Identification of novel antimicrobials using a live-animal infection model - PubMed (original) (raw)

Identification of novel antimicrobials using a live-animal infection model

Terence I Moy et al. Proc Natl Acad Sci U S A. 2006.

Abstract

The alarming increase of antibiotic-resistant bacterial pathogens points to the need for novel therapeutic approaches to combat infection. To discover novel antimicrobials, we devised a screen to identify compounds that promoted the survival of the model laboratory nematode Caenorhabditis elegans infected with the human opportunistic pathogen Enterococcus faecalis. E. faecalis colonizes the nematode intestinal tract, forming a persistent lethal infection. Infected nematodes were rescued by antibiotic treatment in a dose-dependent manner, and antibiotic treatment markedly reduced the number of bacteria colonizing the nematode intestine. To facilitate high throughput screening of compound libraries, we adapted a previously developed agar-based C. elegans-E. faecalis infection assay so that it could be carried out in liquid medium in standard 96-well microtiter plates. We used this simple infection system to screen 6,000 synthetic compounds and 1,136 natural product extracts. We identified 16 compounds and 9 extracts that promoted nematode survival. Some of the compounds and extracts inhibited E. faecalis growth in vitro, but, in contrast to traditional antibiotics, the in vivo effective dose of many of these compounds was significantly lower than the minimum inhibitory concentration needed to prevent the growth of E. faecalis in vitro. Moreover, many of the compounds and extracts had little or no affect on in vitro bacterial growth. Our findings indicate that the whole-animal C. elegans screen identifies not only traditional antibiotics, but also compounds that target bacterial virulence or stimulate host defense.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Curing of a nematode E. faecalis infection on solid medium by antibiotic treatment. (A) Kaplan–Meier survival curves of WT N2 C. elegans feeding continuously on lawns of E. faecalis (Efs) V583 (▴) or E. faecium (Efm) DO (▾). Nematodes feeding for 24 h on E. faecalis and subsequently transferred to lawns of E. faecium (□) die with an LT50 of 14.9 days. Error bars equal SEM. (B) Kaplan–Meier survival curves of E. faecalis V583-infected N2 C. elegans transferred to lawns of E. faecium on media containing tetracycline at 10 μg/ml (▴, P < 0.0001), 2 μg/ml (▿, P < 0.0014), 0.4 μg/ml (♦, P < 0.06), 0.08 μg/ml (●, P = 0.56), or no tetracycline (□). (C) Antibiotic concentrations required to promote rescue of E. faecalis VS583 infected N2 nematodes. Survival was measured 13 days postinfection after treatment with tetracycline (Tet), vancomycin (Van), or ciprofloxacin (Cipro). The MICs for the E. faecalis strain VS583 are 0.39 μg/ml Cipro, 0.27 μg/ml Tet, and 3.1 μg/ml Van. Note that VS583 is a vancomycin-sensitive derivative of V583, the well-studied vancomycin-resistant E. faecalis strain. Error bars are standard deviations. (D) Kaplan–Meier survival curve of glp-4;sek-1 nematodes infected for 12 h on E. faecalis OG1RF and transferred to BHI media containing 20 μg/ml tetracycline (▴), 40 μg/ml vancomycin (▾), or no additional antibiotic (□). Error bars equal SEM.

Fig. 2.

Fig. 2.

The liquid infection assay gauges differences in nematode killing due to E. faecalis strains with varying degrees of pathogenicity or due to antibiotic treatment. (A) Kaplan–Meier survival curves of glp-4;sek-1 nematodes that were infected with the cytolysin (Cyl) producing E. faecalis strain MMH594 (▴, ♦) or the E. faecium strain 11M12 (●). Cyl-infected nematodes were treated with 0 (▴) or 20 μg/ml (♦) tetracycline (Tet). Error bars equal SEM. (B) Kaplan–Meier survival curves of glp-4;sek-1 nematodes that were infected with E. faecalis OG1RF (▴, ♦) or the two-component quorum-sensing regulator mutant OG1RF Δ_fsrB_ (●). OG1RF-infected nematodes were treated with 0 (▴) or 20 μg/ml (♦) tetracycline. (C) The bacterial load in the nematode intestinal tract after antibiotic treatment. E. _faecalis_-infected nematodes were treated in liquid media containing 20 μg/ml ampicillin (Amp) or tetracycline, and the number of cfu per worm was determined. Error bars equal standard deviations. (D) Curing kinetics of selected hit compounds. Shown are Kaplan–Meier survival curves of infected nematodes treated with 25 μg/ml compound 4 (♦), 50 μg/ml compound 9 (●), 25 μg/ml compound 10 (□), 2.5 μg/ml tetracycline (▿), 20 μg/ml tetracycline (▴), or mock treatment (■). In pairwise comparisons to mock treatment using log-rank tests, the difference for all of the treatments was significant, with P < 0.0001.

Fig. 3.

Fig. 3.

Scoring live/dead worms in the liquid killing assay. Living nematodes in the liquid infection assay maintain a sinusoidal shape, whereas dead nematodes in the liquid infection assay appear as straight, rigid rods as the corpse becomes filled with bacteria.

Fig. 4.

Fig. 4.

Structures of the compounds that promote the survival of nematodes infected with E. faecalis. The compound number in bold corresponds to the numbering in Table 1.

Similar articles

Cited by

References

    1. Chambers H. F. Emerg. Infect. Dis. 2001;7:178–182. - PMC - PubMed
    1. Zinner S. H. Expert Rev. Anti Infect. Ther. 2005;3:907–913. - PubMed
    1. Molbak K. Clin. Infect. Dis. 2005;41:1613–1620. - PubMed
    1. Wisplinghoff H., Bischoff T., Tallent S. M., Seifert H., Wenzel R. P., Edmond M. B. Clin. Infect. Dis. 2004;39:309–317. - PubMed
    1. Lane H. C., Montagne J. L., Fauci A. S. Nat. Med. 2001;7:1271–1273. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources