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Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

ABSTRACT: 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.

SUBMITTER: Jung HJ

PROVIDER: S-EPMC6414703 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a critical-priority antibiotic resistance threat that has emerged over the past several decades, spread across the globe, and accumulated resistance to last-line antibiotic agents. While CRKP infections are associated with high mortality, only a subset of patients acquiring CRKP extraintestinal colonization will develop clinical infection. Here, we sought to ascertain the relative importance of patient characteristics and CRKP genetic background in determining patient risk of infection. Machine learning models classifying colonization versus infection were built using whole-genome sequences and clinical metadata from a comprehensive set of 331 CRKP extraintestinal isolates collected across 21 long-term acute-care hospitals over the course of a year. Model performance was evaluated based on area under the receiver operating characteristic curve (AUROC) on held-out test data. We found that patient and genomic features were predictive of clinical CRKP infection to similar extents (AUROC interquartile ranges [IQRs]: patient = 0.59 to 0.68, genomic = 0.55 to 0.61, combined = 0.62 to 0.68). Patient predictors of infection included the presence of indwelling devices, kidney disease, and length of stay. Genomic predictors of infection included presence of the ICEKp10 mobile genetic element carrying the yersiniabactin iron acquisition system and disruption of an O-antigen biosynthetic gene in a sublineage of the epidemic ST258 clone. Altered O-antigen biosynthesis increased association with the respiratory tract, and subsequent ICEKp10 acquisition was associated with increased virulence. These results highlight the potential of integrated models including both patient and microbial features to provide a more holistic understanding of patient clinical trajectories and ongoing within-lineage pathogen adaptation.IMPORTANCE Multidrug-resistant organisms, such as carbapenem-resistant Klebsiella pneumoniae (CRKP), colonize alarmingly large fractions of patients in regions of endemicity, but only a subset of patients develop life-threatening infections. While patient characteristics influence risk for infection, the relative contribution of microbial genetic background to patient risk remains unclear. We used machine learning to determine whether patient and/or microbial characteristics can discriminate between CRKP extraintestinal colonization and infection across multiple health care facilities and found that both patient and microbial factors were predictive. Examination of informative microbial genetic features revealed variation within the ST258 epidemic lineage that was associated with respiratory tract colonization and increased rates of infection. These findings indicate that circulating genetic variation within a highly prevalent epidemic lineage of CRKP influences patient clinical trajectories. In addition, this work supports the need for future studies examining the microbial genetic determinants of clinical outcomes in human populations, as well as epidemiologic and experimental follow-ups of identified features to discern generalizability and biological mechanisms.

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: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.

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Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

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