In Vivo Selection of Reduced Susceptibility to Carbapenems in Acinetobacter baumannii Related to ISAba1-Mediated Overexpression of the Natural blaOXA-66 Oxacillinase Gene

Two clonally related Acinetobacter baumannii isolates, A1 andA2, were obtained from the same patient. Isolate A2, selectedafter an imipenem-containing treatment, showed reduced susceptibilityto carbapenems. This resistance pattern was related to insertionof the ISAba1 element upstream of the naturally occurring bla_OXA-66carbapenemase gene as demonstrated by sequencing, reverse transcription-PCRanalysis, and inactivation of the bla_OXA-66 gene.

INTRODUCTION

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INTRODUCTION

Acinetobacter baumannii is a gram-negative nonfermenting coccobacilluswith a noticeable increase in resistance to carbapenems dueto various and combined mechanisms, but mostly related to carbapenemases(11, 14). These carbapenemases are mostly carbapenem-hydrolyzingclass D β-lactamases (CHDLs) which have been identifiedworldwide in Acinetobacter spp. The four groups of CHDLs identifiedin A. baumannii are the naturally occurring OXA-51-like β-lactamaseand the acquired OXA-23-like, OXA-24/OXA-40-like, and OXA-58-likeβ-lactamases (14). Whereas the contribution of acquiredCHDLs in carbapenem resistance is known (5), despite their weakability to hydrolyze carbapenems (4), the naturally occurringOXA-51-like enzymes may be involved in resistance or decreasedsusceptibility to carbapenems (4, 8, 15, 16). The presence ofthe ISAba1 element upstream of bla_OXA-51-like genes might providepromoter sequences enhancing expression of these genes, as observedwith the naturally occurring bla_ampC gene (named bla_ADC) ofA. baumannii.

We report here the clinical, microbiological, and genetic featuresassociated with the isolation of isogenic A. baumannii strains,with or without reduced susceptibility to carbapenems, recoveredafter an imipenem-containing treatment.

Patients and strains.

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Patients and strains.

Carbapenem-susceptible A. baumannii isolate A1 was recoveredfrom rectal and wound swabs from a 62-year-old patient hospitalizedfor severe trauma in June 2007 in the intensive care unit ofGrenoble Hospital, France. After 3 weeks of an imipenem-containingtreatment (500 mg three times daily together with ciprofloxacinand linezolide), a carbapenem-nonsusceptible A. baumannii isolate,A2, was recovered from the same patient from an endotrachealaspirate (Table 1). Both isolates were identified with the API32GN system (bioMérieux SA, Marcy-l'Etoile, France),and identification was confirmed by sequencing of 16S rRNA genesas described previously (3). During the following months, anoutbreak of A. baumannii A1 was identified at Grenoble Hospitalinvolving 36 patients (eight infections and 28 colonizations)(data not shown).

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TABLE 1. MICs of β-lactams for the A. baumannii strains

Susceptibility testing.

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Susceptibility testing.

Antibiotic susceptibilities of A. baumannii isolates A1 andA2 were determined and interpreted as described previously (1,13). Production of extended-spectrum β-lactamase and overexpressionof the AmpC cephalosporinase were evaluated as described previously(13). Metallo-β-lactamase production was evaluated by usingEtest strips with imipenem and EDTA (AB Biodisk, Solna, Sweden).MICs of β-lactams were determined by using Etest stripson Mueller-Hinton agar plates at 37°C. A. baumannii isolateA1 was susceptible to imipenem, meropenem, and colistin. A.baumannii isolate A2 had a resistance profile similar to thatof isolate A1 but with reduced susceptibility to imipenem andmeropenem (Table 1). Antimicrobial susceptibility testing performedwith cloxacillin-containing plates suggested overexpressionof the naturally occurring gene bla_ADC in both isolates. Neitherextended-spectrum β-lactamase nor metallo-β-lactamaseproduction was detected in either isolate.

Molecular investigations.

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Molecular investigations.

Pulsed-field gel electrophoresis was performed as describedpreviously (13) using restriction enzyme ApaI (GE Healthcare,Orsay, France) for genotyping A. baumannii isolates and showedindistinguishable patterns between isolates A1 and A2 (datanot shown). Preliminary PCR experiments followed by sequencingwith published primers (9) revealed that both isolates possesseda bla_TEM-1 gene. PCR analysis and sequencing performed as describedpreviously (2, 6) identified ISAba1 9 bp upstream of the bla_ADCgene in isolates A1 and A2, further confirming overexpressionof AmpC.

