extfieldsofstudy: Medicine Biology

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Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment

Background The Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia. Results Five of the seven B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms. Conclusions This study showed that the B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0377-9) contains supplementary material, which is available to authorized users.

Background
The Bacillus cereus s. l. species are Gram-positive, facultative anaerobic, spore-forming rods. As soil-associated ubiquitous organisms, B. cereus s. l. are commonly found in food products where they can cause spoilage [1]. In addition, pathogenic strains can be responsible for two different types of foodborne illness in humans. Diarrheal disease has been associated with three toxins: a single protein named cytotoxin K (CytK), and two heat-labile protein complexes: hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe). Emetic syndrome is caused by the heat-resistant toxin cereulide. Emetic syndrome is usually associated with starchy food, such as rice or pasta, whereas diarrheal syndrome is caused by consumption of contaminated sauces, vegetables or dairy products [2]. The incidence of Bacillus cereus on dairy farms has been extensively studied, particularly in Scandinavia and the Netherlands [3,4].
The taxonomy of B. cereus s. l. is complex and has been regularly updated by new findings. To date, seven members have been described: Bacillus cereus sensu stricto, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus anthracis and most recently Bacillus cytotoxicus [5]. These strains share a highly conserved genome, and their 16S rRNA gene sequences show high levels of similarity [6]. Moreover, horizontal transfer of plasmidcarried genes is widespread among B. cereus s. l. strains [7]. These features led studies to question the relevance of the taxonomic segregation of B. cereus s. l. species on the basis of genetic closeness [8,9]. However, it is still possible to discriminate the B. cereus group strains, on the basis of various phenotypical traits. B. mycoides and B. pseudomycoides exhibit a typical rhizoid growth whereas the rest of the species have round to irregular colonies [10]. B. mycoides strains are able to grow at 8°C but not at 40°C. B. weihenstephanensis strains are psychrotolerant and can thus grow at temperatures as low as 7°C but not higher than 43°C [11]. On the contrary, B. cytotoxicus strains have a growth temperature range of 20°C to 50°C [5]. B. thuringiensis strain produce parasporal toxin crystals with insecticidal properties [12]. Finally, B. anthracis strains are non-hemolytic on Sheep Blood Agar (SBA) plates [13].
Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium commonly found in the environment and in animal intestinal tracks. It is also a human pathogen and has been identified as the second most common bacterial source of foodborne illness in the United States [14]. C. perfringens strains are classified into toxigenic types A, B, C, D and E, based on the combination of four different toxins that they can produce (alpha-, beta-, epsilon-and iota-toxins). Moreover, the C. perfringens strains can also produce a diversity of other toxins, such as the beta2 enterotoxin and Clostridium perfringens enterotoxin (CPE).
The isolates in this study were obtained from samples from various sources (e.g., soil, water, faeces, animal feed, milk filter and raw milk) collected on a mixed group of bovine, caprine and ovine dairy farms in Victoria, Australia [15]. The diversity of B. cereus s. l. isolates was evaluated, in terms of taxonomy and toxin production potential at different temperatures in nonagitated reconstituted milk and broth. Molecular ecology of the B. cereus s. l. population was investigated using PFGE. C. perfringens isolates were subtyped using a toxingene-targeted multiplex PCR method. This study provided extensive data about the diversity of the on-farm populations of Bacillus cereus s. l. and C. perfringens in Victoria, Australia. The results regarding the growth conditions and toxin production potential of these pathogens provides information on the potential associated public health risk, and for the design of tailored bacterial control methods on the Victorian dairy farms.

Results
Identification of Bacillus cereus s. l. species A total of 50 isolates were obtained from soil, faeces, feed (grain), raw milk and milk filter samples (27 samples total) collected on seven dairy farms. Where possible, two B. cereus s.l. isolates were taken from each sample to yield a total of 50 isolates from 27 positive samples (i.e. 4 samples only yielded a single, confirmed B. cereus s.l. isolate).
All the isolates exhibited hemolytic activity on SBA plates and were able to grow at a temperature of 10°C, indicating that no B. anthracis (non-hemolytic) or B. cytotoxicus (minimum growth temperature of 20°C) species were among the isolate pool.
Fourteen isolates could not grow at 8°C, identifying them as B. cereus s. s., B. thuringiensis, or B. pseudomycoides. One of these exhibited a rhizoid colony morphology, and was classified as B. pseudomycoides. Of the remaining 13 isolates, three produced bipyramidal or round toxin crystals on sporulation, and were identified as B. thuringiensis (Figure 1). The remaining 10 isolates that were unable to grow at 8°C were classified as B. cereus s. s.
Another five isolates were incapable of growth at 40°C, implicating them as either B. mycoides or B. weihenstephanensis. Colony morphology was then used to differentiate these into B. mycoides (n = 3) showing rhizoid growth, and B. weihenstephanensis, with round to irregular colonies (n = 2).
The remaining 31 isolates were capable of growth at both 8°C and 40°C, exhibited a round to irregular colony morphology, and were implicated as either B. cereus s.s or B. thuringiensis. One of these isolates produced toxin crystals and was designated as B. thuringiensis; the other 30 isolates were identified as B. cereus s. s.

