Comparative evaluation of phenotypic methods to detect methicillin resistance with mecA gene detection by PCR in Staphylococcus aureus

Introduction

Staphylococcus aureus is a frequently encountered bacterial agent in clinical settings, causing a variety of suppurative infections. It can thrive in diverse environments and, as a component of the normal human microbiota, establishes itself in regions such as the nostrils, perineum, underarms, and groin. With the potential to spread, particularly in hospitals, it has developed resistance to antimicrobials such as methicillin.1

Methicillin-resistant Staphylococcus aureus (MRSA) remains a substantial infection risk in both hospital and community settings. Over time, it has grown into a global problem, with prevalence ranging between 13% and 74%.2 In India, the overall prevalence of MRSA stood at 37% with a combined prevalence of 49% in hospital environments and 27% in community settings from 2015 to 2019.3 Infected patients in hospitals serve as conduits for MRSA strains to spread.

The main factor contributing to methicillin resistance is the presence of an altered penicillin-binding protein (PBP2a) that exhibits reduced affinity for most semi-synthetic penicillins Various phenotypic methods, such as the cefoxitin disc diffusion test, are used to detect MRSA in the laboratory. Cefoxitin is preferred over oxacillin as it is a potent activator of the mecA regulatory system. It is also highly effective in identifying borderline oxacillin-resistant Staphylococcus aureus (BORSA) strains and exhibits accuracy comparable to PCR.4, 5, 6 This test, along with other methods such as oxacillin screen agar, oxacillin MIC, cefoxitin MIC, latex agglutination tests, CHROM agar, and automated systems like VITEK, aids in the accurate detection of MRSA.7, 8

Although phenotypic methods are simpler, genotypic methods, such as the mecA polymerase chain reaction assay, are considered as the reference standard due to their 100% sensitivity. However, the genotypic method is costly and restricted to reference centres.9 Errors in methicillin resistance detection can have serious clinical consequences, highlighting the importance of accurate and definitive methods.

The objective of this study was to evaluate and compare conventional methods for MRSA detection with the molecular approach of detecting mecA gene through PCR for accuracy, sensitivity and specificity. The main aim was to identify the best method or combination suitable for regular use in clinical laboratories. Additionally, the secondary objective was to determine the proportion of MRSA isolates originating from hospital and community sources. 

Materials and Methods

A cross-sectional study was carried out in the Department of Microbiology at a tertiary care hospital in Thiruvananthapuram, spanning a duration of a year and a half, commencing in May 2021, after receiving ethics committee approval.

MecA gene detection by polymerase chain reaction 6, 10, 13, 14, 15

The PCR assay to detect the mecA gene was conducted at the Division of Pathogen Biology, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, Kerala.

The positive control was Staphylococcus aureus ATCC 43300 (MRSA) while the negative control was ATCC 25923 (MSSA). The chromosomal DNA of the isolates was extracted by a simple lysis method. An isolated single colony was inoculated in 1 ml of Luria Bertani Broth (LB Broth Difco) in small Eppendorf tubes and incubated overnight at 37°C. The tubes with turbid broth are centrifuged at 6000 rpm for 5 minutes. The supernatant is discarded, and the cell pellet is obtained. The pellet is then resuspended in 400 μl of sterile nuclease-free water. This is heated at 85 for 15 minutes in a water bath and immediately transferred to -20, which causes cell lysis.

Master mix of a single reaction: A 20μL PCR reaction consisted of PCR buffer (2μL), MgCl₂ (2μL), dNTPs (1.5μL), MecA F primer (0.5μL), MecA R primer (0.5μL), Taq polymerase (0.3μL), Template DNA (1μL), and MilliQ H₂O (12.2μL).

The mecA F primer (5'-AAAATCGATGGTAAAGGTTGGC-3'), which corresponds to nucleotides 1282 to 1303, and the mecA R primer (5'-AGTTCTGCAGTACCGGATTTGC-3'), which is complementary to nucleotides 1581 to 1598 within the coding frames, were used for the amplification of the 533-base pair (bp) fragment of the mecA gene.(Figure 1) These primers were taken from a published sequence by Nam et al.15 The PCR technique included a 5-minute denaturation stage at 95°C, followed by 30 amplification cycles. Each cycle consisted of a 60-second denaturation at 94°C, a 60-second annealing at 55°C, and a 90-second extension at 72 °C. The programme ended with a 10-minute extension at 72 degrees Celsius. The PCR products are observed on a 1% agarose gel containing ethidium bromide dye under a UV transilluminator (Gel Doc, Bio-Rad US). A 100-base-pair DNA ladder serves as the molecular-weight size marker.

