Major outbreaks of multidrug-resistant Acinetobacter baumannii associated with nosocomial infections have been increasingly reported worldwide (1, 10, 12). The endemicity of an OXA-24/40-producing A. baumannii clone associated with mortality events in Portugal has been observed at numerous hospitals within the Iberian Peninsula (5, 6, 10). Inversely, Acinetobacter haemolyticus, isolated only occasionally from clinical samples (9), usually presents susceptibility to different antibiotics, including β-lactams (13). The isolation of two carbapenem-resistant A. haemolyticus strains prompted us to assess the relative contribution of clonal spread to the observed high rate of carbapenem-resistant Acinetobacter spp. in a general hospital in Porto, Portugal.
Between January 2001 and October 2004, 224 imipenem-resistant Acinetobacter spp. were collected from several specimen sources and different hospital wards, where A. baumannii was associated with nosocomial infections and colonizations for several months (Table 1). Imipenem resistance significantly increased from 2001 to 2002 and from 2002 to 2003. Macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis (5) and 16S rRNA gene sequencing, performed for each clone and species representative, showed that, with the exception of two clonally related A. haemolyticus isolates, the remainder were A. baumannii isolates, distributed among three different pulsotypes. Clonal dissemination of two major pulsotypes (A and B), widespread throughout the hospital, contributed to the observed A. baumannii imipenem resistance, which has persisted since at least 2001 despite several elimination attempts, including the use of polymyxin. Pulsotype B was predominant from 2001 to 2002, after which clone A emerged as the dominant type (Table 1). This clone was found to be identical to the previously described Iberian OXA-24/40-producing clone (5). Pulsotype C, with only two isolates, seemed to represent a sporadic event within the observed prevalence of clones A and B. Antimicrobial susceptibilities varied among isolates according to clones (Table 2). A. haemolyticus isolates presented resistance to all β-lactams, with the exception of cefepime, ceftazidime, and aztreonam. All Acinetobacter sp. isolates were resistant to ciprofloxacin, whereas susceptibility to aminoglycosides was variable. Only 11 isolates (including the two A. haemolyticus isolates) showed a colistin MIC of ≥4 μg/ml (2). However, when the recently updated CLSI susceptible interpretative criterion of ≤2 μg/ml (3, 8) was applied, the susceptibility rate dropped from 96.1% to 92.1%. Detection of carbapenemase production, ulteriorly identified as an OXA-24/40 enzyme, was performed as previously described (5) and was positive only for clone A A. baumannii isolates and, for the first time, A. haemolyticus isolates. Hybridization assays after both S1 nuclease digestion and I-CeuI digestion, performed as previously described (7), revealed that although some clone A A. baumannii isolates showed a chromosome-positive signal (ca. 150 kb) for the blaOXA-24/40 probe, most also presented a positive hybridization in plasmidic bands of ca. 180 kb and ca. 30 kb. Similar hybridization signals were observed in the A. haemolyticus isolates. Further studies on plasmid characterization, assessing the homology among different plasmids, are ongoing.
TABLE 1.
Clinical data for imipenem-resistant Acinetobacter spp.a
| Yr | % Imipenem resistance (no. of isolates)d | Clone (no. of isolates)b | Ward(s) (no. of isolates) | Main specimen source(s) (no. of isolates)c |
|---|---|---|---|---|
| 2001 | 32 (47) | A (14) | ICU (8), ICU-P (2), ICU-S (1), NC (2), NK (1) | Respiratory tract (12), urine (1), NK (1) |
| B (33) | ICU (6), ICU-P (14), CET (3), surgery 12B (3), Med A, B, and D (3), OBS (2), neurology (1), orthopedics (1) | Respiratory tract (13), urine (9), pus (4), catheter (3), blood (3), CSF (1) | ||
| 2002 | 53 (31) | A (6) | ICU (4), Med B and C (2) | Respiratory tract (5), urine (1) |
| B (22) | Med B and D (7), ICU (5), ICU-P (6), neurology (2), orthopedics (1), urology (1) | Respiratory tract (8), urine (10), pus (1), blood (2), catheter (1) | ||
| A. haemolyticus (2) | Endocrinology (1), Med B (1) | Pus (1), urine (1) | ||
| 2003 | 97 (104) | A (53) | ICU (25), CET (2), ICU-P (1), ICU-S (4), Med D (2), OBS (1), orthopedics (1), NC (5), surgery (9), urology (2), GIN (1) | Respiratory tract (27), pus (4), CSF (4), blood (6), urine (6), ascitic liquid (1), catheter (3), peritoneal liquid (2) |
| B (9) | Surgery 2 (2), PED (1), Med (5), ICU (1) | Respiratory tract (3), urine (5), blood (1) | ||
| C (2) | Med D (1), ICU-P (1) | Catheter (1), urine (1) | ||
| 2004 | 98 (42) | A (9) | ICU (4), surgery (1), NC (1), Med (1), OBS (1), oncology (1) | Respiratory tract (6), blood (1), NK (2) |
| B (1) | GIN (1) | Urine (1) |
ICU, intensive care unit; Med, medical unit(s); ICU-P, polivalent ICU; ICU-S, postsurgical ICU; OBS, observation; NC, neurosurgery; PED, pediatrics; GIN, gynecology; CET, cranium-encephalic traumatism; CSF, cerebrospinal fluid; NK, origin not known.
Number of pulsotyped isolates (in 2003 and 2004, only representative isolates from the different hospital units were included). Clones are designated by capital letters and refer to A. baumannii isolates.
The respiratory tract includes sputum, bronchial secretions, and tracheal aspirate.
