Galleria mellonella infection model demonstrates high lethality of ST69 and ST127 uropathogenic E. coli

Abstract

Galleria mellonella larvae are an alternative in vivo model for investigating bacterial pathogenicity. Here, we examined the pathogenicity of 71 isolates from five leading uropathogenic E. coli (UPEC) lineages using G. mellonella larvae. Larvae were challenged with a range of inoculum doses to determine the 50% lethal dose (LD50) and for analysis of survival outcome using Kaplan-Meier plots. Virulence was correlated with carriage of a panel of 29 virulence factors (VF). Larvae inoculated with ST69 and ST127 isolates (10(4) colony-forming units/larvae) showed significantly higher mortality rates than those infected with ST73, ST95 and ST131 isolates, killing 50% of the larvae within 24 hours. Interestingly, ST131 isolates were the least virulent. We observed that ST127 isolates are significantly associated with a higher VF-score than isolates of all other STs tested (P≤0.0001), including ST69 (P<0.02), but one ST127 isolate (strain EC18) was avirulent. Comparative genomic analyses with virulent ST127 strains revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae infection model. Virulence in the larvae was not correlated with serotype or phylogenetic group. This study illustrates that G. mellonella are an excellent tool for investigation of the virulence of UPEC strains. The findings also support our suggestion that the incidence of ST127 strains should be monitored, as these isolates have not yet been widely reported, but they clearly have a pathogenic potential greater than that of more widely recognised clones, including ST73, ST95 or ST131.

Figure 1. Kaplan-Meier survival analysis of G. mellonella larvae following injection of UPEC cells (2.87×10⁶ cfu/ml) of different sequence types (ST).

Data presented are the mean of three independent assays with each UPEC isolate. Low larvae mortality was recorded following injection of DH5α. Non-injected larvae and PBS injected larvae showed no mortality. Survival outcome of larvae injected with ST69 and ST127 isolates recorded the highest mortality compared to ST73 (P≤0.248), ST95 (P≤0.303) and ST131 (P≤0.054).

Figure 2. Correlation analysis between the five UPEC sequences types: (A) Low LD₅₀ shows significant lethal effects with larvae inoculated with ST69 and ST127 strains.

(B) ST127 shows high virulence capacity compared to that of other STs and ST131 shows relatively low virulence capacity.

Figure 3. BLASTn comparison of O-antigen gene cluster (green) and colanic acid gene cluster (red) from UPEC ST127 strains 536, EC18 and EC41.

The gray shading indicates high nucleotide identity between the sequences (99–100%). In EC18, there is an insertion-sequence (IS1) mediated deletion of most of the O-antigen gene cluster and the colanic acid gene cluster (vertical dotted line denotes contig boundaries in EC18). Figure was prepared using Easyfig .

Figure 4. Virulence profile of ST127 strains based on PCR detection of 29 uropathogen associated virulence factors.

EC18 (avirulent strain) shows a similar profile compared to other ST127 virulent strains. Black blocks represent positive PCR results and strain numbers are in the left hand column.