In Vitro Characterization of Chicken Gut Bacterial Isolates for Probiotic Potentials
Lactobacillus Strain Ecology and Persistence within Broiler Chickens Fed Different Diets: Identification of Persistent Strains
ABSTRACT
Lactobacilli are autochthonous residents in the chicken gastrointestinal tract, where they may potentially be used as probiotics, competitive exclusion agents, or delivery vehicles. The aim of this study was to use an in vivo model to investigate the effect of diet and competing lactic acid bacteria on the colonization of inoculated Lactobacillus strains, with the goal of identifying strains which can consistently colonize or persist for an extended period of several weeks. Chicken-derived Lactobacillus strains were genetically marked with rifampin resistance and administered on day 0 to chickens fed either a normal commercial diet or a specially formulated high-protein diet. Chickens fed the high-protein diet were also coinoculated with two different mixes of additional lactic acid bacteria. Enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) was used to identify rifampin-resistant isolates recovered from chickens. Three strains, belonging to the species Lactobacillus agilis, Lactobacillus crispatus, and Lactobacillus vaginalis, were commonly reisolated from the chickens on both diets at days 21 and 42. The ability of these strains to persist was confirmed in a second chicken trial. All three strains persisted throughout the production period in the chickens fed a commercial diet, while only the L. agilis and L. vaginalis strains persisted in the chickens fed the high-protein diet. In both in vivo trials, competing lactic acid bacteria modified representation of the strains recovered, with all three stains capable of competing in the presence of one or both mixes of coinoculated strains. The in vivo model successfully identified three persistent strains that will be characterized further.
The ecology of the chicken gastrointestinal tract (GIT) has been studied in depth using both culture-dependent (5, 7, 21, 40) and -independent methods (2, 3, 7, 33, 54). These studies have revealed that lactobacilli are autochthonous residents in chickens, where they predominate in the proximal GIT and are present but less abundant within the distal GIT (52). The most commonly identified Lactobacillus species are Lactobacillus crispatus, Lactobacillus reuteri, and Lactobacillus salivarius (1, 7, 15, 26, 28). A detailed understanding of the relationship between these bacteria and their host under different dietary and environmental conditions will facilitate the development of lactobacilli for various applications directed toward increasing broiler production efficiency and improving chicken health.
Since the withdrawal of antimicrobial growth promoters from chicken feed in Europe, the incidence of necrotic enteritis (NE) has increased (9, 51). Consequently, there is a need to develop alternative methods for controlling the causative agent of NE, Clostridium perfringens, in the chicken GIT. Lactobacilli are excellent candidates for alternative control methods due to their autochthonous nature and dominance of the upper GIT microbiota, particularly within the small intestine where NE occurs. Their potential utility in the control of NE has been demonstrated, with several strains of Lactobacillus showing some efficacy as probiotics to decrease C. perfringens carriage within the small intestine of chickens (14, 25, 31, 37, 47). Lactobacilli are also excellent candidates as mucosal delivery vectors designed to express bioactive peptides in situ to reduce colonization by C. perfringens. The use of lactobacilli and other lactic acid bacteria (LAB) as live delivery vectors for therapeutic proteins has recently been reviewed (6, 53), but few studies have been conducted in chickens (46, 55, 56). An attenuated Salmonella enterica serovar Typhimurium delivery vector targeting C. perfringens has provided partial protection against experimental NE challenge (35, 57). Identification of Lactobacillus strains for use as delivery vectors, competitive exclusion agents, or probiotics is complicated by the difficulty in selecting truly autochthonous strains capable of reliably and consistently colonizing the chicken GIT upon subsequent inoculation.
Traditionally, strain selection for in vivo applications has involved several in vitro characterization assays, including assays of aggregation, coaggregation, cell wall hydrophobicity, acid tolerance, bile salt tolerance, adhesion to epithelial cell lines, and antimicrobial activity (23, 32, 34, 44, 49). While these assays can be used to reduce the number of strains examined, they may also bias the selection of strains and could potentially overlook strains which may be competitive or have other desirable characteristics in vivo. One of the limitations of in vivo screening of lactobacilli is the need for reliable high-throughput screening techniques to identify and track persistent strains. Recently, our group reported the application of enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) to simultaneously type large numbers of Lactobacillus isolates from the chicken GIT to the species and strain level (48).
