Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most harmful “superbug,” and it is a major bacterial pathogen that causes multiple infectious diseases. It is capable of infecting intact normal skin and causes a wide spectrum of clinical diseases caused by both hospital-acquired as well as community-acquired.[1] MRSA acquired among hospitalized patients is associated with risk factors such as suppression of the immune system, surgery, indwelling medical devices, etc. [2] The hospital-associated MRSA continue to be endemic in hospitals worldwide. Since the 1990s, the epidemiology of MRSA infections has dramatically altered following the emergence and spread of a new lineage of MRSA, i.e. community-associated MRSA CA-MRSA have acquired a competitive advantage over HA-MRSA and they are rapidly replacing the HA-MRSA clones to become established in the community.[3]

Hospital associated MRSA is acquired when one undergoes frequent or prolonged hospitalization while CA MRSA is prevalent in healthy people. The transmission path in CA-MRSA is within the community where the microbe also occurs as a commensal in nature, which is easily transmissible. This is due to this microbe’s causing localized as well as disseminated infection. Superficial skin lesions to deep-rooted infective infections occur in the individuals. It is also a usual cause of local suppurative lesions.[1]

The resistance in MRSA is primarily mediated by the mecA gene, located at the cell wall of bacteria, which encodes a novel penicillin-binding protein (PBP)-2a and reduced affinity for β-lactam antibiotics. Mutation in (PBP)-2a results in failure to bind and action of the antibiotics.[2] The majority of skin and soft tissue infections in healthy and comparatively young patients without a history of hospital contact are caused by newer, more potent strains of MRSA known as CA-MRSA, where, HA-MRSAs are allied with nosocomial infections e.g. endocarditis. CA-MRSA strains are usually resistant to β-lactams but susceptible to other antimicrobial classes (non β-lactams) and Non-β-lactam antimicrobials, including aminoglycosides, macrolides, lincosamides, and fluoroquinolones, are frequently ineffective against HA-MRSA.[4]

In healthcare, the worldwide growth in methicillin-resistant S. aureus has become a major problem over the past few decades. These methicillin resistance staphylococcal strains are still imply great challenges to physicians as they are well resistant to other higher β-lactam groups of antibiotics which significantly affects morbidity, mortality and hospitalization cost. To this end, there is a need to control MRSA spread.[5]

The mobile gene, SCCmec (staphylococcal cassette chromosome mec) primarily confers methicillin resistance to MRSA isolates. It is flanked by terminal inverted and direct repeats.[6] The SCCmec element in addition to the mec gene complex containing the mecA gene with its regulators, mecI and mecR1 also has the ccr gene complex encoding the recombinases which are responsible for making SCCmec movable.[7] In integrating SCC-mec precisely into bacterial chromosome and excising it out of this site, two site-specific cassette chromosomal recombinases (ccr) perform these tasks.[8] The ccr gene complex is an array of seven to eight upstream (open reading frames) ORFs and middle-of-ORF ccr genes. Three ccr genes have been identified so far in staphylococci namely, ccrA, ccrB, and ccrC. Meanwhile at least 85% nucleotide identity among different types of ccr genes groups them under one subgroup within each type. Up to date three subgroups have been established for three types of ccr: 1) for A-type there are seven subtypes from A1 up to A7; 2) B-type consists of six subtypes B1 through B6 while; C-group has only two subtypes namely C1 and C2 respectively. Multiple antibiotic resistant or heavy metal resistant genes can be inserted in SCCmec via site-specific recombination of ccrAB or/and ccrC.[1]

A multiplex PCR strategy for quickly assigning SCCmec types to MRSA strains was described in 2002. SCCmec types I through III, as well as several epidemiologically significant variations, were correctly identified using this technique (e.g., subtypes IA and IIIA).[6] In 2005, a new set of SCCmec type- and subtype-specific primers, as well as unique multiple assays for rapid and simple SCCmec typing of MRSA, were proposed and proved to be practicable, and helpful.[1]

The mec-gene complex and the ccr-gene complex are the two basic components that make up the SCCmec elements. Molecular characterisation using multiplex PCR (mPCR) of the mobile genetic element carrying methicillin resistance gene mecA is essential in SCCmec typing.[6] On the other hand, kinds of HA-MRSA strains have been identified as such which harbor staphylococcal cassette chromosome mec (SCCmec) types that are older like SCCmec I, II and III; whereas most CA-MRSA strains harbour mostly SCCmec type IV, V or VII.[1] MPCR strategy became available for typing of SCCmec and is widely used to study the natural history of MRSA.

