Resistance and virulence features of hypermucoviscous Klebsiella pneumoniae from bloodstream infections: Results of a nationwide Italian surveillance study

Abstract

Among Enterobacterales, Klebsiella pneumoniae (Kp) is one of the major opportunistic pathogens causing hospital-acquired infections. The most problematic phenomenon linked to Kp is related to the dissemination of multi-drug resistant (MDR) clones producing carbapenem-hydrolyzing enzymes, representing a clinical and public health threat at a global scale. Over the past decades, high-risk MDR clones (e.g., ST512, ST307, ST101 producing bla _KPC-type carbepenemases) have become endemic in several countries, including Italy. Concurrently, the spread of highly virulent Kp lineages (e.g., ST23, ST86) able to cause severe, community-acquired, pyogenic infections with metastatic dissemination in immunocompetent subjects has started to be documented. These clones, designated as hypervirulent Kp (hvKp), produce an extensive array of virulence factors and are highly virulent in previously validated animal models. While the prevalence and distribution of MDR Kp has been previously assessed at local and national level knowledge about dissemination of hvKp remains scarce. In this work, we studied the phenotypic and genotypic features of hypermucoviscous (HMV, as possible marker of increased virulence) Kp isolates from bloodstream infections (BSI), obtained in 2016-17 from 43 Italian Laboratories. Antimicrobial susceptibility testing, whole genome sequencing and the use of two animal models (G. mellonella and murine) were employed to characterize collected isolates. Over 1502 BSI recorded in the study period, a total of 19 Kp were selected for further investigation based on their HMV phenotype. Results showed that hvKp isolates (ST5, ST8, ST11, ST25) are circulating in Italy, although with a low prevalence and in absence of a clonal expansion; convergence of virulence (yersiniabactin and/or salmochelin, aerobactin, regulators of mucoid phenotype) and antimicrobial-resistance (extended-spectrum beta-lactamases) features was observed in some cases. Conventional MDR Kp clones (ST307, ST512) may exhibit an HMV phenotype, but with a low virulence potential in the animal models. To the best of our knowledge, this work represents the first systematic survey on HMV and hvKp in Italy, employing a functional characterization of collected isolates. Future surveillance programs are warranted to monitor the threatening convergence of virulence and resistance among MDR Kp and the spread of hvKp.

Keywords: carbapenemases; clones; genome sequencing; multi-drug resistant; virulence factor.

FIGURE 1

Geographic distribution of participating laboratories. Blue dots represent centers with at least one HMV isolate. Empty dots represent centers with absence of HMV or non-confirmed HMV isolates only (number of confirmed HMV isolates per center in brackets).

FIGURE 2

Results of Kleborate pipeline visualized with the Kleborate-viz tool. Detected resistance determinants are in red while virulence factors are in blue. Legend: AGly_acquired (aminoglycosides), Bla_acquired (beta-lactamases), Bla_inhR (beta-lactamases with resistance to beta-lactamase inhibitors), Bla_Carb (carbapenemase), Bla_ESBL (extended spectrum beta-lactamases), Bla_ESBL_inhR (extended spectrum beta-lactamases with resistance to beta-lactamase inhibitors), Fcyn (fosfomycin), Flq (fluoroquinolones), Gly (glycopeptides), MLS (macrolides), Phe (phenicols), Rif (rifampin), Sul (sulfonamides), Tet (tetracyclines), Tmt (trimethoprim), Tgc (tigecycline).

FIGURE 3

Summary of most relevant features of studied strains (ST, virulence score, resistance score, K type and O type) and results obtained in the animal infection models. (A) Results of experiments in the G. mellonella model expressed as mortality rate (percentage of dead over the total of inoculated larvae). Means and SD of three replicate experiments are show at different time points for each strain (after 24, 48, and 72 h). (B) Results of virulence experiments, for representative HMV isolates, in the murine model expressed as Log10CFU/g organ of the studied strains, recovered from kidneys and livers. Each dot represents data obtained from one mouse specimen and the solid line represents the mean value. In both the models, the NTUH-K2044 virulent strain and the KKBO-1 low-virulence strain were included and used as reference for classification of studied isolates in three different virulence categories: low (green zone), intermediate (yellow) and high (red). Mortality rate at 72 h (A) and Log10 counts (B) for the three groups were compared. P-values < 0.05 were considered significant; *< 0.05, **<0.005, ****<0.0001.