First Report of the Emergence of mecC Gene and CC8/ST239 Tigecycline-Resistant Staphylococcus aureus Clonal Lineage Isolated from Chronic Suppurative Otitis Media

Background: Staphylococcus aureus is one of the most significant bacteria involved in ear infections. However, insights into the molecular attributes of S. aureus collected from patients with chronic otitis media have yet to be reported in Iran. Objectives: The objective of this study was to assess the molecular characteristics of S. aureus isolated from patients with chronic otitis media. Methods: Atotalof 55 S.aureus strainsretrievedfrompatientswithchronicotitismediawereanalyzedbythediskdiffusionmethod and polymerase chain reaction (PCR) to identify the nucA gene. Isolates were genetically classified using the coagulase typing method. S.aureus proteinA( spa )typing,staphylococcalcassettechromosome mec (SCC mec )typing,andmultilocussequencetyping (MLST) were performed on isolates with resistance to specific antibiotics. Results: Overall, out of 55 S. aureus isolates, resistance to mupirocin, fusidic acid, and tigecycline was identified in 12.7%, 5.4%, and 3.6% of isolates, respectively. Fusidic acid-resistant isolates belonged to ST5-SCC mec II/t002/ coa II. Two tigecycline-resistant isolates belonged to CC8/ST239-SCC mec III/t234/ coa VIII. One positive mecC isolate belonged to the CC/ST130-SCC mec XI/t843/ coa III clone. Isolates with the iMLSB phenotype belonged to CC/ST80-SCC mec IV/t044/ coa II (4 isolates), CC8/ST239-SCC mec III/t388/ coa VI (3 isolates), and CC8/ST8-SCC mec IV/t008/ coa III (1 isolate). Conclusions: Our results indicated that S. aureus isolated from patients with chronic otitis media possesses a unique molecular profile with a high percentage of resistance to multiple medications. These findings suggest that resuming the molecular analysis to improve the control and prevention of ear infections related to S. aureus is necessary.


Background
Chronic suppurative otitis media (CSOM) is characterized by middle ear effusion without symptoms of acute inflammation.According to the evidence, a high incidence of CSOM was reported in the developing countries.The CSOM is mainly caused by bacterial middle ear infection (1).Some studies indicated Pseudomonas aeruginosa as the prevalent cause of CSOM, while other researchers displayed Staphylococcus aureus as the most important bacteria.In recent decades, simultaneous resistance to multiple drugs in S. aureus isolated from CSOM has become a severe threat to global health (2,3).As methicillin use increases, methicillin-resistant S. aureus (MRSA) is increasingly reported.Methicillin resistance is mediated by mecA and much less by mecC (4).Up to now, several molecular typing methods have been employed for genotyping S. aureus strains, including pulsed-field gel electrophoresis, staphylococcal cassette chromosome mec (SCCmec) typing, agr typing, protein A gene (spa) typing, multilocus sequence typing (MLST), and coagulase gene (coa) typing (4,5).The coa typing is a multiplex polymerase chain reaction (PCR)-based method that is cost-effective, rapid, easily interpretable, and appropriate for identifying the genetic relationships among S. aureus isolates (5).Only a few studies are available worldwide addressing the genotyping of S. aureus isolated from ear infections (3).In Iran, several studies have focused on the occurrence and phenotypic characteristics of S. aureus isolated from ear infections, but little data has been published on the genetic variability of these isolates (6)(7)(8).

Objectives
The current research was designed to evaluate the antimicrobial resistance profile and molecular characteristics of S. aureus isolates for CSOM based on coa gene polymorphism analysis.

S. aureus Identification
This research was carried out during January 2020 to December 2022 in a teaching hospital of Shahid Beheshti University of Medical Sciences, Iran.Informed consent was received from all participants.Purulent discharge was collected from the middle ear via a sterile swab.The patients had not taken any antibiotics for three weeks prior to the visit and had no history of hospitalization.
The collected purulent swabs were immediately cultured on blood agar (HiMedia, Mumbai, India) and preliminarily recognized as S. aureus by routine techniques.
All phenotypically confirmed S. aureus isolates underwent PCR for the nuc gene detection and final confirmation (9).

Detection of Resistance and Toxin-encoding Determinants
After the genomic DNA extraction using the phenol-chloroform technique, isolates were screened for the existence of the toxin genes, including pvl, eta, etb, and tst, by PCR (10,12).The mupA, fusA, mecC, and mecA genes were detected by PCR as described elsewhere (4).

Molecular Typing
A multiplex PCR-based method with four sets (A-D) was used to analyze the coa types (I-X) with specific primers and PCR conditions introduced by Hirose et al. (5).All isolates were characterized by coa typing while S. aureus protein A (spa) typing, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST) was performed on the mecC-positive, fusidic acid, tigecycline, and mupirocin-resistant isolates according to the same conditions published earlier (11).

