First occurrence of blaOXA-58 in Acinetobacter baumannii isolated from a clinical sample in Southern Brazil

MEDICAL MICROBIOLOGY

First occurrence of bla_OXA-58 in Acinetobacter baumannii isolated from a clinical sample in Southern Brazil

Carolina de Souza Gusatti; Lauren Martins Bertholdo; Letícia Muner Otton; Desirée Padilha Marchetti; Alessandra Einsfeld Ferreira; Gertrudes Corção^*

Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

ABSTRACT

This is the first report of an Acinetobacter baumannii from clinical origin carrying the bla_OXA-58 gene in Brazil. The isolate included in this study was from a patient during an outbreak in Porto Alegre, RS, Southern Brazil, in 2007. It was resistant to most of the beta-lactams tested, it has also the bla_OXA-65 gene and the ISAba1 sequence located upstream to both bla_OXA genes detected and it has a MIC of imipenem of 64 μg/mL.

Key words:bla_OXA-58, ISAba1, bla_OXA-65, carbapenemases, Acinetobacter baumannii.

The nosocomial infections caused by A. baumannii have been the target of intense research in recent years due to their great ability to rapidly acquire resistance to the drugs of choice used in antimicrobial therapy. The most common mechanism of drug resistance in these bacteria is the expression of OXA-carbapenemases, a class D carbapenem-hidrolysing-β-lactamase (1, 5, 12). Currently, there are four families of OXA carbapenemases in Acinetobacter sp.: OXA-23-like, OXA-24-like, OXA-58-like and OXA-51-like enzymes, the last being intrinsic to A. baumannii (9, 17). In addition, the presence of the insertion sequence ISAba1 immediately upstream of the bla_OXA-51 gene contributes to increased expression of resistance to carbapenems, since the presence of this gene is not necessarily related to resistance (20, 21).

Only carbapenemases OXA-23-like and OXA-51-like have been found in Brazil, so far (3, 6, 13). Carbapenemases OXA-58 were first described in Europe, where they are widely disseminated (15, 18). In South America, the presence of the gene was described in Argentina, commonly associated with resistance to carbapenems in Acinetobacter sp. outbreaks (5, 17). The occurrence of this carbapenemase has also been observed in Asia, showing that it is widely distributed throughout the world (5, 9, 17). Unlike bla_OXA-23 and bla_OXA-51 genes, broadly found across the country in different strains (13,19), the bla_OXA-58 gene has not been described in Brazil.

In a previous study (8), 74 clinical isolates were tested for their antimicrobial susceptibility and the presence of bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like and bla_OXA-58-like genes. The confirmation of Acinetobacter species was performed by PCR assay and sequencing of the 16S rRNA gene (7). The Minimum Inhibitory Concentration (MIC) of imipenem was determined by broth microdilution according to the guidelines of the Clinical and Laboratory Standards Institute (4). The detection of the OXA-carbapenemase genes (bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like and bla_OXA-58-like) was based on the MIC results. The PCR was carried out using a multiplex assay (22) for the isolates with MIC ≥ 8 μg/mL. The complete ORF of the gene bla_OXA-51 and the insertion sequence ISAba1 were amplified as previously described (20). The complete bla_OXA-51-like and bla_OXA-58-like PCR products were sequenced by ABI-PRISM 3100 Genetic Analyzer and evaluated using BioEdit Version 7.0.5. All the partial sequences were deposited in GenBank under accession numbers (HM 626370, HM 626369 and HM 626368). In order to verify the position of the ISAba1 sequence, PCR mapping experiments using combinations of the ISAbal forward, OXA-51-like and OXA-58-like reverse primers were performed (Figure 1a and 1b).

The IC-09 isolate included in this study showed resistance to imipenem, meropenem, amikacin, ciprofloxacin, gentamicin, cephalothin, ampicillin-sulbactam, trimetropim-sulfameto xazole and ticarcillin-clavulanate. The species of the isolate was confirmed as A. baumannii and the result of the MIC testing showed resistance to imipenem (64 μg/mL). The isolate was negative to the presence of bla_OXA-24-like and bla_OXA-23-like genes. The PCR reaction indicated the presence of OXA-58-like and OXA-51 like carbapenemases and PCR mapping indicated that the ISAba1 sequence is upstream of both genes but in separate positions of the genome (Figure 1a and 1b). Sequencing analysis of the bla_{OXA-51-like-all} PCR product showed 99% homology with bla_OXA-65 gene. The enzyme OXA-65 was first described in two isolates from Argentina and has 98% identity with OXA-51, thus belonging to the same subgroup primarily described in 2005 (2).

