Caracterización molecular de aislados de Staphylococcus aureus resistente a meticilina (SARM) utilizando mecA-PCR y PCR-RFLP

Marilena Calderón González; Carlos Ramos; Nora Ortiz de Moreno

Submitted December 27, 2022

Published 2022-12-27

Artículos

Vol. 6 No. 2 (2022): Visión Antataura

Molecular characterization of methicillin-resistant staphylococcus aureus (MRSA) using mecA-PCR and RFLP-PCR

Marilena Calderón González ⁺⁻
Carlos Ramos⁺⁻
Nora Ortiz de Moreno ⁺⁻

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DOI: ND

Published: 2022-12-27

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Calderón González , M., Ramos, C., & Ortiz de Moreno , N. (2022). Molecular characterization of methicillin-resistant staphylococcus aureus (MRSA) using mecA-PCR and RFLP-PCR. Visión Antataura, 6(2), 45–63. Retrieved from https://revistas.up.ac.pa/index.php/antataura/article/view/3384

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Abstract

With the objective of characterizing methicillin-resistant Staphylococcus aureus that circulates in our country, a total of 88 positive methicillin-resistant Staphylococccus aureus (MRSA) were obtained from the national epidemiological survey library of Instituto Conmemorativo Gorgas de Estudios de la Salud del Laboratorio Central de Referencia en Salud Pública, Republic of Panama, We amplified mecA and demonstrated its presence in 100% of our isolates of the gene mecA. The characterizacion of the isolates was performed by RFLP-PCR of the coa, spa, HVR genes and digestion with Haell. Then amplificated three genes (coa, spa and HVR). The aisolates were grouped into five categories based on the genes for which amplification was obtained. Restriction digestion of a mixture of the three amplicons produced 19 banding patterns; these patterns were reproducible and comparative to previous studies. Category 1 with amplification in the three genes (24 isolates) generated 19 different banding patterns. Category 2 with amplification in the genes (coa and spa) grouped 25 isolates with 17 different patterns. Category 3 with amplification in (coa and HVR) grouped 10 isolates with 6 patterns. Category 4 with amplification only for coa with 11 isolates and 7 patterns. Category 5, only one isolate with amplification for HVR generated only one type of pattern. The band patterns obtained are reproducible and comparable to other previous studies. Our results suggest that RFLP-PCR is a practical, easy, rapid and discriminant screening methodology for epidemiological surveillance of infections caused by methicillin-resistant Staphylococcus aureus (MRSA).

