LETTER
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 β-lactamases, an AmpC-like enzyme (Acinetobacter-derived cephalosporinase [ADC]) (10) and a carbapenem-hydrolyzing class D β-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 β-lactamases, including metallo-β-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.
Table 1.
Molecular characteristics and susceptibility of the study isolates
| Strain no. | City | Material | PFGE typea | ST | PCR result for: |
MIC (μg/ml)d |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| blaADC and ISAba1b | blaTEM-1b,c | blaOXA-23-likec/plasmid size | blaOXA-24-likec | blaOXA-58-likec | blaOXA-51-likec | blaOXA-51 and ISAba1b | PIP | TZP | SAM | TIM | CAZ | CTX | FEP | IPM | MEM | CST | AMK | GEN | CIP | SXT | |||||
| 1 | Warsaw | Blood | C | 1 | + | TEM-1d | blaOXA-248 | + | >256 | 48 | 24 | >256 | >256 | >256 | 32 | 8 | 8 | 0.25 | >256 | >256 | >32 | 8 | |||
| 2 | Kołobrzeg | Blood | D | 1 | + | blaOXA-69 | >256 | 48 | 3 | 48 | 128 | >256 | 16 | 1.5 | 0.75 | 0.5 | 12 | 4 | >32 | 12 | |||||
| 3 | Rzeszów | Blood | E1 | 1 | + | blaOXA-69 | + | >256 | 48 | 2 | 32 | 24 | 256 | 8 | 1.5 | 1.5 | 0.19 | >256 | >256 | >32 | >32 | ||||
| 4 | Jelenia Góra | Blood | E2 | 1 | + | blaOXA-250 | + | >256 | 96 | 2 | 96 | 16 | 256 | 6 | 4 | 6 | 0.19 | 12 | >256 | >32 | 8 | ||||
| 5 | Jelenia Góra | Blood | E3 | 1 | + | blaOXA-69 | + | 48 | 12 | 2 | 32 | 48 | 24 | >256 | 0.75 | 3 | 0.25 | 6 | >256 | >32 | >32 | ||||
| 6 | Rzeszów | Blood | E4 | 1 | + | blaOXA-69 | + | >256 | 128 | 2 | 32 | 24 | 256 | 48 | 2 | 1.5 | 0.25 | >256 | >256 | >32 | >32 | ||||
| 7 | Kołobrzeg | Blood | B1 | 2 | + | + | blaOXA-66 | >256 | 128 | 16 | >256 | 128 | >256 | 24 | 1.5 | 2 | 0.25 | 96 | 64 | >32 | >32 | ||||
| 8 | Kołobrzeg | Brain | B2 | 2 | + | + | blaOXA-66 | >256 | 256 | 24 | >256 | 128 | >256 | 24 | 1.5 | 2 | 0.25 | 128 | >256 | >32 | >32 | ||||
| 9 | Kołobrzeg | Blood | B2 | 2 | + | + | blaOXA-66 | >256 | 128 | 16 | >256 | 128 | >256 | 24 | 1.5 | 2 | 0.38 | 128 | >256 | >32 | >32 | ||||
| 10 | Bytom | Blood | B3 | 2 | + | + | blaOXA-66 | >256 | 128 | 16 | >256 | 256 | >256 | 24 | 1.5 | 2 | 0.25 | 96 | 256 | >32 | >32 | ||||
| 11 | Zabrze | Blood | B4 | 2 | + | + | blaOXA-23/∼80 kb | blaOXA-66 | >256 | >256 | 48 | >256 | 256 | >256 | 64 | >32 | >32 | 0.25 | >256 | >256 | >32 | >32 | |||
| 12 | Zabrze | Blood | B4 | 2 | + | + | blaOXA-23/∼80 kb | blaOXA-66 | >256 | >256 | 48 | >256 | 256 | >256 | 48 | >32 | >32 | 0.25 | 128 | >256 | >32 | >32 | |||
| 13 | Zabrze | Blood | B4 | 2 | + | + | blaOXA-23/∼80 kb | blaOXA-66 | >256 | >256 | 32 | >256 | 256 | >256 | 64 | >32 | >32 | 0.25 | >256 | >256 | >32 | >32 | |||
