Carbapenem-Resistant Acinetobacter baumannii Isolates Expressing the bla OXA-23 Gene Associated with IS Aba4 in Belgium

Abstract

Carbapenems are among the drugs of choice for treating nosocomial infections due to multidrug-resistant Acinetobacter baumannii strains (4). However, their efficacy is being increasingly compromised by the spread of carbapenem-resistant isolates, mostly following acquisition of carbapenemases of class D (OXA-23, OXA-40-like, and OXA-58 oxacillinases) (1, 6, 8, 9). Outbreaks of OXA-23-producing A. baumannii isolates are increasingly being reported worldwide (7, 8). In these isolates, blaOXA-23 is associated with an upstream-inserted insertion sequence, ISAba1, which is responsible for its mobilization and expression. In two unrelated A. baumannii isolates from France, the blaOXA-23 gene was associated with another insertion sequence, ISAba4 (3). Here, we describe the first ISAba4-blaOXA-23-expressing A. baumannii isolates in Belgium. In July 2007, a 69-year-old Belgian resident was admitted to the intensive care unit (ICU) of a general hospital in Morocco with a diagnosis of pneumonia and cardiac failure. The patient was transferred to the ICU of Saint-Joseph Hospital (Gilly, Belgium), and culture of respiratory secretions grew a carbapenem-resistant and multidrug-resistant A. baumannii strain, GIL1. Despite prompt treatment with colistin, the patient died. The A. baumannii isolate was identified with the API 32GN system (BioMerieux, Marcy-l'Etoile, France) and confirmed by detection of the intrinsic blaoxa-51/69-like gene (10). According to MIC determination by Etest (AB Biodisk, Solna, Sweden), A. baumannii GIL1 was resistant to all β-lactams (2), including imipenem and meropenem (MIC, >32 μg/ml), sulbactam (MIC, 48 μg/ml), ciprofloxacin (MIC, >32 μg/ml) and amikacin (MIC, >256 μg/ml); borderline susceptible to tigecycline (MIC, 6 μg/ml) and rifampin (MIC 3 μg/ml); and fully susceptible to colistin only (MIC, 0.5 μg/ml). PCR analysis followed by sequencing revealed that A. baumannii GIL1 harbored the blaOXA-23 gene, along with the naturally and chromosomally located blaOXA-69 gene and a novel blaADC allele (5), blaADC-39 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU652244,term_id:186969836,term_text:EU652244}}EU652244). blaADC-39 differed from the A. baumannii AYE blaAmpC gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CT025798,term_id:88758819,term_text:CT025798}}CT025798) by only two nucleotide substitutions (A1022C and G1135A) leading to two amino acid changes (N351T and D379N). Isoelectric focusing confirmed the expression of OXA-23 (pI 6.3 to 6.5) and ADC-39 β-lactamase (pI >8.5) (not shown). Using a previously described PCR approach (3), ISAba1 was found inserted upstream of blaADC-39 gene but not upstream of the blaOXA-23 or blaOXA-69 gene. Instead, another insertion sequence, ISAba4, was found upstream of the blaOXA-23 gene, with a genetic organization similar to that described for Tn2007: i.e., orf3 and mobA genes surrounding the ISAba4-blaOXA-23-ATPase fragment inserted in orf1 (3) (Fig. ​(Fig.11). FIG. 1. Genetic organization of the three β-lactamase genes found in A. baumannii GIL1. (A) ISAba4-blaOXA-23. (B) ISAba1-blaADC-39. (C) blaOXA-69. Horizontal arrows represent open reading frames and their transcriptional orientations. Promoters brought ... The blaOXA-23 gene was encoded on a 25-kb plasmid that could be electroporated into rifampin-resistant A. baumannii CIP7010 but was not self-transferable by conjugation. Only the blaOXA-23 gene was transferred in the electroporants, as revealed by susceptibility testing. Since the initial detection of this strain, six other patients of the ICU have been identified as carriers or infected with ISAba4-blaOXA-23-expressing A. baumannii isolates. These isolates were indistinguishable by pulsed-field gel electrophoresis, suggesting clonal diffusion. Furthermore, pulsed-field gel electrophoresis analysis revealed that A. baumannii GIL1 presented identical banding patterns to one ISAba4-blaOXA-23 A. baumannii isolate reported in France, thus illustrating its propensity to disseminate and underscoring the need for epidemic surveys geared to estimate the true prevalence of these ISAba4-OXA-23 producers. ISAba1 and ISAba4 have been suggested to be at the origin of blaOXA-23 gene mobilization and to provide strong promoter sequences for their expression (3). Whether ISAba4 may procure a fitness advantage over ISAba1 in A. baumannii strains in terms of transposition frequency or level of expression needs to be further investigated.