Characterization of pre-antibiotic era Klebsiella pneumoniae isolates with respect to antibiotic/disinfectant susceptibility and virulence in Galleria mellonella

Abstract

The EGD Murray collection consists of approximately 500 clinical bacterial isolates, mainly Enterobacteriaceae, isolated from around the world between 1917 and 1949. A number of these "Murray" isolates have subsequently been identified as Klebsiella pneumoniae. Antimicrobial susceptibility testing of these isolates showed that over 30% were resistant to penicillins due to the presence of diverse blaSHV β-lactamase genes. Analysis of susceptibility to skin antiseptics and triclosan showed that while the Murray isolates displayed a range of MIC/minimal bactericidal concentration (MBC) values, the mean MIC value was lower than that for more modern K. pneumoniae isolates tested. All Murray isolates contained the cation efflux gene cepA, which is involved in disinfectant resistance, but those that were more susceptible to chlorhexidine were found to have a 9- or 18-bp insertion in this gene. Susceptibility to other disinfectants, e.g., H2O2, in the Murray isolates was comparable to that in modern K. pneumoniae isolates. The Murray isolates were also less virulent in Galleria and had a different complement of putative virulence factors than the modern isolates, with the exception of an isolate related to the modern lineage CC23. More of the modern isolates (41% compared to 8%) are classified as good/very good biofilm formers, but there was overlap in the two populations. This study demonstrated that a significant proportion of the Murray Klebsiella isolates were resistant to penicillins before their routine use. This collection of pre-antibiotic era isolates may provide significant insights into adaptation in K. pneumoniae in relation to biocide susceptibility.

FIG 1

Genetic diversity in the Murray collection Klebsiella strains. The midpoint-rooted phylogenetic tree shows the phylogenetic relationships of taxa based on core genome. Taxon names of isolates noted as colony variants are colored similarly (noncolony variants are in black). The adjacent columns show the presence (red) and absence (blue) of bla_SHV genes and virulence-associated genes (black fields denote untested isolates). The presence of K1 (orange) and K3 (purple) capsular types is shown adjacent, and known sequence types (STs) are also shown.

FIG 2

Virulence of K. pneumoniae in G. mellonella after 24 h. Groups of 10 larvae were challenged with 1 × 10⁵ (a) and 1 × 10⁴ (b) CFU of different strains of Murray and modern Klebsiella spp. The proportion of larvae alive at 24 h postinfection is shown, with individual points being the mean of independent triplicates and error bars showing the standard deviation of the mean of all points. Data for virulence of M109 and related modern CC23 isolates are highlighted by gray boxes.

FIG 3

Susceptibility to clinical skin and wound disinfectants. MICs were determined for the Murray isolates (white bars) and the modern isolates (black bars) for triclosan (a) and the topical antiseptics chlorhexidine digluconate (b) and two quaternary ammonium cationic disinfectants, benzalkonium chloride (c) and HDPCM (d), and the number of strains plotted against their MIC levels.