Discussion
The present study revealed that the incidence of fusidic acid resistance was 5.4%, harboring fusA, and belonged to the ST5-SCCmec II/t002/coaII clone.This percentage is lower than the reported rates in other countries, such as Ireland (19.9 %) (13), Kuwait (9.3%), and Germany (10.3%) (13,14).In a meta-analysis in 2021, the low prevalence rate of fusidic acid resistance was noted in S. aureus isolates (0.5%).The incidence of fusidic acid-resistant S. aureus isolates was different in the earlier investigations performed in Iran by Zamani et (17).The higher prevalence rate of fusidic acid resistance in the present study compared to the earlier reports in Iran may be related to the unrestricted prescription of fusidic acid, use of this antibiotic during the initial treatment without susceptibility testing, diverse attitudes towards antimicrobial protocols, and the circulation of fusidic acid-resistant types within the hospitals.Similar to our findings, Chen et al. in Taiwan found that the most prevalent type of fusidic acid-resistant S. aureus strains was found to be ST5-SCCmecII/t002 (29%) and ST239-SCCmecIII/t037 (62%) (18).The same research by den Heijer et al. reported the presence of t002 and t005 types carrying fusA in fusidic acid-resistant S. aureus strains from nine European countries (19).It can be concluded that the common lineages of S. aureus strains resistant to fusidic acid may be circulating from country to country.Therefore, a molecular epidemiological map of these isolates should be supervised worldwide.
The present study revealed an occurrence rate of mecC in 1.8% of MRSA isolates.In line with findings from previous research, this gene has been detected in S. aureus in Pakistan (26), Austria (27), Slovenia (28), and Switzerland (29).Similar to our findings, CC/ST130-SCCmecXI/t843 carrying the mecC gene was also reported earlier in the UK and Denmark as the most prevalent carrying mecC clone among clinical strains (30).In addition, Dermota et al. in Slovenia reported a prevalence of 1.5% for this gene in MRSA isolates possessing the mecC gene belonging to CC/ST130 (28).In our earlier research, CC/ST599 was reported as a mecC-positive S. aureus isolate (4).
Different findings were reported in Malaysia (5.5%) (31), Libya (3.6%) (32), and Iran (6.6%) (33).Furthermore, some studies have documented the presence of mecC in MRSA strains recovered from Taiwan, Germany, China, Italy, Canada, France, Nigeria, and Poland (34).It can be inferred that inadequate governance of antibiotic administration strategies, improper policies, and extensive use of antibiotics, which likely increase the chance of genetic variations and acquisition of tigecycline-resistance genes, may be different causes for the emergence of tigecycline-resistant MRSA isolates.For all these reasons, the high prevalence and genetic variability of tigecycline-resistant MRSA isolates might also pose a severe risk to public health, suggesting the need for further attention to the detection and genetic diversity of these isolates.Our results indicated that all MRSA isolates resistant to tigecycline belonged to the CC8/ST239-SCCmecIII/t234/coaVIII clone.In a similar study in Brazil, Dabul et al. identified S. aureus strains resistant to tigecycline belonging to the ST105-SCCmecII clone (35).Nonetheless, CC8/ST239 clone resistance to tigecycline has been reported in S. aureus from Switzerland, Spain, UK, Kuwait, Japan, Australia, and China (34).This study found a low to moderate prevalence of isolates with iMLSB phenotype (14.5%).Different rates have also been reported from S. aureus isolates from Iran (10.9%) (11), Jordan (76.7%) (36), Nepal (21%) (37), and Brazil (7.9%) (38), which suggested that it might be a remarkable phenomenon influenced by the excessive usage of macrolides, regional locations of the study population, infection prevention protocols in healthcare facilities, and the prior history of antibiotic usage in patients.In the present study, the iMLSB phenotype (14.5%) belonged to the CC/ST80-SCCmecIV/t044 (4 isolates), CC8/ST239-SCCmecIII/t388 (3 isolates), and CC8/ST8-SCCmecIV/t008 (1 isolate) clones.The CC8 clone is described to be a prevalent iMLSB phenotype MRSA in Iran (11).In addition, Goudarzi et al. reported that isolates with iMLSB phenotype were observed in CC88/ST239 (13.3%),CC/ST22 (4%), and CC/ST30 (4%) clonal lineages (12).

Conclusions
Our study on the molecular characterization of S. aureus obtained from CSOM cases indicates the occurrence of MDR S. aureus, which significantly limits the availability of effective antimicrobial treatments.These findings confirmed the dissemination of specific clonal lineages in mecC-positive, inducible, mupirocinand tigecycline-resistant S. aureus strains.Further investigation into these emerging clones would improve understanding of the molecular epidemiological map and their resistance profile trends.Future studies that monitor the genetic diversity of lineages and their prevalence among similar populations are required.

Footnotes
Authors' Contribution: CH. A. conceived and designed the study and drafted the manuscript.M. G. participated in designing the study, performed parts of the statistical analysis, and helped to draft the manuscript.Z. R. re-evaluated the clinical data, revised the manuscript, performed the statistical analysis, and revised the manuscript.P. B. collected and interpreted the clinical data and revised the manuscript.All authors read and approved the final manuscript.

Conflict of Interests:
The authors have no conflict of interest.

Data Reproducibility:
The dataset presented in this study is available on request from the corresponding author during submission or after publication.The data are not publicly available due to maintaining confidentiality.

Ethical Approval:
This study was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran, under the ethical code of IR.SBMU.MSP.REC.1401.366.

Funding/Support:
This work was supported by a fund from the Research Deputy of Shahid Beheshti University of Medical Sciences, Tehran, Iran (Grant No. 43003263).The funding agency has no role in the design of the project, work execution, analyses, interpretation of the data, manuscript writing, and submission.Informed Consent: Informed consent was received from all participants.

Table 1 .
Characteristics of the 55 MRSA Strains Obtained from CSOM Cases