PCR-RFLP Methodology to Identify Nocardia Isolates in Cuba

Yaxsier de Armas^1*, Carlos Manuel Fernández¹, Raúl Díaz¹, Nicolas Bioni Stefano², Gustavo Wissmann², Enrique José Calderón³

¹Institute of Tropical Medicine, Pedro Kour, Havana, Cuba.
²Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.
³Department of Internal Medicine, Center of Biomedical Research Network in Epidemiology and Public Health, Virgen del Rocío University Hospital, Seville, Spain.

DOI: 10.4236/aim.2015.510076                

Abstract

Nocardiosis diagnosis is a major challenge. The clinical features and radiological findings are nonspecific. Traditionally, Nocardia identification is based on colonial and microscopical morphology and biochemical tests. However, molecular biology techniques allow a better characterization of species and biotypes. PCR-RFLP of the 65-kDa heat shock protein (HSP) gene provides a rapid, sensitive, and time and labor-efficient method for this proposal. Using this technique, six of eight isolates tested were identified as Nocardia asteroides type VI. PCR-RFLP of the 65-kDa HSP gene could be very useful for determining the incidence of this pathogen in different population groups and its association with susceptibility/resistance profiles to the drugs of choice for treatment. This work is the first molecular detection of Nocardia species in Cuba.

