MRSA Clinical Isolates Harboring mecC Gene Imply Zoonotic Transmission to Humans and Colonization by Biofilm Formation

1Department of Microbiology, Faculty of Life Sciences, University of Central Punjab, Lahore 2Faculty of Sustainable Agriculture, Universiti Malaysia Sabah, Sandakan Campus, Locked Bag No.3, Sandakan 90509, Sabah, Malaysia 3Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150, Malaysia Article Information Received 18 May 2021 Revised 12 December 2021 Accepted 19 January 2022 Available online 08 June 2022 (early access)

S taphylococcus aureus generally colonizes humans and animals. An estimated 25-50% of the general population are nasal carrier and 10-20% are persistent carriers of S. aureus (Parveen et al., 2020). Over the last few years, methicillin-resistant Staphylococcus aureus (MRSA) has increased significantly in community and hospital-related settings. Asia has the highest number of MRSA infections worldwide (Chen and Huang, 2014). MRSA strains are potentially lethal that mediate virulence by adhesions, production of enzymes, immune modulators, and toxins (Watkins et al., 2012). Antibiotics resistance in MRSA isolates is a global concern. It typically takes place as a spontaneous genetic mutation or may acquire a genetic material such as transposon, integron, plasmid, or gene cassette. MRSA isolates are broadly resistant to multiple drugs; aminoglycosides, fluoroquinolones, chloramphenicol, trimethoprim/sulfamethaxazole, macrolides, and β lactamases due to the presence of mecA gene on these isolates (Taj et al., 2010). Molecular epidemiological studies have revealed that community acquired CA-MRSA and hospital-acquired HA-MRSA are phenotypically and genotypically distinct (Watkins et al., 2012). MRSA isolates carry the mecA gene which encodes for methicillin-resistance binding proteins (PBP2a) (Harkins et al., 2017). The mecA gene mediates methicillin resistance that is transmitted by the staphylococcal cassette chromosome mec (SCCmec) with a size of 21-67 kbp (Nezhad et al., 2017). MRSA isolates that colonize and infect various animals are an important cause of zoonotic O n l i n e

F i r s t A r t i c l e
infections in humans (Tariq et al., 2020). Such MRSA strains harbor novel homolog mecC of mecA. S. aureus produces an extracellular thermostable nuclease, encoded by nuc gene, which is one of the most distinguishing and successful characteristics that might be used for distinguishing S. aureus from other Staphylococcus spp. This suggests that nuc gene is a specific marker gene and PCR is a useful method for identifying this gene in S. aureus (Sahebnasagh et al., 2014). nuc gene is strongly associated with the production of enterotoxin and it can be considered as an indicator of infection with enterotoxin producer S. aureus (Karimzadeh and Ghassab, 2020). In 2011, a new methicillin resistance determinant, the mecC gene, was identified in S. aureus isolates recovered from humans and dairy cattle. The mecC determinant was able to produce low-level resistance to β-lactam antibiotics such as cefoxitin and oxacillin.
Staphylococci are associated with biofilm-mediated infections. Biofilms are linked to numerous human diseases such as chronic lungs, skin lesions, and ear infections. Biofilms tend to colonize medical devices such as catheters and implants. According to the National Institute of Health (NIH), more than 80% of all microbial infections develop biofilms. These types of infections are difficult to diagnose and treat as they are the leading cause of increased hospitalization, accelerated healthcare expenses, and expanded mortality and morbidity (Rabin et al., 2015). Within a biofilm, bacteria communicate with each other by the production of chemotactic particles or pheromones, a phenomenon called quorum sensing (Hassan et al., 2011;Javed et al., 2020). These biofilm-forming bacteria can be up to thousand fold more resistant to antibiotic treatment than planktonic bacteria (Gebreyohannes et al., 2019). Due to the complex pathogenicity and therapeutic importance, the present study is conducted for the molecular detection of mecA, mecC, and nuc genes and investigation of biofilm formation among MRSA clinical isolates.

Materials and methods
A total of 208 different samples including pus, wound swabs, throat swabs, fluids, breasts abscess, ear swabs, urine, and blood were collected. The samples were inoculated onto blood, mannitol salt, and CLED agar. The colonies of S.aureus were identified using Gram staining and biochemical tests (catalase, oxidase, DNase, and coagulase). Phenotypic identification of methicillin resistance was achieved by subjecting the isolates to the cefoxitin disk diffusion test with 30 μg disk according to Clinical and Laboratory Standards Institute (CLSI Guidelines, 2017).
Vancomycin E-test was performed using a minimum inhibitory concentration (MIC) strip for vancomycin antibiotics susceptibility (VA .016-256 μg/ml) following CLSI Guidelines (2017). The strip was placed on MHA having lawned growth of MRSA incubated at 37 o C for 24 h with cefoxitin disk.
The phenotypic production of biofilm in all MRSA isolates was determined by culture in Congo red agar (CRA) plates. The MRSA strains were inoculated on the prepared medium and incubated aerobically for 48 h at 37˚C. The morphology and color of the colonies were noted for biofilm formation.
For molecular identification of MRSA resistance genes DNA was extracted from MRSA positive isolates using the standard method described in the Wizprep ™ GDNA mini kit (Seongnam, Korea). DNA extraction results were confirmed by gel electrophoresis. The three resistance genes of mecA, mecC and nuc genes were amplified using primers sequence of mecA