MRSA stains with SCCmec type IV is one of the most significant and challenging type to characterize as it is the shortest and most mobile structural variant of SCCmec.[9] Moreover, another reason why we have chosen this set was based on SCC mec type IV which is also variable than any other scc mec types because it has several subtypes -subtypes: IVa-IVn.[2] In addition to this, a simplified 9-valent mPCR test designed with only two primer pairs for rapid detection of SCCmec subtypes in MRSA.[1] Consequently, we aimed at focusing our study on hospitalised patients’ MRSA isolates including those from community settings to fully understand molecular epidemiology and evolution of MRSA.

Methodology:

This study included 381 S. aureus isolates that were obtained between 2020 and 2022 from the Department of Microbiology at JSS (Jagadguru Sri Shivarathreeshwara) Hospital in Mysore. The clinical specimens included in this study included pus samples, blood, endotracheal swabs, ear swabs, sputum, urine, and other sterile body fluids. The research was a cross-sectional study conducted in a lab. The research facility is accredited by NABL, and all clinical samples were handled in accordance with the facility's normal operating protocols. By using the coagulase and catalase tests, the isolates were later determined to be S. aureus. Using the Vitek 2 system (VK2C21383, Cyrix Health Care Private Ltd., India), the isolates' antimicrobial susceptibility was assessed. The type of MRSA was also identified using the cefoxtin disc diffusion test in accordance with the Kirby-Baeur protocol. The study's approach is summarized as follows.

Ethical approval: The JSS (Jagadguru Sri Shivarathreeshwara) Medical College, Mysore, Karnataka, India, Institutional Ethics Committee (JSSMC/IEC/260822/38NCT/2022-23 dated 01-09-2022) provided ethical approval prior to sample processing.

Patient involvement:

The research did not directly include the patient; instead, it entailed the normal evaluation of the clinical samples obtained in the microbiology laboratory.

Primary identification of methicillin resistant S. aureus:

The phenotypic identification of S. aureus was verified by microscopic and colony morphology analysis (Gram positive cocci grouped in clusters and groups), positive catalase test with 3% hydrogen peroxide, and positive coagulase test. These isolates underwent further testing utilizing the Kirby Bauer method (Kirby Bauer) using 30 μg of cefoxitin (SD041, HiMedia) and 1μg oxacillin (SD088, HiMedia) disk. Mueller–Hinton agar (MHA) was used to lawn cultivate S. aureus isolates, and the plates were incubated at 37°C for the duration of the experiment. As a positive control, S. aureus ATCC 25923 was employed. As per CLSI recommendations 2021, isolates exhibiting a zone of inhibition ≤21 mm surrounding the cefoxitin disk on MHA were classified as methicillin-resistant S. aureus.[10] The antimicrobial susceptibility profile of S. aureus isolates was simultaneously ascertained using GPC AST P628 Vitek cards with respect to Penicillin, Oxacillin, Ciprofloxacin, Levofloxacin, Erythromycin, Clindamycin, Gentamycin, Linezolid, Vancomycin, Daptomycin, Teicoplanin, Tetracycline, Tigecycline, Rifampicin, and Cotrimoxazole.

After preliminary identification, susceptible strains were removed from the research, keeping only methicillin-resistant isolates.

Extraction of DNA from S. aureus isolates:

PCI (Phenol-Chloroform-Isoamyl Alcohol) technique was used to extract DNA from each S. aureus isolate for molecular gene identification. S. aureus colonies (3–4) were removed from a brand-new growth medium plate and put into a 2ml centrifuge tube with a cover. Next, 5μl of proteinase K (cat.No.25530-015) and 500μl of lysis buffer were added. The samples were lysed for three hours at 56°C in a shaker incubator. After the samples were lysed, they were mixed with an equal volume of phenol, chloroform, and isoamyl alcohol in a 24:24:1 ratio. The solution was then centrifuged for 15 minutes at 5000rpm. The aqueous supernatant was collected in a second, 2ml centrifuge tube that was covered. Subsequently, a 24:1 ratio of isoamyl alcohol to chloroform was introduced. The tubes were thoroughly mixed and then centrifuged for 15minutes at 5000rpm. Once more, a 1.5 ml covered centrifuge tube was used to gently aspirate the aqueous supernatant. For the purpose of precipitating DNA, 500μl of a 1:9 sodium-acetate:ethanol combination was added, and the mixture was kept at -20°C. The tubes were centrifuged at 10,000 rpm for 10 minutes at 4°C following an hour of cold incubation. After discarding the supernatant, 70% ethanol was used to rinse the particle. After the pellet was air dried, elusion buffer was also added, depending on the size of the particle. The DNA was quantified using a nanodrop spectrophotometer, and its quality assessed by electrophoresis on an ethidium bromide-stained 0.8% agarose gel.[11]

