# P-1540. Virulence Analysis of Type VI Secretion System in Neonatal Meningitis E. coli using Galleria mellonella as an Infection Model

## TL;DR

This study investigates how the Type VI Secretion System (T6SS) affects the virulence of E. coli causing neonatal meningitis using a waxworm infection model.

## Contribution

The study identifies specific T6SS genes that reduce E. coli virulence in a Galleria mellonella model and compares virulence across different serogroups.

## Key findings

- Deletion of hcp and icmF genes in O18 E. coli reduced virulence, increasing larval survival to 85% and 65%.
- O7 and O45 serogroup strains caused 100% mortality in larvae within 24 hours, suggesting additional virulence factors.
- T6SS+ strains O18, O12, and O78 showed low survival and high disease scores, while O16 and O83 had higher survival and lower scores.

## Abstract

Neonatal meningitis E. coli (NMEC) is a common cause of bacterial meningitis in newborns worldwide. NMEC replicates within macrophages, survives in the bloodstream, and traverses the blood-brain barrier, resulting in meningitis. Most NMEC cases are associated with serogroups O18, O83, O7, O12, O1, and O45. Two clusters of the Type 6 secretion system (T6SS) (T6SS-1 and T6SS-2) were identified in NMEC O18:K1. Studies demonstrated that the T6SS plays a key role in pathogenesis and biofilm formation of NMEC, though the function of specific T6SS genes remains unclear. This study aimed to investigate the T6SS role in NMEC virulence through in vivo infection of Galleria mellonella larvae, hypothesizing that deletion of T6SS genes would reduce strain virulence.Figure 1.Kaplan-Meier survival curve of G. mellonella larvae infected with T6SS mutant strains.Figure 2.Larval Disease Score (LDS) of larvae infected with T6SS mutant strains throughout the 96h observation period.

Kaplan-Meier survival curve of G. mellonella larvae infected with T6SS mutant strains.

Larval Disease Score (LDS) of larvae infected with T6SS mutant strains throughout the 96h observation period.

An NMEC O18 strain was used to construct T6SS mutants lacking the hcp, icmF, impK, evpB, and vasK genes. Strains belonging to eight different serogroups and distinct T6SS operon structures (O1, O12, O16, O18, O78, O83, O7 and O45) were also analysed. Larvae were infected with 106 CFU/mL of each bacterial strain suspension and incubated at 37 °C for 96 hours. PBS was used as a negative control, along with a non-infected group, and a non-pathogenic E. coli strain (DH5α). Larval activity, melanization, and cocoon production were evaluated and used to calculate the larval disease score (LDS).Figure 3.Kaplan-Meier survival curve of G. mellonella larvae infected with NMEC strains of different serogroups.Figure 4.Larval Disease Score (LDS) of larvae infected with NMEC strains of different serogroups throughout the 96h observation period.

Kaplan-Meier survival curve of G. mellonella larvae infected with NMEC strains of different serogroups.

Larval Disease Score (LDS) of larvae infected with NMEC strains of different serogroups throughout the 96h observation period.

Deletion of hcp and icmF genes attenuated the virulence of the O18 strain, promoting larval survival rates of 85% and 65%, respectively. In contrast, only 15% of larvae infected with the wild-type strain survived. These two mutants also presented lower LDS compared to the WT. However, no attenuation was observed in larvae infected with Δevp or ΔvasK strains. In terms of serogroup virulence, the O7 and O45 strains surprisingly promoted 100% mortality after 24h post-infection. Among the strains T6SS+, infections with O78, O18, and O12 resulted in low survival but higher LDS, while infections with O16 and O83 strains showed higher survival rates (55% and 50%, respectively) and lower LDS.

These findings suggest that T6SS contributes significantly to the virulence of NMEC strains belonging to the O18 serogroup but does not play a major role in the virulence of O7 and O45 strains, which may harbor additional virulence factors.

All Authors: No reported disclosures