The primary aim of this study was to use a new, direct, in vivo screening method to examine the ecology of inoculated Lactobacillus strains in chickens fed different diets (high protein versus commercial). A second aim of this study was to determine the ecological effect of coinoculating two different mixes of competing LAB in chickens fed a high-protein diet, which predisposes chickens to develop NE. Inoculated strains were marked with rifampin resistance (Rifr), and ERIC-PCR was used to identify strains isolated from the chickens at days 21 and 42. Three persistent strains were identified in the initial trial and selected for further characterization in a subsequent experiment, in which two strains persisted in chickens fed the high-protein diet and all three persisted in chickens fed the commercial diet. In general, competing LAB modified strain representation and, in some cases, facilitated colonization of some strains. These three persistent strains will be further characterized as potential vectors to be used in the antibiotic-free control of NE.
MATERIALS AND METHODS
Bacterial strain collection and growth conditions.
All LAB cultures were grown in MRS broth (Difco, Detroit, MI) or agar at 37°C for 24 or 48 h, respectively, under anaerobic conditions using a BD GasPAK EZ container (Becton Dickinson, Sydney, Australia) with an AnaeroGen sachet (Oxoid Australia, Adelaide, Australia). The strains used in this study were collected previously (48), and the species distribution of the strain mixes used in this study are listed in Table 1.
Selection of strains for Rifr marking.
The "selected strains" specified in Table 1 were chosen from commercial and NE chicken isolates identified previously (48) and were chosen based upon multiple isolations from at least two chickens and from two or more different regions of the GIT. Strain mixes 1 and 2 were comprised of random collections of strains recovered from chickens fed commercial and high-protein diets. Some of the selected strains were also represented in the two mixes (Table 1). Rifampin-resistant (Rifr) variants of the selected strains and strain mixes 1 and 2 were generated to facilitate subsequent recovery and enumeration of the strains from the chicken GIT with minimal in vitro cultivation. Isolates were inoculated from glycerol stocks into MRS broth and incubated anaerobically overnight at 37°C. Cultures were centrifuged at 5,000 × g for 5 min, the supernatant was removed, and the cell pellets were resuspended in MRS broth containing 25 μg ml−1 rifampin (MRS Rif25) (Sigma-Aldrich, Sydney, Australia) and incubated anaerobically overnight at 37°C. These were subcultured (1%) into MRS broth containing 100 μg ml−1 rifampin (MRS Rif100) broth and incubated anaerobically overnight at 37°C. A sterile 10-μl disposable inoculating loop (Oxoid Australia) was used to streak the cultures onto MRS Rif100 agar plates incubated under the anaerobic conditions outlined above. Individual colonies were inoculated into MRS Rif100 broth and incubated anaerobically overnight at 37°C. Glycerol stocks of the Rifr cultures were made by mixing equal volumes of overnight culture and MRS broth containing 50% glycerol (vol/vol) and were stored at −80°C.
Preparation of strains for chicken trials.
For chicken trial 1, the Rifr isolates were inoculated from glycerol stocks into 2-ml, 96-well plates (with round deep wells) (Axygen Scientific, Inc., Union City, CA) containing 1 ml MRS Rif100 broth. The plates were sealed using breathable sealing film (Axygen) and incubated overnight as described above. The overnight cultures were resuspended by pipetting, subcultured (1%) into MRS Rif100 broth in 96-well plates, and incubated overnight again. The stationary-phase cultures (108 to 109 CFU ml−1) were resuspended and pooled in a sterile container, mixed by inversion, and stored on ice.
For chicken trial 2, each of the three persistent strains (selected based on performance in trial 1) were inoculated from glycerol stocks onto MRS Rif100 agar plates, subcultured into 20 ml of MRS Rif100 broth within a sterile 50-ml centrifuge tube (Becton Dickinson), and incubated overnight. Strain mixes 1 and 2 were each inoculated into separate 96-well plates as outlined above. The cultures of the three persistent strains were combined with either strain mix 1 or strain mix 2, transferred into a sterile container, mixed by inversion, and stored on ice.
Chicken trial 1.
The chicken trials were conducted within the Small Animal Facility at the Australian Animal Health Laboratory (AAHL) and were assessed, approved, and monitored by the AAHL Animal Ethics Committee. Forty Ross 308 chicks obtained from a commercial hatchery were transported to the AAHL within 24 h of hatch (day 0). The chickens were wing tagged for identification purposes, randomly divided into groups of 10, and transferred into four separate positive-pressure isolators. Within the isolators, food and water were available ad libitum, and they were lined with paper as a litter base and contained plastic cylinders for environmental enrichment.
