Globally Expanding Carbapenemase Finally Appears in Spain: Nosocomial Outbreak of Acinetobacter baumannii Producing Plasmid-Encoded OXA-23 in Barcelona, Spain

Abstract

Resistance of Acinetobacter baumannii clinical isolates to carbapenems is on the rise worldwide mainly in association with the production of OXA-23. Until recently, however, OXA-23 was absent in Spain. In this work, we report the molecular characterization of a hospital outbreak of OXA-23-producing A. baumannii in Barcelona caused by a multidrug-resistant (MDR) clone belonging to international clone IC-II/sequence type ST85 between October 2010 and May 2011. bla_OXA-23 was carried in a plasmid of 90 kb and located within the composite transposon Tn2006.

TEXT

Acinetobacter baumannii is considered an important opportunistic pathogen causing pneumonia, bacteremia, and other respiratory and urinary tract infections (1, 2), and it is also responsible for nosocomial outbreaks worldwide, especially in intensive care units (ICUs) (3). Eradication of nosocomial infections caused by A. baumannii is particularly troublesome due to the ability of this bacterium to rapidly acquire antimicrobial resistance (4) and persist in the environment for long periods of time (5). Increased resistance to carbapenems in A. baumannii has raised special concerns during the last decade, especially since it is associated mostly with the production of acquired carbapenemases belonging to either class B metallo-β-lactamases or carbapenem-hydrolyzing OXA-type class D β-lactamases (2).

Outbreak multidrug-resistant (MDR) isolates causing hospital infections across Europe usually belong to one of the three main international clones or lineages (IC-I, IC-II, and IC-III) (6), with recent studies reporting the spread of genetically related epidemic clones of A. baumannii producing OXA-23 and belonging to IC-II within the Mediterranean region (7–11). Until very recently, however, OXA-23-producing A. baumannii was absent in Spain (12). In this work, we report the molecular characterization of an OXA-23-producing A. baumannii strain causing a hospital outbreak in Barcelona between October 2010 and May 2011.

(Some of this work was presented at the 23rd European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], 27 to 30 April 2013, Berlin, Germany.)

In October 2010, the first carbapenem-resistant A. baumannii isolate associated with this outbreak was identified by Vitek (bioMérieux, Marcy l'Etoile, France) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics GmbH, Leipzig, Germany) (13) from a bronchial aspirate from a 36-year-old male patient admitted to the ICU of the Hospital del Mar, Barcelona, Spain, because of cardiac arrest due to cocaine overdose. The patient had a record of multiple psychiatric stays during the previous 2 years and was already colonized upon ICU admission. Until May 2011, subsequent screenings recovered carbapenem-resistant A. baumannii isolates from 17 different patients and 3 environmental dry surfaces within the ICU. In 10 of the 17 patients (59%), A. baumannii was recovered from rectal samples and its presence was considered colonization, whereas in the remaining patients (41%) it was considered infection and was recovered from different sites/sources (bronchial aspirates, blood culture, urine, or samples from skin and soft tissue infections). The implementation of infection control measures, such as reinforcement of hand hygiene, environmental and equipment cleaning, and cohort isolation of patients, led to outbreak containment, and no additional isolates were recovered after May 2011.

Antimicrobial susceptibility testing performed by Vitek (bioMérieux, Marcy l'Etoile, France) and Etest (AB bioMérieux, Solna, Sweden) interpreted according to CLSI guidelines (14) showed all isolates to be resistant to ceftazidime, cefotaxime, (MIC > 256 μg/ml), cefepime, imipenem, meropenem, ciprofloxacin (MIC > 32 μg/ml), and amikacin (MIC > 64 μg/ml), intermediate to tetracycline (MIC, 8 μg/ml), and susceptible to tobramycin (MIC, 1 μg/ml), gentamicin (MIC, 0.38 μg/ml), and colistin (MIC, 0.125 μg/ml). MIC values of kanamycin, aztreonam and chloramphenicol were >256 μg/ml, and those of tigecycline were 1 to 2 μg/ml.

The analysis of the isolates by pulsed-field gel electrophoresis (PFGE) profiles of ApaI-digested genomic DNA (15) revealed that all isolates were clonally related (Fig. 1). Multilocus sequence typing (MLST) following the Pasteur scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html) assigned all isolates to sequence type ST85, and multiplex PCR to identify clonal lineages was positive for group 1 (IC-II) (16). Additional typing following the 3ST scheme (http://www.hpa-bioinformatics.org.uk/AB/ab_type1.php) provided allele sequences matching those of ST85 isolates from France (allele sequence numbers for ompA, csuE, and bla_OXA-51 being 8, 11, and 10, respectively) (17).

Fig 1.

ApaI PFGE patterns of OXA-23-producing A. baumannii isolates. Lanes 1 to 17, isolates recovered from patients; lanes A1 to A3, environmental isolates; MW, lambda ladder PFGE molecular weight marker.

