Nosocomial spread of ceftazidime-resistant Klebsiella pneumoniae strains producing a novel class a beta-lactamase, GES-3, in a neonatal intensive care unit in Japan

Abstract

Klebsiella pneumoniae strain KG525, which showed high-level resistance to broad-spectrum cephalosporins, was isolated from the neonatal intensive care unit (NICU) of a Japanese hospital in March 2002. The ceftazidime resistance of strain KG525 was transferable to Escherichia coli CSH-2 by conjugation. Cloning and sequence analysis revealed that production of a novel extended-spectrum class A beta-lactamase (pI 7.0), designated GES-3, which had two amino acid substitutions of M62T and E104K on the basis of the sequence of GES-1, was responsible for resistance in strain KG525 and its transconjugant. The bla(GES-3) gene was located as the first gene cassette in a class 1 integron that also contained an aacA1-orfG fused gene cassette and one unique cassette that has not been described in other class 1 integrons and ended with a truncated 3' conserved segment by insertion of IS26. Another five ceftazidime-resistant K. pneumoniae strains, strains KG914, KG1116, KG545, KG502, and KG827, which were isolated from different neonates during a 1-year period in the same NICU where strain KG525 had been isolated, were also positive for GES-type beta-lactamase genes by PCR. Pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR analyses displayed genetic relatedness among the six K. pneumoniae strains. Southern hybridization analysis with a GES-type beta-lactamase gene-specific probe showed that the locations of bla(GES) were multiple and diverse among the six strains. These findings suggest that within the NICU setting genetically related K. pneumoniae strains carrying the bla(GES) gene were ambushed with genetic rearrangements that caused the multiplication and translocation of the bla(GES) gene.

FIG. 1.

Comparison of the amino acid sequence of GES-3 with those of the GES-1, GES-2, and IBC-1 β-lactamases. Only the substituted amino acid residues are indicated by the single-letter amino acid code. Dashes represent positions where no amino acid substitution was observed among the four enzymes. The amino acid residues conserved among class A β-lactamases are indicated with asterisks.

FIG. 2.

Schematic comparison of the class 1 integron on pKGB525 with those on pTK1 (GenBank accession number AF156486), pLAP-1 (GenBank accession number AF326355), and pAK33 (34). Filled circles indicate the positions of GTTRRRY (core site) or the 59-base elements around the gene cassettes. pKGM525, which carries the 11.6-kb BamHI insert, was also sequenced; and the nucleotide sequence from intI1 to IS26 was the same as that found in pKGB525.

FIG. 3.

PFGE analysis of K. pneumoniae isolates. Lanes: 1 and 8, PFGE marker; 2, K. pneumoniae KG914; 3, K. pneumoniae KG1116; 4, K. pneumoniae KG525; 5, K. pneumoniae KG545; 6, K. pneumoniae KG502; 7, K. pneumoniae KG827.

FIG. 4.

Plasmid profiles and Southern hybridization analysis. (A) Plasmid profiles of each strain prepared by the method of Kado and Liu (16); (B) hybridization to large plasmids harbored by each strain; (C) hybridization to the chromosomal position of each strain. The photograph of the results of gel electrophoresis of chromosomal DNAs prepared by the method of Stauffer et al. (35) was omitted. The large plasmids and chromosomal DNA were separately extracted by using freshly prepared reagents to avoid cross contamination of nicked or physicochemically amputated DNA fragments. For strains KG545 and KG827, the bla_GES gene was suggested to be encoded by the chromosome. In strain KG525, the bla_GES gene was suggested to be encoded by both the plasmid and the chromosome. Lanes: M, HindIII-digested DNA marker; 1, K. pneumoniae KG914; 2, K. pneumoniae KG1116; 3, K. pneumoniae KG525; 4, K. pneumoniae KG545; 5, K. pneumoniae KG502; 6, K. pneumoniae KG827.

FIG. 5.

IEF. (A) IEF and staining with Coomassie blue. Lanes: M, pI marker; GES-3, purified GES-3 enzyme. (B) IEF and staining with nitrocefin. Lanes: 1, GES-3-producing E. coli transformant that harbors pKGB525 carrying bla_GES-3; 2, K. pneumoniae KG914; 3, K. pneumoniae KG1116; 4, K. pneumoniae KG525; 5, K. pneumoniae KG545; 6, K. pneumoniae KG502; 7, K. pneumoniae KG827. The bands of pI 7.63 (pI 7.6 in the text) are the chromosomally encoded LEN-1 or SHV-1 β-lactamase of K. pneumoniae, and the bands at pI 6.98 (pI 7.0 in the text) are GES-3. Several β-lactamases with activities at pIs lower than 7.0 were speculated to be partially unfolded GES-3 β-lactamase, because these bands were also found in the IEF gels of an E. coli clone harboring only the bla_GES-3 gene (data not shown).