Before strain administration on day 0, cloacal swabs were taken from the chickens and spread on MRS Rif100 agar plates to determine the background Rifr LAB contents of the chickens. Eight of 10 chickens per isolator were inoculated via crop lavage with 500 μl of culture mix (the strain mixes used in each isolator are described in Table 2), using a 1-ml needleless syringe (Becton Dickinson). The remaining two chickens were used as "in-contact" control chickens to determine if marked strains could be transferred among the chickens. The chickens in isolator 1 were fed a commercially available organic broiler feed (Organic Food Chain Pty Ltd., Toowoomba, Australia) which did not contain antimicrobial growth promoters. From days 0 to 13, the chickens were fed Organic Food Chain starter crumbles, and from days 14 to 42 they were fed Organic Food Chain grower pellets. The chickens in isolators 2 to 4 were fed the high-protein diet used in the NE disease induction model (48), which consisted of low-protein starter crumbles (Ridley AgriProducts Pty Ltd., Melbourne, Australia) from days 0 to 13 and high-protein finisher diet from days 14 to 42. On days 21 and 42, five chickens from each isolator were sacrificed by CO2 asphyxiation. The crop, duodenum, jejunum, and ileum were removed from the chickens. Approximately 1 g of tissue and contents was excised from the crop, the middle of the duodenum, the middle of the jejunum, and the terminal end of the ileum. These sections were transferred to sterile twist tie sample bags (Bacto Laboratories Pty Ltd., Sydney, Australia) and were stored on ice until processed.
Chicken trial 2.
The second chicken trial was performed as outlined for chicken trial 1 but with the following modifications. Chickens in isolators 1 and 2 were inoculated with the three persistent strains (La3, Lc9, and Lv5) (Tables 2 and 3) selected from chicken trial 1. Chickens in isolator 1 were fed the commercial diet as outlined above, while those in isolator 2 were fed the high-protein diet as outlined above. Chickens in isolators 3 and 4 were inoculated with the three persistent strains plus either strain mix 1 or 2, respectively, and fed the high-protein diet. Five chickens from each isolator were sacrificed on days 21 and 42, and gut sections were collected as described above.
Processing of chicken gut sections.
Nine milliliters of phosphate-buffered saline (PBS) was added to each of the bags containing gut samples. The tissue and contents were homogenized, and serial 10-fold dilutions (up to 10−6) were made, plated onto MRS and MRS Rif100 agar, and incubated as described above. Colonies were enumerated for each plate type. Approximately 12 colonies were randomly selected from MRS Rif100 agar plates from each gut section of each chicken and were individually inoculated into 2-ml 96-well plates (with round deep wells) (Axygen) containing 1 ml MRS Rif100 broth. The plates were sealed using breathable sealing film (Axygen) and incubated as outlined above. Overnight cultures were resuspended by pipetting, and 80 μl was transferred to half-skirt 96-well PCR plates (Axygen) and mixed with 80 μl of MRS broth containing 50% (vol/vol) glycerol. The glycerol plates were sealed using 96-well PCR AxyMats (Axygen) and stored at −80°C.
Typing of isolates using ERIC-PCR.
The isolates collected from trials 1 and 2 were typed using the ERIC-PCR protocol for high-throughput analysis described previously (48). Briefly, the remaining overnight culture in the 2-ml 96-well plates was centrifuged at 5,000 × g for 5 min. The pellets were washed twice in TN150 buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8). The pellets were resuspended in 500 μl TN150 buffer, transferred to a new 96-well plate containing 0.3 g of sterile 0.1-mm zirconium-silica beads per well, sealed with an Axymat (Axygen), placed within a 96-well plate adapter, and shaken for 3 min at 30 Hz in a Tissue Lyser (Qiagen, Doncaster, Australia). The plates were centrifuged as described above and stored at −20°C. The culture lysates were subjected to ERIC-PCR, gel electrophoresis, and data analysis as described previously (48). Representatives of strains collected were subject to 16S rRNA gene sequencing as described previously (48).
RESULTS
Influence of diet and background LAB on the colonization and persistence of Lactobacillus isolates in chicken trial 1: identification of Rifr background LAB.
Rifr colonies were isolated from several chickens prior to inoculation and identified using ERIC-PCR and 16S rRNA gene sequencing. These Rifr background isolates were identified as Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, Lactobacillus johnsonii, and L. reuteri strains, along with a single L. crispatus strain (data not shown). Several of these strains were identified in multiple isolators, suggesting that they were present in the chickens before transfer into the isolators, most likely entering the chicken GIT at the hatchery or in transit. Several of the background strains had ERIC-PCR profiles similar to several strains inoculated into the chickens (Lc9, Lj11, Lr2, Lr4, and Lr13). ERIC-PCR was unable to discriminate between the background and inoculated strains.
