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. 2023 Jun 29;10(7):420.
doi: 10.3390/vetsci10070420.

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage

Rie Ikeda  1 , Fanny Laforêt  1 , Céline Antoine  1 , Mare Adachi  1 , Keiji Nakamura  2 , Audrey Habets  1 , Cassandra Kler  1 , Klara De Rauw  3 , Tetsuya Hayashi  2 , Jacques G Mainil  1 , Damien Thiry  1
Affiliations

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage

Rie Ikeda et al. Vet Sci. .

Abstract

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

Keywords: Galleria mellonella larvae; O80:H2; O80:non-H2; STX2d phage; Shigatoxigenic Escherichia coli; enteropathogenic Escherichia coli; pS88 plasmid; virulence.

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Conflict of interest statement

The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Kaplan–Meier survival curves of control E. coli strains: (a) K12-DH5α; (b) serotype O80:H26 (EH3161 strain); (c) serotype O78:H4 (pS88++). HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. *** p-value statistically significant at the threshold 0.001. pS88++: pS88 plasmid carrying the etsC/iucC genes.
Figure 2
Figure 2
Kaplan–Meier survival curves of stx1a AE-STEC O80:H2 strains: (a) SES5320 strain (pS88--); (b) SES5363 strain (pS88++); (c) EH2282 strain (pS88++). HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. *** p-value statistically significant at the threshold 0.001. pS88++: pS88 plasmid carrying the etsC/iucC genes. pS88--: pS88 plasmid not carrying the etsC/iucC genes.
Figure 3
Figure 3
Kaplan–Meier survival curves of stx2d AE-STEC O80:H2 strains: (a) EH3160 strain (pS88++); (b) EH3307/SES2959 strain (pS88--); (c) EH3320/SES3090 strain (pS88++). HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. ** p-value statistically significant at the threshold 0.01. *** p-value statistically significant at the threshold 0.001. pS88++: pS88 plasmid carrying the etsC/iucC genes. pS88--: pS88 plasmid not carrying the etsC/iucC genes.
Figure 4
Figure 4
Kaplan–Meier survival curves of EPEC O80:H2 strains: (a) EH3308/SES2973 strain (pS88--); (b) EH3322/SES3122 strain (pS88++). HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. ** p-value statistically significant at the threshold 0.01. *** p-value statistically significant at the threshold 0.001. pS88++: pS88 plasmid carrying the etsC/iucC genes. pS88--: pS88 plasmid not carrying the etsC/iucC genes.
Figure 5
Figure 5
Kaplan–Meier survival curves of: (a) K12-DH10B recipient strain; (b) K12-DH10B pS88 plasmid-conjugated strain (pS88++); (c) stx1a AE-STEC O80:H2 EH2282 pS88 plasmid donor strain (pS88++). HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. ** p-value statistically significant at the threshold 0.01. *** p-value statistically significant at the threshold 0.001. pS88++: pS88 plasmid carrying the etsC/iucC genes.
Figure 6
Figure 6
Kaplan–Meier survival curves of: (a) K12 DH10B recipient strain; (b) K12 DH10B STX2d phage-transduced strain; (c) stx2d AE-STEC O80:H2 EH3160 STX2d phage donor strain. HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. ** p-value statistically significant at the threshold 0.01. *** p-value statistically significant at the threshold 0.001.
Figure 7
Figure 7
Kaplan–Meier survival curves of E. coli O80:non-H2 strains: (a) serotype O80:H6 SES6039 strain; (b) serotype O80:H26 (EH3161 strain); (c) serotype O80:H45 SES5725 strain; (d) serotype O80:H45 SES6156 strain. HPI = Hour Post-Inoculation. * p-value statistically significant at the threshold 0.05. *** p-value statistically significant at the threshold 0.001.
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