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Applied and Environmental Microbiology, August 2008, p. 5254-5258, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00580-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bacillus subtilis Spores Germinate in the Chicken Gastrointestinal Tract{triangledown}

Stephen T. Cartman,* Roberto M. La Ragione, and Martin J. Woodward

Department of Food and Environmental Safety, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Received 11 March 2008/ Accepted 21 June 2008


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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.


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INTRODUCTION
 
Antibiotic feed supplements have been used in commercial poultry farming for over 50 years due to their growth-promoting and prophylactic properties (3, 7, 11). However, their use in the European Union (EU) was banned in 2006 as part of an initiative aimed at promoting the prudent use of antibiotics (1, 18, 24). As a result, there is now a market for alternative growth-promoting and prophylactic products. In general, feed supplements remain the method of choice, due to the scale and economics of modern poultry farming.

Probiotics are one form of alternative feed supplement or "functional food." They can be defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (10). Many probiotic products are already available for commercially farmed poultry, and a number of these contain or consist of bacterial spores, principally of the genus Bacillus (4, 5, 16). Bacterial spores are particularly well suited for use as live microbial products as they are metabolically dormant and highly resilient to environmental stresses. These intrinsic properties are highly desirable from a commercial perspective and mean that spore-based products have a long shelf life and retain their viability during distribution and storage. Despite this, knowledge of how bacterial spores may function as probiotics is severely limited at present.

Considering that bacterial spores are metabolically dormant, it is of particular interest to question whether or not they are able to germinate in the chicken gastrointestinal (GI) tract. This is an important point to address, as it is widely accepted that microorganisms must be in a viable state to function as probiotics, the implication being that one or more metabolic processes are essential for probiotic function in the host GI tract. Indeed, if spores do not germinate in the chicken GI tract, it is difficult to envisage how they may function as probiotics through mechanisms which require them to be metabolically active (e.g., secretion of antimicrobial compounds and competition for essential nutrients).

Early work with mammalian hosts suggested that bacterial spores are able to germinate in the GI tracts of dogs, rabbits, and mice (13, 15). Definitive proof of this was obtained when it was shown that vegetative cells of Bacillus subtilis could be detected in the small intestines of mice after they had received an oral dose of spores (6, 25). More recently, spore germination has been observed in the GI tracts of pigs, using flow cytometry analysis (17). While all of these findings are important in the context of mammalian hosts, it cannot be assumed that they will translate directly to an avian host, primarily due to differences in GI anatomy and physiology. Consequently, in the wake of the EU ban on antimicrobial feed supplements and in view of the fact that a number of spore-based probiotic products are already available for commercially farmed poultry, in this study we addressed the pertinent question of whether or not spores of the model spore-forming bacterium B. subtilis are able to germinate in the chicken GI tract. We used a 1-day-old chick model as this is the approximate age of chickens when they are first housed within broiler sheds and placed on diets supplemented with "functional foods," such as antibiotics or probiotics.


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Spores are transient members of the chick GI tract.
 
To address the fate of orally administered spores, 1-day-old specific pathogen-free White Leghorn chicks were each dosed by oral gavage with 1 x 109 spores of B. subtilis SC2362 (6, 8, 9) suspended in 0.1 ml of sterile distilled water. This strain of B. subtilis harbors a unique rrnO-lacZ fusion gene and a chloramphenicol acetyltransferase (cat) gene in the chromosome. Spores were prepared as described previously (22), including sequential treatment with lysozyme (20 mg/ml) and heat (68°C for 90 min) to ensure there were no contaminating vegetative cells present. Three chicks were selected at random for sampling at 6, 12, 20, 48, and 168 h after spore doses were administered. Sections of duodenum, jejunum, ileum, cecum, and colon were excised from each chick postmortem, and the number of B. subtilis SC2362 spores per gram of tissue (including luminal contents) was determined by heat treatment (to kill vegetative cells and background contaminants), serial dilution, and plating. A mean value was calculated for the number of spores in the duodenum, the jejunum, and the ileum of each chick and reported as the number of spores per gram of small intestine. Numbers of B. subtilis SC2362 spores were found to decrease with time, throughout the GI tract, indicating that spores are transient members of the chick GI microflora (Fig. 1). This may have been due to spore germination in the GI tract and/or shedding of spores in feces. Between 5 x 102 and 5 x 103 spores remained in the GI tract of each chick at the end of the experimental period.


