraction was done using ultraclean microbial DNA extraction kit supplied by MoBio by QIAGEN, then amplification of mecA gene 9 was performed using the primers forward 5'-AAA ATC GAT GGT AAA GGT TGGC-3' and reverse 5'-AGT TCT GCA GTA CCG GAT TTG C-3' and for mecC gene 4 forward 5′-GAA AAA AAG GCT TAG AAC GCC TC-3′ reverse 5′ GAA GAT CTT TTC CGT TTT CAG C-3′ . PCR conditions for mecA gene 9 Thermal cycler was adjusted to 2 minutes at 95°C for primary denaturation followed by 40 cycles of 45 seconds denaturation at 95°C then 45 seconds at 50°C for annealing, and 1 min at 72°C for extension. The amplified product was identified at 530 base pair (bp) by electrophoresis on 1.5% agarose gels and using 100 bp DNA ladder. PCR conditions for mecC gene 4 Thermal cycler was adjusted to 2 minutes at 95°C for primary denaturation followed by 40 cycles of 45 seconds denaturation at 95°C then 45 seconds at 60°C for annealing, and 1 min at 72°C for extension. The amplified product was identified at 137 bp by electrophoresis on 1.5% agarose gels and using 100 bp DNA ladder ( Fig. 1 & 2).

Data analysis
All statistical procedures were carried out using SPSS version 22.0 for Windows (SPSS Inc, Chicago, IL, USA).

ResUlts
In this work, different clinical samples were collected from 163 patients admitted to different ICUs. From these samples, fifty isolates were identified as MRSA in which 25 isolates were collected from males (50%) and 25 from females (50%). Their age ranged from 18-75, and the mean age was 49.6 ± 13.1 years and all patients were under antibiotic therapy.
The results of antimicrobial susceptibility of MRSA revealed that the highest sensitivity values were found with Trimethoprim/ sulfamethoxazole (SXT), linezolid (LZD), and Chloramphenicol (C) (68% for each antimicrobial agent) while lower sensitivity (66%) was found with Rifampin (RA) followed by Levofloxacin (LEV) (62%). Sensitivity values dropped to 38% with Clindamycin (DA) followed by Doxycycline (DO) and Ciprofloxacin (CIP) with sensitivity in 36% of the isolates. The highest resistance pattern was to Erythromycin (E) and Penicillin (P) as 41 isolates (82%) were resistant to erythromycin and 48 isolates (98%) were resistant to Penicillin (P) as shown in Table (1).
Minimal inhibitory concentration (MIC) of MRSA isolates to Oxacillin was done by using E Test and 12 isolates (24%) were sensitive to Oxacillin while there were 38 isolates (76%) resistant to Oxacillin.
As regards PCR results of determining the presesnce of mecA and mecC genes, all isolates (100%) were positive to mecA gene while only 3 isolates (6%) were positive to both mecA and mecC genes and the isolates that carrying both genes were isolated from blood samples The three isolates carrying both mecA and mecC, two of them were resistant to Oxacillin E-test and one was sensitive, while 47 MRSA isolates carrying only mecA gene, 11 (92%) were sensitive to Oxacillin E-test and 36 (95%) were resistant to Oxacillin E-test.
The results of antimicrobial susceptibility of MRSA carrying both mecA and mecC genes revealed that the highest sensitivity values were found with (CIP), (SXT), (LZD) and (C) agents (66.7% for each antimicrobial agent). Lower sensitivity was found with (LEV), (RA) and (DA) which was 33.3% for each. There was no sensitivity towards other antimicrobial agents as shown in Table (2).

DISCUSSION
MRSA infections are well established in both the healthcare setting and in the community. MecC is a new emerging gene responsible for methicillin resistance in staphylococci. The mecC MRSA produces a characteristic antimicrobial susceptibility pattern different from mecA MRSA as mecA MRSA typically displays resistance to both oxacillin and cefoxitin antibiotic. On the other hand, most of mecC MRSA show resistance to Cefoxitin and are therefore reported as MRSA, although it shows susceptibility to Oxacillin 5 .
Regarding the minimal inhibitory concentration (MIC) using E-test, we found that 12 (24%) isolates were sensitive to Oxacillin and there were 38 (76%) isolates resistant to Oxacillin .on the other hand Saeed et al.in 2014 12 reported (1.2%) strains were sensitive to oxacillin using phenotypic methods in the United Kingdom and Cikman et al. in 2019 13 reported that all tested MRSA isolates were phenotypically resistant to oxacillin and Cefoxitin.
In the current study results of PCR showed that all cases (100%) were positive to mecA gene while only three cases (6%) were positive to both mecA and mecC gene, one of the mecC cases was sensitive to Oxacillin E-test with MIC ≤ 2 and two mecC were resistant to Oxacillin E-test with MIC≥ 4.
One of the first papers published to discuss the emergence of mecC gene in MRSA isolates versus mecA gene was conducted in Denmark 2011 by Garcia-Alvarez 4 who found that mecC gene represents 2.8% of MRSA isolates while in the current study we found 6% mecC gene of MRSA isolates, the author of the previous study contributed this result to rural areas as the detection of mecC gene was linked to livestock. On the other hand, Rania et al. in 2017 14 in Egypt conducted a similar study showed that of 150 isolates (110 MRSA + 40 MR-CoNS) all were subjected to PCR where no MRSA isolates carrying mecC gene were reported detected. This was similar to Ganesan 15 who also did not detect mecC gene in his study. Another study done in Egypt by Khairalla et al. in 2017 16 reported that all the MRSA isolates included in the study contain the mecA gene (n = 34, 100%), while mecC was not identified.
On the other hand Khan and co-workers 17 reported that the prevalence of mecA gene was 54% which is less than reported in this study, they also found 3% mecA negative isolates carrying mecC, while only one MRSA isolate carrying both mecA and mecC genes. also, Aklilu and Hui Ying 18 reported first mecC and mecA positive livestockassociated MRSA in Malaysia, they concluded that out of the 15 mecC positive isolates, 12 were positive for both mecA and mecC genes this concedes with results of the current study concerning the 3 mecC positive were mecA positive, so we can assume that we can find both Journal of Pure and Applied Microbiology mecA and mecC genes in same isolate which needs further studies for confirmation of this finding .

CONClUsiON
In conclusion, this work is one of first report of presence of mecC gene in MRSA isolates in Egypt, mecC gene was detected in 6% of MRSA isolates included in this study and this isolates also carry mecA gene, although this percentage is not alarming, further studies testing for mecC gene in larger scale using different primer sets for mecC, may increase the probability to detect mecC gene especially in rural areas as the presence of mecC is linked to livestock.