Screening for mecA and mecC Gene Carriage among Clinical Isolates of Methicillin Resistant Staphylococcus aureus at a Tertiary Care Hospital: A Cross-sectional Study

1. MBBS Student, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India. 2. Senior Resident, Department of Microbiology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India. 3. Professor and Head, Department of Microbiology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India. 4. Associate Professor, Department of Microbiology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India.

Abstract

Introduction: Staphylococcus aureus (S. aureus) has emerged as one of the most important human pathogen, and has been a leading cause of hospital and community acquired infections. Methicillin-Resistant Staphylococcus aureus (MRSA) carrying the mecA gene is resistant to the majority of β-lactam antibiotics. In 2007, a new S. aureus strain harboring mecA gene homologue, mecC, was found in England which posed diagnostic problems. Accurate and rapid detection of MRSA is required for effective treatment.

Aim: To screen for mecA and mecC genes among methicillin resistant isolates of S. aureus using conventional Polymerase Chain Reaction (PCR) and to associate their presence with Kirby-Bauer disc diffusion and automated Vitek 2 methods.

Materials and Methods: This cross-sectional observational study was conducted in the Department of Microbiology, Victoria hospital, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India, from July to October 2019. A total of 60 non duplicate S. aureus samples were obtained from various clinical samples during the study period. Isolates were subjected to antibiotic susceptibility by cefoxitin disc diffusion and automated Vitek-2. Isolates were screened for mecA and mecC gene carriage using conventional PCR. Descriptive statistics were used for the comparison of data, and appropriate statistical charts were used to present the data.

Results: Among 60 S. aureus isolates, 41 (68.33%) were considered MRSA by conventional Disc Diffusion Method (DDM), and 48 (80%) were considered MRSA by automated Vitek-2. By conventional PCR, only 34 (56.67%) isolates carried the mecA gene, and none of the clinical isolates possessed the mecC gene.

Conclusion: An overall MRSA prevalence of 56.67% was observed by PCR in present study. The mecC gene was not detected in any of the S. aureus isolates. The study indicates the presence of mecA and mecC negative phenotypically identified MRSA isolates. Rather than absolute dependence on the mecA gene as the defining standard in determining MRSA, alternative mechanisms of resistance-presence of mecC, mecB genes, hyperproduction of b-lactamase; can potentially be a knowledge trove for researchers to delve into.

Antibiotic resistance, Cefoxitin disc diffusion, Polymerase chain reaction, Vitek-2

S. aureus has emerged as one of the most important human pathogens, and has been a leading cause of hospital and community acquired infections. It is associated with a variety of infections including septicaemia, pneumonia, wound sepsis, septic arthritis, osteomyelitis and postsurgical toxic shock syndrome with substantial rates of morbidity and mortality. Its impact has been enhanced by the development of MRSA that is resistant to virtually all β-lactam antibiotics [1,2]. According to the Indian Network for Surveillance of Antimicrobial Resistance (INSAR) group, overall prevalence of MRSA is about 40% (3).

The majority of research in this field suggests that resistance in MRSA is conferred by the acquisition of a mobile genetic element, the Staphylococcal Cassette Chromosome (SCCmec), carrying the mecA gene, which encodes an altered Penicillin Binding Protein (PBP)- PBP2a/PBP2'- that has reduced affinity for β-lactam antibiotics. As a result, cell wall biosynthesis in MRSA strains continues even in the presence of otherwise inhibitory levels of β-lactam antibiotics [1,2].

Molecular amplification of the mecA gene is recognised as the gold standard to detect MRSA in the community as these genes are highly conserved among staphylococcal species (4).

In 2007, a new S. aureus strain harboring mecA gene homologue, mecC, was found as an isolate from a bulk tank milk sample in Southwest England, which conferred beta lactam resistance with 70% sequence similarity to mecA gene (5). Subsequently, mecC MRSA have been isolated from 14 different domestic and wild animal species and in a range of infections in humans. These are predominantly skin and soft-tissue infections, but include severe bone infections, nosocomial pneumonia and fatal bacteraemia (1).

Previous studies [6-8] have shown that S. aureus carrying mecC gene were identified as MRSA by Vitek 2 automated system but misidentified as mecA-negative, and therefore methicillin sensitive, by routine PCR and commercial slide agglutination assays for mecA-encoded PBP2a. Thus, the presence of the mecC gene is determined only by molecular methods using specific primers for mecC [6-8]. The report of mecC gene has posed diagnostic problems with the potential to be misdiagnosed as methicillin-sensitive S. aureus, with important potential consequences for individual patients and for the surveillance of MRSA (2).

This study seeks to screen for mecA and mecC genes among methicillin resistant isolates of S. aureus using conventional PCR and to associate their presence with Kirby-Bauer disc diffusion and automated Vitek-2 methods.

Material and Methods

This cross-sectional observational study was conducted in the Department of Microbiology, Victoria hospital, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India, from July to October 2019. Institutional Ethical Clearance (IEC) was obtained (No:BMCRI/PS/82/2019-20).

