Post-natal development of the porcine microbiota composition and activities

The current study describes the development of the porcine microbiota and its metabolic activities during the neonatal and weaning period. Using 16S rRNA-based approaches, we first analysed the ileal and colonic microbiota of neonatal piglets at days 2, 5 and 12 after birth. To further investigate the effect of weaning at 3 weeks of age, 19-day-old piglets (n = 64) were randomly allocated into two groups. Half of the piglets remained with their sows throughout the study, while the remaining piglets were weaned. As revealed by sequence analysis of 16S rRNA gene amplicons, the samples of 2-day-old piglets harboured a consortium of bacteria related to Escherichia coli, Shigella flexneri, Lactobacillus sobrius, Lactobacillus reuteri and Lactobacillus acidophilus. Moreover, species-specific real-time polymerase chain reaction assays unveiled that L. sobrius and L. reuteri predominated in the ileal samples of the neonatal and unweaned piglets with population levels up to 7 x 10(8) cells per gram of lumen content. Following weaning, however, these two lactobacilli were detected at significantly lower levels (< 10(3)) in the ileal samples. Furthermore, a shift in composition and metabolic activities of the predominant microbiota, and emergence of clostridia and E. coli, were encountered in the intestinal samples of the piglets after the early post-weaning period.     

Impact of two probiotic Lactobacillus strains feeding on fecal lactobacilli and weight gains in chicken

Two probiotic strains, Lactobacillus agilis JCM 1048 and L. salivarius subsp. salicinius JCM 1230 isolated from chicken intestine, exhibited probiotic characteristics that can be applied for chicken production. After 7 days of probiotic feeding (FD7), the count of intestinal lactobacilli in the probiotic group (group P, n=10) was significantly (p<0.05) higher than that in the control group (group C, n=9). After 40 days of probiotic feeding (FD40), the lactobacilli and enterococci counts were stable but the Enterobacteriaceae number was significantly reduced (p<0.05). A total of 163 isolated lactobacilli were identified as the L. acidophilus/gallinarum group (49.7%), L. agilis (30.7%), L. salivarius (9.2%), L. reuteri (9.2%), and Lactobacillus spp. (1.2%). The probiotic lactobacilli positively affected the Lactobacillus biota in chickens at FD7, with a significant increase in the number (p<0.05) of L. agilis and group P. The viable counts of each Lactobacillus species at FD40, however, showed no differences between two groups. An increasing incidence of L. agilis was also noted with probiotic feeding. The probiotic effect of two strains resulted in significantly increased weight gains (10.7%) of group P in comparison with group C at FD40 (p<0.01).     

Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis

AIMS: To select Lactobacillus strains from laying hens for potential use as probiotic to control Salmonella Enteritidis infection. METHODS AND RESULTS: One hundred and eighty-six lactobacilli were isolated from the cloaca and vagina of laying hens, and identified at the species level by a polyphasic taxonomic approach. All isolates belonged to the Lactobacillus acidophilus, Lactobacillus reuteri or Lactobacillus salivarius phylogenetic groups, with the L. reuteri group being the most predominant group. Based on genetic diversity, about 50 representative strains were selected and tested for in vitro properties that could be predictive for probiotic activity in laying hens. Salmonella inhibition was shown to be species dependent, and correlated to some extent with the production of lactic acid. A selection of strains was evaluated in a S. Enteritidis challenge experiment. Two strains, L. reuteri R-17485 and Lactobacillus johnsonii R-17504 significantly decreased the colonization of chicks by S. Enteritidis in caeca, liver and spleen. CONCLUSIONS: Lactobacilli isolated from laying hens were observed to inhibit Salmonella growth in vitro, most probably through production of lactic acid, and to decrease in vivo the S. Enteritidis colonization of chicks. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that Lactobacillus isolates from laying hens may have probiotic potential in reducing S. Enteritidis infection.     

