MULTIPLEX-PCR FOR DETECTION OF β-LACTAM RESISTANCE IN Staphylococcus spp.

  • Giovana Hashimoto Nakadomari
  • Amanda Carmen Charalo
  • Ana Claudia Lemes Pavan
  • Vanessa Kelly Capoia Vignoto
  • Ricardo Antonio Pilegi Sfaciotte
  • Sheila Rezler Wosiacki Universidade Estadual de Maringá
DOI: https://doi.org/10.4025/revcivet.v6i2.45050

Resumo

A Staphylococcus Multiplex PCR system was developed for the simultaneous detection of the mecA, mecC, blaZ (resistance genes of b-lactam resistance) and PVL (pathogenicity factor gene), associated with an internal reaction control with the 16S rRNA gene. There were used primers described in the literature with and without modification and designed primers to standardize the hybridization and amplification temperature of distinct bands with 139 bp (mecC), 228 bp (16S), 313 bp (mecA), 408 bp (PVL) and 516 bp (blaZ) of molecular weight. The standardization was performed in ATCC strains and Staphylococcus schleiferiand tested in 56 strains of Staphylococcusspp. The 16S gene (internal control) was amplified in all samples, mecA gene was detected in two samples, mecA associated with mecC gene in one sample, mecA associated to the blaZ gene in 14 samples and the blaZ gene in 15 samples. No resistance genes were amplified in 24 samples. The PVL gene was not amplified in any of the samples tested.

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Referências

ALI, A.O. Detection of MecA, MecC and Femb Genes by Multiplex Polymerase Chain Reaction. Journal of Veterinary Advances, v. 5, n. 12, 2015. doi: 10.5455/jva.20151204111125

ASFOUR, H.A.E.; DARWISH, S.F. Phenotypic and genotypic detection of both mecA- and blaZ-genes mediated β-lactam resistance in Staphylococcus strains isolated from bovine mastitis. Global Veterinaria, v.6, n.1, p.39-50, 2011.

BAUER, A.W.; KIRBY, W.M.M.; SHERRIS, J.C; TURCK, M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, v.45, p.493-496, 1966.

BECKER, K.; LARSEN, A.R.; SKOV, R.L.; PATERSON, G.K.; HOMES, M.A.; SABAT, A.J.; FRIEDRICH, A.W.; KÖCK, R.; PETERS, G.; KRIEGESKORTE, A. Evaluation of a Modular Multiplex-PCR Methicillin-Resistant Staphylococcus aureus Detection Assay Adapted for mecC Detection. Journal of Clinical Microbiology, v. 51, n. 6, p. 1917-19, 2013. doi: 10.1128/JCM.00075-13

BRCAST. Brazilian Commitee on Antimicrobial Suscetibilitty testing. Tabela pontos de corte clínicos. Julho, 2018.

CLSI. Performance Standards for Antimicrobial Disk and diluition Susceptibility bactéria isolated from animals. 3th ed. CLSI document VET01S. Pennsylvania: Clinical and Laboratory Standards Institute; 2015.

CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals. 4th ed. CLSI supplement VET08. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.

CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. CLSI document M100-S23. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2013.

GUARDABASSI, L.; LARSEN, J.; WEESE, J.S.; BUTAYE, P.; BATTISTI, A.; KLUYTMANS, J.; LLOYD, D.H.; SKOV, R.L. Public health impact and antimicrobial selection of meticillin-resistant staphylococci in animals. Journal of Global Antimicrobial Resistance, v. 1, p. 55-62, 2013. doi: https://doi.org/10.1016/j.jgar.2013.03.011

KANEKO, J.; KAMIO, Y. (2004). Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes". Bioscience, Biotechnology, and Biochemistry, v. 68, n. 5, p. 981–1003, 2004. doi:10.1271/bbb.68.981

LARSEN, A.R.; STEGGER, M.; SØRUM, M. spa typing directly from a mecA, spa and pvl multiplex PCR assay—a cost-effective improvement for methicillin-resistant Staphylococcus aureus surveillance. Clinical Microbiology and Infection, v. 14, n. 6, p. 611-4, 2008. doi: https://doi.org/10.1111/j.1469-0691.2008.01995.x

LONCARIC, I.; KÜBBER-HEISS, A.; POSAUTZ, A.; RUPPITSCH, W.; LEPUSCHITZ, S.; SCHAUER, B.; FEBLER, A.T.; KRAMETTER-FRÖTSCHER, R.; HARRISON, E.M.; HOMES, M.A.; KÜNZEL, F.; SZOSTAK, M.P.; HAUSCHILD, T.; DESVARS-LARRIVE, A.; MISIC, D.; ROSENGARTEN, R.; WALZER, C.; SLICKERS, P.; MONECKE, S.; EHRICHT, R.; SCHWARZ, S.; SPERGSER, J.; Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals. Veterinary Microbiology, v. 230, p. 138-144, 2019. doi: https://doi.org/10.1016/j.vetmic.2019.02.014