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References

Álvarez, I., Ponce, J. (2014). Staphylococcus aureus, evolución de un viejo patógeno. Revista Cubana de Pediatría, 84(4), 383-391. http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0034-75312012000400007&lng=es&tlng=es.
Adesida, S.A., Likhoshvay, Y., Eisner, W., Coker, O.A., Abioye, O.A.,Ogunsola, F.T., Kreiswirth, B.N. (2006). Repeats in the 3’ region of the protein A gene is unique in a strain of Staphylococcus aureus recovered from wound infections in Lagos. Nigeria. African Journal of Biotechnology, 5 (20), 1858-1863. https://academicjournals.org/journal/AJB/article-full-text-pdf/8D59E858861
Brown, DFI., Edwards, DJ., Hawkey, PM. (2005). Guidelines for the Laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). Journal of antimicrobial chemotherapy, 56 (6), 1000-1018. https://doi.org//0.1093/jac/dki372.
Corente,M., Monno, R., Totaro, M., Martella, V., Buonavoglia, D., Rizzo, G., Buonavoglia, C. (2005). Characterization of methicillin resistant Staphylococcus aureus (MRSA) isolated at the Policlinic Hospital of Bari (Italy). New Microbiol, 28(1), 57-65. https://www.researchgate.net
Faria, N., Carrico, J.A., Oliveira, D.C., Ramirez, M., De Lencastre, H. (2008). Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin susceptible Staphylococcus aureus strains. Journal of Clinical Microbiology, 46, 136-144. https://doi.org/10.1128/jcm.01684-07.
Fenner, L., Widmer, F., Dangel, M., Frei, R. (2008). Distribution of spa types among methicillin-resistant Staphylococcus aureus isolates during a 6-year period at a low –prevalence university hospital. Journal of Medical Microbiology, 57(5), 612-616. https://doi.org/10.1099/jmm.0.47757-0.
Fifteenth International Supplement. Clinical and Laboratory Standard Institute/NCCLS, document M100-S15. CLSI, Wayne, Pa, USA.
Frenay, H.M., Theelen, L., Schouls et al. (1994). Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. Journal of Clinical Microbiology, 32(3), 846-847. https://doi.org/10.1128/jcm.32.3.846-847.1994.
Geha, DJ., Ujl, JR., Gustaferro, CA. (1994). Multiplex PCR for identification of methicillin-resistant Staphylococcus in clinical laboratory. Journal of Clinical Microbiology, 32(7), 1768-1772. https://doi.org/10.1128/JCM.32.7.
Goh, S.H., Byrne, S.K., Zhang, J.L., Chow, A.W. (1992). Molecular Typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. Journal of Clinical Micrtobiology, 30(7), 1642-1645. https://doi.org/10.1128/jcm.30.7.
Hookey, J., Richarson, J., Cookson, B. (1998). Molecular typing of Staphylococcus aureus base on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase Gene [versión electrónica]. Journal of Clinical Microbiology, 36 (4), 1083-1089. https://doi.org/10.1128.JCM.36.4
Ishino, K., Tsuchizaki, N., Isikawa, J., Hotta, K. (2007). Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus stains. Journal of Clinical Microbioly, 45(2), 607-609. https://doi.org/10.1128/JCM.02099-06.
Lange, C., Cardoso, M., Senczek, D., Schwarz, S. (1999). Molecular subtyping of Staphylococcus aureus isolates from cases of bovine mastitis in Brazil. Veterinary microbiology, 67 (2), 127-141. https://doi.org/10.1016/S0378-1135(99)00031-0.
Mahmoudi, H., Arabestani, M., Mousavi, S., Alikhani, M. (2017). Molecular Analysis of the coagulase gene in clinical and nasal carrier isolates of methicillin-resistant Staphylococcus aureus by restriction fragment polymorphisms. Journal of global antimicrobial resistance, (8), 41-45. https://doi.org/10.1016/j.jgar.2016.10.007.
Mitami, N., Ohnishi, M., Murakawa, K., Okamoto, Y. (2002). Molecular typing of methicillin-resistant Staphylococcus aureus by protein A gene sequencing. Jpn. J. Infect. Dis, 55, 179-180. https://www.ncbi.nlm.nih.gov
Mitami, N., Koizumi, A., Sano, R., Masutani, T., Murakawa, K., Mikasa. K., Okamoto, Y. (2005). Molecular Typing of Methicillin-Resistant Staphylococcus aureus by PCR-RFLP and its Usefulness in an Epidemiological Study of an Outbreak. Japanese Journal of Infectious Disease, 58 (4), 250-252. https://www.niid.go.jp
Mirharimi. SF., Hasani. A., Abdinia. B. (2016). High diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolates based on hypervariable region polymorphisms. Archives of Pediatric Infectious Diseases, 4 (4), e36433. https://doi.org/10.5812/pedinfect.36433.
