Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates possessing the plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in intensive care units of a Chinese hospital

Abstract

Twenty-one Serratia marcescens, ten Klebsiella pneumoniae, and one Escherichia coli isolate with carbapenem resistance or reduced carbapenem susceptibility were recovered from intensive care units (ICUs) in our hospital. Enterobacterial repetitive intergenic consensus-PCR and pulsed-field gel electrophoresis demonstrated that all the S. marcescens isolates belonged to a clonal strain and the 10 K. pneumoniae isolates were indistinguishable or closely related to each other. The MICs of imipenem, meropenem, and ertapenem for all isolates were 2 to 8 microg/ml, except for K. pneumoniae K10 (MICs of 128, 256, and >256 microg/ml). Isoelectric focusing, PCRs, and DNA sequencing indicated that all S. marcescens isolates produced KPC-2 and a beta-lactamase with a pI of 6.5. All K. pneumoniae isolates produced TEM-1, KPC-2, CTX-M-14, and a beta-lactamase with a pI of 7.3. The E. coli E1 isolate produced KPC-2, CTX-M-15, and a beta-lactamase with a pI of 7.3. Conjugation studies with E. coli (EC600) resulted in the transfer of reduced carbapenem susceptibility compared to that of the original isolates, and only the bla(KPC-2) gene was detected in E. coli transconjugants. Plasmid restriction analysis showed identical restriction patterns among all E. coli transconjugants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ompK35/36 gene sequence analysis of outer membrane proteins revealed that K. pneumoniae K10 failed to express OmpK36, because of insertional inactivation by an insertion sequence ISEcp1. All these results indicate that KPC-2-producing S. marcescens, K. pneumoniae, and E. coli isolates emerged in ICUs in our hospital. KPC-2 combined with porin deficiency results in high-level carbapenem resistance in K. pneumoniae. The same bla(KPC-2)-encoding plasmid was spread among the three different genera.

FIG. 1.

PFGE patterns of chromosomal DNA restriction fragments from K. pneumoniae isolates. Lanes 1 to 10, K. pneumoniae K1 to K10; lanes 11 and 12, K. pneumoniae control strains that did not produce KPC-2.

FIG. 2.

Restriction patterns of plasmid DNA from E. coli transconjugants of E. coli, partial K. pneumoniae and S. marcescens isolates, and partial original S. marcescens isolates. Lane 1, E. coli transconjugant of E. coli E1; lanes 2 to 4, E. coli transconjugants of K. pneumoniae K1, K8, and K10; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; lanes 8 and 9, original S. marcescens S1 and S10; lane 10, E. coli EC600 as a negative control; M, 1-kb DNA ladder (MBI Fermentas). Plasmid DNA was digested with EcoRI, HindIII, and BcuI endonucleases.

FIG. 3.

IEF patterns of crude β-lactamase extracts from partial S. marcescens, K. pneumoniae, and E. coli isolates and their E. coli transconjugants. (A) Lane 1, E. coli E1; lanes 2 to 4, K. pneumoniae K1, K8, and K10; lane 5, E. coli transconjugant of E. coli E1; lanes 6 and 7, E. coli transconjugants of K. pneumoniae K1 and K10; M, strains producing TEM-1 (pI of 5.4), TEM-28 (pI of 6.1), SHV-7 (pI of 7.6), and ACT-1 (pI of 9.0). (B) Lanes 1 to 4, S. marcescens S1, S5, S10, and S20; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; M, strains producing TEM-1 (pI of 5.4), TEM-28 (pI of 6.1), SHV-7 (pI of 7.6), and ACT-1 (pI of 9.0).

FIG. 4.

SDS-PAGE analysis of OMPs extracted from K. pneumoniae ATCC 13883 and K. pneumoniae K1 and K10. Lane 1, K. pneumoniae ATCC 13883; lane 2, K. pneumoniae K1; lane 3, K. pneumoniae K10; M, protein molecular mass standard (MBI Fermentas).