Enumeration of total and Rifr LAB.
The CFU were enumerated on both MRS and MRS Rif100 agar to compare total and Rifr colony counts, respectively (see Table S1 in the supplemental material). The crop and ileum had higher counts of Rifr colonies at day 21 than the duodenum and jejunum. The Rifr colonies comprised 9 to 55% of the total LAB microbiota at day 21, while the number of Rifr CFU g−1 and percentage of Rifr isolates were greater at day 42 (11 to 84%).
Representation of different genera based on diet.
Isolates were collected from the MRS Rif100 agar plates from chickens from each of the four isolators at days 21 and 42 and were typed using ERIC-PCR to determine their genus, species, and strain identity (Fig. 1). Almost all of the isolates from the chickens inoculated with the selected strains and fed the commercial diet belonged to the Lactobacillus genus (Fig. 1A) with only a few Enterococcus isolates (1%) identified at day 21. The number of lactobacilli identified at day 21 was lower than that at day 42 in the three isolators with chickens fed the high-protein diet. The general trend indicates that the proportion of isolates belonging to the Enterococcus and Pediococcus genera was greater in the high-protein diet isolator chickens, particularly at day 21, suggesting that the diet, strains included in the various mixes, and/or Rifr background members of these genera influenced their representation.
Influence of diet on representation of selected strains.
The two isolators with chickens inoculated with the selected strains directly compared the effect of diet on strain colonization and persistence within the GIT (Fig. 1B and Table 2). Strains present in chickens from multiple isolators are indicated in Table 3. Five strains (Lr4, Lr14, Ls4, Lv5, and Ls12) were present at both time points in the chickens fed the commercial diet; an additional two strains were present at day 21 (Lc9 and Lr13), while one additional strain was present at day 42 (Lc3). Five strains were identified at both time points in the chickens fed the high-protein diet (Lc3, Lc9, Lr13, Ls2, and Ls4); one additional strain was present at day 21 (Lr8), while three additional strains were present at day 42 (La3, Lj11, and Lv5). As indicated in Fig. 1B, several of these strains had ERIC-PCR profiles similar to the Rifr background strains, and the source (background and/or inoculated) cannot be determined at days 21 and 42. Nonetheless, if these strains are the result of background Rifr, they still persisted in the chickens for up to 42 days. Interestingly, several strains were persistent in chickens within both isolators (Lc3, Lc9, Lr13, Ls4, and Lv5), suggesting that diet may not have a major impact on the representation of particular Lactobacillus strains.
Representation of strains in the presence of two different groups of competing LAB.
The two remaining isolators were used to investigate which strains could persist and compete in the presence of other LAB. The chickens were inoculated with either strain mix 1 or 2, both of which consisted of 95 isolates comprised of 55 and 32 ERIC-PCR types, respectively, including some of the same strains inoculated into the chickens in isolators 1 and 2 (Table 1). Both groups were fed the high-protein diet. Interestingly, several of the selected strains were also well represented in these isolators. La3, Lc3, Lc9, and Lv5 were present in chickens within both isolators at day 21 and/or day 42 (Fig. 1B and Table 3). These data suggest that while several strains were able to colonize and persist from the two different mixes, several of the same strains were able to outcompete other strains. These competitive strains therefore are excellent candidates for further characterization.
Strain representation in the in-contact control chickens.
The in-contact chickens harbored a subset of the strains present in the inoculated chickens (Fig. 1C). This indicates that the strains were transferred via the fecal-oral route from the inoculated chickens to the in-contact chickens and subsequently colonized. The strain distribution within inoculated individuals (data not shown) showed trends similar to those observed in the in-contact chickens, with respect to the strain variety and strain distribution.
Selection of strains for further characterization.
Three strains were selected for further characterization based upon the frequency of their recovery, consistency of colonization, and ability to persist for up to 42 days in chickens within the different isolators; they are referred to as the "persistent" strains. The Lc9 and Lv5 strains were selected based upon their isolation from chickens from all of the isolators, while La3 was selected based upon its isolation from the isolators with chickens fed the high-protein diet. Although the source of Lc9 could not be distinguished as background or introduced, isolates of this strain were persistent in the chicken GIT. The identity of all three strains was confirmed using 16S rRNA gene sequencing.
Characterization of strains La3, Lc9, and Lv5 in chicken trial 2.