PCR detection of metallo- and carbapenem-hydrolyzing OXA-type β-lactamase genes (18–20) was positive only for bla_OXA-51 and bla_OXA-23. The insertion sequence ISAba1 was found upstream of both bla_OXA-23 and the chromosomal bla_ADC gene but not upstream of bla_OXA-51. DNA sequencing confirmed that the bla_OXA-23 sequences displayed 100% identity to that of the original bla_OXA-23 (GenBank accession number AJ132105.1). S1 nuclease digestion followed by pulsed-field gel electrophoresis and Southern hybridization with a digoxigenin (DIG)-labeled probe against the bla_OXA-23 gene demonstrated the genetic location of bla_OXA-23 within a plasmid of circa 90 kb. Plasmid replicon typing performed according to the scheme devised by Bertini et al. (21) was positive for the repAci6 gene, in agreement with previous studies showing a positive correlation between the presence of this replicon group (GR6) and bla_OXA-23 carriage (22). Further characterization of the genetic environment of bla_OXA-23 by inverse PCR (12) revealed its inclusion within a canonical Tn2006 composite transposon (23).

PCR amplification and sequencing of the quinolone resistance-determining region (QRDR) also showed missense mutations in both the gyrA (Ser83→Leu) and parC (Ser80→Leu) genes, concomitant with high-level resistance to ciprofloxacin, as previously described (24). PCR detection of aminoglycoside-modifying genes and 16S rRNA methylases was positive only for aphA6, which is commonly associated with high-level resistance to amikacin (25).

Plasmid transfer to a rifampin-resistant A. baumannii ATCC 17978 strain was successfully achieved by both transformation (26) and conjugation (27) upon selection on LB agar plates containing 8 μg/ml imipenem and 75 μg/ml rifampin. Selected colonies were positive for both bla_OXA-23 and aphA6, showing coexistence of the two genes in the same plasmid, as well as for the repAci6 gene. Acquisition of the plasmid containing the bla_OXA-23 and aphA6 genes turned the ATCC 17978 strain resistant to carbapenems (imipenem and meropenem MICs increased from 0.38 and 0.5 μg/ml, respectively, to >32 μg/ml), kanamycin (MIC increased from 6 μg/ml to >256 μg/ml), and amikacin (MIC increased from 3 μg/ml to 48 μg/ml).

Although outbreak MDR A. baumannii isolates are usually included within one of the three major international clones (IC-I, IC-II, and IC-III), published data seem to indicate that isolates belonging to IC-II possess a considerable ability for epidemic clonal spread (28). During the last decade, genetically related OXA-23-producing A. baumannii isolates belonging to IC-II have also been shown to disseminate within the Mediterranean region and other European countries, with a high percentage of isolates also being included within the widespread clonal complex (CC) CC92/CC2 (7, 9–11, 29–32). It has been suggested that such isolates are progressively replacing previously predominant clones carrying alternative OXA-type genes (7, 9, 10, 29). Until recently, however, carbapenem-resistant A. baumannii in Spain was mostly attributed to OXA-40 and, to a lesser extent, OXA-58 (33), whereas the presence of OXA-23-producing A. baumannii was limited to a single IC-II/ST2 isolate described in Mallorca and most likely imported from Portugal (12).

In the present study, we have described an outbreak caused by a single A. baumannii MDR clone producing OXA-23 and belonging to IC-II that rapidly disseminated among inpatients and environmental samples within one hospital in Barcelona. MLST typing assigned this clone to ST85, which is unrelated to CC92/CC2 but included in a different CC together with ST6 (17) and, therefore, cannot be associated with the OXA-23-producing ST2 epidemic clones emerging in Portugal, Italy, or Greece. ST85 has previously been linked to NDM-1 (34) and OXA-23 in France (35), the latter enzyme being responsible for a small outbreak in Marseille, but it has also been reported in carbapenem-susceptible isolates in Greece (17).

Of note, the composite transposon Tn2006, characteristically harboring the bla_OXA-23 gene in A. baumannii isolates belonging to CC92/CC2, has consistently been found in the chromosome, where it might be contained within AbaR4-like resistance islands (8, 10, 12, 23, 31), although IC-II isolates bearing both a chromosomal copy and a plasmid-encoded copy and IC-I isolates containing only a plasmid-encoded OXA-23 have also been described (10, 23). The presence of a plasmid-carried Tn2006 in the present study reflects the potential dissemination of bla_OXA-23 into epidemic clones other than those within CC92/CC2.

These findings reveal that the emergence of bla_OXA-23-positive isolates in the Mediterranean region could be attributed to the horizontal mobilization of Tn2006 among several genetically related outbreak MDR clones belonging to IC-II with an increased capability to disseminate and persist within the hospital setting. The identification of such outbreak lineages in Spain might represent the final expansion of OXA-23 throughout the Mediterranean region and, perhaps, the end of OXA-40 hegemony within the Iberian Peninsula as well.