Figure 1
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  		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 x 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).


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Spore germination in the chick GI tract.
 
To investigate the possibility of spore germination in the GI tracts of chicks, a reverse transcriptase PCR (RT-PCR)-based method was developed for detecting vegetative cells of B. subtilis SC2362. We decided to use a molecular approach, as preliminary work revealed that the process of serial dilution and plating onto medium supplemented with chloramphenicol was an unreliable method for accurately determining total viable counts of B. subtilis SC2362 (i.e., spores and vegetative cells) in samples of gut tissue. Other researchers have reported similar problems (14, 15), the issues being (i) inefficient and nonsynchronous spore germination in the absence of a heat treatment/activation step, which leads to an underestimation of total viable units, and (ii) growth of background contaminants which obscure colonies of B. subtilis on agar plates. Ultimately, our decision to use RT-PCR was based on successful work by researchers elsewhere (6, 25).

In the first instance, a semiquantitative RT-PCR assay was developed for detecting vegetative cells of B. subtilis SC2362. Total RNA was prepared from 30-mg sections of gut tissue (including luminal contents), using an RNeasy mini-kit (Qiagen), and RNA concentrations were determined by spectroscopy prior to RT-PCR. The primers SC2362-F (GGGAGGGTCATTGGAAACTG) and SC2362-R (CCTATATAAAAGCATTAGTGTATCAACAAGC) were designed using Primer Express software (Applied Biosystems) to target the unique rrnO-lacZ sequence of B. subtilis SC2362. The assay was then validated using sections of control tissue spiked with known numbers of vegetative cells. A visible RT-PCR product of the expected size (117 bp) was amplified when total RNA from gut tissue spiked with ≥1 x 104 vegetative cells of B. subtilis SC2362 was used as the template (Fig. 2). The absolute detection limit of the assay was found to be 100 cells or 3.33 x 105 cells per gram of tissue. (Total RNA was extracted from sections of tissue that weighed 30 mg and were eluted in 50 µl. A 0.5-µl volume of eluate was then used as the template in RT-PCR). No RT-PCR product was amplified from RNA extracted from the parental B. subtilis strain PY79 (26) or from spores of B. subtilis SC2362, indicating that the assay was specific for the rrnO-lacZ mRNA of B. subtilis SC2362 vegetative cells (data not shown). Additional RT-PCRs with an initial RT denaturing step (95°C for 30 min) or with primers Bact-F (CTGGTCGTACCACTGGTATTG) and Bact-R (CAGGTGGGGCAATGATCTTG), used to amplify a 550-bp product from chicken β-actin mRNA (2), confirmed that RT-PCR products were not amplified from contaminating DNA and that the RNA preparation procedure was consistent between samples (data not shown).


Figure 2
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  		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 x 103 vegetative cells; 2, 5 x 103 vegetative cells; 3, 1 x 104 vegetative cells; 4, 5 x 104 vegetative cells; 5, 1 x 105 vegetative cells; 6, 5 x 105 vegetative cells; U, unspiked GI tract tissue control; V, total RNA prepared from 1 x 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).

