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 ~  Abstract
 ~ Introduction
 ~  Antimicrobial Su...
 ~ Results
 ~ Discussion
 ~ Acknowledgment
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 296-301
 

A novel multiplex PCR for molecular characterization of methicillin resistant Staphylococcus aureus recovered from Jeddah, Kingdom of Saudi Arabia


1 Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Kingdom of Saudi Arabia
2 Department of Biology, College of Science, King Abdulaziz University, Kingdom of Saudi Arabia
3 Institute of Biomedical Sciences College of Science, King Abdulaziz University, Kingdom of Saudi Arabia
4 Department of Pharmaceutics, College of Pharmacy, King Saud University, Kingdom of Saudi Arabia

Date of Submission04-Mar-2012
Date of Acceptance06-Apr-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
IMI Moussa
Center of Excellence in Biotechnology Research, King Saud University, Riyadh
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.99490

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 ~ Abstract 

Background: Molecular characterization of staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) is very essential for studying the epidemiology of MRSA. Objectives: This study reports two multiplex PCR for molecular typing of MRSA collected from Jeddah, Kingdom of Saudi Arabia. Materials and Methods: A total of 101 clinical isolates of strains were collected from major hospital laboratories and public health centres, Jeddah, Kingdom of Saudi Arabia during the period from August 2009 to May 2011. All the strains were tested phenotypically by conventional methods and genotypically by a novel multiplex PCR targeting at the same time S. aureus 16S rRNA, Panton - valentine leucocidin (PVL) and mecA resistance genes. All the strains were tested also by multiplex PCR for typing of SCC mec types. Results: All the 101 strains previously identified phenotypically as S. aureus with bacteriological examination were positive for amplification of 756 base pair fragments specific for 16S rRNA of S. aureus. Moreover, all the strains were positive for amplification of 1339 base pair fragments specific for mecA gene, while only 38 strains (37.6%) showed positive amplification of 433 base pair fragments specific for PVL gene. The most predominant SCC mec type among the examined isolates is type V 43 (42.5) followed by SCCmec type III 39 (38.6%). Conclusion: The newly modified multiplex PCR is rapid and sensitive method for detection of MRSA. Moreover, the most predominant SCC mec type among the examined isolates from Jeddah, King Saudi Arabia is type V (42.5%), followed by Type III (38.6%).


Keywords: MRSA, multiplex-PCR, PVL gene, Staphylococcus aureus, staphylococcal cassette chromosome mec


How to cite this article:
Moussa I, Kabli S A, Hemeg H A, Al-Garni S M, Shibl A M. A novel multiplex PCR for molecular characterization of methicillin resistant Staphylococcus aureus recovered from Jeddah, Kingdom of Saudi Arabia. Indian J Med Microbiol 2012;30:296-301

How to cite this URL:
Moussa I, Kabli S A, Hemeg H A, Al-Garni S M, Shibl A M. A novel multiplex PCR for molecular characterization of methicillin resistant Staphylococcus aureus recovered from Jeddah, Kingdom of Saudi Arabia. Indian J Med Microbiol [serial online] 2012 [cited 2019 Nov 18];30:296-301. Available from: http://www.ijmm.org/text.asp?2012/30/3/296/99490



 ~ Introduction Top


Staphylococcus aureus constitutes a major public health threat, as it is one of the major pathogens causing nosocomial infection. [1] It is armed with a wide array of virulence determinants that produce a diverse spectrum of clinical presentations, including the common boil, food poisoning, toxic shock syndrome, osteomyelitis, necrotizing pneumonia and endocarditis. [2]

Methicillin-resistant S. aureus (MRSA) strains which are commonly multidrug resistant and may be only susceptible to vancomycin present both a treatment and infection control challenge in the hospital setting. This problem is further confounded by the recent spread of community-acquired MRSA (CA-MRSA) and the identification of vancomycin-resistant MRSA (VMRSA). [3],[4]

Vancomycin and methicillin resistance in S. aureus continues to emerge as a significant public health threat in both the hospital and community. In addition to the limited treatment options, S. aureus strains acquire and express numerous virulence determinants that continue to increase its ability to cause a wide spectrum of human disease. [3]