Growth profiles of B cereus s. l. species at low temperatures
The growth profile of one representative strain from each B. cereus s. l. species identified in this study (i.e. B. cereus s. s., Bc14-005; B. thuringiensis, Bc13-016; B. weihenstephanensis, Bc14-026; B. mycoides, Bc14-009; and B. pseudomycoides, Bc14-006) is shown in Figure 2. At 8°C the B. weihenstephanensis isolate reached exponential and stationary phase in the shortest times, with an average cell density of 5.55 ± 0.2 log(CFU/ml) measured on day 4. This was followed by the B. mycoides isolate, with an average cell density of 4.83 ± 0.09 log(CFU/ml), also measured on day 4. The B. cereus s. s. isolate reached numbers of 5.75 ± 0.17 log(CFU/ml) by day 6, at which time the representative isolates of both other species has reached stationary growth phase. Neither the B. thuringiensis nor B. pseudomycoides representative isolates grew at 8°C.
The B. weihenstephanensis isolate also grew to higher cell densities on days 2, 3 and 4 at 10°C, when compared with the other 4 species. All representative isolates, with the exception of the B. pseudomycoides isolate, reached stationary stage after approximately 5 days at 10°C; this took approximately 7 days for the B. pseudomycoides isolate.
At 12°C, highest cell densities after 2 days were observed for B. weihenstephanensis. Cell growth plateaued after 3 days (all except B. pseudomycoides) or 4 days (for the B. pseudomycoides isolate).
Genotypic diversity of B. cereus s. l.
PFGE subtyping was utilised to understand the population diversity in samples, across each farm environment, and comparatively across all farms ( Figure 3). Two isolates were taken from each positive sample to examine strain diversity among samples. Comparison of both isolates from each sample identified the same strain in only four of the samples; in all other samples, where two isolates were obtained, they were identified as different strains. Where indistinguishable PFGE profiles (pulsotypes) were obtained for both isolates from the same sample, one of these isolates was removed from the study (this identified 4 isolates as duplicated). This resulted in the speciation of the remaining 46 isolates as: 36 B. cereus s. s.; four B. thuringiensis; three B. mycoides; two B. weihenstephanensis; and one B. pseudomycoides. B. cereus s. s. was identified on all 7 farms, B. thuringiensis on 4 farms, and B. mycoides on Farms B and C. Both B. weihenstephanensis strains were recovered from Farm F, and the B. pseudomycoides isolate was from Farm B.
Population analysis of the 46 remaining isolates yielded 44 distinct pulsotypes; two of these pulsotypes were identified in multiple samples (each of these strains were identified in two separate samples). A pulsotype was shared by B. cereus s. s. isolates Bc13-013 (isolated from soil) and Bc13-012 (isolated from a faecal sample); both samples were from Farm F, taken on the first visit (December 2013). The other pair of isolates indistinguishable by PFGE were Bc13-009 and Bc14-016, isolated from soil samples collected from Farm D in December 2013 and again in March 2014.
No pulsotypes identified in this study were shared across multiple Farms, and a diversity of between three and nine unique pulsotypes were identified on individual farms. The PFGE profiles clustered in 3 major groups: Clades 1, 2 and 3. Clade 1 comprised 7 isolates; all the B. mycoides isolates clustered together in this group, together with one B. weihenstephanensis isolate. The rest of the isolates were B. cereus isolates. Eighty-six percent of members of this grouping were psychrotrophic and weakly hemolytic; only Bc14-020 (showing the lowest similarity among clade 1 isolates) did not grow at 8°C, and was also strogly hemolytic.    representative isolates produced Nhe enterotoxin in TSBYE after 48 h, however the B. weihenstephanensis representative isolate (Bc14-026) was the only isolate not to produce detectable Nhe after 24 h in TSBYE at this temperature. The B. mycoides Bc14-009 isolate was the only isolate to produce detectable Hbl enterotoxin under these conditions, after 48 h. With the exception of B. weihenstephanensis, all isolates produced higher amounts of Nhe in TSBYE at 25°C, when compared with production in 10% RSM. No detectable enterotoxin was produced by the B. pseudomycoides isolate in 10% RSM at 25°C, in contrast to strong Nhe production measured in TSBYE.
With the exception of B. cereus s. s. Bc14-005, all strains produced less enterotoxin at 37°C, in comparison with that measured at 25°C. B. thuringiensis was the only species with detectable Hbl toxin production at 37°C. No enterotoxin production was noted for B. weihenstephanensis in either TSBYE or 10% RSM at 37°C; interestingly, although all other representative isolates produced enterotoxin in TSBYE at this temperature, only the B. cereus s. s. and B. thuringiensis isolates produced detectable toxin in 10% RSM at 37°C.
Overall, toxin production was lowest at 12°C. Neither B pseudomycoides nor B. weihenstephanensis produced detectable Nhe or Hbl at this temperature; production of Nhe by representative isolates of the other three species was low, and no Hbl production was detected at 12°C.

Clostridium perfringens toxin subtyping
Among the 26 isolates, two toxin types were detectedtype A and type D ( Figure 4); all isolates contained alpha-toxin (type A isolates), whereas only 6 strains had both the alpha and epsilon-toxins (type D). The Clostridium perfringens enterotoxin and the beta2 toxin were not detected in any of the isolates. Thus, out of the 26 isolates, 77% were identified as type A and 23% as type D. Type A strains were present on every farm (A to G), whereas type D strains were found only on Farms D and F, which are caprine farms. On Farm D, the type D strains were isolated from feed (grain) and raw milk samples only. On Farm F, the type D strains were isolated from soil, milk and milk filter samples only. No type D strain was isolated from the animal faeces on these farms.

