Monitoring and survival of Lactobacillus gasseri SBT2055 in the human intestinal tract

The monitoring and survival of Lactobacillus gasseri SBT2055 in the human intestinal tract was investigated with seven healthy subjects having a low number of fecal lactobacilli. An increase of fecal lactobacilli (10(3.2-5.2) CFU/g feces) was recognized after ingestion of yogurt with SBT2055 by the subjects. A high positive rate of L. gasseri in fecal lactobacilli detected from the subjects (over 70% at 2nd weeks of feeding) was also observed during the ingestion period using the species-specific PCR system. These findings indicate that the SBT2055 strain in yogurt survived in the human intestinal tract and was recovered from human feces.     

Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract

A PCR-based detection system specific for Lactobacillus paracasei LTH 2579 was developed and applied to follow the fate of the strain in complex ecosystems. This strain was isolated from fruit mash and was characterised as being highly resistant to low pH and bile at concentrations as they occur in the human digestive tract. The application of the subtraction hybridisation technique permitted to identify a 235 bp chromosomal DNA fragment of strain LTH 2579. Based on this target sequence a specific PCR system was developed and combined with the species-specific PCR system for L. paracasei. This combination of PCR based detection systems was successfully applied to monitor L. paracasei LTH 2579 in fermented sausages which were inoculated with this strain (2.0 x 10(7) CFU/g) together with the strongly competitive L. sakei LTH 681 (1.0 x 10(6) CFU/g). At the time of consumption of the sausages the respective counts were 1.8 x 10(7) and 1.4 x 10(8) CFU/g. After consumption of the sausages by three volunteers L. paracasei LTH 2579 was recovered from fecal samples. The counts determined for the strain ranged between 1.2 x 10(7) and 1.5 x 10(8) CFU/g of feces. The fortuitous lactobacilli constituted a share of 5-12% of the lactobacilli in the fecal flora.     

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.     

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

Oral administration of Lactobacillus plantarum strain Lq80 to weaning piglets stimulates the growth of indigenous lactobacilli to modify the lactobacillal population

The composition and the number of fecal or intestinal lactobacilli were determined in weaned piglets before (pre-administration and day 0) and during (days 4, 7, and 14) the administration of Lactobacillus plantarum strain Lq80 (daily dose=10(10) cells). Fecal or intestinal lactobacilli were isolated, and the isolates were grouped by RAPD-PCR and further identified by 16S rDNA partial sequences. During administration, the number of lactobacilli in feces and intestinal contents (log cfu/g) increased from day 0 to day 4 (7.96 vs. 10.07, p<0.05), to day 7 (7.96 vs. 10.18, p<0.05), and to day 14 (7.96 vs. 9.02, p=0.07) in the administered group, although L. plantarum was isolated from the feces of piglets in the administered group at 5.0x10(6 cfu/g. The level of culturable lactobacilli was significantly higher on day 7 in the administered group than in the control group (8.93 vs. 10.18, p)=0.0002). Furthermore, the minor species in the lactobacillal population under the control condition become detectable with the administration of strain Lq80. The oral administration of L. plantarum strain Lq80 was effective to stimulate the development of the lactobacillal population in the intestine of post-weaning piglets.     

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53

The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous Lactobacillus reuteri strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n = 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 x 10(10) CFU of antibiotic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus counts for the three groups ranged from 1 x 10(9) to 4 x 10(9) CFU/g of feces. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 x 10(3) and 5 x 10(6) CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus assemblage (band V1-6).     

Dominance of Lactobacillus acidophilus in the Facultative Jejunal Lactobacillus Microbiota of Fistulated Beagles

Lactobacilli were isolated from jejunal chyme from five fistulated beagles. Cultivable lactobacilli varied from 10(4) to 10(8) CFU/ml. Seventy-four isolates were identified by partial 16S rRNA gene sequencing and differentiated by repetitive element PCR (Rep-PCR), Lactobacillus acidophilus was dominant, and nearly 80% of 54 isolates shared the same DNA fingerprint pattern.     

Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10(2) to 10(6) CFU/g) than in feces (range, 10(8) to 10(11) CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.     