The prevalence of hospital acquired methicillin-resistant S. aureus (HA-MRSA) differs among different countries and different hospitals, but once MRSA strains are introduced into a hospital they may become endemic. [5] MRSA acquired an integrated sequence into their genome (21-67-kb mobile genetic element), termed staphylococcal cassette chromosome mec (SCCmec), which harbours the methicillin resistance and other resistance gene. [6],[7],[8] SCCmec genetic element characterized by the presence of two essential genetic components (the mec gene complex and the ccr gene complex), and the junkyard (J) regions. SCCmec elements are currently classified into types I, II, III, IV and V according to the nature of the mec and ccr gene complexes, and are further classified into subtypes according to differences in their J region DNA . [6],[7],[8] Complete identification of bacterial genetic background and the SCCmec element is very important for molecular typing of MRSA, also its very essential for understanding of the molecular epidemiology of MRSA to easily detect and control the infections with MRSA.

Molecular typing techniques have been used with increasing frequency in studies of the epidemiology of MRSA and also for rapid detection of MRSA clones. [1],[9]

The purpose of this study was, firstly, characterization of the recovered MRSA from different hospitals located in Jeddah, King Saudi Arabia, phenotypically by conventional methods and genotypically by a novel multiplex PCR for direct detection of the S. aureus 16S rRNA gene (which serves as an internal control), the mecA gene, and the Panton - valentine leucocidin (PVL) gene. Secondly, using multiplex PCR reported by Zhang et al., [9] for subtyping of SCCmec elements for molecular characterization of MRSA recovered from outpatient clinic located in Jeddah, Kingdom of Saudi Arabia.

Bacterial isolates

A total of 101 clinical isolates of methicillin-resistant S. aureus (MRSA) strains were collected from the major hospital laboratories and public health centres, Jeddah, Kingdom of Saudi Arabia during the period of August 2009 to May 2011

All the strains were tested phenotypically by conventional methods and genotypically by PCR for direct detection of S. aureus 16S rRNA and mecA genes of S. aureus, including 46 strains recovered from surgical wound infection, 2 strains recovered from abscess and 28 strains recovered from pneumonia and lung abscess, 9 strains from burn and blood stream infection, 4 strains from pus, 6 strains from urinary tract infection, 3 strains from breast while the other 3 strains recovered from bone fracture with surgery. All isolates were identified according to colonial and microscopical morphology, catalase and coagulase production. The 101 strains were tested also by multiplex PCR targeting at the same time S. aureus 16S rRNA, (PVL) and mecA resistance genes.


 ~ Antimicrobial Susceptibility Test Top


Detection of oxacillin resistance by phenotypic method

Cefoxitin and oxacillin disk diffusion methods

Cefoxitin (30 μg) and oxacillin (1 μg) discs were used. The cefoxitin discs diffusion and oxacillin discs diffusion test were performed using the routine discs diffusion procedure, and the results were evaluated according to the interpretive criteria of CLSI, 2006.

Antimicrobial susceptibility test to non-beta-lactam drugs

The antimicrobial susceptibility test to a range of antimicrobial agents was performed using disks (contain penicillin, augmantin, cefazolin, cefuroxime, ceftriaxone, gentamicin, ciprofloxacin, moxifloxacin, clindamycin, erythromycin, azithromycin, linezolid, fusidic acid, mupirocin, nitrofurantoin, rifampin, synercid, teicoplanin, tetracycline, vancomycin) as adopting the Kirby-Bauer disk diffusion method using Muller-Hinton broth and agar and antibiotics disks (Oxoid Limited, Hampshire, England) according to the recommendations of Clinical Laboratory Standards Institute (CSLI) formally National Committee for Clinical Laboratory Standards, (NCCLS), 2006.

Extraction of DNA from bacterial isolates

The bacterial isolates were re-suspended in 400 μl Tris-EDTA buffer (pH 8.0) and heated in heat block at 105°C for 25 min and the genomic DNAs were extracted according to the method described by Moussa and Shibl. [10]

Multiplex PCR for detection of 16S rRNA gene specific for S. aureus, (mecA) gene and PVL genes.

