Abstract
A carbapenem-resistant Acinetobacter baumannii expressing blaOXA-23 was recovered from an intensive care unit patient in a third-level hospital from Spain. Genetic analysis showed the association of this carbapenemase with the transposon Tn2007 located in a plasmid of 10 kb. The isolate was classified as ST-1. This strain has shown a potential ability to displace other endemic strains in the hospital and is the first reported identification of A. baumannii carrying blaOXA-23 related to Tn2007 in Spain.
Introduction
R
Production of acquired carbapenem-hydrolysing class D β-lactamases of the subgroups OXA-23, OXA-24/40, and OXA-58 represents the main mechanism of carbapenem resistance in A. baumannii. Metallo-β-lactamases (MBLs), particularly those related to imipenem-hydrolyzing β-lactamases (IMP) or verona integron-eucoded metallo-β-lactamases (VIM), have also been described in A. baumannii, although with much less frequency.
Materials and Methods
Bacterial strain
In May 2010, a 74-year-old Spanish male, was admitted to the cardiac surgery unit of the Puerta del Mar University Hospital (Cádiz, Spain) for a mitral valve replacement. In the postoperative period, the patient was admitted to the intensive care unit (ICU) and after 10 days, an isolate of A. baumannii (HUPM06), resistant to carbapenems, was isolated from the tip of a central catheter and tracheal aspirate samples.
The isolate was preliminary identified as A. baumannii by using the Wider I system (Soria Melguizo, Madrid, Spain) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonics GmbH, Bremen, Germany). Partial DNA sequencing of rpoB 9 confirmed the identification as A. baumannii.
The isolate was assigned to the ST-1 by multilocus sequence typing (MLST), following the Pasteur scheme (www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html).
Antimicrobial susceptibility
Antimicrobial susceptibility testing was performed by broth microdilution following the CLSI guidelines. 3 The control strains were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and A. baumannii ATCC 19606.
β-Lactamase and insertion sequence screening
Multiplex PCR assays were used to detect the following β-lactamase genes: blaOXA-51-like, blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, 15 blaKPC-like, blaGES-like, blaIMP-like, blaVIM-like, and blaNDM-like.5,6
The presence of the insertion sequences (ISs) ISAba1, 13 ISAba2, 13 ISAba3, 13 ISAba4, 9 and ISAba1813 was determined by PCRs using specific primer pairs of each IS. To determine the upstream and/or downstream location of each IS in relation to a β-lactamase gene, the appropriate primers and PCR conditions described by Woodford et al. 15 were used.
Sequencing and data analysis
The PCR product obtained was purified using the QIAcube (Qiagen GmbH, Hilden, Germany). Sequences were determined by bidirectional DNA sequencing (Macrogen Europe, Amsterdam, The Netherlands). The sequence was aligned with amino acids using the ClustalW program on the EMBL-EBI web server (www.ebi.ac.es/clustalw/).
Genetic location of blaOXA-23
The genetic location of blaOXA-23 was determined by S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot analysis with a digoxigenin-labeled probe (DIG DNA Labeling and Detection Kit; Roche Diagnostic, S.L. Barcelona, Spain) specific to blaOXA-23.
Results
Susceptibility
The MICs (mg/L) were >512 (piperacillin), >128 (ceftazidime, amikacin), >64 (gentamicin, tobramycin, meropenem), >32 (imipenem), >16 (ciprofloxacin), >8 (tetracycline), >1 (rifampin), 0.125 (minocycline), 0.5 (doxycycline, tigecycline), and 0.25 (colistin). According to the CLSI interpretive breakpoints, 3 the isolate was resistant to piperacillin, ceftazidime, amikacin, gentamicin, tobramycin, meropenem, imipenem, ciprofloxacin, and tetracycline, and susceptible to minocycline, doxycycline, tigecycline, and colistin. Following the Comitée de l'Antibiogramme de la Societé Française de Microbiologie (CA-SFM), the isolate was also resistant to rifampin.
MBL genotype
The presence of genes encoding MBLs was not detected by PCRs using primers specific to blaKPC-like, blaGES-like, blaIMP-like, blaVIM-like, and blaNDM-like.
CHO analysis
The strain HUPM06 was positive for blaOXA-23-like and blaOXA-51-like and negative for blaOXA-24/40-like and blaOXA-58-like.
