Escherichia coli Producing an ACC-1 Class C β-Lactamase Isolated in Barcelona, Spain

Abstract

Class C β-lactamases, primarily chromosomally encoded (AmpC), were first described to occur in a plasmid in 1988. Since then, these plasmid-mediated β-lactamases have been identified throughout the world. In the Mediterranean area, most belong to a homogeneous group, CMY-2 to CMY-5 and LAT-1 to LAT-4, related to the chromosomally encoded AmpC-type β-lactamase of Citrobacter freundii (9). In Spain, the first plasmidic AmpC enzyme described was FOX-4, found in an Escherichia coli strain isolated in the Canary Islands in 2000 (2). A CMY-2 enzyme was later detected in Proteus mirabilis, Klebsiella oxytoca, and Salmonella enterica serovars Mikawasima and Montevideo in Barcelona (Catalonia) and Guipuzkoa (Basque Country) (7). To date, no other ampC genes have been described in Spain. We report the first plasmid-encoded ACC-1 β-lactamase, related to the chromosomally encoded AmpC-type β-lactamase of Hafnia alvei (5), isolated in Barcelona from an E. coli strain. The E. coli strain (U49G11) was isolated from the urine of a 55-year-old female with Hodgkin's disease treated with chemotherapy and allogeneic peripheral stem cell transplantation. During her illness, she presented with various infections, including oral candidiasis, human herpesvirus 1 infection, disseminated human herpesvirus 5 (cytomegalovirus) infection, urinary sepsis due to E. coli, and, 1 month later, a Klebsiella pneumoniae bacteremia of unknown origin. Several antimicrobial agents were administered, including meropenem, cefepime, and cefuroxime as β-lactams. Nevertheless, during the month prior to the isolation of the U49G11 strain, she did not receive antimicrobial therapy. U49G11 showed a particular β-lactam-resistant phenotype, which suggested the presence of an AmpC-type enzyme. Using the disk diffusion test according to NCCLS guidelines (6), this strain was resistant to ampicillin, cefazolin, cefuroxime, cefotaxime, ceftazidime, amoxicillin-clavulanate, kanamycin, tobramycin, tetracycline, and trimethoprim-sulfamethoxazole and was susceptible to cefoxitin, cefepime, aztreonam, imipenem, gentamicin, quinolones, chloramphenicol, and nitrofurantoine. The MICs of cefoxitin, cefotaxime, ceftazidime, and cefepime, determined by Etest (AB Biodisk, Solna, Sweden), were 8, >16, >32, and 0.75 mg/liter, respectively. When clavulanate was associated with these cephalosporins, no synergistic effect was observed. The resistance pattern was successfully transferred by conjugation (frequency of 10−6) on solid media using E. coli HB101 (Nalr) as the recipient strain (7). Transconjugants have the same resistance pattern (although MICs of cephalosporins for them are lower) as that of the donors except where trimethoprim-sulfamethoxazole is concerned. The analytical isoelectric focusing (7) performed on the donor and transconjugant strains showed a band with a pI of 7.7, which, together with the susceptibility to cefoxitin, suggested the presence of an ACC-type enzyme. A positive PCR was obtained by using the ACCMF and ACCMR primers, which amplify ACC-type enzymes (8). In order to sequence the gene, an additional PCR was performed using the ACC-1up (5′-TGC GTA AAA AAA TGC AGA A-3′) and ACC-1dn (5′-CTA CTT ATT CCC TTC CA-3′) primers. The deduced amino acid sequence of the obtained product showed 100% homology with the ACC-1 enzyme (1). ACC-1 was first described in Germany (1) and later in France during a multiresistant-K. pneumoniae outbreak in an intensive care unit following the admission of a patient transferred from Tunisia (5); it has also been shown to be present in E. coli and P. mirabilis (3). Studies at Sfax Hospital (Tunisia) reveled the ACC-1 enzyme in K. pneumoniae, P. mirabilis, and S. enterica serovars Livingstone and Mbandaka (4, 10). This is the first time that the AAC-1 enzyme has been reported in Spain, now the fourth country in the world where strains carrying this enzyme have been isolated. Three of these four countries (Spain, France, and Tunisia) are in the Mediterranean area. Such findings suggest that the ACC-1 enzyme is spreading in Europe, especially in the Mediterranean area.