Sample size calculation: Was done using the formula with

n=z²*p*(1-p)/e²;

n=required sample size; p=prevalence of study (40%); e=margin of error, 12%; z=1.96 at 95% CI.

A total of 60 S. aureus strains isolated from various clinical samples such as pus, blood etc., were used in the study. Clinical specimens were as follows: Pus- 47 (78.33%), Blood- 8 (13.33%), Others- Corneal swab 1 (1.67%), Cerebrospinal Fluid (CSF)- 1 (1.67%), Urine- 1 (1.67%), Lacrimal sac abscess- 1 (1.67%), Nasal swab- 1 (1.67%). Confirmation of strains was done using standard tests like catalase, slide and tube coagulase, and growth on Mannitol salt agar (9). Antibiotic susceptibility testing was carried out using cefoxitin disc diffusion test (9) and Automated Vitek-2 methods (10).

Cefoxitin disc diffusion test: All 60 S. aureus isolates were subjected to a cefoxitin disc diffusion test using a 30 μg disc. A 0.5 Mc Farland standard suspension of the isolate was made and lawn culture was done on Muller Hinton Agar (MHA) plates. Plates were incubated at 37°C for 18 hours and zone diameters were measured (9). Zone size was interpreted according to Clinical Laboratory Standard Institute (CLSI) guidelines 2019 criteria. Isolates with an inhibition zone diameter of ≤21 mm were reported as MRSA and ≥22 mm were considered Methicillin-Susceptible S. aureus (MSSA) (11).

Automated Method: Antibiotic susceptibility to oxacillin and cefoxitin among clinical isolates of S. aureus was carried out using Vitek-2- AST-P628 susceptibility cards (bioMerieux). Suspensions of cultures were made and loaded into the test cards according to manufacturer’s instructions (12),(13). As per CLSI 2019 isolates with cefoxitin screen positive and oxacillin MIC ≥4 μg/mL were considered as MRSA and isolates with cefoxitin screen negative and oxacillin MIC ≤2 μg/mL were regarded as MSSA (11).

DNAExtraction by boiling lysis method: A fresh 24 hour bacterial culture on chocolate agar was taken. Only 5-6 bacterial colonies were picked using a straight wire and were suspended in 50 μL nuclease free water. They were then boiled at 99°C for 10 minutes and centrifuged at 13000 rpm speed for one minute and 5 μL of the supernatant was used as template DNA.

Detection of mecA and mecC genes using conventional PCR:

The primer sequences and product length for mecA and mecC are as follows:

mecA-- Forward 5’- TCCAGATTACAACTTCACCAGG-3’ and

Reverse- 5’-CCACTTCATATCTTGTAACG-3’ (162 bp) (8);

mecC- Forward -5’-GAAAAAAAGGCTTAGAACGCCTC-3’ and

Reverse-5’-GAAGATCTTTTCCGTTTTCAGC-3’ (138 bp) (8)

Reaction conditions for both the genes: Amplification was performed with the following program: 15 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, one minute at 59°C, and one minute at 72°C, with a final 10 minutes elongation step at 72°C (8). PCR products (5 μL) were analysed using 2% agarose gel electrophoresis with ethidium bromide dye under ultraviolet transilluminator.

Statistical Analysis

Descriptive statistics was used for the comparison of data and appropriate statistical charts were used to present the data.

Results

A total of 60 S. aureus strains were isolated from various clinical specimens. By conventional DDM, 41 isolates (68.33%) were regarded as MRSA and 19 (31.67%) of them were regarded as MSSA (Table/Fig 1). By automated Vitek-2 method, 48 (80%) isolates were regarded as MRSA and 12 (20%) isolates were regarded as MSSA. Conventional PCR is considered as the gold standard method of detection of MRSA [Table/Fig-2,3]. Among 60 clinical isolates of S. aureus 34 (56.67%) were positive for mecA gene, remaining 26 (43.33%) isolates were mecA negative.

Out of 41 isolates identified as MRSA by DDM, only 33 (80.48%) of them possessed mecA gene. Also, one isolate, phenotypically identified as MSSA possessed mecA gene. Out of 48 isolates identified as MRSA by automated Vitek 2 only 34 (70.88%) of them possessed the mecA gene. Among 60 clinical isolates of S. aureus, none of them possessed the mecC gene which includes the category of mecA negative phenotypically identified MRSA isolates (Table/Fig 4). Comparative data and diagnostic performances of different methods in detection of MRSA have been shown in (Table/Fig 5),(Table/Fig 6).

Discussion

The MRSA is a major pathogen associated with severe nosocomial infections and due to its multidrug resistance, the treatment options are limited. Accurate and early detection of methicillin resistance is of immense importance in the prognosis of infections caused by S. aureus.