The second chicken trial was used to further characterize the three persistent strains identified in chicken trial 1. The effect of diet on the representation and distribution of the three persistent strains within individual chickens was determined in isolators 1 and 2 (Table 2). The ability of the three persistent strains to compete with the strains in mix 1 or 2 (Table 1) in chickens fed the high-protein diet was also assessed in isolators 3 and 4 (Table 2).
Identification of Rifr background LAB.
Before culture administration, none of the persistent ERIC-PCR types were detected in the crops of the chickens, although Rifr background LAB were detected in some chickens (data not shown). ERIC-PCR and 16S rRNA gene sequencing revealed that these bacteria consisted primarily of Pediococcus strains, several Enterococcus strains, and a small number of Lactobacillus reuteri isolates with profiles similar to that of strain Lr12.
Enumeration of total and Rifr LAB.
The Rifr and total LAB CFU g−1 counts were similar in each gut section at days 21 and 42 in chickens inoculated with the three persistent strains in isolators 1 and 2 (see Table S2 in the supplemental material). The Rifr LAB counts were consistently lower than the total LAB counts but were usually within the same log10 CFU g−1 and comprised 23 to 93.3% of the total LAB within the GIT sections (see Table S2 in the supplemental material). The colony counts indicate that the Rifr colonies are competitive and comprise a large proportion of the total LAB in the GIT. The crop had consistently higher numbers of Rifr LAB CFU g−1 (107 to 108) than the rest of the GIT (105 to 107). The number of CFU g−1 in the duodenum was typically 101 to 103 lower than that in the crop.
Representation of different genera based on diet.
The genus distribution (Fig. 2A) and Lactobacillus strain distribution (Fig. 2B) were examined using ERIC-PCR in order to identify which inoculated strains persisted and in what proportions. The genus distribution in the isolators was similar to that observed in trial 1, supporting the previous observation: the Rifr colonies recovered from chickens fed the commercial diet consisted almost entirely of lactobacilli, while several Rifr enterococci and pediococci were recovered from chickens fed the high-protein diet.
Lactobacillus strain distribution within the two diets.
The distributions of the three persistent strains differed between chickens fed the commercial and high-protein diets. All three of the inoculated strains persisted at both time points in the commercially fed chickens, although the distribution of each varied between the two time points, with the Lc9 strain dominating at day 21 (57%) and occurring less frequently at day 42 (20%). The isolation of both the La3 and Lv5 strains increased from day 21 (26 and 6%, respectively) to day 42 (45 and 18%, respectively). The background Rifr Lr12 strain was also present at both time points. These results indicate that all three of the persistent strains were able to colonize well throughout the production period after only a single inoculation at day 0 in chickens fed a commercial diet.
Only La3 and Lv5 strains were persistent throughout the trial in chickens fed the high-protein diet, while the background Lr12 strain was identified only at day 42. The La3 strain dominated and comprised 96% and 76% of the isolates at day 21 and day 42, respectively. In contrast to the results for the high-protein diet in trial 1, the Lc9 strain was not recovered at either time point in chickens in this isolator in trial 2.
Ability of the three persistent strains to persist and compete with other LAB.
The remaining two isolators were used to determine if the three persistent strains could colonize in the presence of competing LAB. The La3 and Lv5 strains were recovered at days 21 and 42, respectively, when inoculated with strain mix 1 (Fig. 2B), while all three persistent strains were recovered at day 42 only when inoculated with strain mix 2 (Fig. 2B). Presumably, the strains must be present at day 21 but below the detection limit of the sampling that was carried out.
Representation of the persistent strains within the GIT sections of individual chickens fed either the commercial or high-protein diet.
The representation of the persistent strains within individual chickens on both diets was examined (Fig. 3). Both the Lc9 and Lr12 strains were present within all the chickens and within most gut sections at day 21 in the commercially fed chickens. La3 and Lv5 were both isolated from two chickens, one of which contained all four strains. At day 42, all four strains were present within all of the chickens, although the strain representations differed among the different gut sections. The La3 strain dominated the gut sections of all chickens fed the high-protein diet at day 21. At day 42, the La3 strain was present in all the gut sections of all the chickens, while Lv5 and Lr12 were isolated predominantly from the small intestine of 4 of 5 and 3 of 5 chickens, respectively.
