An Outbreak of Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae in an Intensive Care Unit of a Major Teaching Hospital in Wenzhou, China

Abstract

Carbapenem-resistant, hypervirulent Klebsiella pneumoniae (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 K. pneumoniae isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), XbaI-macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a Galleria mellonella larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% (n = 8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase bla _KPC-2. Most isolates had hypervirulent genotypes being positive for 19 virulence-associated genes, including iutA (25/29, 86.2%), rmpA (27/29, 93.1%), ybtA (27/29, 93.1%), entB (29/29, 100%), fimH (29/29, 100%), and mrkD (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the Galleria mellonella model with the highest LD₅₀ values for three isolates (<10⁵ CFU/mL) demonstrating the degree of hypervirulence of these CR-hvKP isolates, and is discussed relative to previous outbreaks of CR-hvKP.

Keywords: Klebsiella pneumoniae; ST11; carbapenem-resistance; epidemiology; hypervirulent.

Figure 1

Epidemiology of the K. pneumoniae outbreak cases. Each horizontal line represents the timeline of one patient, with respect to treatments and isolation of tissue or fluid samples, with the abscissa axis showing the relevant dates. Isolates and the treatments of those patients are shown by colored dots. Different specimens are shown by colored lines. TGC, tigecycline; AMK, amikacin; IMP, imipenem; MEM, meropenem; LZD, linezolid; CPZ, cefoperazone; FOS, fosfomycin; TZP, piperacillin-tazobactam; LVFX, levofloxacin; PLB, polymyxin B; PICC, peripherally inserted central catheter.

Figure 2

PFGE results and MLST typing for 29 K. pneumoniae isolates. Genomic DNA from each of the 29 K. pneumoniae isolates was digested using XbaI and the digests subjected to pulsed-field gel electrophoresis to generate diagnostic genomic DNA fragmentation fingerprints. The dendrogram of the PFGE profiles is clustered by the UPGAMA on the basis of the Dice similarity by the Quantity One software package 4.6. The sequences of seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were analyzed for the purpose of MLST typing. PFGE results showed that 29 isolates were divided into five different PFGE clusters (A–E) as represented by different colors. Cluster D and E accounted for 20.6% (6/27) and 65.5% (19/27) of ST11 isolates, respectively, indicating that those strains were highly homologous. The two ST37 isolates belonged to the same cluster (cluster A). Kp6 (ST375) and Kp7 (ST11) formed separate monotypic clusters, cluster B and C, respectively. Asterisk indicates the string test of isolate was positive.

Figure 3

Gene profiles of K. pneumoniae isolates. Virulence genes and drug-resistance genes identified by PCR-based profiling are shown by colored dots, plotted against the phylogeny of the strains from the whole genome PFGE analysis shown in Figure 2. Asterisk indicates the capsule serotype was determined to be of K2 type. The relevant abbreviations are: bla, beta-lactamases (specific isoforms bla_KPC−2 , bla_SHV−11, bla_CTX−M−2 group, bla_CTX−M−9 group, bla_CMY−2, bla_TEM−1, bla_DHA); mutations in gyrA (S83I, D87G) and parC (S80I), and the presence of qnrB, qnrS, and oqxA all reduce susceptibility to fluoroquinolones (42); rmtB, rmtC (encoding 16S rRNA methylases), aac(3')-Ia, aac(6')-Ib, and ANT(3')-Ia (encoding aminoglycoside-modifying enzymes) are all aminoglycoside resistance genes.

Figure 4

K. pneumoniae infection of G. mellonella larvae induces time-dependent lethality. The effect of 1 × 10⁶ colony-forming units (CFU) of each K pneumoniae strain on survival was observed in G. mellonella. The strains were divided into three groups according the hypermucoviscous and resistance phenotypes. High-level drug resistant and hypermucoviscous (Kp1, Kp15). High-level drug resistant and non-hypermucoviscous (Kp4, Kp21). Drug sensitive but hypermucoviscous (Kp6). Larvae were injected with PBS or with 10⁶ CFU of Kp1, Kp4, Kp6, Kp15, and Kp21. The survival was monitored over 72 h post-infection. Mortality of larvae infected with Kp1, Kp4, Kp6, Kp15, and Kp21 were time-dependent.