To determine whether or not orally administered spores of B. subtilis SC2362 germinated in the GI tracts of chicks, RT-PCR was carried out using RNA template from sections of gut tissue which had been sampled from chicks at 6, 12, 20, 48, and 168 h after they had been dosed with spores. The rrnO-lacZ RT-PCR product was amplified from RNA template prepared from the ceca and colons of two of the three chicks sampled at 12 h and all three of the chicks sampled 20 h after spore doses were administered (Fig. 3). Repeat reactions with an initial RT denaturing step or with the primers Bact-F and Bact-R confirmed that these were true positives (i.e., not amplified from contaminating DNA) and that the RNA preparation procedure was consistent between all samples (data not shown). As samples from the 12- and 20-h time points were the only ones to test positive for B. subtilis SC2362 vegetative cells (indicating the presence of at least 3.33 x 105 cells per gram of tissue), we proceeded to analyze these samples by TaqMan real-time quantitative RT-PCR (qRT-PCR) to estimate the numbers of vegetative cells present.


Figure 3
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  		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 x 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 x 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 x 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).

For qRT-PCR, the primers SC2362-F and SC2362-R were used in conjunction with the fluorescently labeled TaqMan probe, SC2362-P ([FAM]-TTCCACTCTCCTCTTCTGCACTCAAGTT-[TAMRA]). The probe was designed, using Primer Express software (Applied Biosystems), to lie across the splice junction of the unique rrnO-lacZ sequence, thus ensuring the assay was specific for vegetative cells of B. subtilis SC2362. The qRT-PCR assay was validated using the same RNA preparations as those used to validate the semiquantitative RT-PCR assay (i.e., those from control tissue spiked with known numbers of vegetative cells). Results were plotted to obtain a calibration curve for the qRT-PCR assay (Fig. 4). The efficiency of the qRT-PCR was low (55%) as an unfortunate consequence of having to specifically target the splice junction of the rrnO-lacZ sequence. However, considering that the qRT-PCR assay was used for absolute quantification of B. subtilis SC2362 vegetative cells (as opposed to relative quantification by comparison to an endogenous housekeeping gene) and that there was a good linear relationship between the log10(number of vegetative cells) and qRT-PCR signal strength (R2 = 0.9970), the assay was fit for the purpose of this study. The absolute detection limit of the qRT-PCR assay was just 2 cells or 667 cells per gram of tissue. (Total RNA was extracted from sections of tissue weighing 30 mg and eluted in 50 µl. A 5-µl volume of eluate was then used as template in qRT-PCR.) Control reactions with no RNA template or RNA template prepared from vegetative cells of the parental B. subtilis strain PY79, spores of B. subtilis SC2362, or from unspiked (negative control) chick tissue did not yield any qRT-PCR signal, confirming that the assay was both robust and specific for the rrnO-lacZ mRNA of B. subtilis SC2362 vegetative cells.


Figure 4
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  		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 x 109 spores of B. subtilis SC2362, total RNA from 1 x 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.

Analysis of experimental samples from the 12- and 20-h time points by qRT-PCR enabled us to estimate the numbers of B. subtilis SC2362 vegetative cells present (Table 1). Furthermore, the enhanced sensitivity of the qRT-PCR assay enabled us to estimate the numbers of vegetative cells in samples which did not test positive by semiquantitative RT-PCR, most notably, samples of small intestine. Considering that spore doses were administered in sterile distilled water, which lacks any of the chemical or nutrient signals associated with spore germination (12, 23), this suggests that spore germination was induced either prior to or upon entry into the chick small intestine. Interestingly, the number of vegetative cells was found to surpass the number of spores between 12 and 20 h postdosing in all regions of the GI tract (Table 1). Based on the results of the present study, it is not possible to comment on whether this was due to spore germination alone or whether cell replication within the host GI tract was also a contributing factor.


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  		TABLE 1. Numbers of B. subtilis SC2362 spores and vegetative cells recovered from GI tracts of chicks


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Retention of B. subtilis in the chick GI tract.
 