To our knowledge, for the first time in Brazil, a positive result was obtained for bla_OXA-58 gene and the sequencing analysis showed 100% homology with the bla_OXA-58 gene of A. baumannii. This enzyme was first described in France in an outbreak of hospital infection in 2003 and since then it has been found around the world. Corroborating with previous studies, this finding confirms the worldwide spread of the OXA-58 carbapenemase (15, 16). In South America this enzyme is prevalent and of public health concern in Argentina, a neighboring country to Rio Grande do Sul State, where the present study was conducted. In Argentina, in contrast to our findings, the MICs observed for imipenem were from 8 to 32µg/mL, indicating a low level of resistance to this antibiotic (5, 14). However, our results suggested that additional mechanisms of resistance may be present, since the MIC was higher. In the present study, the ISAba1 sequence is located upstream to both bla_OXA-65 and bla_OXA-58 genes (Figure 1a and 1b), therefore, its presence may be associated with the increased level of resistance observed (MIC = 64 μg/mL). As previously described, this IS, when located upstream to bla_OXA-51-like, may play a role of promoter and enhance resistance to carbapenems (10, 20, 21). Overexpression of the AdeABC efflux pump may be also associated with this resistance phenotype (11). In conclusion, our study reported the first occurrence of an A. baumannii from clinical origin, carrying the bla_OXA-58 gene in Brazil and suggests that further studies are needed to identify if the expression levels of the bla_OXA-65 and bla_OXA-58 genes and/or efflux pumps may be responsible by the carbapenem resistance level observed.

ACKNOWLEDGEMENTS

Our thanks to the Clinical Pathology Laboratories of the studied hospitals in Porto Alegre, who kindly provided us the Acinetobacter strains and to Dr. Ana Cristina Gales and Danilo Elias Xavier (Laboratório Alerta and Laboratório Especial de Microbiologia Clínica, Division of Infectious Diseases, Universidade Federal de São Paulo) who kindly provided the Acinetobacter strains used as positive controls. This study was supported by CAPES-PROF grant from Brazilian Government.

Submitted: October 05, 2010; Approved: January 16, 2012.

