Heteroresistance to carbapenems in Acinetobacter baumannii

S. Pournaras1, A. Ikonomidis1, A. Markogiannakis2, A. N. Maniatis1 and A. Tsakris2,*

1 Department of Microbiology, Medical School, University of Thessalia, Mezourlo, 41110 Larissa, Greece; 2 Department of Basic Sciences, School of Health Sciences, University of Athens, 123 Papadiamantopoulou Street, 11527 Athens, Greece


* Corresponding author. Tel: +30-210-746-1483; Fax: +30-210-746-1489; Email: atsakris{at}med.uoa.gr

Keywords: imipenem , meropenem , Etest , PFGE , resistance , heterogeneity

Sir,

Acinetobacter baumannii is an opportunistic pathogen with increasing relevance in hospital infections. The organism is particularly associated with nosocomial invasion of burn wounds, pneumonia, bacteraemia, post-neurosurgical meningitis and infections of the urinary tract. Recent data suggest that in several hospital settings large percentages of A. baumannii isolates are resistant to carbapenems.1 Thus, treatment of carbapenem-resistant A. baumannii has become a clinical challenge, since this microorganism is usually resistant to almost all available antimicrobial agents.

Resistance to carbapenems among acinetobacters as well as among other bacteria is known to be homogeneous within a culture. In a previous study,2 as is typical for ß-lactam antibiotics, heteroresistance to meropenem has been detected in population studies among methicillin-resistant staphylococci. The present report describes the spread of hetero-carbapenem-resistant A. baumannii isolates.

In our region, carbapenem-resistant A. baumannii are being isolated with increasing frequency from clinical sources.3 Moreover, several Gram-negative species producing VIM-type metallo-ß-lactamases have been detected in Greek hospitals since 1997. Therefore, clinical isolates of A. baumannii are routinely screened by Etest (AB Biodisk, Solna, Sweden) for imipenem and meropenem, in addition to automated or disc diffusion susceptibility testing, in order to detect carbapenem-resistant isolates. Mueller–Hinton agar plates are inoculated with 0.5 McFarland standard suspension test organisms and the relevant Etest strips are added. Plates are incubated at 37°C for 18 h. During this procedure, eight clinical isolates of A. baumannii that showed subcolonies present in the clear zone of inhibition were recovered from separate patients. The isolates exhibited resistance to all other available antimicrobials except colistin; three of them were recovered from urine specimens, three from bronchial secretions and two from blood specimens. They were provisionally identified to genus species by the API 20NE system (bioMérieux API, Marcy l'Étoile, France) according to the manufacturer's instructions. The identification of A. baumannii was performed by a simplified identification scheme.

Imipenem and meropenem MIC determinations for the eight acinetobacters were repeated by Etest according to the manufacturer's specification; their MICs were in the range 3–12 mg/L and 2–8 mg/L, respectively. However, when the Etest MICs took into account colonies growing within the apparent inner zone of inhibition a subpopulation of resistant cells was observed in the Etest zone of inhibition with an imipenem or meropenem MIC of >= (Figure 1). The same results were obtained when the assay was repeated by using plastic strips from a fresh cartridge and by testing a susceptible control organism in tandem. When the resistant colonies were re-tested the imipenem and meropenem MICs remained in the above ranges and again a subpopulation of resistant isolates was grown in the zone of inhibition. Resistant subpopulations were also grown within the zone of inhibition around imipenem and meropenem discs in the disc diffusion test (data not shown).



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Figure 1. Imipenem and meropenem Etests on A. baumannii showing resistant subpopulation. In this particular instance, imipenem and meropenem MICs were 12 and 6 mg/L, respectively, but a subpopulation of resistant cells was grown at up to an imipenem or meropenem concentration of

 
The MICs of imipenem and meropenem were determined by the agar dilution method according to the NCCLS guidelines.4 The assay was performed by inoculating 104 cfu/spot onto cation-supplemented Mueller–Hinton agar plates containing antibiotic dilutions in the range 0.12–512 mg/L. The MICs for both the original isolates and the subcolonies from within the Etest zone of inhibition were evaluated. Testing of the eight A. baumannii isolates or their resistant colonies by this method demonstrated imipenem MICs of 8–32 mg/L and meropenem MICs of 4–16 mg/L. The MIC for the original isolates was also re-assessed after serial passages in broth containing a subinhibitory concentration (1 mg/L) of imipenem. The imipenem MIC for the clinical isolates did not increase more than one two-fold dilution after eight serial passages in imipenem-containing broth.

The eight original isolates belonged to different PFGE types or subtypes. Subcolonies present in the zone of inhibition belonged to the same puslotype as their respective original isolates. Original isolates and resistant colonies did not carry blaIMP, blaVIM, blaOXA-23, blaOXA-24 and blaOXA-58 as judged by PCR amplification. The exact mechanism of the organisms' reduced susceptibility to carbapenems remains to be determined but an intrinsic mechanism attributed to the presence of a few small-sized porins cannot be excluded.5 Further work on the population analysis and mechanisms of resistance in these strains is proceeding.

In our region, A. baumannii isolates have demonstrated a gradual increase in carbapenem MICs over the last few years.3 The emergence of A. baumannii with heteroresistance to carbapenems, while a significant threat, also poses interesting challenges in regard of definition and detection. It seems that heteroresistant A. baumannii is a phenotypic manifestation within a genetically homogeneous strain that is expressed as population heterogeneity in susceptibility to carbapenems.6 Since heteroresistance to carbapenems in A. baumannii has not been reported previously, it is not known what percentage of A. baumannii isolates are heteroresistant and what clinical impact this heteroresistance may have. Should the use of carbapenems lead either to the selection of resistant subpopulations that subsequently cause infections by imipenem-resistant A. baumannii isolates or to treatment failure, cautious evaluation of Etests on clinically significant isolates may be appropriate.

References

1 . Heritier, C., Dubouix, A., Poirel, L. et al. (2005). A nosocomial outbreak of Acinetobacter baumannii isolates expressing the carbapenem-hydrolysing oxacillinase OXA-58. Journal of Antimicrobial Chemotherapy 55, 115–8.[Abstract/Free Full Text]

2 . Kayser, F. H., Morenzoni, G., Strassle, A. et al. (1989). Activity of meropenem against Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 101–12.

3 . Tsakris, A., Tsioni, C., Pournaras, S. et al. (2003). Spread of low-level carbapenem-resistant Acinetobacter baumannii in a tertiary care Greek hospital. Journal of Antimicrobial Chemotherapy 52, 1046–7.[Free Full Text]

4 . National Committee for Clinical Laboratory Standards (2003). Performance Standards for Antimicrobial Susceptibility Testing: Thirteenth International Supplement M100-S13, Table 2D. NCCLS, Wayne, PA, USA.

5 . Sato, K. & Nakae, T. (1991). Outer membrane permeability of Acinetobacter calcoaceticus and its implication in antibiotic resistance. Journal of Antimicrobial Chemotherapy 28, 35–45.[Abstract]

6 . Rinder, H. (2001). Hetero-resistance: an under-recognised confounder in diagnosis and therapy? Journal of Medical Microbiology 50, 1018–20.[Free Full Text]





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