VEB-1 in Achromobacter xylosoxidans from cystic fibrosis patient, France
Multidrug-resistant Achromobacter xylosoxidans was recovered from the sputum of a patient with cystic fibrosis. The VEB-1 extended-spectrum [beta]-lactamase was detected on a class 1 integron. This first report of a VEB-1–producing isolate in this population requires further investigation to determine its distribution.
Achromobacter (formerly Alcaligenes) xylosoxidans is a newly emerging microorganism isolated with increased frequency from the lungs of patients with cystic fibrosis (CF), but information about its clinical relevance is limited (1). A. xylosoxidans is innately resistant to many antimicrobial drugs (2), except piperacillin, piperacillin-tazobactam, and imipenem, and moderately susceptible to ceftazidime (45% of susceptible isolates), which is widely used to treat infection due to Pseudomonas aeruginosa (3,4). The mechanisms involved in cases of high-level resistance to ceftazidime have not been described for A. xylosoxidans. Possible mechanisms for ceftazidime resistance among gram-negative bacilli are alterations in outer membrane proteins, overproduction of cephalosporinase, or production of an extended-spectrum [beta]-lactamase (ESBL). ESBLs are enzymes distributed worldwide (5) that hydrolyze oxyimino-cephalosporins and monobactams and are susceptible to [beta]-lactamase inhibitors such as clavulanic acid and tazobactam. We report on the isolation from a CF patient of A. xylosoxidans that produced the VEB-1 ESBL. This is the first report of ESBL production in A. xylosoxidans and the first report of a VEB-1–producing isolate from a CF patient.
During the past 10 years in our 1,600-bed university hospital, 37 CF patients had [greater than or equal to] 1 respiratory tract specimen that contained A. xylosoxidans. Preliminary pulsed-field gel electrophoresis of these strains has failed to identify shared isolates among the patients, but studies are ongoing. In November 2003, A. xylosoxidans 476 (AX476) was isolated from the sputum of a 17-year-old male CF patient. This patient had good pulmonary function (forced expiratory volume 1 = 99% of predicted value), had never been colonized or infected by P. aeruginosa, and therefore never received ceftazidime. The strain was identified with the Api 20NE system (bio-Merieux, Marcy-l’Etoile, France), and antimicrobial susceptibility testing was performed and interpreted as recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) (6).
The antibiogram, which was performed by a disk diffusion method, showed AX476 to be highly resistant to ceftazidime, aminoglycosides, sulfonamides, trimethoprim, and ciprofloxacin but fully susceptible to tetracyclines, piperacillin/tazobactam, ticarcillin/clavulanic acid, and imipenem. Because of an unusual synergy between ticarcillin and ticarcillin/clavulanic acid (Figure), we compared the inhibition zones of third-generation cephalosporin disks with and without clavulanic acid (BioRad, Marnesla-Coquette, France). The zones were 7 mm for ceftazidime and 19 mm for ceftazidime plus clavulanic acid (Figure), which strongly indicated production of an ESBL. Isoelectric focusing showed that AX476 produced a [beta]-lactamase with an isoelectric point of 7.4. A large plasmid of [approximately equal to] 200 kb (pJDB 1) was easily transferred by conjugation to Escherichia coli K-12 C600. The transconjugants, E. coli (pJDB1) sorbitol-fermenting, which were selected on MacConkey agar that contained 4 [micro]g/mL of ceftazidime, were resistant to sulfonamides and trimethoprim, had reduced susceptibility to aminoglycosides, and harbored a [beta]-lactamase with an isoelectric point of 7.4. The resistance phenotype of the isolate and the value of the isoelectric point of the enzyme suggested the production of the ESBL VEB-1 (7).
The MICs for [beta]-lactams for AX476 and its transconjugant, determined by Mueller-Hinton broth dilution method, are shown in Table 1. By using [blav.sub.VEB-1]-specific primers, a positive PCR result was obtained on total DNA from AX476 and the transconjugants. All genetic analyses of [bla.sub.VEB-1] published so far have identified either its chromosome (8) or its plasmid (9) location and mostly its integration within class 1 integrons of variable structure. Integrons are potentially mobile genetic elements that comprise conserved sequences that flank a variable region and may contain inserted antimicrobial drug resistance gene cassettes (10). The 5′-conserved segment includes the gene intI1 that encodes an integrase, the cassette integration site attI1, and a promoter responsible for the expression of the genes located downstream within the variable region. The 3′-conserved region contains either a qacE[DELTA]1 gene that encodes resistance to quaternary ammonium compounds or a combination of 3 genes: qacE[DELTA]1, sulI (which encodes resistance to sulfonamides), and orf5 (an open reading frame of unknown function).
To search for the presence of such a class 1 integron in AX476 and its transconjugant, we performed PCR on total DNA of AX476 and E. coli (pJDB 1) by using the primers L1 and R1 specific for the detection of class 1 integrons (11). We obtained a fragment of 2.3 kb in the clinical strain and its transconjugant, which was sequenced on both strands. By using a set of primers, we deduced the structure of this integron (Table 2). Three gene cassettes have been identified. The first, dhfr (dihydrofolate reductase), encoded a putative trimethoprim-resistance protein. This dhfr was identical to that reported in Salmonella enterica serovar Typhi (GenBank accession no. AL513383) and to the dhfr gene cassette contained in a class 1 integron from Klebsiella pneumoniae not yet published (GenBank accession no. AJ971342). The second cassette, [blav.sub.VEB-1], encoded the ESBL VEB-1 first described in E. coli (7). The third and last gene cassette was aadB. It encoded an aminoglycoside adenyltransferase that conferred resistance to kanamycin, gentamicin, and tobramycin and was identical to other sequenced aadB gene cassettes located on integrons containing [bla.sub.VEB-1] gene (7,8). VEB-1 has been detected in Enterobacteriaceae and P. aeruginosa isolates from Southeast Asia (9) but never in A. xylosoxidans. In France, VEB-1-producing isolates of Acinetobacter baumannii have been involved in several outbreaks of nosocomial infection in intensive care units (12,13); however, we have not yet detected a VEB-1–producing isolate in our hospital.