Figure 1

Result of PCR amplification of mecA gene

L – 100bp Ladder

Lane 1 – S. aureus ATCC 43300 (mecA positive)

Lane 2 to 12 – MRSA strains (533bp product)

Lane 13 to 16 – MSSA strains (mecA negative)

Lane 17 – S. aureus ATCC 25923 (mecA negative)

Results

The study included 112 MRSA isolates detected by cefoxitin disc diffusion (CDD) in a variety of clinical specimens. These isolates were mostly from male patients (64.3%), with the majority falling into the 41–50 age range (25.9%). Of the total 112 isolates detected by CDD, 48.7% were obtained from the department of surgery, 23.9% from the orthopaedics department, and 10.6% from ENT. Thus, these three surgical specialties contributed to 83.1% of the total number of isolates included in the study. 61 (54.5%) isolates were from patients admitted to the hospital and 51 (45.5%) from patients consulted in the outpatient department. Notably, pus samples accounted for 79.49% of all MRSA isolates, followed by tissue samples (10.7%). (Table 1)

Out of 112 cefoxitin-resistant S. aureus isolates, 100 isolates had a MIC of ≥4 μg/ml with the Oxacillin E test, and 99 showed greenish-yellow colonies on MRSA CHROM agar. PCR assay for mecA gene identified 101 (90.2%) mecA positive and 11 (9.8%) mecA negative isolates. Out of 101 mecA-positive isolates, the Oxacillin E strip and MRSA CHROM agar accurately identified 99 and 97 isolates as MRSA, respectively. Each test had a sensitivity of 98% and 96.03%. The positive predictive value, negative predictive value and accuracy of Oxacillin E strip were found to be 99%,83.3% and 97.3% respectively and that of MRSA CHROM agar were found to be 98%, 69.23% and 95% respectively.(Table 2, Table 3)

Among 11 mecA negative isolates, one was incorrectly identified as MRSA by the Oxacillin E test and two by MRSA CHROM agar. Consequently, the specificity of each of the test were 91% and 82%.

Susceptibility to other class of antibiotics

Maximum resistance was seen to Erythromycin (83.9%), followed by Ciprofloxacin (72.3%). All 112 isolates (100%) demonstrated sensitivity to vancomycin and linezolid. Vancomycin sensitivity was demonstrated using vancomycin screen agar.11 Additionally, 97.3% of isolates were sensitive to rifampicin, 95.5% to tetracycline, 93% to clindamycin, and 88.4% to cotrimoxazole.11.6% of isolates were D test positive (inducible MLS_B (iMLS_B) phenotype).

Table 4

Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of phenotypic methods

Test	Sensitivity%	Specificity%	PPV%	NPV%	Accuracy%
Oxacillin E strip	98	91	99	83.3	97.3
MRSA CHROM agar	96.03	82	98	69.23	95

Table 5

Distribution of HA- MRSA cases based on predisposing factors

Predisposing factors	Frequency (Out of 45 HA MRSA isolates)	Percentage%
H/O surgery recently	41	91.1
Hospital admission within one year	15	33.3
Indwelling medical devices or catheters	26	57.7
Chronic renal/liver disease	3	6.6
Haemodialysis	1	2.2

Figure 2

MRSA detection by Cefoxitin disc diffusion method

Figure 3

Oxacillin E strip: MIC 12 μg/ml

Figure 4

MRSA CHROM agar

Discussion

Over the past three decades, MRSA has become a prevalent nosocomial pathogen and a significant contributor to infections in both healthcare facilities and the community. MRSA strains commonly exhibit resistance not only to beta-lactams and cephalosporins but also to a broad spectrum of antibiotics.