PCR amplification for MRSA detection:

Primers targeting the mecA gene were used in PCR to detect methicillin-resistant S. aureus; these primers were manufactured commercially by Bioserve Biotechnologies Pvt. Ltd. in Hyderabad.[12] (Table 1)

The mecA gene's ideal annealing temperature was determined by using the temperature gradient PCR experiment with positive control DNA from ATCC 43300. In summary, a 25μl reaction mixture including the following components was produced for the PCR: 1μl of template DNA (100-200ng/μl), 1μl of each forward and reverse primer (10pmol each), 12.5μl of master mix (Product No. MPG-6RS1), and 9.5μl of nuclease-free water. The PCR amplification was performed in a Biorad, T100 automated thermal cycler. The first denaturation was cycled for five minutes at 95°C, then for 30 seconds at 95°C, thirty seconds at 56.2°C, and forty-five seconds at 72°C. The last extension was cycled for five minutes at 72°C. The 2% agarose gel electrophoresed the PCR amplified products, which were then stained with ethidium bromide. Syngene's Gbox gel documentation system was used to record the photos. By visually comparing each band to a molecular weight ladder, the identities of each band were established.

PCR amplification for SCCmec-typing and ccr-typing:

The multiplex PCR reactions were used for the typing of SCCmec element, wherein, 12 different primer sets were used as presented in Table 1.[13] Eight sets of primers were utilized in the multiplex PCR for the detection of markers including SCCmec-typesand 4 different types of ccr-gene including ccr type1, 2, 3, and 5.[14] PCR solution mixture preparation and PCR cycling conditions were same as MRSA detection in all the PCR amplification process only except the annealing temperature.

Results

A total of 381 S. aureus were isolated from various clinical specimens, such as pus samples (86.35%), endotracheal 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.

Phenotypic detection of MRSA:

Out of the 381 samples, 246 (64.56%) isolates were found to be methicillin-resistant Staphylococcus aureus utilizing the disk diffusion technique, which involved employing 30μg of cefoxitin and 1μg of oxacillin disk. By using the vitek2 technique, all of the isolates showed 100% sensitivity to linezolid, vancomycin, and daptomycin, and then rifampicin, teicoplanin, tetracycline, tigecycline, clindamycin, and gentamycin.

Molecular detection of mecA-genes for MRSA:

In order to detect the mecA gene for MRSA molecularly, all 246 MRSA isolates (identified by phenotypic technique) were amplified. A total of 162 (66%) isolates had the mecA gene, with a 162 bp product size; 84 isolates did not yield a positive PCR result.(Figure 1)

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

SCCmec-typing and ccr-typing:

All the 162 mecA-positive MRSA isolates were subjected to multiplex PCR method for the detection of SCCmec- type I, II, III, IVa, IVb, IVc, IVd, V and IX. Total 86 (53%) isolates were typed for different SCCmec elements and 76 isolates were non-typeable. Types of SCCmecelements found in the studied MRSA isolates were type IV (53.48%), and type-V (44.18%), type-III (9.30%), type-II (3.04%), and type I (1.16%) respectively. SCCmec type-IV was the most prevalent type detected in the isolates of MRSA enrolled in the given study followed by prevalence of type-V, and type-III. Among SCCmec type IV, type IVa was the most prevalent (54.34%), followed by IVc (37%) and IVd (9%). SCCmec type I and IVb was not found in any of the isolate. Nine (10.46%) isolates were detected with multiple SCCmec-types. Ccr-typing method confirmed only 69 (80.23%) isolates out of 86 SCCmec typed isolates. Others were no-typeable by ccr-typing. (Table 2, Gel Image Figure: 2 and 3). Clinical backgrounds of all the SCCmec-typeable isolates collected from different clinical sites were mentioned in the Table 2.