Montesinos, I., Salido, E., Delgado, T., Cuervo, M., Sierra, A. (2002). Epidemiologic Genotyping of Methicillin-Resistant Staphylococcus aureus by pulsed Gel Electrophoresis at a University Hospital and comparison with Antibiotyping and Protein A and coagulase gene Polymorphisms. Journal of clinic Microbioly, 40 (6), 2119-2125. https://doi.org/10.1128/JCM.40.6.
Omar, NY, Ali, HAS., Harfoush, R. (2014). Molecular typing of methicillin-resistant Staphylococcus aureus clinical isolates on the basis of protein A and coagulase gene polymorphisms. International Journal of microbiology, Article ID 650328. https://doi.org/10.11.1155/2014/650328.
Parviz, M., Samira, A., Abbas, F., Baharak, N. (2016). Genotyping of coa and aroA genes of methicillin-resistant Staphylococcus aureus strains isolated from nasal samples in Western Iran. Jundishapur J. Microbiol, 9 (1), 1-6. https://doi.org/10.5812/jjm.26480.
Palavencino,E., (2004). Community-acquired methicillin-resistant Staphylococcus aureus infections. Clinics in laboratory medicine, 24(2), 403-418. https://doi.org/10.1016/j.cll.2004.03.007.
Salmenlinna, S., Vuopio-Varkila, J. (2001) Recongnition of two groups of methicillin-resistant- Staqphylococcus aureus strains based on epidemiology, antimicrobial susceptibility, hypervariable-region type, and ribotype in Finland. Journal of Clinical Microbioly, 39 (6), 2243-2247. https://doi.org/10.1128/JCM.39.62243-2247.2001.
Senna, J.P., Pinto, C.A., Carvalho, L.P. y Santos, D.S. (2002). Comparison of pulsed-field gel electrophoresis and PCR analysis of polymorphisms on the mec hypervariable region for typing methicillin-resistant Staphylococcus aureus. Journal of Clinical Microbiology, 40 (6), 2254-2256. 10.1128/jcm.40.6. https://doi.org/10.1128/JCM.40.6.2254-2256.2002
Schwarrzkopf, A. (1995). Coagulase gene polymorphism in Staphylococcus aureus a new epidemiologic marker. Inmunitat und infection, 23(1), 2407-2412. https://doi.org/10.1128/JCM.32.10.
Shakeri, F., Shojai, A., Golalipour, M., Alang, S., Vaez, H., Ghaemi, E. (2010). Spa Diversity among MRSA and MSSA Strains of Staphylococcus aureus in North of Iran. International Journal of Microbiology, 2010, (ID 351397), 1-5. https://doi.org/10.1155/2010/351397.
Schmitz, M., Steiert, M., Tichy, HV., Hofmann, B., Verhoef, J., Heinz, K., Kohrers, K., Jones, E. (1998). Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods. J. Med. Microbiol, 47(4) 341-351. https://doi.org/10.1099/00222615-47-4.
Strommenger, B., Braulke, C., Heuck et al. (2008). “spa typing of Staphylococcus aureus as a frontline tool in epidemiological typing.” Journal of Clinical Microbiology, 46 (2), 574-581. https://doi.org/10.1128/jcm.01599-07.
Teruyo, I., Kuwahara, K., Hiramatsu, K. (2007). Methods in Molecular Biology. Yinduo J (Eds).Methicillin-resistant Staphylococcus aureus (MRSA) Protocols. Chapter 7. Humana Press inc.
Witt, R., Van Belkum, A., Van Leeuwen W. (2011). Molecular diagnostics and genotyping of methicillin-resistant Staphylococcus aureus (MRSA) and update. Clinical Microbiological Diagnostics, 4(10), 375-380. https://doi.org/10.1586/ERM.10.34.
Whichelhaus, T.A., Hunfeld P., Boddinghanus, B., Kraiczyz, P., Schafer, V., Brade, V. (2001). Rapid molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP. Infection & Control Hospital Epidemiology, 22 (5), 294-298. https://doi.org/10.1086/501903.
Wilailuckana, C., Tribuddharat, C., Tiensasitorn, Pongpech, P., Naenna, P., Rugdeekha, S., Dhiraputra, Ch., Danchaivijitr, S. (2006). Discriminatory powers of molecular typing techniques for methicillin-resistant Staphylococcus aureus in a University Hospital, Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 32(2), 327-334. https://pubmed.ncbi.nlm.nih.gov
Yazdchi, B., Pourmand, MR., Hajiabdolgaghi, M., Hoseini, M., Mardani, N. (2008). Molecular characterization of hypervariable region (hvr) and antibiotic susceptibility patterns of Staphylococcus aureus strains isolates collected from Tehran University of medical sciences hospitals. Journal of school of Public Health and Institute of Public Health Research, 2 (6), 39-48. http://sjsph.tums.ac.ir/article-1-143-en.html.
Yinduo, J. (2007). Methods in Molecular in Molecular Biology.Methicillin-resistant Staphylococcus aureus (MRSA) Protocols. New York, Humana Press.
Zamani, A., Sadeghian, S., Ghaderkhani, J., Alikhani, MY., Najafimosleh, M., Goodarzi MT et al. (2007). Detection of methicillin-resistance (mecA) gene in Staphylococcus aureus strains by PCR and determination of antibiotic susceptibility. Ann Microbiol, (57), 273-276. https://doi.org/10.1007/BF03175219.

