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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 155-160

Detection of mecA and staphylococcal cassette chromosome mec gene isolated from Northeast Part of Karnataka Staphylococcus aureus isolates


1 Department of Microbiology, Post Graduate Studies and Research in Microbiology, Gulbarga University, Kalaburagi, Karnataka, India
2 Central Research Laboratory, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, Belgavi, Karnataka, India
3 Department of Biomedical Science, SDM Research Institute for Biomedical Sciences, Shri Dharmasthala Manjunatheshwara University, Dharwad, Karnataka, India
4 Department of Biotechnology, Gulbarga University, Kalaburagi, Karnataka, India

Date of Submission15-Mar-2021
Date of Acceptance24-Apr-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Y M Jayaraj
Department of Post Graduate Studies and Research in Microbiology, Gulbarga University, Kalaburagi - 585 106, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_35_21

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  Abstract 


Background: Methicillin- resistant Staphylococcus aureus (MRSA) strains are predominantly significant because they are leading healthcare- associated infections worldwide and even majorly emerged as community associated infections. Methods: A total of 40 S. aureus isolates were collected from Navodaya Institute of Medical Sciences, Raichur Institute of Medical Sciences, and Mid Tech Diagnostic Laboratory, Raichur, and analyzed for mecA, Panton–Valentine leukocidin (PVL), and staphylococcal cassette chromosome mec (SCCmec) typing by gradient and Multiplex polymerase chain reaction (PCR). Results: Out of 40 S. aureus isolates, 7 (17.8%) were resistant and 33 (82.8%) were sensitive for methicillin antibiotic. The seven isolates showed positive for Vancomycin /cefoxitin Ezy MIC strip and oxacillin antibiotic MIC was ≥ 8μg/ml respectively PCR detection has revealed, seven isolates were positive for mecA and SCCmec typing, but none of the isolates has shown positive for PVL genes. The study shows the presence of SCCmec Type II and III was confirmed by multiplex PCR in hospital-acquired MRSA isolate and mecA gene positive of seven strains of MRSA Conclusion: The study demonstrated high specificity and positive prediction of the detection of MRSA from clinical samples.

Keywords: Hospital acquired/community acquired methicillin-resistant Staphylococcus aureus, mecA, MRSA, PVL, Staphylococcus aureus, staphylococcal cassette chromosome mec


How to cite this article:
Kulkarni V, Kumbar VM, Oli AK, Kambar R, Shivannavar CT, Jayaraj Y M. Detection of mecA and staphylococcal cassette chromosome mec gene isolated from Northeast Part of Karnataka Staphylococcus aureus isolates. Biomed Biotechnol Res J 2021;5:155-60

How to cite this URL:
Kulkarni V, Kumbar VM, Oli AK, Kambar R, Shivannavar CT, Jayaraj Y M. Detection of mecA and staphylococcal cassette chromosome mec gene isolated from Northeast Part of Karnataka Staphylococcus aureus isolates. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:155-60. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/155/318429




  Introduction Top


Methicillin-resistant Staphylococcus aureus (MRSA) has become a major pathogen in hospital-acquired as well as a community-acquired infection.[1],[2] The resistance to methicillin and other β-lactam antibiotics is caused by the mecA gene, which is situated on the staphylococcal cassette chromosome mec (SCCmec). The mecA gene encodes the 78 kDa penicillin-binding protein (PBP) 2a or PBP2.[3]

SCCmec is a mobile genetic element characterized by the presence of terminal inverted and direct repeats, two essential genetic components (the mec gene complex and the ccr gene complex), and the junkyard (J) regions.[4],[5] Different combinations of these complex classes and allotypes generate various SCCmec types. SCCmec elements are currently classified into types I, II, III, IV, and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to the differences in their J region DNA.[6] On the basis of SCCmec elements, MRSA strains were recently classified into hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). HA-MRSA isolates contain mainly type I, II, and III SCCmec elements, while CA-MRSA contains type IV and V SCCmec elements, each of which has several variants.[7]

For instance, majority of Indian HA-MRSA collected between 2002 and 2006 contained type III or IIIA SCCmec elements, while S. aureus isolates collected from 2007 onward from community and hospital settings in India have shown SCCmec type IV and V cassettes. However, in 2010, studies of D'Souza et al.[8] reported the combination of both CA-MRSA (IV and V) and HA-MRSA (III) in the hospital environment. In view of the nosocomial mode of infection and emergence of multiple antibiotic resistances MRSA and their molecular typing, there is a need for periodic evaluation of their prevalence and antibiogram and molecular analysis. Hence, the present study was carried for the detection mecA and SCCmec gene from clinical isolates.