Discussion
Species of B. cereus s. l., along with C. perfringens, may cause illness in both humans and animals. These bacteria are commonly found in many environments, including soil. As such, there is potential for both infection of animals and entry in food chains through contamination of crops, raw milk, or food animals themselves. These organisms are of particular importance in food safety, as their spores may survive thermal treatments, such as pasteurization [16]. Characterization of their populations can thus provide insights into the biology of these organisms, along with associated risks of cross-contamination and infection. This study characterized isolates obtained from seven farms throughout Victoria, Australia, to understand aspects such as population structure, virulence traits, and toxin production.
The most common species identified among B. cereus s. l. isolates collected was B. cereus s. s., which comprised 78% of the population. Species diversity across different farm environments was varied; Farms A and E showed the lowest diversity, where only B. cereus s. s. was identified; all other farms harboured either two or three species.
Comparative analysis of the growth profiles of representative isolates for each of the 5 species at low temperatures suggested B. weihenstephanensis possessed the highest growth rates at all 3 temperatures examined (8°C, 10°C, and 12°C). This characteristic is reflected in the psychrotolerant nature of the organisms, which is known to grow at temperatures as low as 5°C [11,17]. The B. pseudomycoides isolate showed the slowest growth rates at 10°C and 12°C, and did not grow at 8°C. Taken together, these results suggest that toxigenic B. weihenstephanensis isolates may be of higher risk in cases of contaminated refrigerated food products that have experienced temperature adulteration; conversely, the comparative risk may be lower in the case of B. pseudomycoides. Although this data provides an indication of the growth performance of these isolates at low temperature, the duration of lag phase, or the exact maximum growth rate, would require additional bacterial counts in the early growth phase.
PFGE analysis was utilised to examine population diversity among B. cereus s. l. isolates. Of the 23 samples where 2 isolates were taken, only 4 contained the same pulsotype (i.e. the pair of isolates from the other 19 samples possessed different pulsotypes). This indicated a high diversity of B. cereus s. l. among samples. Indeed, it is probable the actual population diversity of B. cererus s. l. in a proportion of the positive samples would have been found to be greater than was identified in this study, had more isolates been taken from those samples. That withstanding, this analysis identified 46 distinct isolates from the original pool of 50.
Two Farm F isolates, Bc13-013 isolated from soil and Bc13-012 isolates from faeces, were indistinguishable by PFGE. Contaminated soil may lead to contamination of the feeding pasture, which in turn may lead to passage to the animal intestinal tract, this may be transient, or lead to colonization of the gut. Either scenario may account for this observation. In the case of the other two non-distinguishable profiles (Bc13-009 and Bc14-016), these were identified in soil samples collected from Farm D in December 2013 and again in March 2014. Both samples were collected from the same pasture field, although not collected at the same area of the field. This suggests a persistent pattern of colonization by this pulsotype in this particular field. Although this pulsotype was not identified in faecal samples from animals on the farm, further insights into its colonization across the farm, and in the herd, would necessitate more extensive sampling. Nonetheless, taken together, both instances where an individual pulsotype was identified in multiple samples highlights the contamination risk associated with resident strains in the farm environment; such bacteria will conceivably be found at higher frequency around the farm environment and in faecal shedding of the herd, and thus at higher risk of contamination of milk, and entry to the dairy food chain.
Further examination of the remaining 42 PFGE profiles indicated a diverse population, not only around individual farm environments, but across the farm environments at large. Each farm had its own distinct community of B. cereus s. l. isolates, with no shared PFGE profiles observed between farms; this observation should be contextualised in that the complete population diversity is likely more complex than this study identified. Farms had a minimum of between three and nine pulsotypes among samples taken, as determined by PFGE analysis. B. cereus s. s. dominated all farms, comprising a minimum of 63% of species in the case of Farm C, up to 100% of all species identified (Farms A and E). This suggests this species is the dominant member of these B. cereus s. l. populations. There was no apparent correlation with farm herd type (i.e. bovine, caprine or ovine) to a higher or lower diversity of isolate pulsotypes or number of species. Since isolation of the organisms does not involve an enrichment step, this does not introduce the possibility of certain isolates out-competing others in   Figure 4 Distribution of toxin types across all seven farms. Samples containing C. perfringens isolates included: milk filters, raw milk, faeces, soil and animal feed. the enrichment process, nonetheless possible competition between strains on the Mannitol Egg Yolk Polymyxin Agar (MYPA) medium used for isolation, or indeed the medium itself, may affect their isolation. Certain B. cereus s. l. isolates may produce bactericidal toxins that inhibit other isolates [18,19], and this may impact the presence of other isolates in localised environments, or the isolation on solid media such as MYPA. Interestingly, these toxins may be directed at inter-strain and -species competition [19]; which may impart a significant competitive advantage to colonisation of an environment that is potentially the primary reservoir of the organism. Such inhibition was observed among isolates in this study (Additional file 1: Figure S1). Enterotoxin production by B. cereus s. l. isolates is central to the manifestation of clinical illness. The emetic form of illness is attributed, in a large part, to the heat-stable cereulide toxin [20]; in cases of diarrheal illness, two of the important pore-forming toxins involved in this pathogenesis are Hbl and Nhe. Although some evidence suggests Nhe is the key toxin in cases of diarrheal illness [21], it is generally thought that this may not necessarily be true for all strains, and the toxins may in fact play a combined role in disease outcome [22]. To understand the implications of milk contaminated with B. cereus s. l., one representative isolate for each of the species identified on farms was grown in either 10% RSM or TSBYE medium at various temperatures (12°C, 25°C, or 37°C). Interesting, all isolates showed a trend of reduced toxin production in milk, when compared with TSBYE; the extent of which varied depending on the combination of temperature and time measured (24 h or 48 h). This may indicate a reduced risk of toxin production in milk and/or dairy products, relative to other food matrices, and thus for certain forms of B. cereus s. l.-induced illness as a result of ingestion of contaminated foodstuffs. Of the five species, the B. weihenstephanensis isolate produced the lowest detected Nhe toxin level under all conditions tested, when compared with other isolates. Production of Hbl toxin was lower than that of Nhe under all experimental conditions; indeed Hbl production was only detected for the B. mycoides and B. thuringiensis isolates. Contrary to this, all isolates produced Nhe. This suggests that all of these isolates may illicit diarrheal disease in human hosts under favourable conditions. Although production of enterotoxins was greatest at 25°C, production of Nhe was identified at 37°C for all isolates, with the exception of B. weihenstephanensis. This again implies the capacity of these isolates to cause diarrheal illness in humans, as this reflects the temperature conditions in vivo. That withstanding, based on the observation that no enterotoxin was detected in 10% RSM at 37°C, the food vector involved in the infection may play some role in the disease outcome. This may be reflected in the low prevalence of dairy products among foods linked to B.cereus s. l. food-poisoning outbreaks [23,24]; however, this does not preclude illness through consumption of contaminated foods, as entertoxin production may also occur in the gut. Toxin production was lowest at 12°C, suggesting that temperature-controlled contaminated food products, such as those refrigerated, may also have a lower associated risk of causing illness. Overall, the B. cereus s. s. and B. thuringiensis isolates showed highest levels of toxin production across the range of conditions (medium, temperature, time) tested.
Two toxin types were identified among the C. perfringens isolates: type A, which may cause foodborne illness in humans [25], however can often be part of the normal intestinal microflora of humans and animals [26]; and type D, which are particularly associated with disease in caprine and ovine animals [27]. Type D isolates were only identified on caprine farms. These results are consistent with the finding of previous studies that showed that C. perfringens type D strains are predominantly associated with goats and sheep, rather than cattle [28]. None of the type D isolates identified in this study were found among faecal samples tested (Figure 4), however milk and/or milk filter samples on both farms harbouring type D strains were positive. These results indicate that transmission may have occurred through another route, without intestinal carriage by, or infection of the animals. Type A strains are more ubiquitous and have been found in cattle, as well as caprine and ovine populations [28]. Type A isolates were found on all seven farms in this study, in a diverse range of samples ( Figure 4). All type A isolates (along with all type D isolates) did not contain either the C. perfringens enterotoxin gene (cpe), or the beta2 toxin gene (cpb2), as determined by the PCR screening method (data not shown) [29]. Beta2 toxin has been implicated in bovine enteritis cases [28,29], suggesting the isolates in this study may be of lower risk of disease to the herds on the associated farms.

Conclusion
This study provides insights to the diversity of the Bacillus cereus group of bacteria and Clostridium perfringens on farm populations in Victoria. First of all, both species were isolated from all seven farms and from the same types of samples. On each farm, the B. cereus s. l. population was found to be diverse and distinct to each localised environment. Enterotoxin production in milk was limited compared to broth in the same conditions, suggesting that enterotoxin production by B. cereus s. l. may be reduced for dairy products. Type A isolates were dominant among the C. perfringens population; however the presence of type D isolates on two of the three caprine farms may be a potential concern with regards to the risk of enterotoxemia in goats.