• 1. Brown, S.; Amyes, S. (2006). OXA β-lactamases in Acinetobacter: the story so far. J. Antimicrob. Chemother 57,1-3.
• 2. Brown, S.; Amyes, S. G. B. (2005). The sequences of seven class D β-lactamases isolated from carbapenem-resistant Acinetobacter baumannii from four continents. Clin. Microbiol. Infect 11, 326-329.
• 3. Carvalho, K.R.; Carvalho-Assef,  A.P.; Peirano, G.; Santos, L.C.; Pereira, M.J.; Asensi, M. D. (2009). Dissemination of multidrug-resistant Acinetobacter baumannii genotypes carrying bla_OXA-23 collected from hospitals in Rio de Janeiro, Brazil. Int. J. Antimicrob Agents. 34, 25-28.
• ⁴
  Clinical and Laboratory Standards Institute. (2010). Performance standards for antimicrobial susceptibility testing: twentieth informational supplement M100-S20. CLSI, Wayne, PA.
• 5. Coelho, J.; Woodford, N.; Afzal-Shah, M.; Livermore, D. M. (2006). Occurrence of OXA-58-like carbapenemases in Acinetobacter spp. collected over 10 years in three continents. Antimicrob. Agents Chemother 50,756-758.
• 6. Dalla-Costa, L. M.; Coelho, J. M.; Souza, H. A.; Castro, M. E.; Stier, C. J.; Bragagnolo, K. L.; Rea-Neto, A.; Penteado-Filho, S. R.; Livermore, D. M.; Woodford, N. (2003). Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J. Clin. Microbiol 41, 3403-3406.
• 7. Ferreira, A.E.; Cunha, G.R.; Fuentefria, D. B.; Corçăo, G. (2007). Perfil de suscetibilidade a antimicrobianos em cepas de Acinetobacter spp isoladas de efluente hospitalar em Porto Alegre-RS. Cad. Farm. 23, 9-14.
• 8. Ferreira, A.E. (2010). Caracterizaçăo molecular e detecçăo de genes de resistęncia em isolados de Acinetobacter sp. de amostras clínicas e de efluente hospitalar. Porto Alegre, Rio Grande do Sul, Brasil, 119 p. (Dr.Thesis, Instituto de Ciencias Básicas da Saúde, UFRGS).
• 9. Higgins P.G.; Dammhayn, C.; Hackel, M.; Seifert, H. (2010). Global spread of carbapenem-resistant Acinetobacter baumannii J Antimicrob Chemother 65, 233-238.
• 10. Higgins, P.G.; Poirel, L.; Lehmann, M.; Nordmann, P.; Seifert, H. (2009). OXA-143, a novel carbapenem-hydrolyzing class D β-lactamase in Acinetobacter baumannii Antimicrob Agents Chemother 53, 5035-5038.
• 11. Lee, Y.; Yum, J.H.; Kim, C.K.; Yong, D.; Jeon, E.H.; Jeong, S.H.;  Ahn, J.Y.; Lee, K.  (2010). Role of OXA-23 and AdeABC efflux pump for acquiring carbapenem resistance in an Acinetobacter baumannii strain carrying the bla_OXA-66 gene. Annals  Clin. Laborat. Sci 40, 43-48.
• 12. Marti, S.; Sanchez-Cespedes, J.; Blasco, M.D.; Ruiz, M.; Espinal, P.; Alba, V.; Fernandez-Cuenca, F.; Pascual, A.; Vila, J. (2008). Characterization of the carbapenem-hydrolyzing oxacillinase OXA-58 in an Acinetobacter genospecies 3 clinical isolate. Antimicrob. Agents Chemother 52, 2955-2958.
• 13. Martins, A.F.;  Kuchenbecker, R.; Sukiennik, T.; Boff, R.; Reiter, K.C.; Lutz, L.; Machado, A.B.M.P.; Barth. A.L. (2009). Carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme. Infection.  37 (5), 474-476.
• 14. Merkier, A.K.; Catalano, M.;  Ramírez, M.S.; Quiroga, C.; Orman, B.; Ratier, L.; Famiglietti, A.; Vay, C.; Di Martino, A.; Kaufman, S.; Centrón, D. (2008). Polyclonal spread of bl_aOXA-23 and bl_{aOXA-58 in} Acinetobacter baumannii isolates from Argentina. J Infect Dev Ctries 2, 235-240.
• 15. Poirel, L.; Marqué, S.; Héritier, C.; Segonds, C.; Chabanon, G.; Nordmann, P. (2005). OXA-58, a novel class D β-lactamase involved in resistance to carbapenems in Acinetobacter baumannii Antimicrob. Agents Chemother 49, 202-208.
• 16. Poirel, L.; Nordmann, P. (2006). Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene bla_OXA-58 in Acinetobacter baumannii Antimicrob. Agents Chemother. 50, 1442-1448.
• 17. Peleg, A. Y.; Seifert, H.; Paterson, D. L. (2008). Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev 21, 538-582.
• 18. Pournaras, S.; Markogiannakis, A.; Ikonomidis, A.; Kondyli, L.; Bethimouti, K.; Maniatis, A. N.;. Legakis, N. J; Tsakris, A. (2006). Outbreak of multiple clones of imipenem-resistant Acinetobacter baumannii isolates expressing OXA-58 carbapenemase in an intensive care unit. J. Antimicrob. Chemother 57, 557-561.
• 19. Takagi, E.H., N. Lincopan, V.C. Cassettari, L.F. Passadore, E.M. Mamizuka, and M.B. Martinez. (2009). Carbapenem-resistant Acinetobacter baumannii outbreak at university hospital. Braz. J. Microbiol 40, 339-341.
• 20. Turton, J. F.; Ward, M. E.; Woodford, N.; Kaufmann, M. E.; Pike, R.; Livermore, D. M.; Pitt, T. L.. (2006). The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii FEMS Microbiol. Lett 258, 72-77.
• 21. Segal, H.; Garny, S.; Elisha, B. G. (2005). Is ISAba1 customized for Acinetobacter? FEMS Microbiol. Lett 243, 425-429.
• 22. Woodford, N.;  Ellington, M. J.; Coelho, J. M.; Turton, J. F.; Ward, M. E.; Brown, S.; Amyes, S. G. B.; Livermore, D. M. (2006). Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 27, 351-353.

Publication Dates

• Publication in this collection
  02 May 2012
• Date of issue
  Mar 2012

History

• Received
  05 Oct 2010
• Accepted
  16 Jan 2012