Three primer sets previously used as a single primer for detection of such genes were used together after adjusting the annealing temperature. The first primer pair was (mecA F: GTG GAA TTG GCC AAT ACA GG and mecA R: TAG GTT CTG CAG TAC CGG AT primers) which can amplify 1399 base pair fragments specific for the mecA gene and its annealing temperature is 58°C according to Weller [11] (1999). The second primer pair was (Staph756F and Staph750R primers) which could amplify 756 base pair fragments specific for 16S rRNA of S. aureus, the third one was (Luk-PV-1 and Luk-PV-2 primers) which could amplify 433 base pair fragments specific for lukS/F-PV genes which encode the PVL S/F bicomponent proteins according to Jo-Ann McClure et al. [12]

The reaction mixtures consisted of 5 μl of the extracted DNA template of the bacterial isolates, 5 μl 10× PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl 2 , 50 mM KCl, 20 mM (NH 4 )2SO 4 ), 1 μl dNTPs (40 μM), 1 μl (1U Ampli Taq DNA polymerase), 1 μl (50 pmol) from the forward and reverse primers. The three sets of primer pairs were used in each reaction mixture and the volume of the reaction mixture was completed to 50 μl using DDW. 40 μl paraffin oil was added and the thermal cycler was adjusted as follows: 94°C for 100 min, followed by 10 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, and 25 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1.5 min, followed by final extension at 72°C for 1.5 min, and the PCR products were stored in the thermal cycler at 4°C until they were collected.

Multiplex PCR for typing of sccmec elements for molecular characterization of the recovered MRSA

The multiplex PCR reported by Zhang et al. [9] for typing and subtyping of SCCmec elements for molecular characterization of MRSA was used. The SCCmec M-PCR typing assay contained eight pairs of primers including the unique and specific primers for SCCmec types and subtypes I, II, III, IVa, IVb, IVc, IVd and V. These primers and their respective concentrations used in the PCR are listed in [Table 1]. All PCR assays were performed directly from bacterial suspensions obtained after the rapid DNA extraction method reported by Zhang et al. [13] The reaction mixtures consisted of 5 μl of the extracted DNA template of the bacterial isolates, 5 μl of 10× PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl 2 , 50 mM KCl, 20 mM NH 4 )2SO 4 ), 1 μl dNTPs (40 mM), 1 μl (1U Ampli Taq DNA polymerase), 1 μl (50 pmol) from the forward and reverse primers. The three sets of primer pairs were used in each reaction mixture and the volume of the reaction mixture was completed to 50 μl using DDW. 40 μl of paraffin oil was added and the thermal cycler was adjusted as follows: beginning with an initial denaturation step at 94°C for 5 min followed by 10 cycles of 94°C for 45 s, 65°C for 45 s, and 72°C for 1.5 min and another 25 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1.5 min, ending with a final extension step at 72°C for 10 min and followed by a hold at 4°C.
Table 1: Primer sequences, amplicon size, concentrations and specifi city of the primers used for SCCmec typing

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Agarose gel electrophoresis

The PCR products were tested for positive amplification by agarose gel electrophoresis previously reported by Sambrook et al. [14] using suitable molecular weight markers.


 ~ Results Top


Antimicrobial susceptibility patterns

Susceptibility to methicillin and oxacillin

All the strains were resistant phenotypically to methicillin and oxacillin which was confirmed by PCR using the specific primers of the mecA resistance gene.

Susceptibility to other non-beta-lactam drugs

All the strains were resistant to penicillin, augmantin, cefazolin, cefuroxime and ceftriaxone (100%). Moreover, the percentages of resistance to gentamicin, ciprofloxacin, moxifloxacin, clindamycin, erythromycin, azithromycin, linezolid, fusidic acid, mupirocin, rifampin, synercid, teicoplanin and tetracycline were 28.7%, 37.6%, 37.6%, 32.8%, 43.5%, 61.4%, 16.8%, 28.7%, 11.9%, 12.9%, 14.9%, 2.0% and 42.6%, respectively. While all isolates were susceptible to vancomycin and nitrofurantoin.