Bacterial isolates in this study
Isolates were obtained from soil, faeces, feed (grain), milk and milk filter samples from 7 dairy farms across Victoria, Australia (3 bovine, 3 caprine, and 1 ovine). Growth of B. cereus s. l. isolates at 8°C and 40°C Isolates were streaked on in Brain-Heart Infusion (BHI) agar plates and incubated overnight at 30°C. For each isolate, one colony was picked from the BHI plate with an inoculating loop and mixed into 5 ml of BHI broth. After overnight incubation at 30°C the cultures reached a concentration of approximately 10 8 -10 9 CFU/ml. For growth assessment at 8°C, serial dilutions were prepared and 3 ml cell suspensions were inoculated with the test isolate at a concentration of 10 2 CFU/ml. Growth was assessed after 14 days by measuring the optical density of the culture at 610 nm. For growth assessment at 40°C, test isolates were streaked on BHI agar plates and incubated at 40°C. Growth was examined after four days. All growth/ no growth determinations were repeated in duplicate to confirm the final result.
Hemolysis testing of B. cereus s. l. isolates Trypticase Soy agar plates with 5% of Sheep Blood were divided in 6 equal sections and inoculated with 24 h BHI broth culture suspensions by touching the surface of the agar with a 1 μl loop. Plates were incubated for 24 h at 35°C and examined for hemolytic activity. Hemolytic strains were identified by a translucent halo of 2 to 4 mm of hemolysis.

Production of toxin crystals by B. cereus s. l. isolates
Analysis of toxin production was carried out based on the FDA standard method, which minimised sequential culturing, and utilises media without curing agents, to reduce the chance of plasmid curing [30]. Nutrient agar slopes were inoculated with a 1 μl loopful of a fresh colony. The slants were incubated for 24 h at 30°C then 4 to 5 days at room temperature, until all the strains sporulated. Smears were prepared with sterile distilled water, air-dried then heat-fixed by passing the slide through the flame of a bunsen burner. The slides were then flooded with methanol. After 30 s the methanol was poured off and the slides air dried. The slides were then flooded with 0.5% basic fuchsin and gently heated from below with a small Bunsen burner until steam was seen. The slides were let to stand for 1 to 2 min before repeating this last step. The slide was then rinsed thoroughly with distilled water, and a cover slip was mounted. The slides were examined for the presence of toxins under 100X magnification using a Leica DM6000B microscope (Leica Microsystems, Germany) with immersion oil.
Growth kinetics of B. cereus s. l. isolates at low temperatures Isolates were streaked onto BHI agar plates and incubated overnight at 30°C. For each isolate, one colony was picked from the BHI plate with an inoculating loop and mixed into 5 ml of BHI broth. After overnight incubation at 30°C the cultures reached a concentration of approximately 10 8 -10 9 CFU/ml. After serial dilutions, 3 ml cell suspensions with an approximate concentration of 10 2 CFU/ml were set in 12-well plates. Sterile BHI broth was used as a negative control and a blank to standardise the data.
The plates were incubated at 8°C, 10°C and 12°C over a period of 14 days. Growth was monitored by taking optical density measurements at 600 nm with a platereader spectrophotometer. Growth curves for each strain and temperature were then plotted, using standard curves generated for each isolate to equate absorbance readings to log(CFU/ml) (data not shown). All growth curves and standard curves were measured in triplicate, with each replicate performed on separate occasions. PFGE subtyping of B. cereus s. l. isolates An in-house method was developed for PFGE subtyping of B. cereus s. l. isolates. Test isolates were streaked on BHI agar plates and incubated for 14-18 h at 30°C. Cell suspensions were then prepared in 2 ml TE buffer (10 mM Tris:1 mM EDTA, pH 8.0), to a final cell optical density of 1.00 at 600 nm, as measured on a spectrophotometer. 400 μl of the cell suspension was mixed with 20 μl of a lysozyme solution (20 mg/ml) and incubated for 2 h at 56°C. Following 1% PFGE-grade agarose (SeaKem Gold; Lonza, Switzerland) was prepared with TE buffer and equilibriated at 55°C. 20 μl of a Proteinase K solution (20 mg/ml) was added to the cell suspensions, followed immediately by 400 μl of the 1% SKG agarose and gently mixed. The plugs were then cast in disposable plug molds (Bio-Rad, UK). After 10 to 15 min at room temperature the plugs were transferred in a Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) containing 25 μl Proteinase K solution (20 mg/ml). The plugs were lysed overnight in a 56°C shaker water bath at 160 rpm. Prior to plug washing, the water bath temperature was reduced to 50°C and the wash solutions were pre-warmed before use. To wash the plugs, the lysis buffer was drained from the tube, and replaced with 5 ml sterile molecular grade water. The tube was then replaced in the water bath at 50°C, and shaken at 160 rpm for 10-15 min. One more wash with molecular grade water was repeated as before, followed by four washes with TE buffer. Plugs were then transferred to 1.5 ml centrifuge tubes filled with 1 ml sterile TE buffer and stored at 4°C until used.
For PFGE analysis, DNA plugs were restricted using either SbfI or NotI endonucleases (New England Biolabs, US). For both enzymes, 40U was incubated into a 200 μl reaction mix at 37°C for 2 h. After restriction, the plugs were equilibrated for 10-15 min in 0.5X Tris Borate EDTA buffer (40 mM Tris-HCl: 45 mM boric acid: 1 mM EDTA ) at room temperature.
DNA fragments were separated on a 1% SeaKem Gold agarose gel using a CHEF-Mapper (Bio-Rad, UK) with the following program: auto algorithm; low molecular weight = 20 kb; high molecular weight = 700 kb; select default values except where noted by pressing "enter"; run time = 21 h; initial switch time = 5 s; final switch time = 80 s; tank temperature = 14°C. The Saccharomyces cerevisiae CHEF DNA Size Marker (Bio-Rad, UK), already restricted, and Salmonella ser. Braenderup H9812 chromosome, prepared according to the Standard Operating procedure for PusleNet PFGE for Salmonella [31] and restricted with 50 U XbaI restriction endonuclease (New England Biolabs, US), were used simultaneously as DNA-size standards for analysis. Gels were observed after staining 30 min in a 300 ml 1% GelRed (Biotium, US) solution and de-staining for 40 min in a 300 ml distilled water bath. Cluster analysis was performed with BioNumerics v6.5 software (Applied Maths, Belgium). Similarity matrixes were produced using the Dice coefficient for the two experiments (digestion with NotI and SbfI), then a combined analysis was performed using the unweighted pair group method with arithmetic mean (UPGMA), with optimisation and tolerance settings of 1%.