  Materials and Methods Top


Samples

A total of 40 S. aureus isolates were collected from the Navodaya Institute of Medical College (NMC); Raichur, Raichur Institute of Medical College (RIMS), Raichur; and Mid-Tech Diagnostic Laboratory, Raichur. The isolates were collected on nutrient agar slants (transport medium) and were transferred to the laboratory within 6 h of collection under aseptic conditions. The S. aureus isolates were further identified by conventional diagnostic procedures (cultural, morphological, and biochemical characters). The present study was approved by the institutional Ethical clearance committee (IECC) of Gulbarga University, Kalaburagi Ref.No.GUG/Dev-I/IECC/2014-15/290 dated:23.10.2014.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing (AST) of isolates of S. aureus was performed by Kirby–Bauer disc diffusion method according to the M02-A11 and M100–S22 Clinical Laboratory Standards Institute (CLSI) January 2012 guidelines and procedures on Mueller–Hinton agar.[9] The antimicrobial agents tested included penicillin-G (10 units), amikacin (30 μg), clindamycin (2 μg), erythromycin (15 μg), tetracycline (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefepime (30 μg), methicillin (5 μg), linezolid (30 μg), co-trimoxazole (trimethoprim/sulfamethoxazole - 1.25/23.75 μg), and imipenem (10 μg). S. aureus ATCC 43300 and MTCC 96 (ATCC 9144) were used as standard strains in the present study.

Oxacillin-cefoxitin confirmation test

To confirm MRSA, oxacillin-cefoxitin disc diffusion method as per the M02-A11 and M100-S22 Clinical Laboratory Standards Institute (CLSI)[9] January 2012 guidelines and procedures on Mueller–Hinton agar was followed (CLSI 2012).[9] S. aureus ATCC 43300 and MTCC 96 were used as standard strains.

Minimum inhibitory concentration tests for methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus

The Vancomycin/Cefoxitin Dual Ezy MIC Strips (cefoxitin: 0.5–64 μg/ml and vancomycin: 0.19–16 μg/ml) from Hi-Media Laboratories Pvt. Ltd, Mumbai (India), were used for further confirmation of vancomycin and methicillin resistance of S. aureus isolates, and M100-S22 CLSI guidelines 2012 were followed (CLSI 2012).[9] S. aureus strain ATCC 43300 and S. aureus strain MTCC 96 were used as the methicillin-resistant and methicillin-sensitive control test standard organisms, respectively.

Preparation of chromosomal DNA

The DNA extraction is done by modified proteinase K method using nonionic detergents. The S. aureus isolates were grown in Brain Heart Infusion (BHI) broth for overnight and were suspended in lysis buffer (phosphate-buffered saline containing 0.5% sodium dodecyl sulfate and 100 μg/ml proteinase). Further, the cells from the culture were collected by centrifugation, the cell suspension was incubated at 37°C for 1 h, and an equal volume of phenol: chloroform (1:1) mixture was added to the cell suspension and vortexed. The aqueous phase of the centrifuged samples was transferred to a fresh tube. The DNA was precipitated by the addition of 30 μl of 3 M sodium acetate and three volumes of cold 99% ethanol. The DNA pellet was washed twice with cold 99% alcohol, air-dried, and suspended in 500 μl of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8]).[10] The bacterial DNA isolation was carried out to study the mecA and SCC mec genes.

Polymerase chain reaction studies

Gradient polymerase chain reaction assay for mecA gene detection

The primers of mecA genes were obtained from Zhang et al.,[11] and the specificity of primers was confirmed by blast analysis at NCBI website. The temperature gradient PCR assays were performed to optimize the annealing temperature using positive control DNA from MRSA (ATCC 43300). The presence of the mecA (gene coding for penicillin-binding protein 2A) was used as internal controls for the detection of MRSA, and the genes were detected by PCR using the forward primer 5' GTG AAG ATA TAC CAA GTG ATT 3' and reverse primer 5' ATG CGC TAT AGA TTG AAA GGA T 3'. The size of the amplified product for mecA gene was 147 base pairs. 1 μl of total DNA template (100 ng) was prepared for 20 μl PCR amplifications as follows: 15 pmol of each respective primer; Taq DNA Polymerase 2x Master Mix Red (Ampliqon) as per the manufacturer's instructions. The PCR conditions were: 5 min DNA denaturing step at 95°C followed by 35 consecutive cycles at 94°C for 30 s; 52°C for 45 s; and 72°C for 45 s.

