Acinetobacter baumannii virulence assay in Caenorhabditis elegans

Abstract

Acinetobacter baumannii is a pathogen that infects hospitalized patients and evades antibiotics by acquiring resistances. It is unclear how A. baumannii infects its host. We used two different strains of A. baumannii, one is the wild type (wt) and the other is a ΔRecA, which we have shown form prominent biofilms. These are multicellular microbial communities encased in protective bacterial produced substances that protects them from antibiotics. We hypothesized that ΔRecA is more virulent than the wt since biofilms increase virulence. To test our hypothesis and better understand the toxicity of A. baumannii, we are developing an assay to test the virulence of the bacteria on the survival of the model organism Caenorhabditis elegans. The assay tests the virulence of A. baumannii using adult C. elegans that have exhausted a lawn of E. coli, picked and placed in a liquid culture containing ~1×108A. baumannii cells and a food dye. The C. elegans are left in this mix for a set amount of time and then deposited onto empty agar plates to allow for easier worm counting. After the liquid from the C. elegans mix has dried out, the survival rates for C. elegans is found. Through these tests, the assay has shown that there is approximately an 80% survival rate for C. elegans picked into an E. coli liquid mix. This result was expected as E. coli does not kill C. elegans. The assay showed there was a 28% survival rate for C. elegans picked into an A. baumannii liquid mix, and a 4% survival rate for C. elegans picked into an A. baumannii ΔRecA liquid mix. These data are in agreement with our hypothesis. To probe on the C. elegans side for what would be the genes responsible for detecting A. baumannii, or other harmful bacteria, we used a daf 7 C. elegans mutant. This strain has a loss of function in a neuron based environment sensing signaling pathway. It has been shown that daf 7 are continuously in a state similar to that of starved wt worms, where feeding rate is slowed down and fat stores are grown. Daf 7 are shown to grow to smaller sizes and to live longer than wt worms. This assay has been repeated with the clean knockout of daf 7 C. elegans, which remarkably and unexpectedly has increased survival rates, by approximately 16‐fold for ΔRecA, when compared to the parental worm. By further understanding the mechanisms underlining A. baumanii virulence, we hope to provide innovative strategies to eliminate this robust bacterium from clinical wards.Support or Funding InformationFunding Provided By Northeastern URFThese photos show C. elegans after the blue dye assay. The blue strips in the C. elegans are their intestines. A: This C. elegans was from a well containing E. coli. B: This C. elegans was from a well containing Ab Wt. C: This C. elegans was from a well containing Ab delta recA.Figure 1The survival rates of C. elegans in the blue dye assay with wt C. elegans in the darker shade and Δdaf 7 C. elegans in the lighter shade. Each bar represents the survival rates for approximately 100 C. elegans. Each survival rate for the wt C. elegans is statistically significant from the other survival rates at a p‐value of 0.05. In Δdaf 7, E. coli and Ab ΔrecA are statistically significant from Ab wt, but not from each other.Figure 2