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[1] Álvarez, I., Ponce, J. (2014). Staphylococcus aureus, evolución de un viejo patógeno. Revista Cubana de Pediatría, 84(4), 383-391. http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0034-75312012000400007&lng=es&tlng=es.

[2] Adesida, S.A., Likhoshvay, Y., Eisner, W., Coker, O.A., Abioye, O.A.,Ogunsola, F.T., Kreiswirth, B.N. (2006). Repeats in the 3’ region of the protein A gene is unique in a strain of Staphylococcus aureus recovered from wound infections in Lagos. Nigeria. African Journal of Biotechnology, 5 (20), 1858-1863. https://academicjournals.org/journal/AJB/article-full-text-pdf/8D59E858861

[3] Brown, DFI., Edwards, DJ., Hawkey, PM. (2005). Guidelines for the Laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). Journal of antimicrobial chemotherapy, 56 (6), 1000-1018. https://doi.org//0.1093/jac/dki372.

[4] Corente,M., Monno, R., Totaro, M., Martella, V., Buonavoglia, D., Rizzo, G., Buonavoglia, C. (2005). Characterization of methicillin resistant Staphylococcus aureus (MRSA) isolated at the Policlinic Hospital of Bari (Italy). New Microbiol, 28(1), 57-65. https://www.researchgate.net

[5] Faria, N., Carrico, J.A., Oliveira, D.C., Ramirez, M., De Lencastre, H. (2008). Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin susceptible Staphylococcus aureus strains. Journal of Clinical Microbiology, 46, 136-144. https://doi.org/10.1128/jcm.01684-07.

[6] Fenner, L., Widmer, F., Dangel, M., Frei, R. (2008). Distribution of spa types among methicillin-resistant Staphylococcus aureus isolates during a 6-year period at a low –prevalence university hospital. Journal of Medical Microbiology, 57(5), 612-616. https://doi.org/10.1099/jmm.0.47757-0.

[7] Fifteenth International Supplement. Clinical and Laboratory Standard Institute/NCCLS, document M100-S15. CLSI, Wayne, Pa, USA.

[8] Frenay, H.M., Theelen, L., Schouls et al. (1994). Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. Journal of Clinical Microbiology, 32(3), 846-847. https://doi.org/10.1128/jcm.32.3.846-847.1994.

[9] Geha, DJ., Ujl, JR., Gustaferro, CA. (1994). Multiplex PCR for identification of methicillin-resistant Staphylococcus in clinical laboratory. Journal of Clinical Microbiology, 32(7), 1768-1772. https://doi.org/10.1128/JCM.32.7.

[10] Goh, S.H., Byrne, S.K., Zhang, J.L., Chow, A.W. (1992). Molecular Typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. Journal of Clinical Micrtobiology, 30(7), 1642-1645. https://doi.org/10.1128/jcm.30.7.