Gradient polymerase chain reaction assay for Panton-Valentine leukocidin gene detection

The gradient PCR assays were performed by optimizing the annealing temperature. The primers of Panton-Valentine leukocidin (PVL) genes were obtained from Gerard et al.,[12] and the specificity of primers was confirmed by blast analysis at NCBI website. The confirmed primer sequences were then sent to Bioserve Biotechnologies Pvt. Ltd, Hyderabad, for synthesis. The genes were detected by PCR using the forward primer 5' ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A 3' and the reverse primer 5'GCA TCA AGT GTA TTG GAT AGC AAA AGC 3'. The size of the amplified product for mecA gene was 433 base pairs. 1 μl of total DNA template (100 ng) was prepared for 25 μl PCR amplifications as follows: 10 pmol of each respective primer; Taq DNA Polymerase ×2 Master Mix Red (Ampliqon) as per the manufacturer's instructions. The PCR conditions were: 3 min DNA denaturing step at 95°C followed by 35 consecutive cycles at 94°C for 30 s; 54°C for 45 s; and 72°C for 45 s.

Multiplex polymerase chain reaction assay for staphylococcal cassette chromosome mec typing studies

The primers of SCC mec type I, SCC mec type II, SCC mec type III, SCC mec type IVa, SCC mec type IVb, SCC mec type IVc, SCC mec type IVd, and SCC mec type V genes were obtained from Zhang et al.[11] The primers were designed by using Primer 3 software, and the specificity of primers was confirmed by blast analysis at NCBI website. The designed primer sequences were ordered from Bioserve Biotechnologies Pvt. Ltd, Hyderabad, for synthesis and were diluted according to the manufacturers' instruction, i.e., dilute the primers with distilled water to obtain required picomolar concentration (1000 pmol). The stock of 1000 pmol was prepared and diluted to required concentrations.

Thus, obtained PCR amplification products were analyzed through the use of electrophoresis in a 2% agarose gel and stained with 0.5 μg/ml ethidium bromide solutions. PCR products were visualized under Gel Documentation (Syngen). The 100 bp DNA ladder served as the molecular weight marker, and DNA from MRSA strain ATCC-43300 used as the positive control. The identity of each band was determined by visual comparison with a molecular weight ladder.


  Results Top


Age- and gender-wise distribution of the samples

It is evident that maximum numbers of S. aureus isolates were from up to 30 years of age group (29 out of 40). The S. aureus isolates were from all the age groups in male cases but were up to 40 years of age group in female cases. Out of 40 S. aureus isolates obtained from Raichur region, 26 (65%) were from pus samples, 6 (15%) were from blood samples, 5 (12.5%) were from burn wound swabs, 2 (5%) were from sputum samples, and 1 (2.5%) S. aureus isolated from urine samples. The details are mentioned in [Table 1].
Table 1: Age and gender wise distribution of Staphylococcus aureus isolated from clinical sample

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Antibiogram studies

The antibiotic resistance pattern of S. aureus isolates are shown in [Table 2]. It is evident that among 40 isolates of S. aureus, 7 (17.5%) were resistant to methicillin antibiotic and 33 (82.5%) were sensitive to the same as per the Kirby–Bauer Disc diffusion method. Out of 7 MRSA, 4 (57.14%) were from pus samples, 2 (28.57%) were from burn wound swabs, and 1 (14.29%) isolate was from blood sample. 1 out of 40 isolates of S. aureus was found to be vancomycin-resistant S. aureus as per Kirby–Bauer disc diffusion method.
Table 2: Antibiotic resistant profile of 40 Staphylococcus aureus isolated from clinical sample

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Minimum inhibitory concentration tests for methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus

In the present study, among 40 isolates, 07 MRSA isolates were confirmed by Vancomycin/Cefoxitin Dual Ezy strip method as shown in the [Figure No.1] and these seven isolates has showed resistance for oxacillin antibiotic with concentration of ≥8 μg/ml by agar dilution method. None of the isolates showed resistance to vancomycin antibiotic.
Figure 1: The Vancomycin/Cefoxitin Dual Ezy MIC tests of Staphylococcus aureus isolate 4

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Polymerase chain reaction studies (mecA gene and staphylococcal cassette chromosome mec typing studies)

It is striking to note that all 7 MRSA isolates showed the presence of mecA gene [Figure 2] while none showed the presence of PVL gene. Among the 7 mecA-positive MRSA isolates, 4 (57.14%) were from pus samples, 2 (28.57%) were from burn wound swabs, and 1 (14.29%) isolate was from blood sample. The SCCmec typing studies revealed that, out of 7 mecA positive MRSA isolates of Raichur region, 1 (14.29%) was type II isolate, 5 (71.43%) were type III isolates, and 1 (14.29%) was nontypable isolate [Figure 3]. Thus, all mecA-positive MRSA isolated from Raichur region were HA-MRSA as showed in the [Table 3].
Table 3: The details of mecA gene and staphylococcal cassette chromosome mec typing of 7 methicillin-resistant Staphylococcus aureus isolated from Raichur region

