Introduction

Staphylococcus aureus is one of the most often isolated bacteria and a human pathogen that causes illnesses like sepsis, bacteraemia, pneumonia, and skin infections.[1] S. aureus is a member of the normal flora, but over time it has transformed into an opportunistic pathogen that causes a variety of infections that are challenging to treat because they contain virulence and drug resistance genes.[2] Among these bacteria Methicillin-resistant Staphylococcus aureus (MRSA) are the most important as they cause significant treatment related complications.[3]

Methicillin, a new semi-synthetic penicillin or penicillinase-resistant drug, was chosen as the drug of choice for penicillin resistant S. aureus due to the widespread occurrence of this bacteria.[4] The structural gene mecA is found in the methicillin resistant strains of S. aureus inserted into the chromosomal element, but not present in the susceptible ones. This accomplishment has made it possible to create a different technique for determining methicillin-resistant S. aureus by searching for the mecA gene.[5]

The divergent mecA homologue mecA_LGA251 or mecC was first discovered in MRSA isolates from human samples in 2011. Since then, it has been intermittently discovered in MRSA strains from 13 other European nations and 14 different host species. Initially, mecC was identified in an MRSA isolate associated with livestock, but reports of its occurrence have been done in numerous countries across all continents, including Denmark, Europe, and many other Asian nations. These reports have come from a variety of sources, including human, animals, and the environment. mecC-MRSA is frequently discovered in domestic and other free-living animals, raising the possibility that these animals may serve as a reservoir for mecC-MRSA and infect humans.[6]

The use of genomics and genetic markers allows a deeper comprehension of the pathogen's biology.[7] The genotypic approach most frequently employed nowadays to detect and confirm MRSA in clinical samples or culture media is the polymerase chain reaction (PCR). This is performed by selecting a pair of closely spaced oligonucleotides to amplify internal regions of the mecA or mecC genes. There are several PCR amplification techniques available, however this process continues to be confirming reference method for MRSA.[8] Therefore, in this study conventional PCR- a cost effective gold standard method for detection of MRSA isolates was employed.

Subjects and Methods:

Settings:

This study comprised a total of 381 clinical samples such as pus samples, blood, endotracheal swabs, ear swabs, sputum, urine, and other sterile body fluids etc., collected in the department of Microbiology, JSS Hospital, Mysore, from 2020 to 2022. All the clinical samples were processed and identified by standard laboratory procedures to detect S. aureus. Molecular detection of genes was done by PCR method.

Ethical approval: Ethical approval was obtained from the IEC (Institutional Ethics Committee, JSS (Jagadguru Sri Shivarathreeshwara) Medical College, Mysore, Karnataka, India) (JSSMC/IEC/260822/38NCT/2022-23 dated 01-09-2022) before processing the samples.

Patient’s involvement:

The study involved with the samples that were received in the microbiology laboratory for routine investigation, and no patient interference was involved.

Primary identification of S. aureus:

Based on colony morphology and microscopic morphology (grape-like Gram-positive cocci) and positivity in catalase and coagulase tests, S. aureus was confirmed and identified in all clinical samples. Next, the samples identified as Staphylococcus aureus were subjected to disc diffusion method (Kirby Bauer) using 30μg cefoxitin (SD041, HiMedia). Briefly, S. aureus isolates were lawn cultured onto Muller Hinton agar (MHA) and the plates were incubated at 37^ºC overnight. S. aureus ATCC 25923 was used as positive control. Isolates with zone of inhibition ≤21mm on MHA around cefoxitin disk was considered as methicillin resistant S. aureus, as per CLSI guidelines 2021. Only methicillin resistant isolates were included in the study and sensitive strains were excluded after initial identification. The antimicrobial susceptibility profile of S. aureus isolates was determined by Vitek2 system.

