ACI-1 from Acidaminococcus fermentans: Characterization of the First β-Lactamase in Anaerobic Cocci

Acidaminococcus fermentans belongs to the group of strictly anaerobic gram-negative cocci. All previously described Acidaminococcus strains are susceptible to b-lactam antibiotics. An A. fermentans strain (RYC-MR95) resistant to penicillin and expanded-spectrum cephalosporin (amoxicillin and cefotaxime MICs, 64 mg/ml) was isolated from a human perianal abscess. A fragment encoding a b-lactamase from genomic DNA was cloned in Escherichia coli K-12 strain HB101, and the recombinant strain expressed resistance to amoxicillin (MIC, 1,024 mg/ml) and cefotaxime (MIC, 4 mg/ml). Clavulanic acid decreased the MICs to 8 and 0.03 mg/ml, respectively. Analysis of the nucleotide sequence revealed a new class A b-lactamase, ACI-1. In accordance with its biochemical properties, we propose to assign ACI-1 to functional group 2be. The ACI-1 enzyme (estimated pI 4.3) had <50% amino acid identity with any other class A b-lactamases, the closest being ROB-1 from Haemophilus influenzae (44%). ACI-1 was closer to class A b-lactamases from some gram-positive organisms (41 to 44% amino acid identity with Bacillus b-lactamases) than to most class A enzymes from gram-negative organisms (TEM-1, 24.6%). The aci1 gene had a G1C content of 42.1%, in contrast with 56% G1C content for genomic DNA from A. fermentans, thus suggesting that aci1 may have been obtained by horizontal gene transfer. b-Lactamase-mediated resistance to b-lactams in anaerobic bacteria has been known since the early 1950s (14). During the last two decades, an increasing number of b-lactamases from anaerobes have been described, in particular among gramnegative rods (11, 20, 28, 33). b-Lactamases have been characterized for the genera Bacteroides (30, 34, 35, 38) and Fusobacterium (40). In Prevotella and Porphyromonas, as well as in Bilophila (12, 21, 29), the presence of b-lactamases is known only by positive nitrocefin reactions. Among gram-positive anaerobic bacteria, b-lactamases have been found in Clostridium (3, 17, 19). b-Lactamases from Bacteroides are cephalosporinases and/or penicillinases; all clostridial and fusobacterial b-lactamases are penicillinases. No b-lactamase has been described previously for anaerobic gram-negative cocci (including Veillonella, Acidaminococcus, and Megasphaera). Indeed, all strains described so far are susceptible to b-lactam antibiotics. In this work, cloning and sequencing of the aci1 gene and molecular characterization of the new b-lactamase ACI-1 from a b-lactam-resistant Acidaminococcus fermentans clinical isolate are reported. To our knowledge, this is the first b-lactamase found in anaerobic gram-negative cocci.