Real-time PCR assay for detection of <em>Staphylococcus aureus</em>, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Liliana Galia; Marco Ligozzi; Anna Bertoncelli; Annarita Mazzariol; Liliana Galia; Marco Ligozzi; Anna Bertoncelli; Annarita Mazzariol

doi:10.3934/microbiol.2019.2.138

AIMS Microbiology

2019, Volume 5, Issue 2: 138-146. doi: 10.3934/microbiol.2019.2.138

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Research article

Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Department of Diagnostics and Public Health, University of Verona, Verona, Italy

Received: 25 November 2018 Accepted: 26 March 2019 Published: 21 May 2019

Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an ‘in house’ real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies.
- Staphylococcus aureus,
- Real-time PCR,
- Panton Valentine Leucocidin,
- MRSA,
- nuc gene,
- coagulase negative staphylococci
Citation: Liliana Galia, Marco Ligozzi, Anna Bertoncelli, Annarita Mazzariol. Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples[J]. AIMS Microbiology, 2019, 5(2): 138-146. doi: 10.3934/microbiol.2019.2.138

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Abstract

Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an ‘in house’ real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies.

Abbreviation MRSA: Methicillin resistant ; MSSA: Methicillin susceptible ; MRCoNS: Methicillin reistant Coagulase Negative Staphilococci; PVL: Panton valentine leucocidin; PCR: Polymerase chain reaction; CFU: Colonies forming unit; PBP2a: Penicillin binding protein 2a;

Conflicts of interests

All authors declare no conflicts of interest in this paper.

References

[1]	Gordon RJ, Lowy FD (2008) Pathogenesis of Methicilln-Resistant Staphylococcus aureus Infection. Clin Infect Dis. 46: S350–S359. doi: 10.1086/533591
[2]	Meyer F, Girardot R, Piemont Y, et al. (2009) Analysis of the specificity of Panton-Valentine leucocidin and gamma-hemolysin F component binding. Infect Immun 77: 266–273. doi: 10.1128/IAI.00402-08
[3]	Vandenesch F, Lina G, Gillet Y, et al. (2009) The end of the controversy: Panton Valentine is the culprit. Med Sci 25: 984–986.
[4]	McDonald RR, Antonishyn NA, Hansen T, et al. (2005) Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 43: 6147–6149. doi: 10.1128/JCM.43.12.6147-6149.2005
[5]	Shallcross LJ, Fragaszy E, Johnson AM, et al. (2013) The role of Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. Lancet Infect Dis 13: 43–54. doi: 10.1016/S1473-3099(12)70238-4
[6]	Dohin B, Gillet Y, Kohler R, et al. (2007) Pediatric bone and joint infections caused by Panton- Valentine leukocidin-positive Staphylococcus aureus. Pediatr Infect Dis J 26: 1042–1048. doi: 10.1097/INF.0b013e318133a85e
[7]	Gillet Y, Issartel B, Vanhems P, et al. (2002) Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet 359: 753–759. doi: 10.1016/S0140-6736(02)07877-7
[8]	Lina G, Piémont Y, Godail-Gamot F, et al. (1999) Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 29: 1128–1132. doi: 10.1086/313461
[9]	Calfee DP, Salgado CD, Milstone AM, et al. (2014) Strategies to Prevent Methicillin-Resistant Staphylococcus aureus Transmission and Infection in Acute Care Hospitals. Infec Cont Hosp Epidemiol 35: 772–796. doi: 10.1086/676534
[10]	Al-Talib H, Yean CY, Al-Khateeb A, et al. (2014) Rapid detection of methicillin-resistant Staphylococcus aureus by a newly developed dry reagent-based polymerase chain reaction assay. J Microbiol Immunol Infection 47: 484–490. doi: 10.1016/j.jmii.2013.06.004
[11]	Johnsson D, Molling P, Stralin K, et al. (2004) Detection of Panton-Valentine leukocidin gene in Staphylococcus aureus by Light Cycler PCR: clinical and epidemiological aspects. Clin Microbiol Infect 10: 884–889. doi: 10.1111/j.1469-0691.2004.00976.x
[12]	Llarrull LI, Fisher JF, Mobashery S (2009) Molecular basis and phenotype of methicillin resistance in Staphylococcus aureus and insights into new β-Lactams that meet the challenge. Antimicrob Agents Chemother 53: 4051–4063. doi: 10.1128/AAC.00084-09
[13]	Teng CS, Lo WT, Wang SR, et al. (2009) The role of antimicrobial therapy for treatment of uncomplicated skin and soft tissue infections from community-acquired methicillin-resistant Staphylococcus aureus in children. J Microbiol Immunol Infect 42: 324–328.
[14]	Boyle-Vavra S, Daum RS (2007) Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton-Valentine leukocidin. Lab Invest 87: 3–9. doi: 10.1038/labinvest.3700501
[15]	Becker K, Heilmann C, Peters G (2014) Coagulase negative staphylococci. Clin Microbiol Rev 27:870–926. doi: 10.1128/CMR.00109-13
[16]	Stegger M, Andersen PS, Kearns A, et al. (2012) Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harboring either mecA or the new mecA homologue mecA(LGA251). Clin Microbiol Infect 18: 395–400. doi: 10.1111/j.1469-0691.2011.03715.x
[17]	Söderquist B, Neander M, Dienus O, et al. (2012) Real-time multiplex PCR for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture. APMIS 120: 427–432. doi: 10.1111/j.1600-0463.2011.02849.x
[18]	Okolie CE, Wooldridge KG, Turner DP, et al. (2015) Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection so staphylococcal virulence and methicillin resistance markers. Mol Cell Probes 29: 144–150. doi: 10.1016/j.mcp.2015.03.002

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