DISCUSSION
In the current study, we selected strains originally isolated from chickens fed either a commercial or high-protein diet and assessed their ability to colonize and persist in the GIT of chickens fed either of these diets. The results indicate that while most strains could not be recovered by use of the procedures outlined, a few strains were able to repeatedly colonize chickens fed either diet. The ability of these strains to compete when inoculated with other strains was also confirmed. In a subsequent experiment where three strains were reinoculated, all three strains colonized chickens fed the commercial diet, two strains colonized chickens fed the high-protein diet, and all three were recovered and competitive when inoculated with large mixes of LAB. The results suggest that diet and competing LAB do not affect the colonization of competitive strains which are ecologically fit for a particular niche. Based upon the consistent colonization of the three persistent strains, they appear to be good candidates for further characterization as live delivery vectors and/or probiotics.
One of the limitations of the in vivo approach was the presence of Rifr background LAB. This complicated the interpretation of the data in two ways. First, the ERIC-PCR profiles of the background Rifr isolates in the chickens fed the commercial diet in trial 1 matched some of the introduced strains, making it impossible to determine if it was the introduced or background isolates that persisted throughout the trial period. One of these strains, Lc9, persisted in relatively high numbers in chickens throughout multiple isolators in trial 1 and was selected for further characterization. The Lc9 strain was not part of the Rifr background in trial 2, where it persisted in the chickens fed the commercial diet, and was recovered from chickens at day 42 in one of the high-protein diet isolators. This confirmed the ability of this strain to persist regardless of its source. The second problem encountered due to the Rifr background was its potential effect on the recovery of Lactobacillus strains from chickens fed the high-protein diet. Rifr background enterococci and pediococci were frequently isolated from chickens fed the high-protein diet in both trials. Although these genera were not inoculated in trial 2, at day 21 in the "persistent strains with strain mix 1" isolator, for example, 95% of the isolates recovered were enterococci (Fig. 2A). All of the Lactobacillus isolates recovered from this isolator represented the same strain. If the number of enterococci recovered had been decreased, greater Lactobacillus strain diversity may have been revealed.
Rifr marking to recover introduced Lactobacillus strains from the indigenous GIT microbiota has been successfully used in chickens (23, 43), ducks (16), pigs (22, 39), mice (19, 41), and humans (20, 45), so a Rifr background was unexpected in this study. Rif MIC levels within several Lactobacillus and Pediococcus species have been reported to range between ≤0.016 and 4 μg ml−1 (12, 13). da Costa et al. (10) investigated the level of antimicrobial resistance in enterococci in broiler feed and broiler feed ingredients and found 58 and 60%, respectively, of enterococcal isolates were resistant to 5-μg Rif discs. Therefore, the level of Rif used in the current study (100 μg ml−1) is well above the MICs reported in the literature for these LAB and is the same level used in other chicken studies (23, 43). Marking lactobacilli with multiple antibiotics for in vivo testing has been performed previously using ducks (16) and humans (20). Increasing the level of antibiotic (e.g., 200 μg ml−1) or marking test strains with resistance to multiple antibiotics are two different strategies that could be implemented in future experiments to resolve the Rifr background issue.
One of the interesting results of the current study was the predominance of enterococci and pediococci among the isolates recovered from chickens fed the high-protein diet. This occurred regardless of the source, i.e., whether these isolates were inoculated into the chickens or acquired from the other background sources. These data indicate that the high-protein diet supports the survival and persistence of these two genera. Interestingly, Apajalahti (4) found that corn- and sorghum-based diets increased the number of enterococci isolated from the cecum of broiler chickens, suggesting that dietary factors may influence the recovery of these genera. While the predominance of these genera suggests that they could be promising candidates as probiotics or delivery vectors against C. perfringens, they were not investigated because these genera did not persist within chickens fed the commercial diet and because of the potential association of animal sources of antibiotic-resistant enterococci with human infections (18).
Our experiments were conducted under controlled conditions and were designed to specifically determine the effect of diet and other LAB on the persistence of the bacteria. Our results show that certain strains are able to persist and compete regardless of diet and competing LAB, and, in some cases, competing LAB can fortuitously support persistence of some strains. Hautefort et al. (29) found that the presence of a resident microbiota had a positive effect on the adhesion capacity of inoculated strains. The inoculation of, or background presence of, a mixed microbiota may create a synergistic environment that assists some strains in becoming established.
Interestingly, the three competitive strains examined here were originally isolated from chickens fed a high-protein diet, yet they were able to persist and compete in chickens fed both the high-protein and commercial diets in the present study. This suggests that regardless of microbiota changes during the development of chickens (1, 27, 28, 33, 38), or different diets and additives (11, 15, 17, 24, 27, 54), it may be possible to identify particular strains which can persist over time and under different conditions.
