Identification of a New Integron Harboring bla_IMP-10 in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

LETTER

We describe herein the characterization of a novel class 1 integron harboring the bla_IMP-10 gene in Acinetobacter baumannii clinical isolates. The imipenem resistance rates in A. baumannii increased from 12.6% to 71.4% in Brazilian centers between 1997 to 1998 and 2008 to 2010 (1). The increase was associated mainly with the spread of clonally related OXA-23-producing A. baumannii isolates and, to a lesser extent, with the spread of OXA-143-producing A. baumannii isolates that may represent diverse clonal lineages (2, 3). In contrast, IMP-1-producing A. baumannii isolates have been restricted to a few hospitals located in the São Paulo state (4, 5).

Two carbapenem-resistant A. baumannii isolates were recovered from blood (Acb3035) and bronchoalveolar lavage fluid (Acb3101) cultures of distinct patients admitted to the pneumology unit of a tertiary-care teaching hospital located in the city of São Paulo, Brazil, in August 2000. The clinical specimens were collected 3 days apart. The species identification was confirmed by rpoB sequencing (6). Antimicrobial susceptibility was determined by CLSI broth microdilution (7), except that for tigecycline, which was determined by Etest (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's recommendations. The two A. baumannii isolates were resistant to ampicillin-sulbactam (MICs, 32/16 μg/ml), ceftriaxone (MICs, 256 μg/ml), ceftazidime (MICs, 32 μg/ml), cefepime (MICs, 128 μg/ml), imipenem (MICs, 32 μg/ml), meropenem (MICs, 64 μg/ml), ciprofloxacin (MICs, 2 μg/ml), amikacin (MICs, 128 μg/ml), and gentamicin (MICs, 64 μg/ml). In contrast, both isolates were susceptible to polymyxin B (MICs, 1 μg/ml) and minocycline (MICs, 0.5 μg/ml) but showed high MICs of tigecycline (MICs, 4 μg/ml) (8).

The presence of β-lactamase genes was investigated by PCR followed by DNA sequencing using specific primers (9, 10). Both A. baumannii isolates carried the bla_IMP-10, bla_OXA-100 (a bla_OXA-51-like gene), and bla_TEM-1 genes. The sequencing of bla_IMP-10 genetic context (5, 11) showed that bla_IMP-10 was harbored by a new class 1 integron designated In990 (GenBank accession number KP177455) (Fig. 1A). The In990 5′ coding sequence (5′-CS) was composed of intI1, the attI1 recombination site, and a promoter region constituted by a PcS promoter ([TTGACA](N)17[TAAACT]) (Fig. 1B). The first gene cassette of In990 was bla_IMP-10, followed by two aminoglycoside-modifying enzyme-encoding genes (aacA31 and aadA1a) and the qacEΔ1 and sul1 genes at the 3′-CS. The cassette arrangement of In990 was very similar to that of In86, which carries bla_IMP-1 in the Gram-negative bacilli, including A. baumannii, isolated within the same period of time and at the same institution (4, 5, 12) (Fig. 1A). However, a rare variation of PcS, PcS_TGN-10, was found in In86 (5), and a 19-bp duplication (CAACGATGTTACGCAGCAG) upstream of ORF11 was observed only in In990 (Fig. 1B). ORF11 encodes a 12-amino-acid peptide because a stop codon was created at the very beginning of the first gene cassette (13). Although the 19-bp duplication resulted in a suppression of this stop codon, another stop codon is present at the junction with the coding sequence of bla_IMP-10. Such duplication might affect the translation level of bla_IMP-10 since this gene already displays a well-conserved ribosomal binding site. Southern blotting/hybridization experiments revealed that the bla_IMP-10 gene was located on a plasmid of ∼146 kb in both isolates (14). Conjugation experiments failed to transfer bla_IMP-10 (15). The clonal relatedness of the two IMP-10-producing A. baumannii isolates was assessed by pulsed-field gel electrophoresis (PFGE) using the ApaI restriction enzyme as described previously (16) and revealed that the two strains were identical. Multilocus sequencing typing (MSLT) was performed according to the Institut Pasteur scheme (17). Both the Acb3035 and Acb3101 strains belonged to the new sequence type, 400 (ST400), which is not related to those of main clonal complexes known to have spread worldwide (Fig. 1C).

FIG 1.

(A) Comparison of class 1 integrons In86 and In990, carrying bla_IMP-1 (5, 12) and bla_IMP-10, respectively, in different Brazilian Gram-negative bacillus isolates. Arrows designate transcription directions of genes. The figure is not to scale. (B) Schematic representation of partial nucleotide sequence of the 5′-CS region, from the Pc promoter to the beginning of bla_IMP-10. The 19-bp duplication upstream of ORF11 is shown in bold. The asterisks show the end of attI1, the integron-associated recombination site recognized by IntI1. The black circles show the Shine-Dalgarno sequence. (C) Clustering of IMP-10-producing A. baumannii STs (MLST) by eBurst (http://eburst.mlst.net/), with 634 MLST profiles representing 634 isolates from the database (http://pubmlst.org/abaumannii/; last accessed 12 November 2014). ST400, determined for the studied isolates, is indicated by a black arrow.

Initially reported in 1997 (18), bla_IMP-10 has been harbored by distinct integrons in Gram-negative bacilli, especially in Japan (18, 19). Hu and Zhao reported Serratia marcescens isolates carrying bla_IMP-1 or bla_IMP-10 genes in class 1 integrons with identical cassette arrangements (19), which is similar to our findings. IMP-10 differs from IMP-1 by a single base replacement of G→T at nucleotide 145, leading to the amino acid substitution Val49Phe and a change to a β-lactam hydrolysis profile. Previous studies demonstrated that IMP-10, in comparison with IMP-1, lacks activity against extended-spectrum penicillins, the fourth-generation cephalosporin cefpirome, and monobactams but that it kept its activity against third-generation cephalosporins, cefepime, and carbapenems (19, 20). However, in the studied A. baumannii isolates, this phenotype was masked by the overexpression of bla_ADC and bla_OXA-100 by ISAbaI.

This work describes a new class 1 integron, In990, harboring bla_IMP-10 and reports for the first time, to our knowledge, IMP-10 among A. baumannii isolates. The natural occurrence of bla_IMP-10 seems to be inherently associated with previous bla_IMP-1 spread in the nosocomial environment, since both genes, in general, share the same backbone integrons. The events that promote the selection of bla_IMP-10 are still unknown but may have been influenced by antibiotic pressure exerted by a patient's therapy or local prescribing antibiotic practices.

(This work was presented in part as an oral presentation [O078] at the 24th European Congress of Clinical Microbiology and Infectious Diseases-ECCMID in Barcelona, Spain, in 2014.)