| 14 | Sosnowiec | Blood | B4 | 2 | + | blaOXA-66 | >256 | >256 | 3 | 96 | 128 | >256 | 24 | 1 | 3 | 0.25 | 96 | >256 | >32 | >32 | |||||
| 15 | Warsaw | Blood | B5 | 2 | + | blaOXA-58 | blaOXA-66 | >256 | 256 | 8 | >256 | 192 | >256 | 24 | 16 | >32 | 0.25 | >256 | >256 | >32 | >32 | ||||
| 16 | Cracow | Blood | B6 | 2 | + | + | blaOXA-23/∼70 kb | blaOXA-66 | + | >256 | >256 | 48 | >256 | 128 | >256 | 48 | >32 | >32 | 0.25 | >256 | >256 | >32 | >32 | ||
| 17 | Wejherowo | Blood | B6 | 2 | + | + | blaOXA-66 | >256 | 98 | 16 | >256 | 96 | >256 | 24 | 1.5 | 2 | 0.25 | 96 | >256 | >32 | >32 | ||||
| 18 | Bytom | Blood | B7 | 2 | + | + | blaOXA-66 | >256 | 64 | 16 | >256 | 96 | >256 | 24 | 1.5 | 1.5 | 0.25 | 96 | 256 | >32 | >32 | ||||
| 19 | Bytom | Blood | B8 | 2 | + | + | blaOXA-66 | >256 | 128 | 24 | >256 | 192 | >256 | 24 | 2 | 2 | 0.19 | 96 | >256 | >32 | >32 | ||||
| 20 | Rzeszów | Blood | B9 | 2 | + | + | blaOXA-66 | >256 | 32 | 24 | >256 | 64 | >256 | 12 | 1 | 2 | 0.25 | 96 | 256 | >32 | >32 | ||||
| 21 | Cracow | Blood | B10 | 2 | + | blaOXA-23/∼80 kb | blaOXA-66 | >256 | >256 | 24 | >256 | 64 | >256 | 48 | >32 | >32 | 0.5 | >256 | 192 | >32 | >32 | ||||
| 22 | Jelenia Góra | Blood | B11 | 2 | + | + | blaOXA-66 | >256 | 64 | 24 | >256 | 256 | >256 | 16 | 1 | 2 | 0.25 | 128 | >256 | >32 | >32 | ||||
| 23 | Jelenia Góra | Blood | B11 | 2 | + | + | blaOXA-66 | >256 | 96 | 16 | >256 | 96 | >256 | 16 | 1.5 | 1.5 | 0.38 | 128 | >256 | >32 | >32 | ||||
| 24 | Inowrocław | Blood | B12 | 2 | + | blaOXA-66 | >256 | 128 | 3 | 64 | 128 | >256 | 24 | 2 | 2 | 0.19 | 64 | >256 | >32 | >32 | |||||
| 25 | Suwałki | Blood | B13 | 2 | + | blaOXA-72 | blaOXA-66 | >256 | >256 | 6 | >256 | 128 | >256 | 24 | >32 | >32 | 0.38 | 96 | >256 | >32 | >32 | ||||
| 26 | Cracow | CSFe | B14 | 2 | + | blaTEM-1d | blaOXA-23/∼70 kb | blaOXA-66 | >256 | >256 | 32 | >256 | 64 | >256 | 48 | >32 | >32 | 0.19 | >256 | >256 | >32 | >32 | |||
| 27 | Pisz | CSF | B15 | 2 | + | + | blaOXA-66 | >256 | 64 | 16 | >256 | 24 | >256 | 12 | 1 | 1 | 0.125 | 12 | 6 | 0.38 | 0.19 | ||||
| 28 | Kołobrzeg | Blood | A1 | 5 | + | blaOXA-130 | + | >256 | >256 | 8 | 96 | 16 | >256 | 4 | 2 | 1.5 | 0.125 | 48 | 1.5 | >32 | 0.5 | ||||
| 29 | Kołobrzeg | Blood | A2 | 5 | + | blaOXA-249 | + | >256 | >256 | 8 | >256 | 24 | >256 | 8 | 6 | 6 | 0.19 | >256 | 1 | >32 | 0.75 | ||||
| 30 | Katowice | Blood | F | 193 | blaOXA-120 | 3 | <0.016 | 0.5 | 1.5 | 0.75 | 1.5 | 0.5 | 0.25 | 0.25 | 0.19 | 4 | 1.5 | 0.19 | 0.19 | ||||||
PFGE types are designated by capital letters; numbers following these specify subtypes, distinguished according to Tenover et al. (17).
+, positive result of PCR amplification. A blank field means that the PCR was negative.
The full name of a gene means that the gene was sequenced for an isolate.
