Dataset Information

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.

SUBMITTER: Kobayashi SD

PROVIDER: S-EPMC4837910 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Kobayashi Scott D SD Porter Adeline R AR Dorward David W DW Brinkworth Amanda J AJ Chen Liang L Kreiswirth Barry N BN DeLeo Frank R FR

The Journal of infectious diseases 20160114 10

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 prima ...[more]

PMID: 26768252

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Project description:Carbapenem-resistant Klebsiella pneumoniae ST258 (CRKP-ST258) are a global concern due to their rapid dissemination, high lethality, antibiotic resistance and resistance to components of the immune response, such as neutrophils. Neutrophils are major host mediators, able to kill well-studied and antibiotic-sensitive laboratory reference strains of K. pneumoniae. However, CRKP-ST258 are able to evade neutrophil phagocytic killing, persisting longer in the host despite robust neutrophil recruitment. Here, we show that neutrophils are unable to clear a CRKP-ST258 isolate (KP35). Compared to the response elicited by a prototypic K. pneumoniae ATCC 43816 (KPPR1), the neutrophil intracellular response against KP35 is characterized by equivalent production of reactive oxygen species (ROS) and myeloperoxidase content, but impaired phagosomal acidification. Our results ruled out that this phenomenon is due to a phagocytosis defect, as we observed similar efficiency of phagocytosis by neutrophils infected with KP35 or KPPR1. Genomic analysis of the cps loci of KPPR1 and KP35 suggest that the capsule composition of KP35 explain the high phagocytosis efficiency by neutrophils. Consistent with other reports, we show that KP35 did not induce DNA release by neutrophils and KPPR1 only induced it at 3 h, when most of the bacteria have already been cleared. l-arginine metabolism has been identified as an important modulator of the host immune response and positively regulate T cells, macrophages and neutrophils in response to microbes. Our data show that l-arginine supplementation improved phagosome acidification, increased ROS production and enhanced nitric oxide consumption by neutrophils in response to KP35. The enhanced intracellular response observed after l-arginine supplementation ultimately improved KP35 clearance in vitro. KP35 was able to dysregulate the intracellular microbicidal machinery of neutrophils to survive in the intracellular environment. This process, however, can be reversed after l-arginine supplementation.

Project description:A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-Kp IMPORTANCE Klebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine.

Project description:The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains belonging to sequence type 258 (ST258) are frequent causes of hospital-associated outbreaks and are a major contributor to the spread of carbapenemases. This genetic lineage emerged several decades ago and remains a major global health care challenge. In this study, genomic epidemiology was used to investigate the emergence, evolution, and persistence of ST258 carbapenem-resistant K. pneumoniae outbreak-causing lineages at a large tertiary care hospital over 8 years. A time-based phylogenetic analysis of 136 ST258 isolates demonstrated the succession of multiple genetically distinct ST258 sublineages over the 8-year period. Ongoing genomic surveillance identified the emergence and persistence of several distinct clonal ST258 populations. Patterns of multidrug resistance determinants and plasmid replicons were consistent with continued evolution and persistence of these populations. Five ST258 outbreaks were documented, including three that were caused by the same clonal lineage. Mutations in genes encoding effectors of biofilm production and iron acquisition were identified among persistent clones. Two emergent lineages bearing K. pneumoniae integrative conjugative element 10 (ICEKp10) and harboring yersiniabactin and colibactin virulence factors were identified. The results show how distinct ST258 subpopulations have evolved and persisted within the same hospital over nearly a decade.IMPORTANCE The carbapenem class of antibiotics is invaluable for the treatment of selected multidrug-resistant Gram-negative pathogens. The continued transmission of carbapenem-resistant bacteria such as ST258 K. pneumoniae is of serious global public health concern, as treatment options for these infections are limited. This genomic epidemiologic investigation traced the natural history of ST258 K. pneumoniae in a single health care setting over nearly a decade. We found that distinct ST258 subpopulations have caused both device-associated and ward-associated outbreaks, and some of these populations remain endemic within our hospital to the present day. The finding of virulence determinants among emergent ST258 clones supports the idea of convergent evolution of drug-resistant and virulent CRKP strains and highlights the need for continued surveillance, prevention, and control efforts to address emergent and evolving ST258 populations in the health care setting.

Project description:Carbapenem-resistantKlebsiella pneumoniae(CR-Kp) is significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract, where the bacterium is usually present at low density, but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood. Nevertheless, gaining such knowledge could lead to innovative intervention strategies for addressing colonization and infection. Here, we adopted anin vivomodel to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis, and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, the concentration of oxidative species and amino acids was increased in the mouse intestine following treatment. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were particularly upregulated, however their deletion did not impair CR-Kp fitness in vivoupon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein CRP as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, CRP deletion in CR-Kp resulted in strongly impaired gut colonization, but this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance towards bacterial fitness remains cryptic. Further analyses utilizing this model may identify key factors to tackle the intestinal stage of CR-Kp colonization.

Dataset Information

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

Publications

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

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