Keywords

Nocardia, PCR-RFLP, Cuba, Molecular Identification, 65-kDa HSP Gene

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Changmin, Y., Min-Jung, K., Chang-Seok, K., Nam Yong, L., Eun-Jeong, J. and Joon-Sup, Y. (2014) Necrotizing Pneumonia and Empyema in an Immunocompetent Patient Caused by Nocardia cyriacigeorgica and Identified by 16S rRNA and secA1 Sequencing. Annual Laboratory Medicine, 34, 71-75. http://dx.doi.org/10.3343/alm.2014.34.1.71
[2]	Baio, P.V.P., Ramos, J.N., Santos, L.Sd., Soriano, M.F., Ladeira, E.M. and Souza, M.C. (2013) Molecular Identification of Nocardia Isolates from Clinical Samples and Overview of Human Nocardiosis in Brazil. PloS Neglected Tropical Diseases, 7, e2573. http://dx.doi.org/10.1371/journal.pntd.0002573
[3]	Brown-Elliott, B.A., Brown, J.M., Conville, P.S. and Wallace, R.J. (2006) Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy. Clinical Microbiology Review, 19, 259-282. http://dx.doi.org/10.1128/CMR.19.2.259-282.2006
[4]	Steingrube, V.A., Wilson, R.W., Brown, B.A., Jost Jr., K.C., Blacklock, Z., Gibson, J.L. and Wallace Jr., R.J. (1997) Rapid Identification of Clinically Significant Species and Taxa of Aerobic Actinomycetes, Including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella Isolates, by DNA Amplification and Restriction Endonuclease Analysis. Journal of Clinical Microbiology, 35, 817-822.
[5]	Bonifaz, J.A. (2012) Micología Médica Básica. 4th Edition, McGraw-Hill, New York.
[6]	Berd, D. (1973) Laboratory Identification of Clinically Important Aerobic Actinomycetes. Apply Microbiology, 25, 665-681.
[7]	Condas, L.A.Z., Garcia, M., Domingues, M., de Vargas, A.P.C., Matsuzawa, T., Yazawa, K., Siqueira, A.K., Salerno, T., Lara, G.H.B., Risseti, R.M., Ferreira, K.S. and Gonoi, T. (2014) Molecular Identification and Antimicrobial Resistance Pattern of Seven Clinical Isolates of Nocardia spp. in Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo, 56, 397-401.
[8]	Saubolle, M.A. and Sussland, D. (2003) Nocardiosis: Review of Clinical and Laboratory Experience. Journal of Clinical Microbiology, 41, 4497-4501. http://dx.doi.org/10.1128/JCM.41.10.4497-4501.2003
[9]	Santamaria Saber, L.T., Figueiredo, J.F. and Santos, S.B. (1993) Nocardia Infection in Renal Transplant Recipient: Diagnostic and Therapeutic Considerations. Revista do Instituto de Medicina Tropical de Sao Paulo, 35, 417-421.
[10]	Boiron, P., Provost, F., Chevrier, G. and Dupont, B. (1992) Review of Nocardial Infections in France 1987 to 1990. European Journal Clinical Microbiology Infectious Disease, 11, 709-714. http://dx.doi.org/10.1007/BF01989975
[11]	Kageyama, A., Yazawa, K. and Taniguchi, H. (2005) Nocardia concava Sp. Nov., Isolated from Japanese Patients. International Journal Systematic Evolutionary Microbiology, 55, 2081-2083. http://dx.doi.org/10.1099/ijs.0.63280-0
[12]	Amin, A., Mahmood, S.F., Anis, M., Adhi, F., Ahmad, S., Ali, F. and Khan, E. (2012) Pulmonary Nocardiosis: A Comparative Analysis of Nocardia asteroides and Non-Asteroides Species. Tropical Doctor, 42, 94-96. http://dx.doi.org/10.1258/td.2011.110311
[13]	Morán, Y., Chacón, O., Córdoba-Selle, M.C., Domínguez-Larrinaga, R., Herrera, L. and Borrás-Hidalgo, O. (2013) Identification and Molecular Characterization of Nocardia sp. as a New Causal Agent of Tobacco False Broomrape. Journal of Phytopathology, 161, 86-91.
[14]	Salazar, D., Reyes, T.M., Rodríguez, F., Bandera, F., Reyes, A., Medina, V.Z. and de Armas, Y. (2011) Rhodococcus equi en paciente VIH/Sida: Primera detección molecular en Cuba. Revista Cubana de Medicina Tropical, 63, 253-256.
[15]	Steingrube, V.A., Brown, B.A., Gibson, J.L., Wilson, R.W., Brown, J., Blacklock, Z., Jost, K., Locke, S., Ulrich, R.F. and Wallace Jr., R.J. (1995) DNA Amplification and Restriction Endonuclease Analysis for Differentiation of 12 Species and Taxa of Nocardia, including Recognition of Four New Taxa within the Nocardia asteroides Complex. Journal of Clinical Microbiology, 33, 3096-3101.
[16]	Wilson, R.W., Steingrube, V.A., Brown, B.A. and Wallace Jr., R.J. (1998) Clinical Application of PCR-Restriction Enzyme Pattern Analysis for Rapid Identification of Aerobic Actinomycete Isolates. Journal of Clinical Microbiology, 36, 148-152.
[17]	Plikaytis, B., Yakrus, M., Butler, W., Woodley, C., Silcox, V. and Shinnick, T. (1992) Differentiation of Slowly Growing Mycobacterium Species, Including Mycobacterium tuberculosis, by Gene Amplification and Restriction Fragment Length Polymorphism Analysis. Journal of Clinical Microbiology, 30, 1815-1822.
[18]	Telenti, A., Marchesi, F., Balz, M., Bally, F., Bottger, E.C. and Bodmer, T. (1993) Rapid Identification of Mycobacteria to the Species Level by Polymerase Chain Reaction and Restriction Enzyme Analysis. Journal of Clinical Microbiology, 31, 175-178.
[19]	Rodriguez-Nava, V., Couble, A., Devulder, G., Flandrois, J.P., Boiron, P. and Laurent, F. (2006) Use of PCR-Restriction Enzyme Pattern Analysis and Sequencing Database for hsp65 Gene-Based Identification of Nocardia Species. Journal of Clinical Microbiology, 44, 536-546. http://dx.doi.org/10.1128/JCM.44.2.536-546.2006
[20]	Kasai, H., Ezaki, T. and Harayama, S. (2000) Differentiation of Phylogenetically Related Slowly Growing Mycobacteria by Their gyrB Sequences. Journal of Clinical Microbiology, 38, 301-308.
[21]	Wallace, J.R., Steele, L.C., Sumter, G. and Smith, J.M. (1988) Antimicrobial Susceptibility Patterns of Nocardia asteroides. Antimicrobial Agents Chemotherapy, 2, 1776-1779. http://dx.doi.org/10.1128/AAC.32.12.1776