[11] Hookey, J., Richarson, J., Cookson, B. (1998). Molecular typing of Staphylococcus aureus base on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase Gene [versión electrónica]. Journal of Clinical Microbiology, 36 (4), 1083-1089. https://doi.org/10.1128.JCM.36.4

[12] Ishino, K., Tsuchizaki, N., Isikawa, J., Hotta, K. (2007). Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus stains. Journal of Clinical Microbioly, 45(2), 607-609. https://doi.org/10.1128/JCM.02099-06.

[13] Lange, C., Cardoso, M., Senczek, D., Schwarz, S. (1999). Molecular subtyping of Staphylococcus aureus isolates from cases of bovine mastitis in Brazil. Veterinary microbiology, 67 (2), 127-141. https://doi.org/10.1016/S0378-1135(99)00031-0.

[14] Mahmoudi, H., Arabestani, M., Mousavi, S., Alikhani, M. (2017). Molecular Analysis of the coagulase gene in clinical and nasal carrier isolates of methicillin-resistant Staphylococcus aureus by restriction fragment polymorphisms. Journal of global antimicrobial resistance, (8), 41-45. https://doi.org/10.1016/j.jgar.2016.10.007.

[15] Mitami, N., Ohnishi, M., Murakawa, K., Okamoto, Y. (2002). Molecular typing of methicillin-resistant Staphylococcus aureus by protein A gene sequencing. Jpn. J. Infect. Dis, 55, 179-180. https://www.ncbi.nlm.nih.gov

[16] Mitami, N., Koizumi, A., Sano, R., Masutani, T., Murakawa, K., Mikasa. K., Okamoto, Y. (2005). Molecular Typing of Methicillin-Resistant Staphylococcus aureus by PCR-RFLP and its Usefulness in an Epidemiological Study of an Outbreak. Japanese Journal of Infectious Disease, 58 (4), 250-252. https://www.niid.go.jp

[17] Mirharimi. SF., Hasani. A., Abdinia. B. (2016). High diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolates based on hypervariable region polymorphisms. Archives of Pediatric Infectious Diseases, 4 (4), e36433. https://doi.org/10.5812/pedinfect.36433.

[18] Montesinos, I., Salido, E., Delgado, T., Cuervo, M., Sierra, A. (2002). Epidemiologic Genotyping of Methicillin-Resistant Staphylococcus aureus by pulsed Gel Electrophoresis at a University Hospital and comparison with Antibiotyping and Protein A and coagulase gene Polymorphisms. Journal of clinic Microbioly, 40 (6), 2119-2125. https://doi.org/10.1128/JCM.40.6.

[19] Omar, NY, Ali, HAS., Harfoush, R. (2014). Molecular typing of methicillin-resistant Staphylococcus aureus clinical isolates on the basis of protein A and coagulase gene polymorphisms. International Journal of microbiology, Article ID 650328. https://doi.org/10.11.1155/2014/650328.

[20] Parviz, M., Samira, A., Abbas, F., Baharak, N. (2016). Genotyping of coa and aroA genes of methicillin-resistant Staphylococcus aureus strains isolated from nasal samples in Western Iran. Jundishapur J. Microbiol, 9 (1), 1-6. https://doi.org/10.5812/jjm.26480.

[21] Palavencino,E., (2004). Community-acquired methicillin-resistant Staphylococcus aureus infections. Clinics in laboratory medicine, 24(2), 403-418. https://doi.org/10.1016/j.cll.2004.03.007.

[22] Salmenlinna, S., Vuopio-Varkila, J. (2001) Recongnition of two groups of methicillin-resistant- Staqphylococcus aureus strains based on epidemiology, antimicrobial susceptibility, hypervariable-region type, and ribotype in Finland. Journal of Clinical Microbioly, 39 (6), 2243-2247. https://doi.org/10.1128/JCM.39.62243-2247.2001.

