Screening for bacillus isolates in the broiler gastrointestinal tract

Abstract

Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.

FIG. 1.

Morphological diversity of fecal sporeformers from poultry. Phase-contrast microscopy of poultry gut sporeformers grown in Difco sporulation medium (DSM). All microphotographs are at the same scale. Isolates represented in the different panels are as follows: A, 3; B, 10; C, 37; D, 52; E, 53; F, 56; G, 62; H, 197; I, 200; J, 210; K, 241; and L, 259. Scale bar, 2 μm. The presence of vacuoles or inclusions of undefined nature is indicated by an arrowhead in panels A and K.

FIG. 2.

Plasmid profile of poultry fecal Bacillus isolates. Agarose gel electrophoresis of representative examples of plasmids isolated from poultry isolates by the method of Anderson and McKay with the small-scale procedure. Lanes: 1, 268; 2, 278; 3, 285; 4, 287; 5, 306; 6, 369; L, molecular size markers (the sizes of some of the bands are indicated for reference). The positions of chromosomal and plasmid DNAs are indicated by arrows on the left side of the figure.

FIG. 3.

Poultry fecal Bacillus spore morphological diversity. Thin-section electron micrographs of spores produced by selected gut Bacillus isolates, collected and processed from 24-h cultures in DSM. The following spore structures are indicated for a typical spore of B. subtilis (isolate 200, panel E): Cr, spore core; Cx, spore cortex; Uc, Ic, and Oc, under, inner, and outer spore coats, respectively. Other panels represent spores from the following isolates: A, 3; B, 37; C, 56; D, 197; F, 210; G, 259; and H, 259. An asterisk indicates the hair-like structures visible with spores of isolate 259. Scale bar: 0.1 μm in panels A to G; 0.2 μm in panel H.

FIG. 4.

Representative examples of antimicrobial activity among poultry fecal Bacillus isolates. Antimicrobial activity was screened by a colony overlay assay where producing strains (clockwise starting on the bottom left: 259, 200, 210, and the B. clausii strain present in the probiotic Enterogermina, which was tested in parallel) grown as spots for 24 h on LB agar plates were overlaid with an indicator strain (A, E. coli O78:K80; B, B. cereus Biosubtyl Da Lat; C, C. perfringens fD00385; D, L. innocua).