IS detection
The PCRs were positive for ISAba1 and ISAba4. The relative position of ISAba1 and ISAba4 with respect to blaOXA-23 was determined using separate independent PCRs containing all the possible primer pair combinations between these two ISs and blaOXA-23. The unique positive PCR was that containing the reverse primer used for ISAba4 and the reverse primer of blaOXA-23, indicating that ISAba4 is located upstream of blaOXA-23. As this association between ISAba4 and blaOXA-23 has been observed in the Tn2007 transposon,2,4 a set of internal primers covering the complete Tn2007 (GenBank: EF059914.1) was designed for PCR amplification and DNA sequencing. The nucleotide sequences obtained were aligned, confirming the situation of ISAba4 upstream of blaOXA-23 and their localization within a Tn2007 (GenBank: EF059914.1).
Genetic location blaOXA-23-like
No hybridization was observed between the probe and chromosomal DNA. In contrast, the probe hybridized with a small plasmid band of ca. 10 kb.
Discussion
Resistance to carbapenems in A. baumannii is increasing worldwide and complicating the treatment of severe infections caused by this microorganism. At this moment, production of OXA-23 is the most frequently observed mechanism of carbapenem resistance in A. baumannii. 1 Nosocomial outbreaks of carbapenem-resistant A. baumannii carrying blaOXA-23 have been reported in many countries. Some studies performed in Spain, before 2010, showed that blaOXA-40 and, to a lesser extent, blaOXA-58 was the most prevalent carbapenemase in A. baumannii. 14 This situation appears to be changing, particularly in Spain, as indicated by our study and other studies.7,10,11 The first blaOXA-23 detected in Spain was described by Espinal et al. in an isolate of A. baumannii ST-2 from a male patient in February 2010 in Mallorca. 7 In a more recent study, Mosqueda et al. 11 have described a nosocomial outbreak caused by a clone of A. baumannii ST-85 carrying blaOXA-23 within Tn2006 and in a large plasmid of 90 kb.
Our results and those reported in other studies suggest that the transmission or dissemination of blaOXA-23 appears to be occurring through the acquisition of transposons rather than by clonal dissemination.
The isolate of A. baumannii described in our study differs from the isolates of A. baumannii carrying blaOXA-23 from other studies in the type of ST, the type of transposon containing blaOXA-23, and in the size of the plasmid where it locates blaOXA-23. A. baumannii HUPM06 is probably nonclonally related with those isolates of A. baumannii previously described in Spain and France. A. baumannii HUPM06 showed an ST-1, in contrast with the STs described in Spain (ST-2, ST-58, and ST-118)7,10,11 and in France (ST New). 12 However, isolates classified as ST-1 have been described in Belgium, Algeria, Reunion, and United Arab Emirates. 12
The blaOXA-23 of A. baumannii HUPM06 was located within Tn2007, as has been described in Belgium, Algeria, and France, 12 whereas in the previous studies performed in Spain, the transposon carrying blaOXA-23 was Tn2006. In our strain, blaOXA-23 was located in a small plasmid of 10 kb, whereas in other studies it was located in a plasmid of 90 kb7,10 or in the chromosome. 11
After the first detection of the isolate of A. baumannii HUPM06, there have been 37 other patients in the same ICU identified as carriers or infected (considering colonized patients as those in whom positive cultures were obtained in the absence of clinical infection) with blaOXA-23-producing isolates of A. baumannii indistinguishable by PFGE (data not shown), suggesting clonal diffusion. Before the introduction of this clone of OXA-23-producing A. baumannii, there has been an endemic setting of nosocomial transmission of blaOXA-58-producing A. baumannii in this ICU. During the period from May to December 2010, both clones (OXA-23 and OXA-58-producing) of A. baumannii have coexisted in the same ICU and, at this moment, it appears that the OXA-23 clone has displaced the OXA-58 clone. MLST of A. baumannii OXA-58 showed a single-locus variant of ST-80, while A. baumannii OXA-23 was assigned to the ST-1, discarding any link between the two clones. Other molecular and epidemiological studies should confirm this hypothesis.
In conclusion, this is the first report describing blaOXA-23 located within Tn2007 in a clinical isolate of A. baumannii from Spain, showing the potential ability to displace other clones of A. baumannii.
Footnotes
Disclosure Statement
No competing financial interests exist.