Genes coding for the acquired CHDLs were searched out by usingPCR analysis as described previously (5, 12), but results remainednegative for both isolates. However, the same bla_OXA-66 genecorresponding to the intrinsic bla_OXA-51-like gene of A. baumanniiwas identified in isolates A1 and A2 by using primers PreOXA-Ab-1and PreOXA-Ab-2 (Table 2). ISAba1 was identified 7 bp upstreamof the bla_OXA-66 gene in isolate A2 but not in isolate A1. Thisinsertion element belonging to the IS4 family (10) was insertedin such a way that its transposase gene was in the oppositeorientation with respect to bla_OXA-66. That discrepancy betweenisolates A1 and A2 might explain the different resistance profilestoward carbapenems. Noteworthy is that a PCR experiment confirmedthat isolate A1 originally possessed ISAba1 in its genome.

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TABLE 2. Primers used in this study

In order to investigate the possible indirect involvement ofthat ISAba1-bla_OXA-66 structure in reduced susceptibility tocarbapenems, quantitative reverse transcription-PCR (RT-PCR)experiments were performed to measure the bla_OXA-66 expression.Bacterial strains were grown aerobically in Luria-Bertani brothuntil mid-log phase; aliquots were removed and added to RNAprotect(Qiagen, Courtaboeuf, France), and total cellular RNA was extractedusing the RNeasy mini kit (Qiagen) according to the manufacturer'sprotocol. One-step RT-PCR was performed using a LightCycler(Roche, Mannheim, Germany) with the QuantiFast SYBR green RT-PCRkit (Qiagen). A post-PCR melting curve analysis was performedas described elsewhere (7). The bla_OXA-66 transcript levelswere normalized against the 16S RNA gene, and quantificationswere repeated in triplicate (primers used are listed in Table2 and referenced elsewhere [5]). Transcriptional profile analysisindicated a 50-fold increased expression of bla_OXA-66 in isolateA2 compared with that of isolate A1 (mean ± the standarddeviation, 50 ± 6.7).

In order to better precisely identify the role of OXA-66 expressionin reduced susceptibility to carbapenems in isolate A2, a knockoutmutant for the bla_OXA-66 gene was constructed as described previously(5). Briefly, plasmid pACYC184 unable to replicate in A. baumanniiand harboring a chloramphenicol resistance gene was used asa suicide vector. The arr-2 gene conferring resistance to rifampinobtained as described previously (5) was inserted into the EcoRV-restrictedplasmid pACYC184, giving rise to pACYC184-RA. An internal fragmentof the bla_OXA-66 gene was generated using primers OXA-Abint-BamHI-Aand OXA-Abint-BamHI-B (Table 2) and inserted into the BamHI-restrictedplasmid pACYC184-RA, giving rise to plasmid pACYC184-RA-OXA-66,and then introduced into the rifampin-susceptible A. baumanniiA2 isolate by electrotransformation. Selection of A. baumanniiA2 (pACYC184-RA-OXA-66) was made on plates containing rifampin(25 µg/ml) and chloramphenicol (40 µg/ml). Inactivationof the bla_OXA-66 gene by insertion of pACYC184-RA-OXA-66 wasverified by PCR amplification of the arr-2 gene and nonamplificationof the entire bla_OXA-66 gene under standard PCR conditions.Inactivation of the bla_OXA-66 gene resulted in increased susceptibilityto carbapenems in A. baumannii A2 ( ${Delta}$ OXA-66) compared to thatin A. baumannii isolates A2 and A1 (Table 1), indicating thatthe bla_OXA-66 gene was involved in reduced susceptibility tocarbapenems even when weakly expressed, such as in isolate A1.

Conclusions.

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Conclusions.

This study corresponds to the first report of an in vivo selectionof reduced susceptibility to carbapenems in A. baumannii. Thisreduced susceptibility to carbapenems was related to selectionof the ISAba1-related overexpression of bla_OXA-66. Turton etal. (16) had suggested the involvement of ISAba1 in the overexpressionof bla_OXA-51-like genes, and other studies showed their significantexpression levels by using RT-PCR experiments (8, 15). However,inactivation of the bla_OXA-51-like gene had not been performedto clearly assess the real impact of this mechanism. We demonstratedhere that inactivation of a bla_OXA-66 gene in A. baumannii resultsin a higher susceptibility to carbapenems. Although the ISAba1-bla_OXA-66association may lead to reduced susceptibility to carbapenems,it may constitute a resistance mechanism that could enhanceselection of other mechanisms of resistance, efflux overexpression,impaired permeability, or acquisition of other CHDLs.

ACKNOWLEDGMENTS

This work was supported by a grant from the Ministèrede l'Education Nationale et de la Recherche from France (grantUPRES-EA3539, Université Paris XI, Paris, France) andmostly by a grant from the European Community (6th PCRD, LSHM-CT-2005-018705)and by the INSERM.

S.F. was funded by a grant-in-aid from the Fonds d'Etude etde Recherche du Corps Médical des Hôpitaux de Paris,Paris, France.

FOOTNOTES

* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 Rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.aphp.fr

Published ahead of print on 23 March 2009.

REFERENCES

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