Figure 2: Validation and application of MRSA harbouring different types of SCCmec gene. Lane 1: 100 bp ladder. Lane 2-20: Typeable MRSA isolates (L14,17,19;Non-typeable MRSA isolates).

Figure 3: Validation and application of MRSA harbouring ccr-gene (C5/A5B5). Lane 1: 100 bp ladder. Lane 2,5,6,9,10,11: MRSA isolates with ccr-C5-gene. Lane 3,4,7,8,12,13,14,16,19,20: Non-typeable MRSA isolates.

Discussion:

S. aureus is a currently a significant health risk. Both the general public and the immune-compromised are at considerable risk from methicillin resistant S. aureus (MRSA) associated to the general population as well as MRSA linked to healthcare. Methicillin resistant S aureus (MRSA) must be accurately identified in order to treat the affected patient and stop the ijjnfection from spreading.[15]

The present study comprises of total 381 S. aureus isolates, where 246 (64.56%) isolates were identified as MRSA by Kirby-Bauer method using cefoxitin and oxacillin disk. This outcome suggesting that the prevalence rate was quite high. Similar prevalence rates of MRSA in hospital environments 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).[16-21]

Furthermore, in this study, molecular detection identified a total of 162 (66%) isolates harboring the mecA gene specific for MRSAout of 246 phenotypically confirmed methicillin-resistant isolates (by disk diffusion method). Similar results were established in the study of Yasir Rashid et al., where out of 500 clinical samples, 45 isolates were detected as S. aureus, of which only 12 isolates were found to carry the mecA gene for MRSA.[22] The present study is comparable to the study of Marwa Raad et al.,in which 120 isolates were detected as MRSA by routine microbiological detection out of 231 samples, of which only 76.67% (n=92) of the isolates were resistant to beta-lactams and were also detected as mecA-positive MRSA by PCR.[23] In another study by Feleke et al., only 14 isolates were detected as mecA-positive MRSA out of 139 S. aureus isolates.[24] These findings were concordant with the results observed in the present study.

According to the antibiotic susceptibility profile, all the isolates were sensitive (100%) to linezolid, vancomycin and daptomycin, followed by rifampicin (99%), teicoplanin (99%), tetracycline (98%), tigecycline (96%), clindamycin (85%) and gentamycin (72%). A number of earlier reports from the Indian subcontinent and foreign groups can also be compared to the findings of the present study, where susceptibility was reported to be 86.2% by Brown et al., 87.2% by Adhikari et al., 79.3% by Raut et al., and 92% by Sanjana et al.[25-28] Khanal et al. reported 73.3% gentamicin susceptibility, Sanjana et al. reported 69%, and Rajaduraipandi et al. reported 58.15%.[20,28,29]

In the present study total 86 (53%) isolates were typeable for different SCCmec elements. The prevalence of only five types of SCCmec elements including type I, II, III, IV, and V were found in the studied isolates of MRSA. The prevalence of SCCmec types were type IV (53.48%), and type-V (44.18%), type-III (9.30%), type-II (3.04%), and type I (1.16%). Among SCCmec type IV, type IVa was the most prevalent (54.34%), followed by IVc (37%) and IVd (9%). Similar result found in the study by Naime Kashefi Pasandideh et al., in the year 2022, where they identified 41 MRSA out of 460 clinical isolates, of which 15 isolates (36.5%) carried SCCmec IV, 11 isolates (26.8%) carried SCCmec V, nine isolates (21.9%) carried SCCmec III, two isolates (4.8%) carried SCCmec II, and three isolates (7.3%) carried SCCmecI.[30] In another study by Elisa De Tomi, et al., MRSA strains revealed the acquisition and insertion of SCCmec of class IV (n = 5) was the prevalent followed by class I (n = 1), and V (n = 1).[31]

In the year 2021, a study by Mingbiao et al., reported 45 mecA positive isolates out of 499 clinical samples, of which SCCmec type IV and V was predominant.[32] In another study by Yamuna et al., in the same year, found SCCmec type V was most predominant followed by III, IV, I and II, which is very similar to the present study.[33]