Extraction of DNA from S. aureus isolates:

Extraction of DNA from all the S. aureus isolates was done by PCI (Phenol-chloroform-isoamyl alcohol) method for molecular detection of genes. Colonies (3 to 4) of S. aureus from fresh culture media plate was collected in a 2ml capped centrifuge tube and added with 500μl lysis buffer and 5μl proteinase K. The samples were lysed at 56^oC for 3hrs in a shaker incubator. The lysed samples were added with equal volume of phenol, chloroform and isoamyl alcohol mixture (24:24:1) and centrifuged at 5000rpm for 15minutes. The aqueous supernatant was collected in another 2ml capped centrifuge tube and added with equal volume of chloroform: Isoamyl alcohol in the ratio of 24:1. The tubes were gently mixed and centrifuged at 5000rpm for 15minutes. Again, the aqueous supernatant was carefully aspirated into a 1.5ml capped centrifuge tube. For the precipitation of DNA, 500μl of sodium-acetate: ethanol (1:9 ratios) was added and kept at -20^oc. After cold incubation for 1 hour, the tubes were centrifuged at 10,000rpm for 10minutes at 4^oc. The supernatant was discarded and the pellet was washed with 70% ethanol. Further the pellet was air dried and elusion buffer was added based on the pellet size. The DNA was quantified using a nanodrop spectrophotometer and the quality was assessed by electrophoresis on 0.8% agarose gel stained with ethidium bromide.[9]

Molecular detection of S. aureus by PCR:

The PCR was carried out with primers targeting 16S rRNA gene as a reference for bacteria and nuc- gene specific for Staphylococcus aureus.(Gel Image Figure 1) For the detection of Methicillin resistant S. aureus primers targeting mecA and mecC genes ware commercially synthesized (Bioserve Biotechnologies Pvt. Ltd, Hyderabad). The primer pairs used in the study are listed in Table 1.

Polymerase chain reaction assay for mecA and mecC gene detection:

The temperature gradient PCR assay was performed to optimize the annealing temperature for mecA and mecC gene using positive control DNA from ATCC 43300 and ATCC BAA 2312 respectively. Briefly, 30μl reaction mixture was prepared for the PCR containing the following, template DNA 1μl (100-200ng/μl), primers 0.8μl each of forward and reverse (10pmol each primer), master-mix 7.2μl and nuclease free water 21.8μl. The PCR amplification was carried out in an automated thermal cycler (Biorad, T100). The cycling conditions were 95°C for 5 min for initial denaturation, followed by 34 cycles of 95°C for 30sec, 56.2°C for 30s and 72°C for 45sec, and the final extension at 72°C for 5 min. The PCR amplified products were electrophoresed on 2% agarose gel stained with ethidium bromide and the images were documented using Syngene: Gbox gel documentation system. The identity of each band was determined by visual comparison with a molecular weight ladder.

Figure 1: PCR Amplification of 16s rRNA specific for Staphylococci. Lane 1:50 bp ladder. Lane 2-5:Positive isolates showing bands at 756bp.

Statistical analysis:

Data was analysed using JMPO Pro 17. Sensitivity and specificity of phenotypic methods were calculated using mecA gene PCR as the gold standard. Categorical values were represented by using numbers and percentages. The results were considered significant at p-value ≤0.5 (confidence level of 5%).

Results:

A total of 381 MRSA isolates were isolated from various clinical specimens such as pus samples (86.35%), endo-tracheal aspirants (3.67%), blood (3.41%), ear swabs (3%), sputum (1.57%), urine (1%), and other sterile body fluids (0.78%), during the study period in the Department of Microbiology, JSS Medical College and Hospital.(Table 2)

Primary identification:

Staphylococci were identified by the colony characteristic as 1-3 mm diameter, smooth, low convex, glistening. On blood agar, β-hemolysis was observed. On Gram’s stain, uniformly stained, Gram-positive cocci around 1µm diameter arranged in cluster without any capsule were presumptively considered as Staphylococci. A total of 246 (64.56%) isolates were identified as methicillin-resistant Staphylococcus aureus among 381 samples, by disk diffusion method using cefoxitin 30μg. All isolates were sensitive (100%) to linezolid, vancomycin and daptomycin followed by rifampicin, teicoplanin, tetracycline, tigecycline, clindamycin and gentamycin respectively.

Molecular detection of genes:

All 246 MRSA isolates (detected by phenotypic method) were amplified for molecular detection of nuc gene specific for S. aureus. PCR method identified a total of 162 (65.85%) isolates as S. aureus (nuc-gene positive), and same were subjected to PCR assay for mecA and mecC gene for MRSA detection. mecA gene with a product size of 162bp confirmed a total of 162 (65.85%) isolates as MRSA and none of the isolates were found to carry the mecC gene for MRSA. In 84 (34.14%) isolates, mecA or mecC gene was not detected by PCR method. (Gel Image Figure: 2, 3 & 4) The antibiotic susceptibility profile for all mecA- positive MRSA isolates is tabulated below. (Table 3)

Figure 2: Showing PCR Amplification of nuc-gene for Staphylococcus aureus. Lane 1: 100 bp ladder; Lane 2 and 15: Positive Control and Negative Control.Lane 3-9 and 11-14: Isolates Positive for nuc-gene respectively. Lane 10: Isolates Negative for nuc-gene.

