Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae strain 11978 was isolated in Turkey in 2001 and was found to be resistant to all beta-lactams, including carbapenems. Cloning and expression in Escherichia coli identified five beta-lactamases, including two novel oxacillinases. The beta-lactamase OXA-48 hydrolyzed imipenem at a high level and was remotely related (less than 46% amino acid identity) to the other oxacillinases. It hydrolyzed penicillins and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-48) gene was plasmid encoded and not associated with an integron, in contrast to most of the oxacillinase genes. An insertion sequence, IS1999, was found immediately upstream of bla(OXA-48). Another plasmid that encoded a second oxacillinase gene, bla(OXA-47), located inside a class 1 integron was identified in K. pneumoniae 11978. OXA-47 had a narrow spectrum of hydrolysis activity and did not hydrolyze ceftazidime or imipenem, as is found for the beta-lactamase (OXA-1) to which it is related. In addition, beta-lactamases TEM-1 and SHV-2a were expressed from the same K. pneumoniae isolate. Analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of ca. 36 kDa. Thus, the high-level resistance to beta-lactams of this clinical isolate resulted from peculiar beta-lactamases and modification of outer membrane proteins.

FIG. 1.

Nucleotide sequence of a 2,622-bp fragment of recombinant plasmid pA-1 containing the bla_OXA-48 gene. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The start codons of the ORFs are indicated by horizontal arrows. The left inverted repeat (IRL) of IS1999 is shaded in gray, and the putative −35 and −10 promoter sequences (P_out) provided by the insertion sequence element are underlined. The tnpA transposase gene is indicated. The asterisk indicates the stop codon, and the vertical arrow indicates the Sau3AI cloning insertion site in pVT-1.

FIG. 2.

Comparison of the amino acid sequence of OXA-48 to those of OXA-23, OXA-40, OXA-1, and OXA-10. The shading indicates conserved residues of the oxacillinases, and asterisks indicate highly conserved residues. The numbering of the β-lactamases is according to DBL (8).

FIG. 3.

OMP profiles of K. pneumoniae 11978 (lane A) and reference strain K. pneumoniae CIP53153 (lane B). Lane M, molecular size marker, with sizes (in kilodaltons) indicated on the left. The arrow indicates the position of the 36-kDa protein that was missing from K. pneumoniae 11978, whereas the 35- and 37-kDa proteins likely appeared in the form of a doublet.