MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 106(4) June 2011
PAGES: 505-506 DOI: 10.1590/S0074-02762011000400020 Short communication
Occurrence of blaOXA-23 gene in imipenem-susceptible Acinetobacter baumannii

Karyne Rangel CarvalhoI, +; Ana Paula D'Alincourt Carvalho-AssefI; Lia Galvão dos SantosII; Maria José Félix PereiraIII; Marise Dutra AsensiI

ILaboratório de Pesquisa em Infecção Hospitalar, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21040-360 Rio de Janeiro, RJ, Brasil
IIHospital Federal de Bonsucesso, Rio de Janeiro, RJ, Brasil
IIIRede Labs D'Or, Rio de Janeiro, RJ, Brasil

Abstract

The aim of the current study was to describe the occurrence of the blaOXA-23 gene and the ISAba1 element in imipenem-susceptible Acinetobacter baumannii strains. By performing the polymerase chain reaction mapping using combinations of ISAba1 forward primers and the blaOXA-23-like gene reverse primers, we demonstrated that the ISAba1 element did not occur upstream of the blaOXA-23 gene in five of 31 isolates, which explained the lack of resistance to imipenem despite the presence of the blaOXA-23 gene. All of the blaOXA-23-positive isolates were susceptible to imipenem and meropenem with minimal inhibitory concentration 4 µg/mL. Pulsed-field gel electrophoresis analysis revealed four genotypes among the five blaOXA-23-positive isolates. The current report of the blaOXA-23 gene in imipenem-susceptible isolates provided evidence that this gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes.

Carbapenem resistance in Acinetobacter baumannii is predominantly caused by the carbapenem-hydrolysing class D ?-lactamases (CHDLs). Sequence-based comparisons classify OXA-carbapenemases into eight subgroups as follows: four of the OXA-carbapenemases have been identified in A. baumannii, namely, OXA-23-like, OXA-24-like, OXA-51-like, OXA-58-like and OXA-143, which is a novel plasmid-mediated CHDL (Higgins et al. 2009). OXA-23 was identified as the first member of this enzyme group in 1985 in Scotland, which was before the wide availability of imipenem in clinical practice. Since 1985, a number of outbreaks of OXA-23-producing imipenem-resistant Acinetobacter spp have been reported worldwide (Mugnier et al. 2010). The OXA-23-encoding gene is mainly found on plasmids and has been associated with the ISAba1 or ISAba4 elements (Corvec et al. 2007). The ISAba1 or ISAba4 element that is upstream of blaOXA-23-like genes provide promoter sequences that enhance their expression (Corvec et al. 2007). ISAba1 regulates blaOXA-23 mobility and has two copies that surround the ß-lactamase gene and form a composite transposon (defined as Tn2006) (Mugnier et al. 2010).ISAba4 belongs to the IS982 family, is 975 bp in length, possesses two 18-bp inverted repeats and encodes a 292-amino-acid putative transposase. These insertion elements may be involved in transposition processes at the origin of acquisition of the blaOXA-23gene (Corvec et al. 2007).

The current study described the silent carriage of the blaOXA-23 gene in five imipenem-susceptible A. baumannii isolates.

From April 2005-September 2007, 31 clinical isolates of imipenem-susceptible A. baumannii were collected from non-related patients at five hospitals in Rio de Janeiro, Brazil. The isolates were identified using conventional techniques and confirmed using the amplified ribosomal DNA restriction analysis (Carvalho et al. 2009) and blaOXA-51 polymerase chain reaction (PCR) (Turton et al. 2006b).

Among the 31 imipenem-susceptible isolates, five were positive for blaOXA-23 by PCR and DNA sequencing using the following primers: OXA-23 F (5'-ACTTGCTATGTGGTTGCTTCTC-3') and OXA-23-R (5'-TGTCAAGCTCTTAAATAATATTCAGC-3'), which annealed at the region between 987 and 1,778-bp positions in GenBank isolate AJ132105 (Higgins et al. 2009). In addition, these isolates were positive for ISAba1 (ISAba1 F: 5'-CACGAATGCAGAAGTTG-3' and ISAba1 R: 5'-CGACGAATACTATGACAC-3') (Turton et al. 2006a) but negative for ISAba4 (ISAba4 F 5'-ATTTGAACCCATCTATTGGC-3' and ISAba4 R 5'-ACTCTCATATTTTTTCTTGG-3') (Corvec et al. 2007). The nucleotide sequences of blaOXA-23 and ISAba1 have already been deposited in the GenBank under the accessions JF421124 and JF340121, respectively.

PCR mapping using the combination of the ISAba1 forward primer and the blaOXA-23-like reverse primer were negative. In five carbapenem-resistant control isolates, the ISAba1 element was detected upstream of the blaOXA-23 gene (Carvalho et al. 2009).

According to antimicrobial susceptibility tests, such as the E-test (AB Biodisk, Solna, Sweden) and the agar dilution method, all blaOXA-23-positive imipenem-susceptible isolates were susceptible to imipenem and meropenem [minimal inhibitory concentration (MIC) < 4 µg/mL]. These results may be explained by the absence of the ISAba1 element upstream of the blaOXA-23-like gene.