Figure 3: Showing PCR Amplification of mecA gene for MRSA. Lane 1: 50 bp ladder; Lane 2 and 9: Positive Control and Negative Control for mecA gene.Lane 3-8, 10-15: S. aureus isolates Positive for mecA gene respectively.

Figure 4: Showing PCR Amplification of mecC gene for MRSA. Lane 1: 50 bp ladder; Lane 2 and 15: Positive Control and Negative Control for mecC gene of MRSA isolates. Lane 3-14: S. aureus isolates negative for mecC gene.

The sensitivity of cefoxitin disk diffusion method, Vitek 2 method was 66% and 100%. The sensitivity of oxacillin disk diffusion method, Vitek 2 method was 50% and 95%. The specificity was 66% with both cefoxitin and oxacillin disk diffusion method and 65% with both cefoxitin and oxacillin Vitek 2 method.

Discussion:

Staphylococcus aureus is one of the most important cause of cutaneous or subcutaneous infections and nosocomial infections.[3] Although being a part of the normal flora, S. aureus have evolved into an opportunistic pathogen that causes a number of ailment that are difficult to cure because they include virulence gene.[2]

In the present study, a total 246 out of 381 (64.56%) clinical isolates were identified as methicillin resistant S. aureus by the standard microbiological procedure suggesting the prevalence rate is quite high. Similar prevalence rates of MRSA in hospital environment were previously reported by Agnihotri et al (60.7%; n=31/44), Singh et al (53.6%; n=180/336), Jaiswal et al (72.4%; n=71/98), Dhar et al (79%; n=42/53), Khanal and Jha et al (68.0%; n=408/600) and Tiwari et al (69.1%; n=112/162).[14-19]

Further, in this study the molecular detection identified a total of 162 (66%) isolates by nuc-gene specific for S. aureus, out of 246 methicillin resistant isolates (by cefoxitin disk diffusion method). All the nuc-positive 162 S. aureus isolates were further amplified for mecA/mecC gene responsible for methicillin resistance and detected as MRSA carrying mecA-gene. Only 84 isolates were negative for both nuc-gene and mec-genes. Similar results were established in the study of Yasir Rashid et al where out of 500 clinical samples only 97 isolates revealed homology with Staphylococci by 16S rRNA and 45 isolates were detected as S. aureus, of which only 12 isolates were found to carry mecA gene for MRSA.[20]

The present study is comparable to the study of Marwa Raad et al.,where only 120 isolates were detected as MRSA by routine microbiological detection out of 231 samples, of which only 76.67% (n=92) isolates were resistant to beta-lactams and also detected as mecA positive MRSA by PCR method.[2] In another study by Feleke et al., collected a total of 1365 samples of which only 139 isolates were identified as S. aureus by phenotypic method, and only 14 isolates were detected as mecA positive MRSA.[21] These findings were concordant to the results seen in the present study.

As per the antibiotic susceptibility profile, all isolates were sensitive (100%) to Linezolid, vancomycin and daptomycin followed by rifampicin (99%), teicoplanin (99%), tetracycline (98%), tigecycline (96%), clindamycin (85%) and gentamycin (72%) respectively. A number of earlier reports from the Indian subcontinent and foreign groups also can be compared to the current study where susceptibility reported as 86.2% by Brown et al 87.2% by Adhikari et al, 79.3% by Raut et al and 92% by Sanjana et al.[21-24] Gentamicin susceptibility reported as 73.3% by Khanal et al, 69% by Sanjana et al and 58.15% by Rajaduraipandi et al in their studies.[18,24,25]

The most striking oxacillin susceptibility pattern was discovered in 4.37% of mecA-positive MRSA isolates. Oxacillin MIC for those samples was ≤2µg/ml. Resistant strains are detected with oxacillin MIC ≥4µg/ml. This indicates that some oxacillin susceptible mecA-positive MRSA (OSMRSA) isolates are also present in the study. In a recent investigation by Lorena et al., 26 mecA positive MRSA isolates were identified as oxacillin sensitive MRSA isolates.[26]

