Phagocytosis and Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

Abstract

Carbapenem-resistant Klebsiella pneumoniae strains classified as multilocus sequence type 258 (ST258) are among the most widespread multidrug-resistant hospital-acquired pathogens. Treatment of infections caused by these organisms is difficult, and mortality is high. The basis for the success of ST258, outside of antibiotic resistance, remains incompletely determined. Here we tested the hypothesis that ST258K. pneumoniae has enhanced capacity to circumvent killing by human neutrophils, the primary cellular defense against bacterial infections. There was limited binding and uptake of ST258 by human neutrophils, and correspondingly, there was limited killing of bacteria. On the other hand, transmission electron microscopy revealed that any ingested organisms were degraded readily within neutrophil phagosomes, thus indicating that survival in the neutrophil assays is due to limited phagocytosis, rather than to microbicide resistance after uptake. Our findings suggest that enhancing neutrophil phagocytosis is a potential therapeutic approach for treatment of infection caused by carbapenem-resistant ST258K. pneumoniae.

Keywords: Klebsiella pneumoniae; neutrophil; phagocytosis.

Figure 1.

Phagocytosis of multilocus sequence type 258 (ST258) and Staphylococcus aureus. Human neutrophils were combined with Klebsiella pneumoniae strain NJST258_2 (left panels) or S. aureus strain USA300 (right panels), and binding and phagocytosis were evaluated using immunofluorescence microscopy. Top panels are phase-contrast images (arrowheads indicate selected bacteria), middle panels (green staining) show all bacteria, and bottom panels show extracellular bacteria (red staining). Images are representative of the phagocytosis data quantitated in Figure 2. Abbreviation: PMN, polymorphonuclear leukocyte.

Figure 2.

Phagocytosis of multilocus sequence type 258 (ST258) clinical isolates by human neutrophils. A–D, Human neutrophils were combined with Klebsiella pneumoniae clinical isolates or Staphylococcus aureus strain USA300 (positive control), and synchronized phagocytosis assays were performed as described in “Methods” section. Bacteria were either left unopsonized (Unops; A and B) or were opsonized with 5% normal human serum (NHS; C and D). The association index is the number of bacteria surface bound or ingested per neutrophil. Phagocytic index is the number of ingested bacteria per neutrophil. Data are the mean ± standard error of the mean of 3–5 separate experiments. Abbreviation: PMN, polymorphonuclear leukocyte.

Figure 3.

Production of neutrophil reactive oxygen species (ROS). Human neutrophils were combined with unopsonized (A and B) or normal human serum–opsonized (C and D) Klebsiella pneumoniae clinical isolates, opsonized zymosan (Opz; a positive control), or Staphylococcus aureus strain USA300 (Ops USA300) as indicated, and production of ROS was measured as described in “Methods” section. A and C, Luminol-enhanced chemiluminescence. B and D, Oxidation of 2′-7′dichlorofluorecin diacetate to 2′-7′dichlorofluorescein. Results are the mean of 3–6 separate experiments. Abbreviations: ΔFL/min, change in millifluorescence units per minute; PMN, polymorphonuclear leukocyte; RLU, relative light unit.

Figure 4.

Survival of multilocus sequence type 258 (ST258) during phagocytic interaction with human neutrophils. Neutrophils were combined with unopsonized (Unops; A) or normal human serum–opsonized (Ops; B) Klebsiella pneumoniae clinical isolates, phagocytosis was synchronized, and percentage survival was determined. Results are the mean ± standard error of 9 separate experiments. P < .05 vs the bar with the corresponding asterisk color. Statistics were performed using repeated-measures analysis of variance and the Tukey post hoc test (GraphPad Prism, version 6.05).

Figure 5.

Ultrastructural analysis of ingested Klebsiella pneumoniae. Human neutrophils were cultured with unopsonized K. pneumoniae strain NJST258_2, phagocytosis was synchronized, and samples were analyzed by TEM. A and B, ingested K. pneumoniae 60 minutes after start of the assay. Yellow arrows indicate neutrophil granules, including those that have fused with the K. pneumoniae–containing phagosome. C, Ingested K. pneumoniae 120 minutes after the start of phagocytosis. Destruction of bacteria is evident in panels B and C. Inset, Black square represents the area of the micrograph from which panel C was derived. Abbreviations: K.p., K. pneumoniae; PMN, polymorphonuclear leukocyte.

Figure 6.

Klebsiella pneumoniae alters neutrophil viability and turnover. A and C, Unopsonized (Unop; A and C) or normal human serum (NHS)–opsonized (B and D) K. pneumoniae clinical isolates were cultured for 6 hours with human neutrophils at a 1:1 ratio of K. pneumoniae colony-forming units to neutrophils. C and D, Antibody specific for Fas (αFas; 500 ng/mL) was added 20 minutes following phagocytosis to accelerate polymorphonuclear leukocyte (PMN) apoptosis. Surface association of annexin V–fluorescein isothiocyanate (FITC) only (AnxV+; not PI+) and staining of nuclear DNA with propidium iodide (PI+) were measured by flow cytometry. Data are the mean ± standard error of the mean of at least 3 separate experiments. Statistical analyses were performed using 1-way analysis of variance and the Dunnett post hoc test.