[23] Senna, J.P., Pinto, C.A., Carvalho, L.P. y Santos, D.S. (2002). Comparison of pulsed-field gel electrophoresis and PCR analysis of polymorphisms on the mec hypervariable region for typing methicillin-resistant Staphylococcus aureus. Journal of Clinical Microbiology, 40 (6), 2254-2256. 10.1128/jcm.40.6. https://doi.org/10.1128/JCM.40.6.2254-2256.2002

[24] Schwarrzkopf, A. (1995). Coagulase gene polymorphism in Staphylococcus aureus a new epidemiologic marker. Inmunitat und infection, 23(1), 2407-2412. https://doi.org/10.1128/JCM.32.10.

[25] Shakeri, F., Shojai, A., Golalipour, M., Alang, S., Vaez, H., Ghaemi, E. (2010). Spa Diversity among MRSA and MSSA Strains of Staphylococcus aureus in North of Iran. International Journal of Microbiology, 2010, (ID 351397), 1-5. https://doi.org/10.1155/2010/351397.

[26] Schmitz, M., Steiert, M., Tichy, HV., Hofmann, B., Verhoef, J., Heinz, K., Kohrers, K., Jones, E. (1998). Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods. J. Med. Microbiol, 47(4) 341-351. https://doi.org/10.1099/00222615-47-4.

[27] Strommenger, B., Braulke, C., Heuck et al. (2008). “spa typing of Staphylococcus aureus as a frontline tool in epidemiological typing.” Journal of Clinical Microbiology, 46 (2), 574-581. https://doi.org/10.1128/jcm.01599-07.

[28] Teruyo, I., Kuwahara, K., Hiramatsu, K. (2007). Methods in Molecular Biology. Yinduo J (Eds).Methicillin-resistant Staphylococcus aureus (MRSA) Protocols. Chapter 7. Humana Press inc.

[29] Witt, R., Van Belkum, A., Van Leeuwen W. (2011). Molecular diagnostics and genotyping of methicillin-resistant Staphylococcus aureus (MRSA) and update. Clinical Microbiological Diagnostics, 4(10), 375-380. https://doi.org/10.1586/ERM.10.34.

[30] Whichelhaus, T.A., Hunfeld P., Boddinghanus, B., Kraiczyz, P., Schafer, V., Brade, V. (2001). Rapid molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP. Infection & Control Hospital Epidemiology, 22 (5), 294-298. https://doi.org/10.1086/501903.

[31] Wilailuckana, C., Tribuddharat, C., Tiensasitorn, Pongpech, P., Naenna, P., Rugdeekha, S., Dhiraputra, Ch., Danchaivijitr, S. (2006). Discriminatory powers of molecular typing techniques for methicillin-resistant Staphylococcus aureus in a University Hospital, Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 32(2), 327-334. https://pubmed.ncbi.nlm.nih.gov

[32] Yazdchi, B., Pourmand, MR., Hajiabdolgaghi, M., Hoseini, M., Mardani, N. (2008). Molecular characterization of hypervariable region (hvr) and antibiotic susceptibility patterns of Staphylococcus aureus strains isolates collected from Tehran University of medical sciences hospitals. Journal of school of Public Health and Institute of Public Health Research, 2 (6), 39-48. http://sjsph.tums.ac.ir/article-1-143-en.html.

[33] Yinduo, J. (2007). Methods in Molecular in Molecular Biology.Methicillin-resistant Staphylococcus aureus (MRSA) Protocols. New York, Humana Press.

[34] Zamani, A., Sadeghian, S., Ghaderkhani, J., Alikhani, MY., Najafimosleh, M., Goodarzi MT et al. (2007). Detection of methicillin-resistance (mecA) gene in Staphylococcus aureus strains by PCR and determination of antibiotic susceptibility. Ann Microbiol, (57), 273-276. https://doi.org/10.1007/BF03175219.

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