We used an in vivo approach to successfully identify persistent strains under conditions of interest to the Australian poultry industry. After analyzing data from our previous study (48), we selected strains for further characterization in the present study. We incorporated the following: (i) a rational approach with a "selected strain mix" in trial 1, which was comprised of strains that were isolated multiple times from multiple chickens, and (ii) a random approach, in which different mixes of strains were selected. While some of the strains which were selected via the rational approach were competitive, others were not. Furthermore, as indicated above, strains originally isolated from chickens fed one diet may be able to persist in chickens fed different diets. These results illustrate the difficulty in predicting which strains may persist, even when basing strain selection upon relevant in vivo ecological data. Likewise, basing the selection of strains upon in vitro-assessed characteristics may be just as problematic for predicting strains of interest for in vivo applications. In vitro screening assays based on resistance to bile and low pH, bacteriocin production, and adherence to epithelial cells have been frequently used to select lactobacillus strains for use as probiotics and/or delivery vectors within chickens (30, 32, 36, 42). It can be difficult to predict how these in vitro characteristics translate to the corresponding activity in vivo. For example, Hautefort et al. (29) reported that several strains were able to persist within mice without adhesion to the GIT lining, indicating that in vitro adhesion assays may be redundant. Gardiner et al. (22) argued that performance in the gut can be determined only truly in vivo, suggesting that the time and expense of assays such as those described here may be worthwhile.
One potential advantage of the in vivo assays described here is the ability to track strains following a single inoculum on day 0 throughout the production period. While several articles have described Lactobacillus strains which are able to survive and persist within the GIT of poultry (8, 16, 31, 42, 46), the persistence ranged from 1 day through 4 weeks after cessation of administration. Ehrmann et al. (16) described the persistence of their two selected strains within ducks for up to 28 days using only a single dose. The second chicken trial was used to confirm the persistence of three strains of interest (La3, Lc9, and Lv5), and, as in the first trial, these strains were able to persist throughout the full 42 days. Having probiotic/delivery vector strains persist throughout the full broiler production period (∼42 days) following a single administration is the ideal situation for producers, as the strains do not necessarily need to be used as ongoing in-feed additives. While prolonged administration of the strains would ensure full coverage of a flock, the in-contact chicken data from trial 1 indicate that these strains can be passed from chicken to chicken and subsequently colonize the in-contact chickens, indicating that ongoing administration may not be necessary. In situations such as those observed in trial 2, where the Lc9 strain was able to persist in chickens fed the commercial diet but not those dosed with the three strains on the high-protein diet, it would be beneficial to use multiple delivery vector/probiotic strains to provide full coverage if a single strain did not colonize, as has been suggested previously (22, 50).
In summary, an in vivo chicken model was used to successfully identify three strains that were able to persist in the presence of other LAB in chickens fed two different diets following a single inoculation. It is of great benefit for future studies that the selected strains successfully colonized and persisted in chickens under experimental NE disease induction conditions and under normal commercial feeding conditions. La3, Lc9, and Lv5 are all excellent candidates for further in vitro/in vivo characterization for use as delivery vectors and/or probiotics, as they are able to survive and persist for extended periods in the chicken GIT.
FIG. 1.
FIG. 2.
FIG. 3.
TABLE 1.
| Species | No. (name) of selected strain(s) | No. (name) of strain(s) in: | ||
|---|---|---|---|---|
| Strain mix 1 | Strain mix 2 | |||
| Lactobacillus agilis | 1 (La3) | 1 (La3) | 1 (La3) | |
| Lactobacillus crispatus | 3 (Lc3, Lc5, Lc8) | 4 (Lc2, Lc3, Lc5, Lc8) | ||
| Lactobacillus gallinarum | 2 (Lg4, Lg5) | 3 (Lg2, Lg4, Lg5) | 1 (Lg6) | |
| Lactobacillus johnsonii | 7 (Lj1, Lj2, Lj4, Lj6, Lj10, Lj12, Lj13) | 8 (Lj4, Lj6, Lj8 to Lj13) | 2 (Lj1, Lj2) | |
| Lactobacillus reuteri | 6 (Lr2 to Lr4, Lr8, Lr13, Lr14) | 3 (Lr11to Lr14) | 9 (Lr1 to Lr9) | |
| Lactobacillus salivarius | 4 (Ls2, Ls4, Ls13, Ls14) | 12 (Ls1 to Ls12) | ||
| Lactobacillus saerimneri | 3 (Lsa1 to Lsa3) | |||
| Lactobacillus vaginalis | 1 (Lv5) | 1 (Lv5) | 5 (Lv1 to Lv5) | |
| Enterococcus cecorum | 2 (Ec1, Ec2) | |||
| Enterococcus faecalis | 2 (Efl1, Efl2) | 2 (Efl1, Efl2) | ||
| Enterococcus faecium | 3 (Efm1 to Efm3) | |||
| Enterococcus villorum | 1 (Ev1) | |||
| Enterococcus sp. | 3 (E1, E2, E3) | 3 (E1, E2, E3) | ||
| Pediococcus acidilactici | 3 (Pa1, Pa2, Pa4) | 2 (Pa1, Pa4) | 3 (Pa1 to Pa3) | |
| Pediococcus pentosaceus | 1 (Pp1) | 1 (Pp2) | 1 (Pp1) | |
| Veillonella sp. | 1 (Ve1) | 1 (Ve1) | ||
| Total no. of ERIC-PCR types/species/isolates a | 34/12/34 | 45/14/95 | 27/9/95 | |
a
Some ERIC-PCR types were represented by multiple isolates within mixes 1 and 2.