Multiplex PCR for detection of 16S rRNA gGene specific for S. aureus, mecA and PVL genes.

Multiplex PCR for detection of S. aureus species specific 16S rRNA, mecA resistance gene and PVL gene allow direct detection of MRSA harbouring the PVL gene. All the 101 strains (100%) previously identified phenotypically as S. aureus with bacteriological examination were positive for amplification of 756 base fragments specific for 16S rRNA of S. aureus using Staph756 F and Staph750 R primers. Moreover, all the 101 strains (100%) which appeared methicillin/oxacillin resistant phenotypically with the antimicrobial susceptibility test were positive for amplification of 1339 base pair fragments specific for the mecA gene, while only 38 strains (37.6%) showed positive amplification of 433 base pair fragments specific for lukS/F-PV by using Luk-PV-1 and Luk-PV-2 primers. The majorities of the PVL genes were recorded in soft tissues and wound infections 18 (39.1%) strains. Moreover, the PVL gene was recorded in patients suffering from pneumonia and respiratory infections 7 (25.0%) strains. The distribution of the PVL gene among different samples is illustrated in [Table 2] and [Figure 1].
Figure 1: Multiplex PCR showing positive amplifi cation of 756 base fragments specific for 16S rRNA of S. aureus, 1339 base pair fragments specific for the mecA gene and 433 base pair fragments specific for PVL gene. Lanes 8 and 20 showing methicillin-sensitive S. aureus. Lane 17 showing 100bp ladder.

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Table 2: Distribution of Panton– valentine leucocidin Gene among the tested isolates

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Typing of SCC mec elements of the recovered MRSA using multiplex PCR

All the 101 strains were tested by the multiplex PCR reported by Zhang et al. [9] for typing and subtyping of SCC mec elements for molecular characterization of MRSA using eight pairs of primers including the unique and specific primers for SCC mec types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V. Among them, 43 (42.5%), 39 (38.6%), 16 (15.8%) and 3 (2.9%) isolates belonged to SCC mec type and subtypes V, III, IVa, and IVc but no SCC mec types and subtypes I, II, IVb or IVd were found among the isolates tested, as shown in [Figure 2], [Figure 3], and [Figure 4].
Figure 2: Multiplex PCR showing positive amplification of 147 base fragments specific for the mecA gene and 280 base pair fragments specific for SCC mec Type III. Lane 1 showing 100bp ladder.

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Figure 3: Multiplex PCR showing positive amplification of 147 base fragments specific for the mecA gene and 325 base pair fragments specific for SCC mec Type V. Lane 12 showing 100bp ladder.

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Figure 4: Multiplex PCR showing positive amplification of 147 base fragments specific for the mecA gene and 776 base pair fragments specific for SCC mec Type IVa. Lane 1 showing 100bp ladder.

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 ~ Discussion Top


Infections not acquired at a health care setting or institution, are considered community-associated MRSA (CA-MRSA), these organisms have recently emerged as an important cause of community-associated staphylococcal infections [15],[16] . PCR have been reported for the identification of MRSA [17] , or the identification of specific virulence gene. [13] . McClure et al. [18] developed a new multiplex assay to aid with the early identification of CA-MRSA strains. This assay targets the staphylococcus genus-specific 16S rRNA gene, the luk S/F-PV and the SCC mec type 4a. This assay was shown to be 100% accurate and reliable. Moreover, this assay is easily amenable to routine clinical use in any molecular biology laboratory with PCR capabilities but it could not detect the mecA gene. Therefore, one of the main objectives of this study was the improvement of this multiplex PCR to be able to detect the mecA gene specific for methicillin/oxacillin resistant, for simultaneous detection of the staphylococcus genus-specific 16S rRNA gene and the luk S/F-PV genes. Results observed in [Figure 1] revealed that all the 101 strains (100%) previously identified phenotypically as S. aureus with bacteriological examination were positive for amplification of 756 base pair fragments specific for 16S rRNA of S. aureus using Staph756 F and Staph750 R primers. Moreover, all the 101 strains (100%) which appeared methicillin/oxacillin resistant phenotypically with the antimicrobial susceptibility test were positive for amplification of 1339 base pair fragments specific for the mecA gene, while only 38 strains (37.6%) showed positive amplification of 433 base pair fragments specific for lukS/F-PV by using Luk-PV-1 and Luk-PV-2 primers. The obtained results indicated the higher sensitivity and specificity of such multiplex PCR for rapid detection and identification of MRSA, in addition to the time consuming. Our results confirm the conclusion of Jo-Ann McClure et al. [12] and Moussa and Shibl,. [10] The majorities of the PVL genes were recorded in soft tissues and wound infections 18 (39.1%) which indicated the involvement of the PVL gene with sever skin and soft tissue infections specially, necrotizing skin infection. [9],[15] PVL gene also was found in 7 (25.0%) strains out of 28 strains recovered from pneumonia and pulmonary abscess. The obtained results confirm the conclusion of Bocchini et al. [19] and Gillet et al.; [20] they mentioned that PVL is a cytolytic toxin associated with S. aureus furuncles and necrotizing pneumonia. Moreover, the presences of the PVL gene in such strains decreased survival in patients with community-acquired S. aureus pneumonia. [20]

