Extensively Drug-Resistant Klebsiella pneumoniae Causing Nosocomial Bloodstream Infections in China: Molecular Investigation of Antibiotic Resistance Determinants, Informing Therapy, and Clinical Outcomes

Abstract

The rise in diversity of antimicrobial resistance phenotypes seen in Klebsiella pneumoniae is becoming a serious antibiotic management problem. We sought to investigate the molecular characteristics and clinical implications of extensively drug-resistant (XDR) K. pneumoniae isolated from different nosocomial bloodstream infections (BSIs) patients from July 2013 to November 2015. Even in combination treatment, meropenem did not protect against mortality of BSIs patients (P = 0.015). In contrast, tigecycline in combination with other antimicrobial agents significantly protected against mortality (P = 0.016). Antimicrobial susceptibility tests, molecular detection of antibiotic resistance determinants, conjugation experiments, multilocus sequence typing (MLST), S1-PFGE, Southern blot, SDS-PAGE, immunoblot analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize these isolates. These XDR K. pneumoniae strains were resistant to conventional antimicrobials except tigecycline and polymyxin B and co-harbored diverse resistance determinants. rmtB, bla_KPC-2 as well as bla_CTX-M-9 were located on a transferable plasmid of ~54.2 kb and the most predominant replicon type was IncF. 23 of the 35 isolates belonging the predominant clone were found to incorporate the globally-disseminated sequence type ST11, but others including a unique, previously undiscovered lineage ST2281 (allelic profile: 4-1-1-22-7-4-35) were also found and characterized. The porins OmpK35 and OmpK36 were deficient in two carbapenemase-negative carbapenem-resistant strains, suggesting decreased drug uptake as a mechanism for carbapenem resistance. This study highlights the importance of tracking hospital acquired infections, monitoring modes of antibiotic resistance to improve health outcomes of BSIs patients and to highlight the problems of XDR K. pneumoniae dissemination in healthcare settings.

Keywords: Klebsiella pneumoniae; XDR; antimicrobial resistance determinants; bacteraemia; clinical outcomes.

Figure 1

PFGE analysis of XDR K. pneumoniae isolates. In order to generate diagnostic genomic DNA fragmentation fingerprints, genomic DNA from each of the XDR K. pneumoniae isolates was digested using XbaI and subjected to pulsed-field gel electrophoresis. DNA fingerprints were revealed by Gel Red staining. For MLST-based categorization of the strains, the sequences of seven housekeeping genes (i.e., gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were analyzed, and the PFGE patterns have been organized according to a dendogram of 35 XDR K. pneumoniae isolates based on MLST analysis. In red text are those isolates with no detectable carbapenemase genes. The gray box highlights the prevalence of the ST11 sequence type.

Figure 2

Gel image of S1 PFGE result of all XDR K. pneumoniae isolates. Isolates were digested using S1 nuclease and subjected to pulsed-field gel electrophoresis. The gel was subjected to Gel Red staining and analyzed in a CHEF-Mapper XA PFGE system. H = size marker strain Salmonella enterica ser. Braenderup H9812 digested with XbaI.

Figure 3

Plasmid profiles of E. coli transconjugants. (A) In order to verify plasmids carried by the XDR K. pneumoniae isolates, transconjugation experiments were established using E. coli J53 as a recipient strain. Genomic DNA was isolated from the transconjugants, digested using S1 nuclease and subjected to pulsed-field gel electrophoresis. H = size marker strain Salmonella enterica ser. Braenderup H9812 digested with XbaI; Lanes 2–12 represent transconjugants of the corresponding number of donor isolates, all of the isolates code consistent with Table 3. (B) The corresponding Southern blot, hybridized with a DNA probe to the bla_KPC−2 sequence.

Figure 4

Remodeling of the outer membrane proteome in the XDR K. pneumoniae isolates. (A) Total membranes were isolated from K. pneumoniae ATCC 13883 and subjected to sucrose density fractionation. Membrane fractions were subject to SDS-PAGE, and then Coomassie blue staining (top panel) and immunoblotting (bottom panel) using antisera recognizing known outer (BamB) and inner (F1β) membrane proteins. (B) Whole cell lysates from K. pneumoniae ATCC 13883 and K. pneumoniae clinical isolates FK688 and FK1934 were analyzed by SDS-PAGE and western immunoblotting for OmpK35 and OmpK36.