Characterization of dominant cultivable lactobacilli and their antibiotic resistance profiles from faecal samples of weaning piglets

AIMS: To examine the lactic acid bacteria flora of weaning piglets, to define the distribution of different lactobacilli species in piglet faecal samples, and to determine the susceptibility phenotype to 11 antibiotic of different families. METHODS AND RESULTS: The faecal samples were taken from piglets with good herd status at 11 and 28 days after weaning. The Lactobacillus isolates (n = 129) from 78 animals housed in pairs in 39 pens were preliminarily identified by their morphology and biochemical characteristics. Partial 16S ribosomal DNA (16S rDNA) was used to identify the isolates to the species level, and RAPD (randomly amplified polymorphism DNA) profiles to differentiate Lactobacillus isolates to the strain level. Based on these studies, 67 strains were selected for antibiotic resistant tests. The most numerous Lactobacillus species found in the piglets was Lactobacillus reuteri (n = 43). Other lactobacilli were L. salivarius (n = 15), L. agilis (n = 4), L. johnsonii (n = 2), L. vaginalis (n = 1), L. mucosae (n = 1) and L. gallinarum (n = 1). All the strains were susceptible to chloramphenicol, ampicillin and gentamicin. Two L. salivarius isolates and two L. reuteri isolates were found to be multiresistant. CONCLUSIONS: This study indicates that the faecal Lactobacillus flora in piglets consists mainly of L. reuteri, L. salivarius and L. acidophilus group lactobacilli, and the distribution of lactobacilli is similar between individuals of the same age and with the same diet. Most of the Lactobacillus isolates tested were sensitive to the antibiotics used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable information on Lactobacillus species distribution and their antibiotic resistance profiles in piglets is obtained.     

Relative ability of orally administered Lactobacillus murinus to predominate and persist in the porcine gastrointestinal tract

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at approximately 10(10) CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at approximately 10(7) to 10(8) CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at approximately 10(5) CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 ( approximately 10(6) CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted approximately 10(7) CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by approximately 87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.     

Lactobacilli and enterococci — Potential probiotics for dogs

Forty strains of enterococci and forty strains of lactobacilli isolated from feces of 10 healthy dogs were tested for the antimicrobial activity, tolerance to bile and adhesion activity. The total count of fecal enterococci reached 5.5 log CFU/g and of lactobacilli 7.6 log CFU/g. Screening for production of bacteriocin-like substances showed an to partly inhibit the growth of Enterobacter sp. (hazy zones of inhibition). Ten strains of Enterococcus sp. and nine strains of Lactobacillus sp. were found without any inhibitory activity against all indicators used. Seven enterococcal strains and six lactobacilli with the broadest antimicrobial spectrum were selected for further probiotic assays. In the presence of 1% bile, the survival rate of selected enterococci (71.7-97.5%) was higher than that of lactobacilli (66.7-75.4%). The adhesion of strains to human intestinal mucus (5.1-8.2% by enterococci, 2.7-8.3% by lactobacilli) was found to be similar as adhesion to canine intestinal mucus (3.7-10.6% by enterococci, 2.1-6.0% by lactobacilli). Strain AD1, one lactobacillus isolate, reduced the higher level of serum cholesterol and alanine aminotransferase after oral administration to dogs suffering from diseases of the gastrointestinal tract.     

Modifying effects of fermented brown rice on fecal microbiota in rats

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.     

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt

To date, there is significant controversy as to the survival of yogurt bacteria (namely, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) after passage through the human gastrointestinal tract. Survival of both bacterial species in human feces was investigated by culture on selective media. Out of 39 samples recovered from 13 healthy subjects over a 12-day period of fresh yogurt intake, 32 and 37 samples contained viable S. thermophilus (median value of 6.3 x 10(4) CFU g(-1) of feces) and L. delbrueckii (median value of 7.2 x 10(4)CFU g(-1) of feces), respectively. The results of the present study indicate that substantial numbers of yogurt bacteria can survive human gastrointestinal transit.     

Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 x 10(9) lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10(2) cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.     

Effects of lactobacilli on yeast-catalyzed ethanol fermentations.

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.     

Microbiological characterization of wet wheat distillers' grain, with focus on isolation of lactobacilli with potential as probiotics

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50 degrees C, with L. amylolyticus being able to grow at temperatures as high as 54 degrees C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.     

Comparison between Cultured Small-Intestinal and Fecal Microbiotas in Beagle Dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10² to 10⁶ CFU/g) than in feces (range, 10⁸ to 10¹¹ CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.