Molecular characterization of SCC mec types and subtypes of MRSA is very essential for molecular epidemiology and controlling the human epidemics due to this organism. Moreover, molecular typing of SCC mec is one of the most important tools for studying the epidemiology of the MRSA to determine the genetic relatedness and genetic similarity of the recovered isolates from certain localities, especially with the emerging of community-acquired MRSA worldwide. [9] The multiplex PCR reported by Zhang et al. [9] for typing and subtyping of SCC mec was used in this study for complete identification of the recovered strains. The most predominant SCC mec type among the examined isolates is type V 43 (42.5%), these groups being implicated in currently emerging community MRSA outbreaks. [7],[9],[15] The SCC mec type III was also higher 39 (38.6%), and it has been associated with hospital-acquired MRSA. Our results confirm that MRSA strains associated with community transmission typically carry SCC mec types IV or V, which are smaller and theoretically more easily transferable between organisms than the SCC mec types (I-III), characteristic of MRSA strains that predominate in healthcare settings. [21]

The newly modified multiplex PCR could detect the S. aureus species specific for the 16S rRNA gene, mecA gene specific for the methicillin/oxacillin-resistant and PVL gene. Moreover, the predominant SCC mec type among the examined isolates is type V (42.5%), followed by type III (38.6%).


 ~ Acknowledgment Top


This work was supported by king Abdul-Aziz City for Science and Technology and the author's express also great appreciation to the Deanship of Scientific Research at King Saud University the research group No.: RGP-VPP-162." for the continuous support.

 
 ~ References Top

1.Mehndiratta PL, Bhalla P, Ahmed A, Sharma YD. Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: A reference laboratory perspective. Indian J Med Microbiol 2009;27:116-22.  Back to cited text no. 1
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3.Diep BA, Chambers HF, Graber CJ, Szumowski JD, Miller LG, Han LL, et al. Emergence of multidrug-resistant, community-associated, methicillin-resistant Staphylococcus aureus Clone USA300 in men who have sex with men. Ann Intern Med 2008;148:249-57.  Back to cited text no. 3
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6.Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, et al. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin- resistant Staphylococcus aureus. Antimicrob Agents Chemother 2001;45:1323-36.  Back to cited text no. 6
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8.Ma XX, Ito T, Tiensasitorn C, Jamklang M, Chongtrakool P, Boyle-Vavra S, et al. Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 2002;46:1147-52.  Back to cited text no. 8
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11.Weller TM. The distribution of mecA, mecR1 and mec1 and sequence analysis of mec1 and the mec promoter region in Staphylococci expressing resistance to methicillin. J Antimicrob Chemother 1999;43:15-22.  Back to cited text no. 11
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14.Sambrook J, Fritsch EF, Maniatis T. Molecular coloning a laboratory manual. Vol. 1. New York: Cold Spring Harbor Laboratory press; 1989.  Back to cited text no. 14
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]

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1 Corrections in molecular characterization of S. aureus
Kumar, V.
Indian Journal of Medical Microbiology. 2013; 31(1): 92
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