Extended-spectrum β-lactamase-producing strain of Acinetobacter baumannii isolated from a patient in France

Abstract

Sir, Acinetobacter spp., especially Acinetobacter baumannii, are common opportunistic pathogens in immunocompromised patients and currently cause 10% of nosocomial infections in intensive care unit patients. They tend to be resistant to multiple antibiotics and to produce cephalosporinases. Indeed, hyperproduction of cephalosporinases, together with decreased outer membrane permeability, are the predominant mechanisms of resistance to ceftazidime amongst A. baumannii isolates. Most extended-spectrum -lactamases (ESBLs) are the result of mutations that alter the hydrolytic properties of the restricted spectrum penicillinases, TEM-1 and -2 and SHV-1. These enzymes are principally mediated by plasmids which spread readily amongst Enterobacteriaceae and their presence is detected by the demonstration of synergy between clavulanic acid, which inhibits the lactamase, and a third-generation cephalosporin (most effectively ceftazidime) with the double-disc diffusion test. In the course of routinely assessing all ceftazidime-resistant Acinetobacter spp. isolates by the double-disc diffusion test, we identified a strain of A. baumannii (Ama-1) exhibiting only slight synergy that was most evident when the ceftazidime disc was placed 2 cm from the clavulanic acid disc. The MICs of amoxycillin, ticarcillin, piperacillin, ceftazidime and imipenem for the isolate were determined by the agar dilution method according to recommendations of a working party of the British Society for Antimicrobial Chemotherapy. Ama-1 was resistant to all of the -lactams tested, with the exception of imipenem. In the presence of clavulanic acid at a fixed concentration of 2 mg/L, the MIC of ticarcillin was reduced from 512 mg/L to 256 mg/L and that of ceftazidime from 512 mg/L to 128 mg/L. Similarly, in the presence of sulbactam at a concentration of 4 mg/L, the MIC of ceftazidime fell from 512 mg/L to 256 mg/L. The activity of any -lactamase(s) produced by this isolate is therefore inhibited only minimally by either -lactamase inhibitor. In an attempt to determine the molecular basis of this resistance phenotype, we assayed for a putative ESBL gene by PCR analysis with TEM-specific primers (5 -GTATGGATCCTCAACATTTCCGTGTCG-3 and 5 -ACCAAAGCTTAATCAGTGAGGCA-3 ) and SHVspecific primers (5 -TTATCTCCCTGTTAGCCACC-3 and 5 -GATTTGCTGATTTCGCCG-3 ). Neither gene was demonstrated. However, primers used to detect the gene for the ESBL, PER-1 (5 -ATGAATGTCATTATAAAAGC-3 and 5 -AATTTGGGCTTAGGGCAGAA-3 ), yielded a 925 bp PCR product. The sequence analysis of this product revealed total identity with blaPER-1 which was originally detected in a strain of Pseudomonas aeruginosa. A survey recently carried out in Turkey showed that 46% of Acinetobacter spp. hospital isolates possessed PER1-type -lactamases. In this study, attempts to demonstrate plasmid carriage of blaPER-1 in Ama-1 and to transfer the ESBL resistance phenotype from Ama-1 to Escherichia coli DH10B by mating-out assays and by electroporation (electrotransformation) were unsuccessful. Ama-1 was isolated following culture of a rectal swab obtained from a 90 year old female patient on the intensive care unit. She had previously been admitted to two other hospitals in Paris but, to the best of our knowledge, had not travelled to Turkey, nor had she been in contact with either Turkish patients or travellers to Turkey. PER-1-positive A. baumannii strains were not isolated from the rectal swabs of 16 other patients on the intensive care unit at the same time as the patient from whom Ama-1 was recovered. Moreover, comparison of ApaI-digested genomic DNA extracted from Ama-1 with DNA from four randomly selected PER-1-type -lactamase-positive and one PER1-type -lactamase-negative A. baumannii strains from Turkey by pulsed-field gel electrophoresis (PFGE) revealed different restriction patterns (Figure). Thus, the French isolate was not clonally related to the Turkish strains. This is the first report from outside Turkey of the detection of an ESBL in an Acinetobacter spp. clinical isolate. We believe that it is also the first description of the sequencing-based identification of an ESBL gene in an