Identification of the mecA gene is the major evidence (gold standard) for the detection of MRSA isolate. This statement was approved by many researchers all over the world: in India, the USA, England, Japan, Spain etc., (4). Phenotypic methods of detection of MRSA like conventional disc diffusion and automated Vitek-2 methods have been compared to the gold standard mecA gene detection by PCR in many studies. A study by Anand KB et al., all the 32 isolates detected as MRSA by cefoxitin disc diffusion possessed the mecA gene (9). A study by Thampi DK et al., in a tertiary care centre highlighted that both the methods gave results in agreement with PCR (12). Another study by Madhavan A et al., reported two false negative isolates by disc diffusion, whereas they were found to be MRSA by Vitek 2 (13). Both the methods reported high concordance with PCR. Most of the studies around the world have reported disc diffusion by cefoxitin as an accurate surrogate marker and automated Vitek 2 method as a time saving approach for early detection [12,13].

However, in present study, only 33 of 41 (80.48%) isolates and 34 of 48 (70.88%) identified as MRSA by cefoxitin disc diffusion and automated Vitek-2 methods respectively tested positive for mecA gene. This indicates the presence of mecA negative phenotypically identified MRSA isolates. One isolate identified as MSSA by cefoxitin disc diffusion possessed mecA gene, whereas, it was reported as MRSA by Vitek-2. The false negative result can be explained by the fact that mecA positive strains differ in their level of resistance, i.e., resistance is usually heterogeneous with only a few cells (one in 104/mm3 or 106/mm3) expressing the phenotype (13). Also expression of drug resistance depends on the other conditions like growth media, temperature, osmolarity (14). Other studies like in Sudan (4), Turkey (15), Egypt (16) have not demonstrated the mecC gene.

A study in Iraq, demonstrated the presence of 22 (30%) out of 27 mecA negative isolates. Many studies around the world have also highlighted mecA negative phenotypically identified MRSA isolates as mentioned in (Table/Fig 7) [4,15-19]. A study by Pal S et al., in Uttarakand, out of 196 isolates determined as MRSA by cefoxitin disc diffusion only 164 showed the presence of mecA gene. 32/196 (16.3%) tested negative for mecA (19). Few studies have demonstrated the mecC gene amongst such isolates. A study by Bali N et al., in Kashmir demonstrated the presence of mecC gene in India for the first time with 3.9% of 102 MRSA isolates having the mecC gene (20). A study by Khan AA et al., in Pakistan also demonstrated the mecC gene in 3% of MRSA isolates and both the genes in one MRSA isolate (21). Also, a previous study in Nigeria reported the complete absence of mecA genes and five major SCCmec types in randomly selected 36 phenotypically identified MRSA (22).

The absence of detection of mecA and mecC genes among phenotypically identified MRSA highlights the presence of other factors contributing to the resistance. Gene instability and primer design could be proposed as one of the reasons [22,23]. Olayinka BO et al., reported the loss of mecA gene during storage at - 80° celsiuslayinka. The study included collecting isolates over a period of time, preliminary characterisation, storing and subculturing over a considerable length of time before final transportation for molecular characterisation (22). It may be related to borderline (low level)- methicillin resistant strains of S. aureus (BORSA) that lack the mecA gene but identified as MRSA by phenotypic methods. This happens generally due to hyper production of Type A β-lactamase (22). Heterogenous methicillin resistant strains with thicker and rougher cell walls and hence more Penicillin-Binding Protein 2 (PBP2) could produce methicillin resistance without the mecA gene (24). Another new discovery includes the mecB gene. The mecB gene has been found as a part of plasmid, distantly related to a macrococcal plasmid which provides MRSA. Routine phenotypic methods for susceptibility testing cannot distinguish between methicillin resistance determinants thus, mecB encoded methicillin resistance can remain undiscovered (25). These findings provide clear evidence of mechanisms other than mecA and mecC genes responsible for beta-lactam, resistance of MRSA and molecular methods alone are not enough for confirmed characterisation of MRSA isolates (5).

Limitation(s)

This study, however, is subject to several limitations. Authors were unable to further research into the rationale behind mecA and mecC negative phenotypically identified MRSA strains due to time and resource constraints.

Conclusion

Present study showed an overall MRSA prevalence of 56.67% by PCR. Phenotypic methods of MRSA detection are good alternatives to molecular methods in resource restrained setup. The mecC gene was not detected in any of the S. aureus isolates. However, authors believe that this study will encourage further research into unanswered questions concerning the origin of the mec homolog and the epidemiology of Staphylococcal isolates harboring mecC in India. It is crucial for diagnostic laboratories to understand, that absolute dependence on the mecA gene as the defining standard in determining resistance of S. aureus, has become the subject of distrust by many researchers. Alternative mechanisms of resistance have to be further explored and taken into consideration while testing.

DOI and Others

DOI: 10.7860/JCDR/2023/62215.17812

Date of Submission: Dec 12, 2022
Date of Peer Review: Feb 04, 2023
Date of Acceptance: Mar 27, 2023
Date of Publishing: May 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: The study was funded by Rajiv Gandhi University of Health Sciences (RGUHS)- Undergraduate Research program.
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. Yes

PLAGIARISM CHECKING METHODS:
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ETYMOLOGY: Author Origin