Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Abstract

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Fig. 1.

(A) Predatory kill curves of E. coli S17-1 and S. Enteritidis P125109 by B. bacteriovorus HD100. Curves show the optical density (at 600 nm) of E. coli with heat-killed Bdellovibrio (solid squares), E. coli with live predatory Bdellovibrio (open squares), Salmonella with heat-killed Bdellovibrio (solid triangles), and Salmonella with live predatory Bdellovibrio (open triangles). Predatory Bdellovibrio was added to late-log-phase prey (either E. coli S17-1 or S. Enteritidis P125109), diluted in Ca-HEPES buffer, and incubated aerobically at 29°C with shaking at 200 rpm. A small amount of prey replication occurred due to the carryover of remaining nutrients within the prey cultures, but this growth was limited due to the dilution of the prey into the nonnutrient Ca-HEPES buffer. The drop in OD₆₀₀ reflects the successful predation on, and lysis of, the prey cells both early (during the small amount of prey growth) and later during the incubation period when the prey cells entered stationary phase. Each point represents the means from three biological repeats, and error bars show the standard errors above and below the means. (B) A B. bacteriovorus HD100 cell entering an S. Enteritidis P125109 prey cell. Cells were stained with 0.5% uranyl acetate (URA), pH 4.0. The scale bar represents 1 μm.

Fig. 2.

(A) Weight gain by control and Bdellovibrio-treated chickens. Birds were dosed with Bdellovibrio (treated) or buffer (control) at 2 days of age and then weighed daily for the next 28 days. •, control; ○, Bdellovibrio treated. (B) Scatter plot showing counts of bacterial populations from the cecal contents of control (C) and Bdellovibrio-treated (T) chickens. A group of 12 Hy-line brown birds each were challenged orally with 100 μl of approximately 1.9 × 10⁷ PFU/ml of B. bacteriovorus HD100 at 2 days of age. An identical control group was challenged with Ca-HEPES buffer. At 28 days postdose, the birds were sacrificed and targeted bacterial populations were enumerated on selective agar. Abbreviations: blood, total anaerobic count on blood agar incubated under anaerobic conditions; bifido, bifidus selective agar; mitis, mitis salivarius agar; Rogosa, Rogosa agar; chromogenic, chromogenic agar. Each data point represents counts of bacterial populations from the cecum of a single bird. The horizontal lines represent the means for each group.

Fig. 3.

Scatter plot showing the effect of Bdellovibrio treatment on colonization of chicken ceca by Salmonella Enteritidis. Ten groups of 18 Hy-line brown chicks each were challenged orally with approximately 3 × 10⁷ CFU of S. Enteritidis P125109 at 2 days of age. Four of these groups subsequently were dosed orally with approximately 9.8 × 10⁷ PFU of Bdellovibrio bacteriovorus HD100 at 6 days of age. Two further groups were dosed with nonpredatory HI ΔpilA Bdellovibrio, matched to the predatory Bdellovibrio number by total protein content. During each of the 3 days following Bdellovibrio treatment, six birds from each group were sacrificed, and the number of Salmonella organisms in the cecal contents of each bird was determined by spread-plating serial dilutions of cecal suspensions onto brilliant green agar. The results from four independent biological repeats were pooled from each day. Each data point represents the number of Salmonella organisms in the cecal contents of a single bird. The horizontal line represents the means for each group.

Fig. 4.

Representative ceca from treated birds. (A) Predatory HD Bdellovibrio-treated bird; (B) nonpredatory HI ΔpilA Bdellovibrio-treated bird; (C and D) control birds. Scale bars, 5 cm. Normal ceca contain dark free-flowing fecal material, but Salmonella infection is widely reported (1, 2, 16, 36, 42) to cause pale ceca with dense non-free-flowing contents due to inflammation and white blood cell infiltration.