Semi-quantitative toxin production assay
The production of the enterotoxins Nhe and Hbl was investigated with five isolates, representing each of the Bacillus cereus s. l. species isolated in this study: B. cereus s. s. (Bc014-005), B. pseudomycoides (Bc14-006), B mycoides (Bc14-009), B. weihenstephanensis (Bc14-026), and B. thuringiensis (Bc13-016). Toxin production was measured at three temperatures: 12°C, measured at 48 h; and 25°C and 37°C, both measured after 24 h and 48 h. Toxin production was assayed in two test media: 10% reconstituted skim milk (RSM) or tryptic soy broth with yeast extract (TSBYE), separately for each representative isolate. Overnight cultures grown at 30°C were diluted to a concentration of approximately 10 6 CFU/ml in the test medium. A final concentration of 10 4 CFU/ml in 10 ml of the test medium was used for each test. The tubes were incubated without agitation. The Nhe and Hbl toxins were detected using the Duopath Cereus Enterotoxins detection kit (Merck, US). For 10% RSM cultures, 100 μl was added to 900 μl sterile molecular grade water; for TSBYE broth cultures, 100 μl of sample was added to 100 μl 10% RSM in 800 μl sterile molecular grade water. A volume of 150 μl was the applied to the toxin test chip and results were assessed after 30 min, according to the product datasheet. The intensity of the line was scored as -(no line), + (faint line), ++ (clear line), +++ (thick clear line), to achieve a semi-quantitative measure of toxin production.


extfieldsofstudy: Biology Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2015.jsonl.gz.gz

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Intestinal Microbiota Succession and Immunomodulatory Consequences after Introduction of Lactobacillus reuteri I5007 in Neonatal Piglets

Seventy-two, suckling piglets, obtained from 9 litters standardized to 8 piglets, were assigned to 1 of 3 treatments (n = 24) to compare short-term, early administration with intermittent, longer-term administration of Lactobacillus reuteri I5007. The treatments were a control (given a placebo of 0.1% peptone water from day 1 to 5) or treatments in which 1.7 × 1010 CFU L. reuteri was administrated either daily for 4 days starting on day 1 or every 4th day from day 1 to 17. Five piglets per treatment were killed at 3 time points (day 7, 14 and 21). Denaturing Gradient Electrophoresis of ileal digesta revealed an increase in the presence of L. reuteri I5007 and Clostridium lentocellum (on day 14 and 21) in the every 4th-day treatment and Actinobacillus porcinus (on day 7 and 14) in both L. reuteri treatments, while reducing the abundance of E. coli on day 21 in the every 4th-day treatment. Real-time qPCR of ileal digesta showed an increase in Bifidobacterium spp. on day 14 for both L. reuteri I5007 treatments. An increase in the concentration of lactic acid and a lower pH was observed in the first 4-day treatment on day 7 and the every 4th day treatment on day 14. The relative abundance of mRNA for TGF-β was increased while that for IFN-γ was decreased in the mesenteric lymph nodes of piglets treated with L. reuteri every 4th day. In conclusion, early intervention with L. reuteri increases the presence of beneficial bacteria and decreases the presence of undesirable microbes in the lower gastrointestinal tract. The changes appear to be mediated by altering the intestinal pH through lactic acid production resulting in favorable bacterial species colonization. A prolonged duration of treatment (i.e. every 4th day) would appear to be superior to treatment only during the first 4 days.

Introduction
The presence and composition of the microbiota in the intestine in early life makes a key contribution to the establishment of gut homoeostasis and maturation of the immune system [1][2][3]. Microbial colonization is a complex process made more challenging by the low diversity of intestinal microbes and the under developed immune system of neonates [4,5]. However, a large number of studies have demonstrated that early intervention with desirable microbes may help to establish a stable bacterial ecology and enhanced immunological development [5][6][7][8]. Thus, strategies resulting in the establishment of an effective gut ecosystem should be implemented in early life.
Lactobacilli are normal inhabitants of the gastrointestinal tract in both humans and animals and have been intensively researched during the past several decades for their specific probiotic properties and effects on the intestinal microbiota [9,10]. Lactobacillus reuteri (L. reuteri) I5007, initially known as L. fermentum I5007, was isolated in our laboratory from the colonic mucosa of healthy, weaned piglets. It has been shown to possess many excellent probiotic characteristics, including digestive enzyme tolerance, high adhesion to Caco-2 cells, competitive exclusion against pathogen invasions and alleviation of weaning stress in piglets [11][12][13].
Research has shown that oral administration of L. reuteri I5007 can improve performance, reduce the incidence of diarrhea and improve the intestinal health of suckling piglets [14]. However, detailed knowledge about the mechanism through which L. reuteri mediates its effects is not yet available. In addition, there is limited information available about the most appropriate dosing regimen. The only published research in which L. reuteri has been administered to suckling piglets involved daily administration for the first 14 days of life [14]. From a management perspective, this is very labor intensive. It is not known whether treatment for a shorter period of time or the use of an intermittent treatment regimen can also be effective in establishing an effective gut ecosystem.
The aim of the current study was to investigate the effects of L. reuteri I5007 administration on the microbial populations in the lower gastrointestinal tract, short chain fatty acid (SCFA) and pH levels in the colon, and cytokine and transcription factor expression in the mesenteric lymph nodes of suckling pigs. In addition, a comparison was made of short-term administration (i.e. daily for 4 days starting on day 1) with longer term intermittent administration (i.e. every 4th day from day 1 to 17). Our hypothesis was that intermittent treatment for a longer period of time would be superior to short-term daily administration.

Ethics statement
The procedures used in this experiment were approved by the China Agricultural University Institutional Animal Care and Use Committee (Beijing, China).
Growth and storage conditions for L. reuteri I5007 L. reuteri I5007 was cultivated in sterile De Man Rogosa Sharpe media for 24 h at 37°C in an anaerobic environment, followed by centrifugation at 5000 x g for 10 min at 4°C. The cells were re-suspended in reconstituted skim milk (20% wt/vol) and immediately freeze-dried. The freeze-dried powder containing 1.7 x 10 10 colony forming units (CFU)/g was stored in sealed packets at a temperature of 4°C until needed.

Animals and treatments
Nine, Large White x Landrace, crossbred sows were artificially inseminated by one Duroc boar to minimize the genetic variation among their off-spring. The sows were allowed to farrow in concrete-floored farrowing pens at the China Agricultural University Swine Research Facility located in Beijing, China (non-SPF facility) and litter size was standardized to 8 piglets. The piglets which remained in the litter were selected so that each litter was balanced for average weight and sex. All surplus piglets were cross fostered to sows not on the experiment.
One day 1, the litters were allocated to 1 of 3 treatments with 3 litters of 8 piglets assigned to each treatment (n = 24). The treatments comprised a control treatment (piglets given a placebo of 4 mL of 0.1% peptone water on days 1 to 5) or two experimental treatments in which 1.7 × 10 10 CFU L. reuteri was administrated either daily from day 1 to 5 or every 4th day from day 1 to 17 (i.e. days 1, 5, 9, 13 and 17). One gram of L. reuteri was dissolved in 4 mL of 0.1% peptone water for oral administration to each pig using a 5 mL syringe.
Piglets were given iron supplementation and had their teeth and tails cut within the first three days life, but had no access to creep feed during the experiment and therefore relied on sow's milk as their sole source of nutrients. On days 7, 14 and 21, five piglets closest to the average weight for each treatment (regardless of litter) were euthanized with electricity, and the luminal contents from the ileum and proximal distal colon were collected and then stored at -80°C . On day 21, mesenteric lymph node samples, were excised from the ileum of each piglet, and stored at -80°C until total RNA was extracted.