The two most common SCCmec types found in the isolates of MRSA included in the study were type-IV (53.48%), and type-V (44.18%). As per Classification of staphylococcal cassette chromosome mec (SCCmec), SCCmec types IV and V belong to CA-MRSA. Our study also revealed that CA-MRSA infections were more prevalent in our hospital than HA-MRSA infections. SCCmec type IV and V elements (CA-MRSA) were found in 37 in-patients and 27 out-patients isolates. Furthermore, HA-MRSA was present in 8 in-patient isolates and 4 out-patient isolates. In a previous study conducted at the same study centre by Madhuri Kulkarni et al., similar findings were also found. Among the 58 mecA positive MRSA, isolated from hospitalised patients, 45 isolates belonged to SCCmec type IV, and 5 isolates belonging to type V. [34] Epidemiological changes in MRSA isolates also found in the study of Jennifer et al., where they have identified 43% CAMRSA among hospitalized patients.[35] In addition, El-Baghdady K et al. discovered 77 CAMRSA out of 108 MRSA clinical isolates recovered from hospitalised patients with various illnesses or who had undergone various surgeries.[36]

In the current study, two isolates were detected with multiple SCCmec-types, isolate 1 revealed the acquisition of type III and V, and isolate 2 revealed the type II, IVc and V. Similar result found the study of Yamuna et al., where 18 isolates out of 32 showed multiple SCCmec types.[37]

The present study also found 76 isolates which were non-typeable for SCCmec types which is similar to the previous study done by Madhuri kulkarni et al. at the same tertiary care hospital.[34] According to the studies of Mariem et al., Ghanbari et al., and Mahtab et al., SCCmec typing do not show 100% type ability and have poor discriminatory power also.[34,38,39] Non-typeablity result was also found in the study of Vivek Kulkarni et al, where out of 7 isolates, 1 isolate was non-typeable.[40] In the study of Mahtab et al., 64% MRSA isolates were non-typeable and in the study of Mohammad et al., 9 MRSA isolates were non-typeable.[39]

Ccr-typing method amplified only 69 isolates out of 86 SCCmec typed isolates. Others were no-typeable by ccr-typing. Similar result found in the study of Mohammad et al., where 43 isolates out of 53 MRSA were typeable only.[41]

Several studies worldwide have used SCCmec typing by PCR to identify SCCmec types in their regions. However, these studies reported varying degrees of typeability, less than 100%. For example, a study in Palestine, 96.4% typeability was reported, while in Alexandria, Mansoura, and Cairo, Egypt, 90%, 94%, and 88.8% were reported.[42-45] Shahinda Rez et al reported a lower percentage of typeability at 75%.[46]

The present study also determined on the drug resistant pattern of MRSA isolates with SCCmec types. SCCmec type IVc and V showed the highest resistance to antibiotics (10nos), while SCCmec type I and II displayed least resistance to antibiotics (4nos). A study by A. Japoni et al also reported MRSA resistant to eight different antibiotics.[47] Multidrug resistant MRSA with SCCmec type IV was also observed in the study of Christiana Rezk et al.[48] Majority of the MRSA isolates (31nos) showed resistant to five different antimicrobial drugs, followed by 19 MRSA isolates were resistant to 4 different antimicrobials and 10 MRSA isolates were resistant to 6 different antimicrobials. Only 3 MRSA isolates were resistant to 8 different antimicrobials. The most common resistant pattern in our isolates were; resistant to levofloxacin (95%), followed by ciprofloxacin (93%), erythromycin (65%), Trimethoprim/Sulfamethoxazole (51%), and tigecyclin (21%). A Hungarian study found resistance to erythromycin, clindamycin, and ciprofloxacin, while a Kuwait study found a high proportion of isolates resistant to tetracycline, erythromycin, ciprofloxacin, and trimethoprim/sulfamethoxazole, contrary to our findings.[49,50]

Conclusion:

SCCmec gene plays a core role in antibiotic resistance, MRSA evolution and molecular epidemiology of staphylococcal diseases. In order to stop the spread of antibiotic resistance in hospitals, screening for MRSA among both in-patients and out-patients is required. A decreased sensitivity of MRSA to ten commonly used antibiotics has been observed, with SCCmec type IVc and V being the predominant isolate. This result suggests that by improving antibiotic prescribing, it can also lessen the selective pressure for the establishment and persistence of MRSA associated with antibiotic misuse. The option to actively monitor the healthcare environment is made possible by the identification of CA-MRSA in hospitals, which is a crucial step. This surveillance can lessen the spread of CA-MRSA in the hospital environment by preventing the favourable conditions needed for the bacteria's proliferation. The significance of hospital infection control procedures and their strict application in hospitals would be enhanced as a result of this.

Conflict of interest

The authors declare no conflicts of interest.

Financial Disclosure

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

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