All 31 isolates were susceptible to polymyxin B (MIC < 1 µg/mL). Using the disk diffusion method, four isolates were non-susceptible (i.e., intermediate or resistant) to aztreonam, ceftazidime, cefepime and ciprofloxacin, whereas three isolates were susceptible to ampicillin-sulbactam and amikacin. These findings suggest the presence of another resistance mechanism that has not been previously investigated, such as extended spectrum beta lactamases, the modification of penicillin-binding proteins and porins or the upregulation of the efflux system (Zarrilli et al. 2009).

Macrorestriction with ApaI-digestion following pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the blaOXA-23-positive isolates. The blaOXA-23-positive isolates were clustered into four different genotypes (B, C, G and I). The criteria for the classification of DNA band patterns included the following: undistinguishable (clonally related isolates), closely related (clonal variants, < 3 different bands), possibly related (4-6 different bands) and unrelated (> 6 different bands) (Tenover et al. 1995). Two isolates proved to be genetically related (genotype C). Except for genotype I, all of the genotypes were detected among blaOXA-23-negative isolates. Genotypes B and C were detected among imipenem-resistant A. baumannii isolates as described previously (Carvalho et al. 2009). Chromosomally encoded blaOXA-23-like genes have been described in carbapenem-susceptible Acinetobacter radioresistens isolates, suggesting that this species is a source of these genes (Poirel et al. 2008, Boo & Crowley 2009, Mendes et al. 2009). Poirel et al. (2008) has identified a similar plasmid backbone in several blaOXA-23-positive A. baumannii and A. radioresistens isolates. These results suggest that a plasmid-mediated ISAba1 element originating from A. baumannii may have entered A. radioresistens to be transposed and targeted to the regions that are upstream and downstream of the chromosomal blaOXA-23-like genes. A transposon-like structure is then formed, which enhances the expression of the blaOXA-23-like genes. This structure may transpose and target a plasmid inside the A. radioresistens genome and, finally, this plasmid conjugates into A. baumannii to spread the resistance determinant in the latter species (Poirel et al. 2008).

The current report of blaOXA-23 in imipenem-susceptible A. baumannii isolates provided evidence that the blaOXA-23 gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes. The undetected carba-penemase genes are challenging, because the laboratory detection of such genes and the subsequent measures for infection control in hospital settings generally target only phenotypically multidrug-resistant organisms. The future control of multidrug resistance may require the identification of multidrug-resistant isolates and their reservoirs using molecular-based techniques.

 

ACKNOWLEDGEMENTS

To all hospital participants who collected isolates for the study, and to PDTIS-IOC platform, for DNA sequencing.

 

REFERENCES

Boo TW, Crowley B 2009. Detection of blaOXA-58 and blaOXA-23-like genes in carbapenem-susceptible Acinetobacter clinical isolates: should we be concerned? J Med Microbiol 58: 839-841.

Carvalho KR, APD'A Carvalho-Assef, Peirano G, dos Santos LCG, Pereira MJF, Asensi MD 2009. Dissemination of multidrug-resistant Acinetobacter baumannii genotypes carrying blaOXA-23 collected from hospitals in Rio de Janeiro, Brazil. Int J Antimicrob Agents 34: 25-28.

Corvec S, Poirel L, Naas T, Drugeon H, Nordmann P 2007. Genetics and expression of the carbapenem-hydrolyzing oxacillinase gene blaOXA-23 in Acinetobacter baumannii. Antimicrob Agents Chemother 51: 1530-1533.

Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H 2009. OXA-143, a novel carbapenem-hydrolyzing class D ?-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 53: 5035-5038.

Mendes RE, Bell JM, Turnidge JD, Castanheira M, Deshpande LM, Jones RN 2009. Codetection of blaOXA-23 gene (blaOXA-133) and blaOXA-58 in Acinetobacter radioresistens: Report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 53: 843-844.

Mugnier PD, Poirel L, Naas T, Nordmann P 2010. Worldwide dissemination of the blaOXA-23 carbapenemase gene of Acinetobacter baumannii. Emerg Infect Dis 16: 35-40.

Poirel L, Figueiredo S, Cattoir V, Carattoli A, Nordmann P 2008. Acinetobacter radioresistens as a silent source of carbapenem resistance for Acinetobacter spp. Antimicrob Agents Chemother 52: 1252-1256.

Tenover FC, Arbeit RD, Goering RV, MicKelsen PA, Murray BE, Pearsing DH, Swaminathan B 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial isolate typing. J Clin Microbiol 33: 2233-2239.

Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM, Pitt TL 2006a. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett 258: 72-77.

Turton JF, Woodford N, Glover J, Yarde S, Kaufmann ME, Pitt TL 2006b. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. J Clin Microbiol 44: 2974-2976.

Zarrilli R, Giannouli M, Tomasone F, Triassi M, Tsakris A 2009. Carbapenem resistance in Acinetobacter baumannii: the molecular epidemic features of an emerging problem in health care facilities. J Infect Dev Ctries 5: 335-341.

Received 30 September 2010
Accepted 5 May 2011
Financial support: FAPERJ, IOC-FIOCRUZ

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