Determination of colonic short chain fatty acid (SCFA) concentrations and pH
The SCFA in the colonic digesta were measured using a gas chromatographic method following the preparation procedures of Franklin et al. [15] using a HP 6890 Series Gas Chromatograph (Hewlett Packard, Palo Alto, CA) equipped with a HP 19091N-213 column with 30.0 m × 0.32 mm i.d. (Agilent, Palo Alto, CA). The pH of the digesta samples was measured using a digital IQ150 pH meter (Spectrum, Chicago, IL).

DNA isolation
To improve the amount and quality of DNA, the luminal samples (approximately 0.2 g) were beaten with glass beads, using a Mini Bead Beater (BioSpec Products, Bartlesville, OK) according to Zoetendal et al. [16]. The DNA was then extracted using a QIAamp Stool Mini-Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The concentration of extracted DNA was measured using a NanoDrop Spectrophotometer (P330, Implen, Germany).

Denaturing gradient gel electrophoresis (DGGE)
The luminal contents (ileal and proximal colon) from individual piglets (n = 4 per treatment) were used to analyze the profile of the gut microbiota using DGGE methodology. Due to lack of space on the gels, only 4 piglets per treatment were analyzed. The 4 piglets chosen for DGGE analysis were randomly selected from the 5 pigs slaughtered at each time period.
Amplification programs were set using a Bio-Rad Thermocycler (Bio-Rad, Hercules, CA) with an initial denaturation at 94°C for 3 min followed by 35 cycles consisting of denaturation at 94°C for 30 s, annealing at 56°C for 20 s, and extension at 68°C for 40 s. The final extension was set at 68°C for 7 min. The amplicons were checked by electrophoresis on 1% (wt:vol) agarose gel in 1% SYBR Green I (Sigma, St, Louis, MO) to verify that a single product of the expected size was obtained.
The target amplicons were separated by DGGE. Electrophoresis was performed on 8% (wt: vol) polyacrylamide gels using a vertical gradient of denaturants between 45 and 58% (a 100% denaturing solution was defined as 40% (vol:vol) formamide and 7 M urea). For each amplicon, the same concentration of DNA was loaded in one lane of the gel.
Electrophoresis was initiated by pre-running for 10 min at 200 V and then for 16 h at 85 V in 0.5 x Tris-acetate-EDTA buffer at a constant temperature of 60°C in a DCode System Apparatus (Bio-Rad, Hercules, CA). The DGGE gels were stained by SYBR Green I (Sigma, St, Louis, MO) while being agitated for 15 min in an orbital shaker (Qilinbeier, Haimen, China) at room temperature, and then photographed with a Gel Doc XR + System (Bio-Rad, Hercules, CA), and analyzed with Quantity One Basic 4.6.6 (Bio-Rad, Hercules, CA).
The total number of bands and the Shannon-Weaver index were used to quantify microbial diversity as described by Gafan et al. [18]. Predominant bands (five bands) in pre-run DGGE gels were excised, purified and amplified. Amplicons of the V6-V8 regions of 16S rRNA from the pure strain of L. reuteri I5007 were prepared by mixing equal amounts and used as the marker for gel to gel comparison.
Bands of interest were excised from the original gels and their DNA fragments were reamplified under the same conditions using corresponding primers without the GC Clamp. The PCR products were purified and concentrated using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and then transformed into E. coli JM109 using the pGEM-T vector system (Promega, Madison, WI) according to the manufacturer's instructions. The transformants were grown on Luria-Bertani agar in the presence of 100 μg/mL ampicillin and 20 μg/mL X-Gal. The positive recombinants (3 clones on each band) were screened and identified by PCR and sent for sequencing (Shanboyuanzhi Service, Beijing, China). Homology searches were performed using the BLAST program in the NCBI database (http://blast.ncbi. nlm.nih.gov/).

Nucleotide sequence accession numbers
The sequences presented in Table 1 were deposited in the EMBL databases under the accession numbers HF952840-HF952849, HF952850-HF952858, and HF952859-HF952864 for the ileal profile, colonic profile and the marker respectively.

Quantification of targeted genus groups in the luminal contents by realtime qPCR
The abundance of targeted bacteria in the intestinal luminal contents were analyzed by realtime qPCR. Concentrations of Bifidobacterium spp., Lactobacillus spp. and total bacteria were determined in both ileal and colonic digesta on days 7, 14 and 21. The primers used are listed in Table 2.
Standard curves were constructed using the PCR products of Lactobacillus acidophilus ATCC 4356, Bifidobacterium longum ATCC 15707 and Escherichia coli ATCC K88. Bifidobacterium longum ATCC 15707 was grown in trypticase-peptone-yeast extract-glucose media and  15 pmol of each primer, and 2 μL of DNA template was used. PCR protocols for the above bacterial genus were obtained by a linear regression curve and then log copy numbers were plotted against C T values. The standard curves had correlation coefficients of 0.994 for Bifidobacterium, 0.991 for Lactobacillus, and 0.993 for total bacteria. The linear range of the standard curves for Bifidobacterium spp., Lactobacillus spp., and total bacteria was the same and ranged from 2 to 9 log (copy/g).

Real-time PCR analysis of the targeted genes in the mesenteric lymph nodes
Total RNA was isolated from the mesenteric lymph nodes using a RNeasy Mini Kit (Qiagen, Hilden, Germany). The purity was determined by the ratio of A 260 :A 280 using a NanoDrop Spectrophotometer (P330, Implen, Germany) and then the quality was checked with 1% agarose gel electophoresis following the procedures outlined by Aranda et al. [28]. The extracted RNA was converted into first-strand cDNA by reverse transcription of 1 μg of total RNA using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer's protocol and stored at -80°C. Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Singapore) using SYBR Green PCR Master Mix (Takara, Dalian, China). All reactions were run in triplicate. The mRNA expression is expressed as the ratio between the targeted gene and β-actin gene expression.

Data analysis
Statistical comparisons of measured indices among the three treatments were performed by Analysis of Variance followed by Student Neuman Keuls's multiple-comparison test using the Statistical Analysis Systems Statistical Software Package Version 8.02 (SAS Institute, Cary, NC). Individual pig served as the experimental unit. Differences at P 0.05 were considered significant.

