Comprehensive Arrayed Transposon Mutant Library of Klebsiella pneumoniae Outbreak Strain KPNIH1

Abstract

Klebsiella pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of K. pneumoniae, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of the predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. On the basis of an independent transposon sequencing assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity by using custom phenotypic microarray plates. This screening identified several mutations increasing sensitivity to β-lactams (in acrB1, mcrB, ompR, phoP1, and slt1) and found that two-component regulator cpxAR mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone, and several β-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism.IMPORTANCE K. pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are recognized as a top public health threat by the Centers for Disease Control and Prevention. The analysis of these major nosocomial pathogens has been limited by the experimental resources available for studying them. The work presented here describes a sequence-defined mutant library of a K. pneumoniae strain (KPNIH1) that represents an attractive model for studies of this pathogen because it is a recent isolate of the major sequence type that causes infection, the epidemiology of the outbreak it caused is well characterized, and an annotated genome sequence is available. The ready availability of defined mutants deficient in nearly all of the nonessential genes of the model strain should facilitate the genetic dissection of complex traits like pathogenesis and antibiotic resistance.

Keywords: Cre; KPC; ST258; cpxR; essential gene; phenotypic microarray.

FIG 1

Transposon T30. (A) Overall structure. The transposon is a 1,271-bp Tn5 derivative carrying mosaic ends (ME) and loxP recombination sites flanking the chloramphenicol resistance determinant (cat). The resistance marker can be eliminated by Cre recombination at the loxP sites, leaving a 219-bp insertion “scar” carrying both mosaic ends and one loxP site. (B) Whole transposon sequence. (C) Insertion scar sequence. The 219-bp sequence remaining after loxP × loxP recombination is shown. The sequence encodes stop codons in all except the +2 reading frame.

FIG 2

Mutant browser screenshot. The locations of transposon insertions in the two-allele library are shown for one of two lac regions in the KPNIH1 genome. The searchable browser (https://tools.uwgenomics.org/tn_mutants/index.php) provides linked information about each insertion and can be used to create lists of mutants for request.

FIG 3

Essential genes unrepresented in the two-allele library. (Top) Overlap between K. pneumoniae genes unrepresented in the two-allele library and identified as essential by Tn-seq. (Bottom) Overlap of E. coli essential genes with K. pneumoniae genes.