PATERSON, G.K.; HARRISON, E.M.; HOLMES, M.A. The emergence of mecC methicillin-resistant Staphylococcu aureus. Trends in Microbiology, v. 22, n. 1, p. 42-47, 2014. doi: 10.1016/j.tim.2013.11.003

PICHON, B.; HILL, R.; LAURENT, F.; LARSEN, A.R.; SKOV, R.L.; HOMES, M.; EDWARDS, G.F.; TEALE, C.; KEARNS, A.M. Development a real-time quadruplex PCR assay for simultaneous detection of nuc, PVL, mecA and homologue mecALGA251. Journal of Antimicrobial Chemotherapy, v. 67, n. 10, p. 2338–2341, 2012. doi: 10.1093/jac/dks221

RALLAPALLI, S.; VERGHESE, S.; VERMA, R.S. Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus. PLos One, v. 9, n. 5, e97617, 2014. doi: 10.1371/journal.pone.0097617

ROCCHETTI, T.T.; MARTINS, K.B.; MARTINS, P.Y.F.; OLIVEIRA, R.A.; MONDELLI, A.L.; FORTALEZA, C.M.C.B.; CUNHA, M.L.R.S. Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction. Brazilian Journal of Infection Disease, v. 8, n. 2, p. 99-105, 2014. doi: 10.1371/journal.pone.0097617

RUDKIN, J.K.; EDWARDS, A.M.; BOWDEN, M.G.; BROWN, E.L.; POZZI, C.; WATERS, E.M.; CHAN, W.C.; WILIAMS, P.; O’GARA, J.P.; MASSEY, R.C. Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum Sensing System. The Journal of Infectious Diseases, v.205, n.5, p.798–806, 2012. doi: 10.1093/infdis/jir845

SFACIOTTE, R. A. P.; BORDIN, J. T.; VIGNOTO, V. K. C.; MUNHOZ, P. M.; PINTO, A. A.; BARBOSA, M. J. B.; CARDOZO, R. M.; OSAKI, S. C.; WOSIACKI, S. R. Antimicrobial Resistance in Bacterial Pathogens of Canine Otitis. American Journal of Animal and Veterinary Sciences, v.10, n.3, p.162-169, 2015. doi: 10.3844/ajavsp.2015.162.169

SOLANKI, G. Polimerase Chain Reaction. International Journal of Pharmacological Research, v. 2, n. 3, 98-102, 2012.

STEGGER, M.; ANDERSEN, P.S.; KEARNS, A.; PICHON, B.; HOLMES, M.A.; EDWARDS, G.; LAURENT, F.; TEALE, C.; SKOV, T.; LARSEN, A.R. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251. Clinical Microbiology and Infection, v. 18, p. 395-400, 2012. doi: https://doi.org/10.1111/j.1469-0691.2011.03715.x

SZMIGIELSKI, S.; PRÉVOST, G.; MONTEIL, H.; COLIN, D.A.; JELJASZEWICZ, J. Leukocidal toxins of staphylococci. Zentralbl Bakteriol. v. 289, n. 2, p. 185–201, 1999. doi:10.1016/S0934-8840(99)80105-4

TABATABAEI, S.; NAJAFIFAR, A.; BADOUEI, M.A.; SAKEHI, T.Z.; TAMAI, I.A.; KHAKSAR, E.; ABBASSI, M.S.; GHAZISAEEDI, F. Genetic characterization of methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in pets and veterinary personnel in Iran: new insights into emerging methicillin-resistant S. pseudintermedius (MRSP). Journal of Global Antimicrobial Resistance, v. 16, p. 6-10, 2019. doi: https://doi.org/10.1016/j.jgar.2018.08.022

VELASCO, V.; SHERWOOD, J.S.; ROJAS-GARCÍA, P.P; LOGUE, C.M. Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecAand Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat. PLos One, v. 9, n. 5, e97617, 2014. doi: 10.1371/journal.pone.0097617

Publicado
2019-08-03
Como Citar
Nakadomari, G. H., Charalo, A. C., Pavan, A. C. L., Vignoto, V. K. C., Sfaciotte, R. A. P., & Wosiacki, S. R. (2019). MULTIPLEX-PCR FOR DETECTION OF β-LACTAM RESISTANCE IN Staphylococcus spp. Revista De Ciência Veterinária E Saúde Pública, 6(2), 262-275. https://doi.org/10.4025/revcivet.v6i2.45050
Edição
v. 6 n. 2 (2019)
Seção
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Declaro que o presente artigo é original, não tendo sido submetido à publicação em qualquer outro periódico nacional ou internacional, quer seja em parte ou em sua totalidade. Declaro, ainda, que uma vez publicado na revista Arquivo de Medicina veterinária e Saúde Pública da UEM, editada pela Universidade Estadual de Maringá, o mesmo jamais será submetido por mim ou por qualquer um dos demais co-autores a qualquer outro periódico. Através deste instrumento, em meu nome e em nome dos demais co-autores, porventura existentes, cedo os direitos autorais do referido artigo à Universidade Estadual de Maringá e declaro estar ciente de que a não observância deste compromisso submeterá o infrator a sanções e penas previstas na Lei de Proteção de Direitos Autorias (Nº9609, de 19/02/98).