Analysis of the microbiota populations using PCR-DGGE analysis
To compare the different species of bacteria present in the gastrointestinal tract among the treatments, the V6-V8 regions of 16S rRNA PCR amplicons were analyzed and are presented in Figs. 1 and 2 respectively. Each profile had thirty-six sample lanes. The bands marked in the profiles represent microbial populations of interest in the luminal contents, and their presence or absence, reflect changes in microbial community succession.
From the profiles (Fig. 1), we observed that L. reuteri I5007 administration successfully altered the microbiota composition of the ileal digesta. The main effects of L. reuteri I5007 on the bacterial communities were in increasing the abundance of Clostridium lentocellum (on day 14 and 21) in the every 4th-day treatment and Actinobacillus porcinus (on day 7 and 14) in both L. reuteri treatments, while reducing the relative abundance of E. coli on day 21 in the every 4th-day treatment. Lactobacillus vaginalis was observed to increase in abundance with both L. reuteri I5007 treatments on day 7 while the L. reuteri I5007 strain was also increased at this time point compared with the control treatment. We also observed that Clostridium perfringens, Clostridium spp., Streptococcus henryi and Escherichia coli were the predominant inhabitants in the ileal contents during the experiment, with an increase in the proportions of Streptococcus henryi on day 14 and 21 while there was a reduction in Clostridium perfringens on day 21.
Compared with the bacterial composition in the ileal contents, the bacteria in the colonic samples were more diversified (Fig. 2, Tables 3 and 4). By day 21, there was approximately a 2-fold difference in the number of bands found in the colonic digesta compared with the ileal  digesta. In addition, the Shannon diversity index was higher in the colonic digesta than in the ileal digesta. At day 21, the number of bands in the ileal digesta in the first 4-day treatment was significantly lower than for the other two treatments. For colonic digesta, there was no difference in the number of bands or in the Shannon diversity index as a result of treatment.
Clostridium clostridioforme, Bacillus weihenstephanensis and Clostridium glycolicum were the predominant species observed in the colon throughout the experiment, which was different from the profile in the ileum. Numbers of Clostridium glycolicum were increased with both L. reuteri I5007 treatments, while the Clostridium clostridioforme like phenotype was decreased in proportion in the every 4th-day treatment on day 7 and 21. Numbers of Clostridium aldenense were decreased on day 21 in the every 4th-day treatment. An increase in the numbers of Clostridium aldenense in the first 4 day treatment was observed compared with the other two treatments on day 14. The L. reuteri I5007 strain (99% identity) was positively detected on day 7 in both ileal and colonic profiles for both L. reuteri I5007 treatments.

Quantification of targeted bacteria in ileal and colonic digesta by qPCR
Analysis of targeted bacteria by qPCR revealed that the abundance of Lactobacillus spp. did not change with either L. reuteri I5007 treatment in either ileal or colonic digesta (P > 0.05. Fig. 3). Numbers of Bifidobacterium spp. in the ileal digesta were significantly increased with both L. reuteri I5007 treatments compared with the control treatment on day 14 (P < 0.05), while the numbers of total bacteria also showed a similar trend (P < 0.05) in the ileal digesta on day 21.

pH and short chain fatty acid concentrations in colonic digesta
On day 7, a lower pH in the colonic digesta, accompanied by an increased concentration of lactic acid was observed in the first 4 days treatment compared with the control treatment (P < 0.05. Table 5) while no significant differences were observed between the control and the every 4th-day treatment (P > 0.05). On day 14, the pH in the colonic digesta of pigs in the every 4th-day treatment was significantly lower compared with the control treatment (P < 0.05), and lactic acid concentrations were observed to be significantly higher (P < 0.05). No significant differences were found between the control and the first 4 days treatment (P > 0.05). On day 21, no differences were observed among the three treatments in either pH or lactic acid concentrations in the colonic digesta (P > 0.05). The concentrations of acetic acid, propionic acid and butyric acid were not altered by treatment on any day.

Relative mRNA expression of cytokine and transcription factors in the mesenteric lymph node
At the end of the experiment on day 21, the relative transcription levels of various cytokines and transcription factors in the mesenteric lymph node were analyzed using real-time qPCR (Fig. 4). The relative abundance of mRNA for TGF-β was increased while that for IFN-γ was decreased in the mesenteric lymph nodes of piglets treated with L. reuteri every 4 th day while the values for piglets treated with L. reuteri in the first 4-day treatment were intermediate to the other two treatments. mRNA levels for IL-1β, IL-4, IL-6 IL-10, IL-17, IL-12p40, TNF-α, and Foxp3 were unaffected by dietary treatment.

