Bacillus subtilis spores germinate in the chicken gastrointestinal tract

Abstract

A number of poultry probiotics contain bacterial spores. In this study, orally administered spores of Bacillus subtilis germinated in the gastrointestinal (GI) tracts of chicks. Furthermore, 20 h after spores were administered, vegetative cells outnumbered spores throughout the GI tract. This demonstrates that spore-based probiotics may function in this host through metabolically active mechanisms.

FIG. 1.

Enumeration of B. subtilis SC2362 spores in chick GI tissues. Tissue samples were taken postmortem at 6, 12, 20, 48, and 168 h after 1 × 10⁹ spores had been administered to each chick. The number of spores in the small intestine (open bars), cecum (solid bars), and colon (cross-hatched bars) of each chick was determined by serial dilution and plating, as described in the text. Bars are mean results for three chicks sampled. Error bars are standard errors of the means. No spore formers were recovered from any tissue excised from three undosed (negative) control chicks (not shown).

FIG. 2.

Validation and sensitivity of the semiquantitative RT-PCR assay for detecting vegetative cells of B. subtilis SC2362. RT-PCR was carried out with the primers SC2362-F and SC2362-R, using template RNA prepared from sections of chick GI tissue (30 mg) spiked with known numbers of vegetative cells. Lanes: 1, 1 × 10³ vegetative cells; 2, 5 × 10³ vegetative cells; 3, 1 × 10⁴ vegetative cells; 4, 5 × 10⁴ vegetative cells; 5, 1 × 10⁵ vegetative cells; 6, 5 × 10⁵ vegetative cells; U, unspiked GI tract tissue control; V, total RNA prepared from 1 × 10⁶ B. subtilis SC2362 vegetative cells; +, RT-PCR control reaction supplied by the manufacturer (550-bp product from rabbit globin mRNA); M, 50-bp ladder (Invitrogen).

FIG. 3.

Detection of B. subtilis SC2362 vegetative cells (rrnO-lacZ) in chick tissues by semiquantitative RT-PCR. Three chicks (1, 2, and 3) were sampled at 6, 12, 20, 48, and 168 h after 1 × 10⁹ spores had been administered. Total RNA was prepared from sections of GI tract tissue (30 mg) and used as template in RT-PCR with the primers SC2362-F and SC2362-R. A 117-base pair product was generated if ≥3.33 × 10⁵ vegetative cells per gram of tissue was present in the sample. U, pooled RNA template from samples of GI tissue excised from the three undosed (negative) control chicks; V, total RNA prepared from 1 × 10⁶ vegetative cells of B. subtilis SC2362; +, RT-PCR control reaction supplied by manufacturer (550-bp product from rabbit globin mRNA); M, 50-bp ladder (Invitrogen).

FIG. 4.

qRT-PCR calibration curve. The equation of the line describes the relationship between the log₁₀(number of B. subtilis SC2362 vegetative cells in a 30-mg section of chick GI tract tissue) and the qRT-PCR signal strength (40 − cycle threshold [Ct] value), when a 0.1 volume of total RNA (i.e., 5 μl) was used as the template. Results are mean (40 − Ct) values calculated for all tissues spiked with equivalent numbers of B. subtilis SC2362 vegetative cells, irrespective of which region of the chick GI tract the tissue was from. No qRT-PCR signal was detected in negative control reactions which contained no template, total RNA from 1 × 10⁹ spores of B. subtilis SC2362, total RNA from 1 × 10⁹ vegetative cells of B. subtilis PY79 or total RNA from unspiked (negative) control chick tissue (not shown). All standards were analyzed in duplicate on four separate occasions.