F -A A A A T C G A T G G T A A A G G T T G G C R-A G T T C T G C A G TA C C G G AT T T T G C , mecC F -G A A A A A A A G G C T T A G A A C G C C T C
R-GAAGATCTTTTCCGTTTTCAGC and nuc gene F -G C G AT T G AT G G T G ATA C G G T T R -A G C -CAAGCCTTGACGAACTAAAGC. The product size of these genes was 533bp, 138bp, and 278bp, respectively.
Sequencing was done using commercial sequencing services from Macrogen (Seoul, Korea). Bioinformatics tools (NCBI-nBLAST) were used to determine the similarity index of sequences. MEGA version X software was used to construct the phylogenetic tree by the neighbor-joining method.

Results and discussion
In the present study out of a total of 208 isolates, 100 isolates were identified as MRSA by phenotypic and genotypic identification methods. The prevalence of MRSA was determined as 48% (100/208). In another study conducted by Khan et al. (2020) in Islamabad, Pakistan MRSA prevalence was 65% which is higher than our O n l i n e
All the 100 MRSA isolates were sensitive to vancomycin antibiotic drug with a mean minimum inhibitory concentration of 54μg/ml (Supplementary Fig.  1).
20% of the isolates indicated brown colonies (weak biofilm producers), 80% isolates showed red colonies (non-biofilm producers) and none of them depicted black colonies (strong biofilm producers). The results of biofilm formation were different from the previous study which was done by Haddad et al. (2018). They reported that 46.5% and 53.5% of isolates were respectively strong and moderately biofilm-formers. Another study reported that 53.8% of MRSA isolates exhibited moderately attached biofilms and 28.5% of isolates were non-biofilm producers (Smith et al., 2008). Based on the findings of the present study it was suggested that the susceptibility to methicillin and biofilm formation is not associated with each other ( Supplementary Fig. 2).
Molecular analysis revealed that all the 100 MRSA isolates were positive for both mecA and nuc genes while only 3% (3/100) isolates were positive for the mecC gene. All mecC genes positive MRSA were also positive for mecA gene and detected in pus samples only. The results of our study were parallel to the study conducted by (Pramodhini et al., 2011;Skov et al., 2013). These studies also conferred 100% sensitivity and specificity of cefoxitin with mecA gene. The detection of the mecA gene is considered as the gold standard for identification of MRSA by PCR and all the isolates carry the mecA gene on the genome as described by Brown et al. (2005). The prevalence of the mecC gene was 3% as only three MRSA isolates showed positive results for the mecC gene by PCR. The results of our investigation are comparable to previous studies on the detection of mecC gene-positive MRSA isolates. A similar prevalence rate of mecC gene was reported in other regions such as 2% in Austria (Kerschner et al., 2015) and 3% in Islamabad Pakistan (Khan et al., 2020). The presence of mecC gene in MRSA highlights the zoonotic transmission of the organisms due to frequent contact of people with animals. The present study indicates that all the 100 MRSA isolates were positive for nuc gene which is parallel to the previous study conducted by Elshimy et al. (2018) and Amin et al. (2020). They confirmed the presence of nuc gene by PCR amplification in all 166 and 50 MRSA isolates, respectively.
NCBI blast analysis showed 96-99% similarities to already submitted sequences in the NCBI data bank. The tree showed an inter-relationship of strain with closely related previously reported S.aureus strains.

Conclusion
Multidrug-resistant and mecC gene-positive MRSA

O n l i n e F i r s t A r t i c l e
isolates are rapidly emerging in Pakistan. Owing to the rapid emergence of mecC gene-positive MRSA isolates with zoonotic transmission to humans and its therapeutic and diagnostic importance; the mecC gene-positive MRSA clinical isolates can pose serious healthcare problems in the future. It needs immediate attention. Therefore, the mecC gene should be detected with the mecA and nuc gene for the identification of MRSA clinical isolates. It also requires early identification of biofilm formation and necessary interventions for its effective treatment and control.

Funding
No funding was obtained for this work

Supplementary material
There is supplementary material associated with this article. Access the material online at: https://dx.doi. org/10.17582/journal.pjz/20210518040546

Statement of conflict of interest
The authors have declared no conflict of interest.