Discussion
Immediately after birth, the gastrointestinal tract of neonates is involved in a process of colonization and succession by a complex and dynamic community of microbiota [29,30]. Accumulated evidence has demonstrated that early exposure with desirable microbiota followed by an appropriate colonization process in early life could alter the pattern of microbial succession as well as immunological maturation [31,32]. This experiment examined the impact of L. reuteri administration on the microbial populations in the gastrointestinal tract, VFA and pH levels in the colon, and cytokine and transcription factor expression in the mesenteric lymph nodes. In order to function as a probiotic and have the ability to alter the microbial populations in the lower gastrointestinal tract, L. reuteri I5007 must be able to survive passage through the upper gastrointestinal tract and colonize in the lower intestinal tract. It did so successfully, as shown by it being positively identified in both the ileal and colonic digesta on day 7. However, we were unable to identify L. reuteri I5007 in either ileal or colonic digesta on day 14 and 21. This can be explained by the fact that bacterial populations can only be detected by DGGE when they comprise more than 1% of the total bacterial community [33]. The microbiota in the gut slowly build up after the birth and L. reuteri I5007 can temporarily dominate an unstable intestinal microbiota (on day 7). As the piglets age, the abundance of other gut bacteria increased. As a result, L. reuteri I5007 only comprised a small proportion of the gut bacteria community on days 14 and 21 and therefore could not be detected by DGGE. Having proven that L. reuteri I5007 was able to survive passage through the upper gastrointestinal tract and colonize in the lower intestinal tract, the next step was to determine what effect it had on the numbers of beneficial bacteria in the lower intestinal tract. Accumulated studies have demonstrated that probiotic exposure could contribute to an increase in the numbers of Bifidobacteria and Lactobacilli in the luminal environment [34,35], and our experiment showed similar findings as evidenced by the increased abundance of L. vaginalis and L. reuteri I5007 shown by DGGE of ileal digesta while real-time qPCR of the ileal digesta showed an increase in the number of Bifidobacterium spp. on day 14 for both L. reuteri I5007 treatments.
The increased abundance of Lactobacilli and Bifidobacteria was accompanied by a decreased abundance of E. coli in the ileal digesta in the every 4th-day treatment throughout the experiment. Researchers have reported that E. coli are normal inhabitants in the intestine of neonates but can also act as opportunistic pathogens, and the reduction of these putrefactive bacteria may reduce the probability of pathophysiology disorders [36,37]. Based on above collective data we conclude that the exogenous strain of L. reuteri I5007 could temporarily dominate in the intestinal communities and manipulate the microbiota profile resulting in an increase in beneficial bacteria and a decrease in pathogenic bacteria.
It is reasonable to speculate that the alterations in the microbial community due to the exposure to L. reuteri I5007 are mediated by the reduced pH observed in the lower gastrointestinal tract as a result of L. reuteri I5007 administration. On day 7, a lower pH in the colonic digesta, accompanied by an increased concentration of lactic acid was observed in the first 4 days treatment compared with the control treatment. On day 14, the pH in the colonic digesta of pigs in the every 4th-day treatment was significantly lower compared with the control treatment and lactic acid concentrations were observed to be significantly higher. This theory is supported by  a previous study which observed that high numbers of lactobacilli could shape the gastrointestinal niche by altering the environmental pH through lactic acid production and favorable bacterial species colonization [32]. Short chain fatty acids are the major products from hindgut fermentation processes and have been considered as contributing an important role in normal gut maintenance [3,38]. Our previous work [15] and that of others [39] indicated that propionic acid is usually the second most prominent VFA in colonic digesta. In the present experiment, the quantity of propionic acid in all samples was very low in comparison to acetic and butyric acid. We have no explanation for this apparent discrepancy.
An important finding of the present study was that the microbial communities found in the ileum were different from those seen in the colon. We observed that Clostridium perfringens, Clostridium spp., Streptococcus henryi and Escherichia coli were the predominant inhabitants in the ileal digesta while Clostridium clostridioforme, Bacillus weihenstephanensis and Clostridium glycolicum were the predominant species observed in the colon.
The major microorganisms detected in the present study are all the part of the normal microbiota of the digestive tract of piglets. Clostridium spp. is a broad genus, and are ubiquitous in the gastrointestinal tract [40]. In addition, previous studies have shown that the intestine of neonatal piglets is initially colonized by large numbers of E. coli and Streptococcus spp. [41], which become the dominant bacteria at the end of the first week of life and remain present for the entire suckling period [42]. We also found these strains in the ileum of neonatal piglets in the present study. Bacteroides and low densities of Eubacterium, Fusobacterium, and Propionibacterium are usually found in the suckling period [42,43], but they were not detected in the present study due to the limitations of the method used.
Not only did the most prevalent microbiota differ between the ileum and the colon but bacterial diversity varied as well. By day 21, there was approximately a 2-fold difference in the number of bands found in the colonic digesta compared with the ileal digesta. In addition, the Shannon diversity index was higher in the colonic digesta than in the ileal digesta. Konstantinov et al. [44] reported that a higher number of DGGE bands were observed in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) of weaning piglets. The present study showed that the number of DGGE bands in the colon was lower than Konstantinov et al. [44] described. However, the number of bands was consistent with what Liu et al. [14] observed in neonatal piglets. Potential reasons for the discrepancy between Konstanintov et al. [44] and the results of the present study and that of Liu et al. [14] were that the samples were collected at different ages and the piglets were fed different diets.
Based on the findings reported above, it is evident that one mechanism through which L. reuteri I5007 functions is by increasing the presence of beneficial bacterial species in the lower gastrointestinal tract and decreasing the presence of undesirable microbes. We also studied the immunological consequences of early intervention with L. reuteri I5007.
A mature intestine harbors millions of commensal bacteria with thousands of species [45,46]. Thus, it is essential that the intestinal mucosa and epithelial cells develop an organized and elaborate mechanism for fighting pathogenic microbes. A beneficial microbial community could contribute to the development and maturation of the immune system [1]. Recently published data have proven that early exposure to beneficial microbial during the crucial stage of immune maturation is essential for the development of an effective immune system [2,7,47], and targeted microbes can be artificially manipulated to favor a beneficial gut colonization and also future immune system responses [6,48].
IFN-γ, which is commonly used as a marker for pro-inflammatory responses [49], was observed to be significantly reduced in the every 4th-day treatment compared with piglets in the control group. In one of our previous studies, L. reuteri I5007 was found to decrease the mRNA expression of the pro-inflammatory cytokine IL-1β in the ileum of neonatal piglets [14]. In contrast, TGF-β, which has generally been regarded as mediating immune tolerance and preventing excessive immune responses [50], had an increased expression in the mesenteric lymph node after treatment with L. reuteri in the every 4 th day treatment. Taken together, these data suggest that one explanation for the improved performance noted in previous studies with piglets treated with L. reuteri [14] could be a mitigation in the magnitude of immune responses thus allowing a greater proportion of the nutrients contained in feed to be directed towards growth rather than the immune system.
Probiotics have been widely studied in humans and animals. However, many questions regarding the use of probiotics remain to be answered. There is very limited information available about appropriate dosing regimens [51].
A previous study conducted in our laboratory showed that oral administration of L. reuteri I5007 improved suckling pig performance. However, in that study, L. reuteri was administered daily to suckling piglets for the first 14 days of life [14]. From a management perspective, this is very labor intensive. It is not known whether treatment for a shorter period of time or use of an intermittent treatment regimen can be effective in establishing an effective gut ecosystem. In the present study, differing durations of treatment (first 4-day treatment and every 4 th day treatment) of L. reuteri I5007 on gut microbial succession as well as immunological consequences in neonatal piglets were assessed. Based on the lower pH and lactic acid concentrations in the colonic digesta on day 14 as well as the increase in the relative abundance of mRNA for TGF-β and the decreased relative abundance of mRNA for IFN-γ observed in the mesenteric lymph nodes of piglets treated with L. reuteri every 4 th day, we suggest that prolonged treatment using the every 4 th day regimen is superior to treatment only during the first 4 days.
A shortcoming of the present study was that only one negative control group was used in the present study design with administration of 0.1% peptone water from day 1 to 4 while there was no similar control group for the pigs in the every 4 th day treatment. The lack of a second control group was a conscious decision due to lack of space on the gels and the general intensity of the work. It was also assumed that the placebo of 0.1% peptone would not affect intestinal microbiota succession and immunomodulatory consequences. However, animals in all three treatments were the same age and weight and the difference in pepton-placebo administration was the only confounding factor in the experiment.
In conclusion, early intervention with L. reuteri I5007 increases the presence of beneficial bacterial species and decreases the presence of undesirable microbes. The changes appear to be mediated by altering the environmental pH through lactic acid production resulting in favorable bacterial species colonization. The changes observed in the levels of the mRNA expression for IFN-γ and TGF-β in the mesenteric lymph nodes suggest that one explanation for the improved performance noted in previous studies with piglets treated with L. reuteri could be a reduction or prevention of an excessive immune response thus allowing a greater proportion of the nutrients contained in feed to be directed towards growth rather than the immune system. A prolonged duration of treatment (i.e. every 4 th day) would appear to be superior to treatment only during the first 4 days of life.