TABLE 2.
| Isolator | Trial 1 | Trial 2 | ||||
|---|---|---|---|---|---|---|
| Diet | Strains administered | Diet | Strains administered | |||
| 1 | Commercial | Selected strains | Commercial | 3 persistent strains | ||
| 2 | High protein | Selected strains | High protein | 3 persistent strains | ||
| 3 | High protein | Strain mix 1 | High protein | 3 persistent strains with strain mix 1 | ||
| 4 | High protein | Strain mix 2 | High protein | 3 persistent strains with strain mix 2 | ||
TABLE 3.
| Species | Strain a | Originally isolated from chickens on indicated diet b | Recovered from chickens inoculated with: | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Selected strains on commercial diet | Selected strains on high protein diet | Strain mix 1 on high protein diet | Strain mix 2 on high protein diet | ||||||||||||||
| Day 21 | Day 42 | Day 21 | Day 42 | Day 21 | Day 42 | Day 21 | Day 42 | ||||||||||
| L. agilis | ANU22-30 (La3) | High protein | + | + | + | + | + | ||||||||||
| L. crispatus | ANU29-31 (Lc3) | Commercial | + | + | + | + | + | + | |||||||||
| L. crispatus | ANU22-31 (Lc9) c | High protein | + | + | + | + | + | + | |||||||||
| L. johnsonii | ANU26-21 (Lj11) c | Commercial | + | + | + | ||||||||||||
| L. reuteri | ANU23-04 (Lr13) c | Commercial | + | + | + | + | |||||||||||
| L. salivarius | ANU27-29 (Ls4) | Commercial | + | + | + | + | |||||||||||
| L. salivarius | ANU24-29 (Ls8) | Commercial | + | ||||||||||||||
| L. salivarius | ANU28-17 (Ls10) | Commercial | + | + | + | ||||||||||||
| L. salivarius | ANU31-15 (Ls12) | Commercial | + | + | + | ||||||||||||
| L. vaginalis | ANU16-27 (Lv5) | High protein | + | + | + | + | + | ||||||||||
| Total no. of species typed | 4 | 4 | 3 | 6 | 7 | 5 | 4 | 7 | |||||||||
| Total no. of strains typed | 8 | 6 | 6 | 8 | 9 | 7 | 5 | 9 | |||||||||
a
Strain names consistent with the report of Stephenson et al. (48) are given along with strain designations used in the present study in parentheses. Strains in bold type were selected for further characterization in the subsequent chicken trial.
b
Source of strains isolated previously (48).
c
Rifr background strain that was present in the preinoculation microbiota.
Acknowledgments
We thank S. Wilson, M. E. Ford, S. A. Sheedy, A. L. Keyburn, T. M. Crowley, and P. A. Cottee of the Commonwealth Scientific and Industrial Research Organization (CSIRO) Livestock Industries Division for their technical assistance during the chicken trials.
This research was supported by the Rural Industries Research and Development Corporation (RIRDC) Chicken Meat Program (project ANU-72A), the Australian National University, and the CSIRO.
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Volume 76 • Number 19 • 1 October 2010
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Received: 12 May 2010
Accepted: 1 August 2010
Published online: 17 December 2020
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David P. Stephenson
Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia
Robert J. Moore
CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia
Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia
ANU Medical School, The Australian National University, Canberra, ACT 0200, Australia
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In Vitro Characterization of Chicken Gut Bacterial Isolates for Probiotic Potentials
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