PIP, piperacillin; TZP, piperacillin-tazobactam; SAM, ampicillin-sulbactam; TIM, ticarcillin-clavulanic acid; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IPM, imipenem; MEM, meropenem; CST, colistin; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole. MICs for nonsusceptible isolates are indicated in bold.
CSF, cerebrospinal fluid.
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 β-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.
ACKNOWLEDGMENTS
We thank A. Baraniak for her very helpful input, I. Łe̢towska, A. Skoczyńska, E. Stefaniuk, and D. Żabicka for kindly providing the isolates, and K. Horowitz for correcting the style and expression.
This work was partially financed by the grant SPUB MIKROBANK from the Polish Ministry of Science and Higher Education.
We acknowledge the use of the A. baumannii MLST database, which is located at Institut Pasteur, Paris, France.
Footnotes
Published ahead of print 12 September 2012
REFERENCES
- 1. Baraniak A, et al. 2011. Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008-2009. Antimicrob. Agents Chemother. 55: 5493–5499 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Bertini A, et al. 2010. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54: 4168–4177 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Bonnin RA, Poirel L, Licker M, Nordmann P. 2011. Genetic diversity of carbapenem-hydrolysing beta-lactamases in Acinetobacter baumannii from Romanian hospitals. Clin. Microbiol. Infect. 17: 1524–1528 [DOI] [PubMed] [Google Scholar]
- 4. Chang HC, et al. 2005. Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J. Clin. Microbiol. 43: 1632–1639 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Clinical and Laboratory Standards Institute 2006. Performance standards for antimicrobial susceptibility testing; 21st informational supplement. CLSI M100-S21 Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
- 6. Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. 2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One 5: e10034 doi:10.1371/journal.pone.0010034 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Dijkshoorn L, et al. 1996. Comparison of outbreak and nonoutbreak Acinetobacter baumannii strains by genotypic and phenotypic methods. J. Clin. Microbiol. 34: 1519–1525 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Evans BA, Hamouda A, Towner KJ, Amyes SG. 2008. OXA-51-like beta-lactamases and their association with particular epidemic lineages of Acinetobacter baumannii. Clin. Microbiol. Infect. 14: 268–275 [DOI] [PubMed] [Google Scholar]
- 9. Heritier C, Poirel L, Nordmann P. 2006. Cephalosporinase over-expression resulting from insertion of ISAba1 in Acinetobacter baumannii. Clin. Microbiol. Infect. 12: 123–130 [DOI] [PubMed] [Google Scholar]
- 10. Hujer KM, et al. 2005. Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 β-lactamase: defining a unique family of class C enzymes. Antimicrob. Agents Chemother. 49: 2941–2948 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Lee K, Lim YS, Yong D, Yum JH, Chong Y. 2003. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-β-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol. 41: 4623–4629 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Mugnier PD, Poirel L, Naas T, Nordmann P. 2010. Worldwide dissemination of the blaOXA-23 carbapenemase gene of Acinetobacter baumannii. Emerg. Infect. Dis. 16: 35–40 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Nemec A, et al. 2008. Emergence of carbapenem resistance in Acinetobacter baumannii in the Czech Republic is associated with the spread of multidrug-resistant strains of European clone II. J. Antimicrob. Chemother. 62: 484–489 [DOI] [PubMed] [Google Scholar]
- 14. Netsvyetayeva I, et al. 2011. Acinetobacter baumannii multidrug-resistant strain occurrence in liver recipients with reference to other high-risk groups. Transplant Proc. 43: 3116–3120 [DOI] [PubMed] [Google Scholar]
- 15. Poirel L, Naas T, Nordmann P. 2010. Diversity, epidemiology, and genetics of class D β-lactamases. Antimicrob. Agents Chemother. 54: 24–38 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Seifert H, et al. 2005. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. J. Clin. Microbiol. 43: 4328–4335 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Tenover FC, et al. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33: 2233–2239 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Turton JF, et al. 2006. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol. Lett. 258: 72–77 [DOI] [PubMed] [Google Scholar]
- 19. van Dessel H, et al. 2004. Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals. Res. Microbiol. 155: 105–112 [DOI] [PubMed] [Google Scholar]
- 20. Woodford N, et al. 2006. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 27: 351–353 [DOI] [PubMed] [Google Scholar]
- 21. Wroblewska MM, Towner KJ, Marchel H, Luczak M. 2007. Emergence and spread of carbapenem-resistant strains of Acinetobacter baumannii in a tertiary-care hospital in Poland. Clin. Microbiol. Infect. 13: 490–496 [DOI] [PubMed] [Google Scholar]