P was <0.01 for the difference between the two values (after Bonferroni's adjustment) for 2001 and 2002, and P was <0.001 for the difference between the two values for 2002 and 2003. No significant differences were observed between 2003 and 2004 (P = 0.73)
TABLE 2.
In vitro susceptibilities of imipenem-resistant Acinetobacter sp. clinical isolates
| Species | Clone | Carbapenemase production | MIC range (μg/ml)a
|
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IPM | MEM | AMX | AMC | CAZ | FEP | FOX | TIC | TIM | PIP | TZP | ATM | CST | |||
| A. baumannii | A | blaOXA-40 | ≥32 | ≥32 | ≥256 | ≥256 | ≥256 | ≥32 | ≥256 | ≥256 | ≥256 | ≥256 | ≥256 | 8->256 | <1-≥16 |
| B | Negb | ≥32 | 2-8 | 64->256 | 64->256 | 4-8 | 8-24 | ≥256 | ≥256 | ≥256 | ≥256 | ≥256 | 16->256 | <1-≥16 | |
| C | Neg | 32 | 4 | 128 | ≥256 | 4 | 2 | ≥256 | ≥256 | ≥256 | 64 | 64 | 8 | 2-4 | |
| A. haemolyticus | blaOXA-40 | ≥32 | ≥32 | ≥256 | ≥256 | 4 | 4 | 32 | ≥256 | ≥256 | ≥256 | ≥256 | 4 | 4 | |
IPM, imipenem; MEM, meropenem; AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; TIC, ticarcillin; TIM, ticarcillin-clavulanic acid; PIP, piperacillin; TZP, piperacillin-tazobactam; ATM, aztreonam; CST, colistin. The MICs of β-lactams were determined by the Etest method and those of colistin by the agar dilution method (11).
Neg, absence of carbapenemase production.
We describe, for the first time, the presence of an OXA-24/40 enzyme in an A. haemolyticus clinical isolate. Although the spread of OXA-24/40, both in the Iberian Peninsula and in France, has been correlated with the progressive dissemination of a single A. baumannii clone, the observation of this enzyme in a different, previously unreported, genomic species, A. haemolyticus, poses new questions on OXA-24/40 dissemination. It now seems reasonable to suspect a horizontal dissemination of the blaOXA-40 gene between different species, an ability supported by the observation of this enzyme, previously described as chromosomally encoded (7), in a plasmid. Notwithstanding, the dissemination of “successful” clones may possibly contribute to the high rates and persistence of imipenem-resistant A. baumannii isolates (4).
Acknowledgments
We are grateful to Nuno Monteiro for helpful discussions and critical review of the manuscript.
Footnotes
Published ahead of print on 2 July 2007.
REFERENCES
- 1.Afzal-Shah, M., and D. M. Livermore. 1998. Worldwide emergence of carbapenem-resistant Acinetobacter spp. J. Antimicrob. Chemother. 41:576-577. [DOI] [PubMed] [Google Scholar]
- 2.Catchpole, C., J. Andrews, N. Brenwald, and R. Wise. 1997. A reassessment of the in vitro activity of colistin sulphomethate. J. Antimicrob. Chemother. 39:255-260. [DOI] [PubMed] [Google Scholar]
- 3.Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement, vol. 26. M100-S16. Clinical and Laboratory Standards Institute, Wayne, PA.
- 4.Coelho, J., J. Turton, M. Kaufmann, J. Glover, N. Woodford, M. Warner, M. Palepou, R. Pike, T. Pitt, B. Patel, and D. Livermore. 2006. Occurrence of carbapenem-resistant Acinetobacter baumannii clones at multiple hospitals in London and Southeast England. J. Clin. Microbiol. 44:3623-3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Da Silva, G., S. Quinteira, E. Bértolo, J. Sousa, L. Gallego, A. Duarte, and L. Peixe. 2004. Long-term dissemination of an OXA-40 carbapenemase-producing Acinetobacter baumannii clone in the Iberian Peninsula. J. Antimicrob. Chemother. 54:255-258. [DOI] [PubMed] [Google Scholar]
- 6.Gallego, L., and K. Towner. 2001. Carriage of class 1 integrons and antibiotic resistance in clinical isolates of Acinetobacter baumannii from northern Spain. J. Med. Microbiol. 50:71-77. [DOI] [PubMed] [Google Scholar]
- 7.Héritier, C., L. Poirel, D. Aubert, and P. Nordmann. 2003. Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-40 of Acinetobacter baumannii. Antimicrob. Agents Chemother. 47:268-273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Jones, R., T. Anderegg, and J. Swenson. 2005. Quality control guidelines for testing gram-negative control strains with polymyxin B and colistin (polymyxin E) by standardized methods. J. Clin. Microbiol. 43:925-927. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lambert, T., G. Gerbaud, M. Galimand, and P. Courvalin. 1993. Characterization of Acinetobacter haemolyticus aac(6′)-Ig gene encoding an aminoglycoside 6′-N-acetyltransferase which modifies amikacin. Antimicrob. Agents Chemother. 37:2093-2100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Lopez-Otsoa, F., L. Gallego, K. Towner, L. Tysall, N. Woodford, and D. Livermore. 2002. Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a hospital in northern Spain. J. Clin. Microbiol. 40:4741-4743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, PA.
- 12.Poirel, L., and P. Nordmann. 2006. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin. Microbiol. Infect. 12:826-836. [DOI] [PubMed] [Google Scholar]
- 13.Visalli, M., M. Jacobs, T. Moore, F. Renzi, and P. Appelbaum. 1997. Activities of β-lactams against Acinetobacter genospecies as determined by agar dilution and E-test MIC methods. Antimicrob. Agents Chemother. 41:767-770. [DOI] [PMC free article] [PubMed] [Google Scholar]