Inconsistencies in the identification of MRSA have a substantial influence on patient care, highlighting the importance of precision in detection. Therefore, techniques employed for detecting MRSA in clinical specimens should demonstrate elevated sensitivity and specificity while ensuring a quick turnaround of results. Presently, mecA amplification using PCR is accepted as the bench mark for detecting methicillin resistance in S. aureus.9, 17 Even though there is growing agreement in the literature about this method, not all clinical laboratories have access to it. For this reason, phenotypic approaches are still preferred in environments with limited resources. However, due to a variety of environmental factors and the presence of different strains within the S. aureus population, the performance of phenotypic methods in methicillin resistance detection is inconsistent, time-consuming, and faces challenges in identifying all resistant isolates.18

In this study, despite the resistance observed in all 112 isolates through the cefoxitin disc diffusion, only 101 isolates exhibited a positive result for mecA. Unlike several studies that reported 100% accuracy for cefoxitin disk diffusion,4, 5, 6 our investigation found a significant number of false positives using this method, consistent with reports published by Bhutia et al. and Jain et al.18, 19

Oxacillin E strip demonstrated a sensitivity of 98% and a specificity of 91% in our study. Among the 101 MRSA isolates positive for mecA, two exhibited inconsistent results for the oxacillin MIC and PCR, indicating mecA positivity but an oxacillin MIC of ≤ 2 μg/ml (1.5 μg/ml, 0.5 μg/ml). This disparity could potentially be clarified by the variation in mecA gene expression among different Staphylococcus isolates.20

Out of 11 mecA-negative isolates, 10 were found to be negative by the oxacillin E strip. One isolate showed false positivity, i.e., mecA negative and oxacillin MIC ≥4μg/ml. This inconsistency is attributed to the varied expression of methicillin resistance, such as modification of existing PBP (MODSA) or low-level resistance demonstrated by penicillinase hyperproducer isolates (BORSA) found in many strains.7

MRSA CHROM agar (Hi-Media), demonstrated a sensitivity of 96.03% and a specificity of 82% in this study. Some studies have assessed this medium, with a few reporting false-positive outcomes with the chromogenic medium. In our study, we had two isolates that produced greenish-yellow colonies on CHROM agar but were negative for the mecA gene, and four isolates were found to be falsely negative. A study by Datta et al.3 found a sensitivity of 98.07% and a specificity of 99.2% for CHROM agar MRSA (Hi-Media), using mecA gene detection through PCR as the reference standard.

In this study, none of the tests, including cefoxitin, the oxacillin E test, or CHROM agar, achieved 100% accuracy when compared to PCR for MRSA detection. These results align with studies conducted by Bhutia et al.18 As a result, a combination of tests is recommended, with the PCR method preferred for confirming resistance due to its ability to detect mecA-mediated resistance quickly and simultaneously.

Of the 45 HA MRSA isolates in our analysis, 33.3% were from patients who had been hospitalized at least once in the previous year. 91.1% of HA MRSA isolates in our study had a history of recent surgery, consistent with reports published by Chatterji et al. and Dhanalakshmi et al.21, 22 Studies indicate that there is variation in the HA-MRSA epidemiology across various regions of India. The biological features of the staphylococcal isolates, variations in patient populations, and infection control techniques can all be attributed to these disparate rates.23

MRSA, once confined to the hospitals, is now a rising community infection. Moreover, recent change in the epidemiological scenario, resulting in the presence of community-acquired MRSA within hospital environments. On occasions, it displaces the conventional MRSA strains isolated from hospitals. This transformation increases the risk of antibiotic resistance and complicates infection management.24

In the present study, 83.9% of all MRSA strains were resistant to erythromycin. This is comparable to the studies of Lohan et al.,25 Mallick et al.,26 and Joshi et al.,27 which showed 76.5%, 74.5%, and 70.8% resistance to erythromycin, respectively. Additionally, higher resistance levels were observed for ciprofloxacin at 72.3% and doxycycline at 58%. Vancomycin and linezolid resistance were not detected.

Conclusion

Cefoxitin serves as a good surrogate marker for methicillin resistance. However, constant surveillance and breakpoint assessments are necessary. Our analysis shows that none of the phenotypic methods were as accurate as PCR for MRSA detection. Despite this, the oxacillin MIC by E test performed better and provided additional information for isolates with discrepant disk diffusion results. According to our results, Cefoxitin disk diffusion method and Oxacillin E strip are an effective combination for detecting MRSA in a resource constraint setting. Efforts should be made to implement regular PCR testing for the mecA gene, which detects MRSA strains from significant clinical specimens or special units like ICUs, given the benefits of timely detection and accuracy.

Ethics Approval

Approval was obtained from Human Ethics Committee, Medical college, Trivandrum, referenced as HEC.No.04/33/2021/MCT. dated 15^th march 2021.

Source of Funding

This study received no external funding.

Conflict of Interest

The authors declare that they have no competing interests.