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Figure 2: mecA gene detection in 7 methicillin-resistant Staphylococcus aureus isolates from clinical samples of Raichur region. Lane M - 100 bp Ladder; Lane C - Positive control methicillin-resistant Staphylococcus aureus strain (ATCC - 43300). Lane 1–7: Detection of mecA gene in 1–7 clinical samples of Raichur region

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Figure 3: SCCmec Typing of 7 mecA-positive methicillin-resistant Staphylococcus aureus isolated from Raichur region. Lane M - 100bp Ladder; Lane C - Positive control methicillin-resistant Staphylococcus aureus strain (ATCC –43300). Lane 1–7: Detection of SCCmec gene types in methicillin-resistant Staphylococcus aureus isolates of Raichur region

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  Discussion Top


The prevalent of MRSA is becoming a grave challenge in public health, and also, it is one of the most worrisome microorganisms encountered in healthcare. Previously thought to be restricted to the hospital environment (HA-MRSA), the present pathogen of extreme importance is increasingly being isolated from the community settings as well (CA-MRSA). Most CA-MRSA strains carry PVL genes and possess small mobile SCCmec elements; the larger SCCmec types I, II, and III encountered in the HA-MRSA as compared to the smaller elements IV, V, or VI which are easily transferable.[13] The present study emphasizes on mecA, PVL, and SCCmec typing of S. aureus isolated from northeast part of Karnataka.

The current study was confined to antibiogram and molecular analysis of S. aureus isolates from the Raichur region (Karnataka, India). Thus, S. aureus isolates were collected from the various hospitals (NMC and RIMS) and diagnostic centers (Mid-Tech Diagnostic Laboratory) of Raichur region (Karnataka).

The antibiogram studies indicate the emergence of multiple drug resistance S. aureus strains. It is significant to note that all 7 MRSA strains were also resistant to penicillin-G (100%), ceftazidime (100%), and ceftriaxone (100%) antibiotics, while majority of them were resistant to other antibiotics in usage (cefotaxime [71.43%], co-trimoxazole [71.43%], cefepime [57.14%], tetracycline [57.14%], and erythromycin [57.14%]). Moreover, they were resistant to vancomycin to imipenem. One of each among the MRSA isolates was resistant to vancomycin and linezolid antibiotic. A number of earlier reports from foreign groups and the Indian subcontinent also showed the emergence of multiple drug resistance S. aureus isolates [Table 4]. The presence of MDR strains in the nosocomial environment leads into the failure of antibiotic therapy and a higher rate of mortality and morbidity.
Table 4: Comparative evaluation of antibiotic-resistant pattern of methicillin-resistant Staphylococcus aureus isolates of clinical samples among various studies

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The prevalence rate of MRSA in the present study was 17.5% (07 out of 40). The low incidence of MRSA may be due to the less number of sample sizes. In contrast to this, Anupurba et al., 2003[14] and Khadri and Alzohairy, 2010[15] showed the prevalence of MRSA around 55% and Subedi and Brahmadathan, 2005,[16] Orrett and Land, 2006,[17] Brown and Ngeno, 2007,[18] Kumari et al., 2008,[19] Ahmad, 2008,[20] Bala et al., 2010,[21] and Onwubiko and Sadiq, 2011[22] showed the prevalence of MRSA in the range of 11%–26%. The reports of Mulla et al., 2007[23] and Sanjana et al., 2010[24] were near to our reports on prevalence rate of MRSA in hospital environment.

The present showed the presence of mecA gene in all the tested MRSA isolates; This substantiates the similar type of results have been reported by many research group.[25],[26],[27],[28],[29],[30]


  Conclusion Top


MRSA isolates require a new policy to control infection. In our study, we found increased sensitivity to commonly used antibiotics. In addition, they showed more resistant to methicillin antibiotic. SCCmec type II and III were recognized as the predominant type and highly positive for mecA gene. Hence, appropriate PCR-based strategies to be used in the epidemiological identification of PVL, mecA, and SCCmec elements are necessary for determining MRSA strains and controlling HA- and CA-MRSA infection.

Statement of ethics

The study was approved by the Department of Microbiology, Gulbarga University Kalaburagi. As per the research ethics requirement, the Institutional Ethical Clearance Committee (IECC) vide its meeting held on October 9, 2014. Approved the Letter. Ref. No. DEV-I/IECC/2014–15/290 dated October 23, 2014.

Acknowledgment

We are thankful to Department of Microbiology, Gulbarga University, Kalaburagi, Karnataka, to complete successful of my research work. Further, I will thank you for Teaching and Nonteaching of NMC Raichur, RIMS, Raichur, and Mid-Tech Diagnostic Laboratory, Raichur, for supporting in collection of the samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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