April 21, 2020  |  

Tracking short-term changes in the genetic diversity and antimicrobial resistance of OXA-232-producing Klebsiella pneumoniae ST14 in clinical settings.

To track stepwise changes in genetic diversity and antimicrobial resistance in rapidly evolving OXA-232-producing Klebsiella pneumoniae ST14, an emerging carbapenem-resistant high-risk clone, in clinical settings.Twenty-six K. pneumoniae ST14 isolates were collected by the Korean Nationwide Surveillance of Antimicrobial Resistance system over the course of 1 year. Isolates were subjected to whole-genome sequencing and MIC determinations using 33 antibiotics from 14 classes.Single-nucleotide polymorphism (SNP) typing identified 72 unique SNP sites spanning the chromosomes of the isolates, dividing them into three clusters (I, II and III). The initial isolate possessed two plasmids with 18 antibiotic-resistance genes, including blaOXA-232, and exhibited resistance to 11 antibiotic classes. Four other plasmids containing 12 different resistance genes, including blaCTX-M-15 and strA/B, were introduced over time, providing additional resistance to aztreonam and streptomycin. Moreover, chromosomal integration of insertion sequence Ecp1-blaCTX-M-15 mediated the inactivation of mgrB responsible for colistin resistance in four isolates from cluster III. To the best of our knowledge, this is the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea. Furthermore, although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics.We describe stepwise changes in OXA-232-producing K. pneumoniae ST14 in vivo over time in terms of antimicrobial resistance. Our findings contribute to our understanding of the evolution of emerging high-risk K. pneumoniae clones and provide reference data for future outbreaks.Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

April 21, 2020  |  

Molecular Characterization of a Multidrug-Resistant Klebsiella pneumoniae Strain R46 Isolated from a Rabbit

To investigate the mechanisms of multiple resistance and the horizontal transfer of resistance genes in animal pathogens, we characterized the molecular structures of the resistance gene-related sequences in a multidrug-resistant Klebsiella pneumoniae strain R46 isolated from a rabbit. Molecular cloning was performed to clone the resistance genes, and minimum inhibitory concentrations (MICs) were measured to determine the resistance characteristics of the cloned genes and related strains. A conjugation experiment was conducted to assess the transferability of the resistance plasmids. Sequencing and comparative genomic methods were used to analyze the structures of the resistance gene-related sequences. The K. pneumoniae R46 genome consisted of a chromosome and three resistance plasmids named pR46-27, pR46-42, and pR46-270, respectively. The whole genome encoded 34 antibiotic resistance genes including a newly identified chromosome-encoded florfenicol resistance gene named mdfA2. pR46-270, besides encoding 26 antibiotic resistance genes, carried four clusters of heavy metal resistance genes and several virulence-related genes or gene clusters. The plasmid-encoded resistance genes were mostly associated with mobile genetic elements. The plasmid with the most similarity to the floR gene-harboring plasmid pR46-27 was pCTXM-2271, a plasmid from Escherichia coli. The results of this work demonstrated that the plasmids with multidrug resistance genes were present in animal-derived bacteria and more florfenicol resistance genes such as mdfA2 could be present in bacterial populations. The resistance genes encoded on the plasmids may spread between the bacteria of different species or genera and cause the resistance dissemination.

April 21, 2020  |  

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

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-KpIMPORTANCEKlebsiella 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. Copyright © 2019 Jung et al.

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