Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis

Abstract

Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the Drosophila melanogaster microflora and examined the occurrence of enterococci in the gastrointestinal consortium of Drosophila. In a survey of laboratory-reared Drosophila and wild-captured flies, we found that Drosophila was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinaraum, and Enterococcus durans, as well as a previously detected but uncultured Enterococcus species. Drosophila could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to Drosophila compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the Drosophila GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies.

FIG. 1.

Phylogenetic tree of the microbial flora identified in four separately reared Drosophila stocks. Phylotypes identified by alignment with the RDP-II database are followed by GenBank accession numbers for previously identified strains. The number of times that a clone of each phylotype occurred is indicated in parentheses. Cultured phylotypes are indicated by bold type, but because plating efficiencies were not determined, numbers are not indicated unless the phylotypes were also observed in the less biased rRNA gene census. The tree was constructed with PAUP by neighbor-joining analysis using a general time-reversible model. Bootstrap values calculated from 1,000 tree iterations are indicated at branch points. The scale bar indicates evolutionary distance (10 substitutions per 100 nucleotides).

FIG. 2.

Individual variation within phylotypes occurring in individual Drosophila stocks. Clones were grouped into bacterial phyla based on their positions in Fig. 1. Relative phylotype frequencies, including the percentages of Enterococcus clones, are shown for individual fly libraries and for the combined data set.

FIG. 3.

Rarefaction analysis of 16S rRNA gene clone libraries. Observed (A) and Chao1 (B) rarefaction curves, plotted using a 97% similarity cutoff, were computed with DOTUR. The error bars in panel B indicate the 95% confidence intervals for Chao1 estimation of species richness after 1,000 randomizations.

FIG. 4.

Venn diagram describing a comparison of phylotype membership in Drosophila microbial consortia for three fly types. Oregon R OU (red), Oregon R Bloomington (blue), and wild-captured MA (yellow) 16S rRNA gene libraries were compared using SONS. The numbers in the circles indicate the estimated numbers of OTUs that are shared or are unique to each fly type. Below each Drosophila stock designation are the Chao1 estimate and 95% confidence interval for richness at a similarity level of 97%.

FIG. 5.

Colonization of Drosophila with E. faecalis FA2-2. One hundred adult Oregon R Bloomington flies were fed cornmeal-molasses food containing erythromycin (50 μg/ml) for 24 h, transferred to food inoculated with 2 ×10⁷ CFU/ml E. faecalis FA2-2 for 24 h (24 h + FA2 - 2), and then transferred to sterile food. Enterococcal colonization was tracked for 7 days. The error bars indicate standard errors of the means for experiments conducted independently three times.

FIG. 6.

Effect of cytolysin-producing E. faecalis on Drosophila survival. One hundred adult Oregon R Bloomington flies were associated with either E. faecalis FA2-2/pAM714 producing cytolysin or the isogenic cytolysin-negative control FA2-2/pAM771. The percentage of survival of colonized flies was determined for 1 week. Controls were placed on sterile food after elimination of native enterococci by erythromycin. The symbols indicate means of experiments conducted three separate times, and the error bars indicate standard errors of the means.

FIG. 7.

Colonization of Drosophila with cytolysin-producing E. faecalis FA2-2. One hundred adult Oregon R Bloomington flies were fed cornmeal-molasses food containing erythromycin (50 μg/ml) for 24 h, transferred to food inoculated with 2 × 10⁷ CFU/ml E. faecalis FA2-2/pAM714 or FA2-2/pAM771 for 24 h (24 hr + bac), and then transferred to sterile food. Enterococcal colonization was tracked for 7 days. The error bars indicate standard errors of the means for experiments conducted independently three times.

FIG. 8.

Immunohistochemical staining of thin sections of Drosophila naturally colonized with Enterococcus. Ten-micrometer cross sections of Oregon R flies either naturally colonized with enterococci or hatched and raised with 50 μg/ml erythromycin (negative control) were stained with affinity-purified rabbit anti-Enterococcus antibodies and visualized with Alexafluor 594-conjugated goat anti-rabbit antibodies. (A) Schematic diagram of Drosophila GI tract. Red indicates portions of the GI tract naturally colonized by Enterococcus. Ph, pharynx (cibarium); Sd, salivary duct; Es, esophagus; Car, cardium; Vent, pro ventriculus; CrStlk, crop stalk; Cr, crop; Sg, salivary gland; Int, intestine; Mal, Malpighian tubule; Rect, rectum. (B) Hindgut section showing colonized rectum. Magnification, ×85. (C) Hindgut negative control. Magnification, ×85. (D) Esophagus and crop stalk of colonized fly. Magnification, ×850. (E) Intestine of colonized fly. Magnification, ×850. (F) Ventriculus of negative control fly. Magnification, ×850. (G) Ventriculus of colonized fly. Magnification, ×850. (H) Rectum of colonized fly. Magnification, ×850. (I) Anus of colonized fly. Magnification, ×850. The diagram (A) was adapted from the study by Hartenstein (32), with permission of the publisher.