The gram-positive tonsillar and nasal flora of piglets before and after weaning

AIMS: To investigate gram-positive nasal and tonsillar microbial flora of piglets before and after weaning. METHODS AND RESULTS: The nasal and tonsillar gram-positive bacterial flora of 20 non-weaned piglets (2 weeks of age) and 20 weaned piglets (6 weeks of age), obtained from four different piggeries, was quantified by culture and identified by tDNA-PCR. The most widely occurring species from nasal conchae before as well as after weaning in the different piglets investigated were Streptococcus suis and Rothia nasimurium. After weaning a wide variety of Lactobacillus species appeared but in low numbers. In the tonsils, Strep. suis, Strep. dysgalactiae, S. hyicus, S. aureus, Arcanobacterium pyogenes and Actinomyces hyovaginalis were the species isolated from the largest number of pigs before and after weaning. S. aureus and most lactobacilli became more prevalent after weaning. Bacteria not known to be associated with pigs found in the present study included R. nasimurium, Strep. gallolyticus, Pediococcus pentosaceus and some Lactobacillus species. CONCLUSIONS: Over 30 different gram-positive bacterial species may occur in nasal conchae and tonsils of unweaned piglets at 2 weeks of age and of 6-week-old weaned piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that weaning is associated with changes in prevalence of only a small minority of the highly diversified bacterial flora of the nares and tonsils of pigs.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.     

Characterization and Transfer of Antibiotic Resistance in Lactic Acid Bacteria from Fermented Food Products

The study provides phenotypic and molecular analyses of the antibiotic resistance in lactic acid bacteria (LAB) from fermented foods in Xi'an, China. LAB strains (n = 84) belonging to 16 species of Lactobacillus (n = 73), and Streptococcus thermophilus (n = 11) were isolated and identified by sequencing their 16S rRNA gene. All strains were susceptible to ampicillin, bacitracin, and cefsulodin, and intrinsically resistant to nalidixic acid, kanamycin, and vancomycin (except L. bulgaricus, L. acidophilus, and S. thermophilus, which were susceptible to vancomycin). Some strains had acquired resistance for penicillin (n = 2), erythromycin (n = 9), clindamycin (n = 5), and tetracycline (n = 14), while resistance to gentamycin, ciprofloxacin, streptomycin, and chloramphenicol was species dependent. Minimum inhibitory concentrations presented in this study will help to review microbiological breakpoints for some of the species of Lactobacillus. The erm(B) gene was detected from two strains of each of L. fermentum and L. vaginalis, and one strain of each of L. plantarum, L. salivarius, L. acidophilus, L. animalis, and S. thermophilus. The tet genes were identified from 12 strains of lactobacilli from traditional foods. This is the first time, the authors identified tet(S) gene from L. brevis and L. kefiri. The erm(B) gene from L. fermentum NWL24 and L. salivarius NWL33, and tet(M) gene from L. plantarum NWL22 and L. brevis NWL59 were successfully transferred to Enterococcus faecalis 181 by filter mating. It was concluded that acquired antibiotic resistance is well dispersed in fermented food products in Xi'an, China and its transferability to other genera should be monitored closely.     

Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the guts of weaning piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 +/- 5.5) than in the ileum (9.7 +/- 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli

AIMS: To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. METHODS AND RESULTS: Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and >25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. CONCLUSIONS: A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level.     

Ecophysiology of the Developing Total Bacterial and Lactobacillus Communities in the Terminal Small Intestine of Weaning Piglets

Weaning of the pig is generally regarded as a stressful event which could lead to clinical implications because of the changes in the intestinal ecosystem. The functional properties of microbiota inhabiting the pig's small intestine (SI), including lactobacilli which are assumed to exert health-promoting properties, are yet poorly described. Thus, we determined the ecophysiology of bacterial groups and within genus Lactobacillus in the SI of weaning piglets and the impact of dietary changes. The SI contents of 20 piglets, 4 killed at weaning (only sow milk and no creep feed) and 4 killed at 1, 2, 5, and 11 days post weaning (pw; cereal-based diet) were examined for bacterial cell count and bacterial metabolites by fluorescence in situ hybridization (FISH). Lactobacilli were the predominant group in the SI except at 1 day pw because of a marked reduction in their number. On day 11 pw, bifidobacteria and E. coli were not detected, and Enterobacteriaceae and members of the Clostridium coccoides/Eubacterium rectale cluster were only found occasionally. L. sobrius/L. amylovorus became dominant species whereas the abundance of L. salivarius and L. gasseri/johnsonii declined. Concentration of lactic acid increased pw whereas pH, volatile fatty acids, and ammonia decreased. Carbohydrate utilization of 76 Lactobacillus spp. isolates was studied revealing a shift from lactose and galactose to starch, cellobiose, and xylose, suggesting that the bacteria colonizing the SI of piglets adapt to the newly introduced nutrients during the early weaning period. Identification of isolates based on partial 16S rRNA gene sequence data and comparison with fermentation data furthermore suggested adaptation processes below the species level. The results of our study will help to understand intestinal bacterial ecophysiology and to develop nutritional regimes to prevent or counteract complications during the weaning transition.     

