Plasmid-mediated AmpC-type β-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum β-lactams, including moxalactam

Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded β-lactamase (MOX-1) which conferred resistance to broad spectrum β-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10^-7. The structural gene of MOX-1 (bla(MOX-1)) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was >512 μg/ml. The apparent molecular mass and pI of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg²⁺ and Cu²⁺ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine β-lactamase inhibitors, inhibited the enzyme activity competitively (K(i)s = 5.60 and 0.35 μM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum β-lactams. A hybridization study confirmed that bla(MOX-1) is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX- 1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type β-lactamase that provides enteric bacteria resistance to broad-spectrum β-lactams, including moxalactam.