Multiple drug resistance is also seen in the current study. Most of the mecA positive MRSA were resistant to ciprofloxacin (90%), oxacillin (95%), trimethoprim/sulfamethoxazole (93%), levofloxacin (94%), and erythromycin (61%) respectively. The antibiotic-resistant pattern of all the MRSA isolates according to the Vitek 2 method can be compared to various studies. The strong resistance pattern of oxacillin can be compared to Khadri et al., Suzanne et al., and Bala et al., (100%).[27-29] Contrarily, the resistant patterns for ciprofloxacin and erythromycin can be compared to the research of Sanjana et al (CIP-71.08%, E-58.06%), Khadri et al (E-83%), Bala et al (CIP-98.68%, E58.63%), Anupurba et al (CIP-84.1%, E-80.1%), Subedi et al (CIP-94.4%, E-83.4%), Kumari et al (E-70.41%, CIP-67.35%), Arora et al (CIP-67.8%, E-61.7%), Orret et al (E86.7%, T-78.7%, CIP-59.1%), and Onwubika et al (E-100%), respectively.[24,27,29-35] The rate of gentamicin resistance in the current study was 20.62%, and similar patterns were established in earlier studies by Suzanne et al. (10.2%) and Eyob Yohanesset al. (1.2%).[28,36]

The study's sensitivity and specificity ratio is comparable to T. Yamazumi et al's (95%:85%) and another trial's (87%:96%) sensitivity and specificity of oxacillin disc and cefoxitin, respectively.[37,38] P-value for both the method was <0.001, which is considered to be highly significant in statistical analysis.

Inter-laboratory variability in isolate identification and testing can impact MRSA reporting, with molecular methods showing lower rates than phenotypic methods, which have been noticed in the present study.[39] The investigation revealed many variations between the frequencies of MRSA identified phenotypically and genotypically. This may be caused by the methicillin resistance's characteristic trait, ie., heterogeneous expression. The change of native PBPs, the overproduction of beta-lactamases, or maybe a methicillinase, all have an impact on the resistance in phenotypes. A possible solution is to detect nuc and orfX Staphylococcus aureus-specific gene markers concurrently. Secondly, for the confirmation of MRSA, methodologies based on the detection of mecA are the most accurate.[40]

Inexpensive methods include bench phenotypic tests, biochemical techniques like mannitol salt agar, chrome agar and DNase, and detection of PBP2a, latex agglutination tests. The controversy on true versus false MRSA identification and reporting can be resolved through manual phenotypic methods and multiple gene sequencing, nucleic acid amplification, which are expensive and unavailable, particularly in developing countries.[41]

Conclusion:

In the conclusion, the prevalence of MRSA in this study is still lower even when using PCR as the gold standard method for determining methicillin resistance in S. aureus isolates, when compared to the cefoxitin disc diffusion approach. The most reliable way to confirm the presence of MRSA is to find the mec-genes or its products (PBP2a). Some recent research works advised that, the disc diffusion testing with cefoxitin discs is superior to the majority of the phenotypic techniques, although it can be employed in situations when molecular resources are not accessible for routine MRSA screening by PCR.

Clinically, it is essential to differentiate mecA-positive resistant isolates from irregular reported isolates that display their resistance in either heterogeneous or homogenous form, which may have an effect on treatment. Therefore, to prevent the spread of resistant strains (superbugs), the study found that the PCR detection approach has the potential to accurately detect MRSA by detecting mec-genes which may also be considered a confirmatory test method for MRSA detection using mec-genes as a genetic marker.

In the present study, molecular method showing lower MRSA rates than phenotypic methods. Addition of some other potential molecular assays may analyse those samples without any error. Lack of alternative phenotypic approaches for MRSA identification is a significant limitation of the study.

Conflict of Interest: Authors declare no conflict of interest.

Financial Disclosure: This current study was funded by JSSAHER, Mysore, Karnataka, India. (JSSAHER/REG/RES/URG/54/2011-12/2267).

Authors' contributions: All the authors were involved in the study conception and design. They contributed to the methodology, writing the manuscript, data acquisition, analysis and interpretation. All authors read and approved the final version of the manuscript.

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