Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) has emerged worldwide as a major pathogen, causing diverse infections that are resistant to most front-line antibiotic classes. The majority of KPC-Kp infections result in poor outcomes such as death or persistent disease, despite treatment with antibiotic regimens that include agents like colistin. Treatment failures are associated with emergence of colistin resistance (or resistance to other agents) in a minority of cases. Despite the clinical importance of KPC-Kp, its virulence attributes and pathogenic mechanisms are poorly understood. A major limitation to studying KPC-Kp pathogenesis has been the need for a facile and clinically-relevant animal model that distinguishes the relative virulence of clinical or mutant isolates. In preliminary studies, we developed a mouse model of KPC-Kp intra- abdominal infection (IAI) that differentiates the relative virulence of paired KPC-Kp isolates recovered from two patients with persistent bacteremia. In one patient, colistin resistance emerged during treatment with this agent; in the other, both initial and follow-up (i.e., persistent) isolates were colistin-susceptible. For each pair, the initial isolate caused significantly greater and more rapid mortality in mice than its persistent partner; however, the persistent isolates were recovered at higher concentrations and for longer durations within the peritoneal fluid and intra-abdominal abscesses. We performed whole genome sequencing (WGS) on the pair of colistin-susceptible and -resistant isolates, and profiled transcriptomes for the pair in the peritoneal cavity by RNA-Seq. Using WGS and KPC-Kp transcriptome data, we identified several genes and biological processes that may be linked to the emergence of colistin resistance and/or persistence in vivo. The goal of this project is to identify KPC-Kp chromosomal and plasmid genes in aims 1 and 2, respectively, that contribute to long-term persistence during IAI.
In aim 1, we will first generate high-quality, complete genome sequences for both pairs of KPC-Kp isolates by using Illumina MiSeq and PacBio platforms, and identify genes that are differentially expressed by each pair at different stages of IAI (peritonitis and intra-adominal abscesses) by RNA-Seq. WGS and RNA-Seq data will be used to prioritize genes for further investigation. We will then study mutant strains for certain chromosomal genes for persistence in the mouse model.
In aim 2, we will use plasmid-cured and complemented KPC-Kp isolates to assess if plasmids that are unique to the latter isolate in a pair contribute to persistence. Thereafter, we will study the persistence of mutant strains for specific plasmid genes. In completing these aims, we will define mechanisms by which KPC-Kp causes disease and evades elimination from the host, and the relationship between colistin resistance and virulence. This project will lead to follow-up studies in which molecular mechanisms of colistin resistance, pathogenesis and persistence in vivo are defined in detail, and exploited as potential therapeutic targets.

Public Health Relevance

In this project, we are studying Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), extremely antibiotic-resistant bacteria that cause serious infections throughout the world. This will be one of the first studies to identify the mechanisms by which KPC-Kp causes infections and becomes resistant to the last-line antibiotic colistin. Data from the study may lead to the development of new therapies against infections due to KPC-Kp and other extremely resistant bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1)
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Ernst, Nancy L
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University of Pittsburgh
Internal Medicine/Medicine
Schools of Medicine
United States
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Shields, Ryan K; Nguyen, M Hong; Chen, Liang et al. (2018) Pneumonia and Renal Replacement Therapy Are Risk Factors for Ceftazidime-Avibactam Treatment Failures and Resistance among Patients with Carbapenem-Resistant Enterobacteriaceae Infections. Antimicrob Agents Chemother 62:
Shields, Ryan K; Nguyen, M Hong; Hao, Binghua et al. (2018) Colistin Does Not Potentiate Ceftazidime-Avibactam Killing of Carbapenem-Resistant Enterobacteriaceae In Vitro or Suppress Emergence of Ceftazidime-Avibactam Resistance. Antimicrob Agents Chemother 62:
Shields, Ryan K; Clancy, Cornelius J; Pasculle, A William et al. (2018) Verification of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Susceptibility Testing Methods against Carbapenem-Resistant Enterobacteriaceae and Pseudomonas aeruginosa. J Clin Microbiol 56:
Rivosecchi, Ryan M; Clancy, Cornelius J; Shields, Ryan K et al. (2017) Effects of Isavuconazole on the Plasma Concentrations of Tacrolimus among Solid-Organ Transplant Patients. Antimicrob Agents Chemother 61:
Shields, Ryan K; Nguyen, M Hong; Press, Ellen G et al. (2017) In Vitro Selection of Meropenem Resistance among Ceftazidime-Avibactam-Resistant, Meropenem-Susceptible Klebsiella pneumoniae Isolates with Variant KPC-3 Carbapenemases. Antimicrob Agents Chemother 61:
Haidar, Ghady; Clancy, Cornelius J; Chen, Liang et al. (2017) Identifying Spectra of Activity and Therapeutic Niches for Ceftazidime-Avibactam and Imipenem-Relebactam against Carbapenem-Resistant Enterobacteriaceae. Antimicrob Agents Chemother 61:
Shields, Ryan K; Nguyen, M Hong; Chen, Liang et al. (2017) Ceftazidime-Avibactam Is Superior to Other Treatment Regimens against Carbapenem-Resistant Klebsiella pneumoniae Bacteremia. Antimicrob Agents Chemother 61: