Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina

Abstract

In recent years, an increase in the prevalence hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with sequence type 25 (ST25) was detected in hospitals of Tucuman (Northwest Argentina). In this work, the virulence and the innate immune response to two K. pneumoniae ST25 strains (LABACER 01 and LABACER 27) were evaluated in a murine model after a respiratory challenge. In addition, comparative genomics was performed with K. pneumoniae LABACER01 and LABACER27 to analyze genes associated with virulence. Both LABACER01 and LABACER27 were detected in the lungs of infected mice two days after the nasal challenge, with LABACER01 counts significantly higher than those of LABACER27. Only LABACER01 was detected in hemocultures. Lactate dehydrogenase (LDH) and albumin levels in bronchoalveolar lavage (BAL) samples were significantly higher in mice challenged with LABACER01 than in LABACER27-infected animals, indicating greater lung tissue damage. Both strains increased the levels of neutrophils, macrophages, TNF-α, IL-1β, IL-6, KC, MCP-1, IFN-γ, and IL-17 in the respiratory tract and blood, with the effect of LABACER01 more marked than that of LABACER27. In contrast, LABACER27 induced higher levels of IL-10 in the respiratory tract than LABACER01. Genomic analysis revealed that K. pneumoniae LABACER01 and LABACER27 possess virulence factors found in other strains that have been shown to be hypervirulent, including genes required for enterobactin (entABCDEF) and salmochelin (iroDE) biosynthesis. In both strains, the genes of toxin-antitoxin systems, as well as regulators of the expression of virulence factors and adhesion genes were also detected. Studies on the genetic potential of multiresistant K. pneumoniae strains as well as their cellular and molecular interactions with the host are of fundamental importance to assess the association of certain virulence factors with the intensity of the inflammatory response. In this sense, this work explored the virulence profile based on genomic and in vivo studies of hypermucoviscous carbapenem-resistant K. pneumoniae ST25 strains, expanding the knowledge of the biology of the emerging ST25 clone in Argentina.

Keywords: Klebsiella pneumoniae; carbapenem resistant; genomic; hypermucoviscous; respiratory infection; sequence type 25.

Figure 1

Lung colonization and damage induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were challenged nasally with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, the bacterial cell counts in lung homogenates, the lung wet:dry ratio, lactate dehydrogenase (LDH) enzyme activity, and the albumin concentration were determined in broncho-alveolar lavage (BAL) samples. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05. Basal levels of BAL albumin were below the detection limit.

Figure 2

Variations in respiratory and blood leukocytes induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were nasally challenged with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, leukocyte, macrophage, and neutrophil counts were performed in broncho-alveolar lavage (BAL) samples, and leukocyte, monocyte, and neutrophil counts were performed in blood. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05.

Figure 3

Variations in inflammatory cytokines in the respiratory tract and blood induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were nasally challenged with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, the TNF-α, IL-1β, and IL-6 levels were determined in broncho-alveolar lavage (BAL) and blood samples. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05.

Figure 4

Variations in the inflammatory chemokines in the respiratory tract and blood induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were nasally challenged with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, the KC and MCP-1 levels were determined in broncho-alveolar lavage (BAL) and blood samples. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05.

Figure 5

Variations in the inflammatory cytokines in the respiratory tract and blood induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were nasally challenged with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, the IFN-γ and IL-17 levels were determined in broncho-alveolar lavage (BAL) and blood samples. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05.

Figure 6

Variations in the regulatory cytokines in the respiratory tract and blood induced by KPC-2-producing hypermucoviscous ST25 strains of Klebsiella pneumoniae. Immunocompetent adult BALB/c mice (six weeks) were nasally challenged with K. pneumoniae LABACER 01 or LABACER 27. Two days after challenge, the IL-10 and IL-27 levels were determined in broncho-alveolar lavage (BAL) and blood samples. Results represent data from three independent experiments. Asterisks (*) indicate significant differences from the uninfected control group (basal control), p < 0.05. The crosses (†) indicate significant differences between the indicated groups, p < 0.05.

Figure 7

Phylogenetic tree constructed with sequences of genes encoding ribosomal protein subunits (rps) of Klebsiella pneumoniae (KP), Klebsiella quasipneumoniae (KQ), and Klebsiella variicola (KV).

Figure 8

Virulence factor genes shared between the Klebsiella pneumoniae LABACER 01 (KP-LABACER 01), LABACER 27 (KP-LABACER 27), 28872, whw, and SWU01 strains.

Figure 9

Venn diagram comparing shared genes and unique genes found in the sequenced genomes of Klebsiella pneumoniae strains LABACER 01, LABACER 27, NTUH-K2044, ATCC43816, and RJF293.

Figure 10

Scanning electron microscopy analysis of the KPC-2-producing hypermucoviscous ST25 Klebsiella pneumoniae strains LABACER 01 and LABACER 27.

Figure 11

Transmission electron microscopy analysis of the KPC-2-producing hypermucoviscous ST25 Klebsiella pneumoniae strains LABACER 01 and LABACER 27.

Figure 12

Virulence factor genes found in KPC-2-producing hypermucoviscous ST25 Klebsiella pneumoniae strains LABACER 01 and LABACER 27. The genes in the purple boxes are shared between the two LABACER strains as well as with other hypermucoviscous and hypervirulent K. pneumoniae strains (28876, SWU01, whw, ATCC 43816, NTUH-K2044, and RJF293). The genes in the pink boxes were detected only in the indicated LABACER strain.