Abstract
BackgroundSeveral strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis.ResultsThe 14.0 kb KpGI-5 island exhibited a genome-anomalous G + C content, possessed near-perfect 46 bp direct repeats, encoded a γ1-chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107∆fim2, KR2107∆fim and KR2107∆fim∆fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis.ConclusionAlthough fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.
Highlights
Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains
The KpGI-5 genomic island codes for a novel predicted chaperone/usher fimbrial system Whilst screening five tRNA gene insertion hotspots in sixteen clinical K. pneumoniae isolates for strain-specific DNA using a technique called tRIP-PCR [13,14], we found that K. pneumoniae KR116 possessed an ‘occupied’ met56 tRNA locus. tRIP-PCR using primers PR601 and PR647, which were designed to amplify across the met56 tRNA locus, failed to amplify a product in KR116
Open reading frame (ORF) and BLAST analyses were performed on the KpGI-5 sequence (Figure 1 and Table 1)
Summary
Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Hypervirulent, pyogenic liver abscess-causing K. pneumoniae strains that infect otherwise healthy individuals have emerged from initial endemic foci in Taiwan and China, and are spreading into North America and Europe [4,5,6]. This highlights the increasing threat that K. pneumoniae poses to public health and the importance of elucidating its mechanisms of pathogenesis. The functions of strain-specific genomic regions in K. pneumoniae strains associated with other types of infection remain poorly studied
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