Finally, estimates were made as to what percentage of the original spore dose (1 x 109) was retained in the GI tract of each chick at 12 and 20 h postdosing. To do this, the GI tissues of 12 1-day-old chicks were excised and weighed immediately postmortem. The mean weight of small intestines was 1.21 ± 0.12 g, that of ceca was 0.25 ± 0.10 g, and that of colons was 0.1 ± 0.01 g. Using these approximations in conjunction with plating (spores) and qRT-PCR (vegetative cells) data, estimates were made as to the numbers of spores and vegetative cells retained throughout the entire GI tract of each chick at 12 and 20 h postdosing. Between 0.13% and 1.23% of the original spore dose was estimated to have been retained in the GI tract of each chick (Table 2). This finding is comparable with that from work conducted in mice (6, 25) and supports the notion that B. subtilis is a transient member of the chick GI microflora, which does not colonize the GI tract. The remainder of the original spore dose may have been shed in feces or germinated in the GI environment and subsequently died due to unfavorable conditions.


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  		TABLE 2. Retention of B. subtilis SC2362 in the GI tracts of chicksa

In conclusion, we have proven for the first time that spores of B. subtilis are able to germinate in the chicken GI tract. This principle finding agrees with studies conducted with a mouse model (6, 25). However, in those studies, vegetative cells were detected only in the small intestines of mice dosed with spores. In contrast, vegetative cells were detected throughout the GI tracts of chicks in the present study. This observation demonstrates that the findings of experiments conducted with a mammalian model may not translate directly to an avian host. Moreover, these findings may reflect genuine physiological and/or physicochemical differences between the GI tracts of chicks and mice. Notwithstanding these differences, it is noteworthy that chicks in the present study were 1 day old at the time of dosing, whereas mice used in the aforementioned studies were 6 weeks old at the time of dosing. Therefore, the state of GI microflora maturity and/or physiological status of the host may have played some part in the apparent disparity between chicks and mice. Nonetheless, the findings of the present study are considered highly relevant, as functional foods (e.g., probiotics) are typically administered to commercially farmed chickens from one day of age.

B. subtilis spore germination occurred rapidly in the GI tracts of chicks, with vegetative cells outnumbering spores 20 h after spore doses were administered. This may have been due to spore germination alone, although cell replication within the GI environment may also have been a contributing factor. Even though B. subtilis is classically considered to be an obligate aerobe, it may well survive and replicate within the anoxic GI environment, as it can use nitrate or nitrite (in place of oxygen) as a terminal electron acceptor during anaerobic respiration (20, 21).

The RT-PCR assays developed in the present study were designed to identify B. subtilis SC2362 vegetative cells based on the presence of rrnO-lacZ mRNA. Considering that spore germination per se does not require de novo protein synthesis (19), the vegetative cells detected in this study must have either entered or completed the process of spore outgrowth, which is characterized by the onset of metabolic processes (23). This proves that although spore-based probiotics are metabolically dormant upon administration, they may germinate in the GI tract of chicks and function through mechanisms which require them to be metabolically active (e.g., secretion of antimicrobial compounds and/or competition for essential nutrients).


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ACKNOWLEDGMENTS
 
We thank Simon M. Cutting for the kind gift of B. subtilis SC2362 and Le Hong Duc for technical assistance. We are also extremely grateful to the Animal Services Unit (ASU) at VLA, particularly Pete Gilmore and Sue Turner.


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FOOTNOTES
 
* Corresponding author. Present address: Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Phone: 44 115 846 7959. Fax: 44 115 846 8002. E-mail: stephen.cartman{at}nottingham.ac.uk Back

{triangledown} Published ahead of print on 27 June 2008. Back


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Applied and Environmental Microbiology, August 2008, p. 5254-5258, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00580-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Flint, J. F., Garner, M. R. (2009). Feeding beneficial bacteria: A natural solution for increasing efficiency and decreasing pathogens in animal agriculture. J. Appl. Poult. Res. 18: 367-378 [Abstract] [Full Text]  

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