Evolution of beta-lactam : Resistance in Klebsiella pneumoniae

Abstract: K. pneumoniae is recognized as a common opportunistic pathogen. Numerous reports have been published worldwide on outbreaks in different healthcare settings. K. pneumoniae is inherently resistant to penicillins, including semisynthetic broad-spectrum penicillins. The drug of choice for empirical treatment is often a cephalosporin. However, the use of cephalosporins is known to select for extended-spectrum beta-lactamase (ESBL)-producing strains. The focus of this thesis is the beta-lactamase gene in K. pneumoniae, and its relationship to beta-lactamase genes present in plasmids in gram-negative bacteria. In Paper I, the intension was to identify presumed beta-lactamase SHV-1-encoding plasmids in fecal Klebsiella isolates from neonates in Swedish special care units. No such plasmids were detected, however. Instead, a chromosomal beta-lactamase gene was identified in all K. pneumoniae, but in none of the Klebsiella oxytoca isolates. This species-specific gene was seen in 10 allelic variants; some closely related to the prototypic plasmid-borne SHV-1 gene, indicating that an allelic variant of the K. pneumoniae chromosomal betalactamase gene is the ancestor of the plasmid-borne SHV-encoding genes. In Paper II, the observed diversity of the chromosomal K. pneumoniae betalactamase gene was further investigated in order to study its evolution in relation to the three phylogenetic groups of K. pneumoniae. Three sequence groups, corresponding to the phylogenetic groups, were identified, blaSHV, blaLEN, and blaOKP. In Paper III, the genetic context of blaSHV in K. pneumoniae chromosomes and plasmids from various gram-negative bacteria was analyzed. Plasmid-borne blaSHV genes were found to be surrounded by DNA highly similar to the K. pneumoniae chromosome. IS26 elements flanked the blaSHV regions. Nine distinct junctions between IS26 and K. pneumoniae chromosomal DNA, and seven different region-lengths were identified. In contrast to a high diversity observed among chromosomal sequences, only two groups of plasmid sequences were seen. This thesis has demonstrated that only one of three ancient K. pneumoniae chromosomal beta-lactamase gene families, blaSHV, is found on plasmids. This is possibly the result from a single IS26 mediated mobilization of blaSHV and surrounding DNA from K. pneumoniae. The two groups of plasmid blaSHV regions seen today could be the result of post-mobilization evolution involving size reductions and nucleotide substitutions. We conclude that mobilization of blaSHV from K. pneumoniae chromosomes is not a driving force in the emergence of resistance in response to beta-lactam therapy. The spread is more likely a consequence of mobilization of IS26 flanked blaSHV regions between plasmids, and mobilization of plasmids between different bacteria.