Development of intestinal lactobacilli in normal piglets

The development of intestinal Lactobacillus spp. in 18 normal piglets from six litters was investigated from 1 to 45 d after birth. Faecal Lactobacillus strains isolated from piglets and their dams were identified at species and biovar levels on the basis of carbohydrate fermentation. A definite pattern was observed in the changes of four species and 20 biovars of lactobacilli appearing in faeces, with growth of the piglets. Lactobacillus reuteri colonized the animals on the first day of birth. The Lact. acidophilus group appeared 1 week after birth and maintained high levels in the intestine. Biovars B, C and E of Lact. reuteri were recovered as major biovars on the first to fourteenth days after birth. In the third and fourth weeks after birth, however, biovars F and G of Lact. reuteri were mainly recovered from piglet faeces. By the seventh week, biovars B, C and E predominated again in the intestine. No change in the composition of species and biovars of the genus Lactobacillus in the dams was detected.     

Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins.     

Distribution of salivary Lactobacillus and Bifidobacterium species in periodontal health and disease

We surveyed the distribution of salivary Lactobacillus and Bifidobacterium species in periodontitis patients and healthy subjects. Approximately 700 lactobacilli and 300 bifidobacterial isolates were obtained from 16 young, orally healthy subjects (mean age +/- standard deviation: 21.0+/-2.0 y), 16 periodontitis patients (51.6+/-13.8 y), and 14 well-maintained former periodontitis patients (60.2+/-9.6 y). Among eleven Lactobacillus species detected in saliva, L. salivarius, L. gasseri, and L. fermentum were prevalent, but no species was specifically associated with periodontal health. In contrast, of four Bifidobacterium species, B. adolescentis was specifically (P<0.05) prevalent in young healthy subjects compared with the other two groups. Furthermore, the bifidobacterial count of the well-maintained subjects was the highest (P<0.05) among the groups. These results suggest that bifidobacterial count and species might be associated with periodontal health status and/or age.     

Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.     

Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli

Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L. crispatus, L. reuteri, and L. agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniques. These lactobacilli associated with the horse, except for L. reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats. A symbiotic relationship of these lactobacilli with the horse is suggested.     

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

Diversity of Lactobacillus and Bifidobacterium in feces of herbivores, omnivores and carnivores

The Lactobacillus and Bifidobacterium population in the feces of 26 animals (16 species) were studied by culture-dependent and culture-independent techniques. Lactobacilli were detected from a few herbivores, all carnivores and some omnivores. Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus vaginalis and Lactobacillus ingluviei were the most dominant lactobacilli in carnivores. These species were, however, not predominant in herbivores and omnivores. Lactobacillus brevis, Lactobacillus casei, Lactobacillus parabuchneri, Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, usually present in raw plant material, were present in omnivores but not in carnivores. Bifidobacteria were detected in only four herbivores and two omnivores. Bifidobacterium pseudolongum was the only Bifidobacterium species detected in herbivores. Bifidobacteria detected in the two omnivores are phylogenetically not closely related to known species and are possible novel species in the genus.     

Oral cavity as natural reservoir for intestinal lactobacilli

Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract are likely to be transient (allochthonous), originating from either the oral cavity or food. In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a 3-months interval. Bacteriological culture, performed by incubation under standard (37 degrees C, anaerobic) and alternative (30 degrees C, microaerobic) conditions, as well as PCR-DGGE with group-specific primers were used to characterize the predominant lactobacilli. Cell counts varied among the subjects and over time, reaching up to 10(7)CFU/ml in saliva and 5 x 10(6)CFU/g in faecal samples. The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. Our results together with recent published data give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity.