Evaluation of the Carba NP test for carbapenemase detection in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., and its practical use in the routine work of a national reference laboratory for susceptibility testing
Acinetobacter baumannii is currently one of the key nosocomial pathogens causing severe infections; of special concern is its resistance to expanded-spectrum cephalosporins (ESCs) and carbapenems, often associated with the few so-called European clones (6, 7, 19). It has two natural -lactamases, an AmpC-like enzyme (Acinetobacter-derived cephalosporinase [ADC]) (10) and a carbapenem-hydrolyzing class D -lactamase (CHDL; the OXA-51 type) (15), which affect susceptibility upon increased expression due to ISAba1 insertion upstream of their genes (9, 18). Moreover, acquired -lactamases, including metallo-lactamases (MBLs) and four CHDL types, the OXA-23, OXA-24/40, OXA-58, and OXA-143 types, are observed (15). Knowledge of A. baumannii in Poland has been limited to single isolates (9, 14, 21); our aim was to analyze a bigger group of A. baumannii strains. (Part of this work was presented at the 22nd European Congress of Clinical Microbiology and Infectious Diseases, London, United Kingdom, 31 March to 3 April 2012.) The study was performed on 30 patient-unique isolates (Table 1) collected during a surveillance of invasive infections in Polish hospitals. These were all A. baumannii isolates from blood (n 27), cerebrospinal fluid (n 2), or brain tissue (n 1) that were received in 2009 from 16 hospitals in 13 cities. The species was identified by Vitek2 (bioMérieux, Marcy l’Etoile, France), followed by sequencing of the 16S-23S rRNA intergenic spacer (4). MICs were evaluated by Etest (bioMérieux) and interpreted according to the EUCAST (www.eucast.org) or CLSI (5) guidelines. All but one of the isolates were multiresistant, including nonsusceptibility to ESCs. Regarding the carbapenems, five isolates were intermediate to at least meropenem and eight isolates were resistant to both imipenem and meropenem. The ADC overexpression was assessed by PCR for ISAba1 upstream of the blaADC genes (9); all of the 29 ESC-nonsusceptible isolates were positive. The isolates were checked by PCR for Ambler class A -lactamase genes, including blaTEM, blaPER-1, blaGES, and blaVEB. Only blaTEM genes were found in 17 isolates, and they were confirmed by sequencing to be blaTEM-1. All of the isolates were negative in the disk assay with EDTA for MBL production (11). The increased expression of blaOXA-51-type genes was analyzed by PCR for ISAba1 upstream of the genes (18). Amplicons were obtained for eight isolates, including most of those with low carbapenem resistance levels. Of the acquired CHDL genes (20), blaOXA-23 was amplified and sequenced in six isolates; blaOXA-24/40 and blaOXA-58 types were each detected once in other isolates and found to be blaOXA-72 and blaOXA-58, respectively. These eight isolates were all the ones that exhibited in vitro high-level resistance to carbapenems. The location of the mobile CHDL genes was studied by hybridization of gene-specific probes to total DNA cut with nuclease S1, as reported previously (1). The blaOXA-23 probe hybridized with plasmids of 70 or 80 kb in the six corresponding isolates, all of which proved positive in plasmid replicon typing, done by PCR of the aci6 replicase gene, originally found on pACICU2 (2). The blaOXA-72 and blaOXA-58 probes hybridized with chromosomal DNA only. The sequence context of blaOXA-23 was analyzed by PCR mapping of the transposons Tn2006, Tn2007, and Tn2008 (12). All six isolates had ISAba1 upstream of blaOXA-23, characteristic of Tn2006 and Tn2008 (9, 20). Then two primers were designed for the 3= and 5= parts of the ATPase associated with diverse cellular activities (AAA) gene (ATPnear, 5=-GAATCTGCCAGCCAATGATG-3=, and ATPfar, 5=-AGTATGTACACATGCCACAC-3=, respectively), which has the 5= part truncated in Tn2008 only (12). The isolates contained the Tn2008-like elements, as indicated by the positive PCR with primers OXA-23-likeF (20) and ATPnear and the negative PCR with ATPfar. The isolates were typed by ApaI pulsed-field gel electrophoresis (PFGE) (16). DNA patterns were analyzed according to the method of Tenover et al. (17). All of the isolates were also characterized by multilocus sequence typing (MLST) (6) and by sequencing the blaOXA-51-like alleles. The PFGE revealed 24 patterns, classified into six types. Type B was the predominant type, with 21 isolates from 14 hospitals, consistent with its being sequence type (ST) 2, which is the European clone II (6). It had the blaOXA-66 allele of the blaOXA-51 gene, as shown in earlier reports (8). All of the eight isolates with acquired CHDLs were classified into ST2. Another PFGE type, type E, consisting of four isolates from two centers plus two isolates of other types, was grouped as ST1, the European clone I (6). As reported (8), all ST1 isolates carried blaOXA-69-related alleles, namely, blaOXA-69, as well as the new blaOXA-248 and blaOXA-250. Their ISAba1-mediated expression was the only mechanism of carbapenem resistance found. The other isolates belonged to ST5 (with blaOXA-130 or new blaOXA-249) or the new ST193 (new blaOXA-120). In general, this first, more extensive analysis of A. baumannii strains in Poland showed results, such as common multiresistance, high predominance of the ST2 clone, and its notable heterogeneity, that were similar to those from other regions (3, 6, 13, 19). However, interesting differences from other Central European countries were also observed; in Romania, ST2 uniformly produced OXA-23, while ST1 had OXA-23 or OXA-58, not overexpressing the blaOXA-51-like genes (3). In the Czech Republic, almost all carbapenem resistance was associated with ST2, mostly due to blaOXA-51 overexpression (13). All these data show the multidirectional evolution of the major A. baumannii clones in neighboring countries. Nucleotide sequence accession numbers. Sequences of the new blaOXA-51 alleles appear in the EMBL database under the following accession numbers: HE963768 for blaOXA-120, HE963769 for blaOXA-248, HE963770 for blaOXA-249, and HE963771 for blaOXA-250.