Bacterial species associated with interdigital phlegmon outbreaks in Finnish dairy herds.

BACKGROUND: Severe outbreaks of bovine interdigital phlegmon (IP) have occurred recently in several free stall dairy herds in Finland. We studied the aetiology of IP in such herds, and the association of bacterial species with the various stages of IP and herds of various morbidity of IP. Nineteen free stall dairy herds with IP outbreaks and three control herds were visited and bacteriological samples collected from cows suffering from IP (n = 106), other hoof diseases (n = 58), and control cows (n = 64). The herds were divided into high morbidity (morbidity ≥50%) and moderate morbidity groups (9-33%) based on morbidity during the first two months of the outbreak. RESULTS: F. necrophorum subspecies necrophorum was clearly associated with IP in general, and T. pyogenes was associated with the healing stage of IP. Six other major hoof pathogens were detected; Dichelobacter nodosus, Porphyromonas levii, Prevotella melaninogenica, Treponema spp. and Trueperella pyogenes. Most of the samples of acute IP (66.7%) harboured both F. necrophorum and D. nodosus. We found differences between moderate morbidity and high morbidity herds. D. nodosus was more common in IP lesion in high than in moderate morbidity herds. CONCLUSIONS: Our result confirms that F. necrophorum subspecies necrophorum is the main pathogen in IP, but also T. pyogenes is associated with the healing stage of IP. Our results suggest that D. nodosus may play a role in the severity of the outbreak of IP, but further research is needed to establish other bacteriological factors behind these severe outbreaks.

Effect of a multispecies probiotic supplement on quantity of irritable bowel syndrome-related intestinal microbial phylotypes.

BACKGROUND: Probiotics can alleviate the symptoms of irritable bowel syndrome (IBS), possibly by stabilizing the intestinal microbiota. Our aim was to determine whether IBS-associated bacterial alterations were reduced during multispecies probiotic intervention consisting of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention has previously been shown to successfully alleviate gastrointestinal symptoms of IBS. METHODS: The faecal microbiotas of 42 IBS subjects participating in a placebo-controlled double-blind multispecies probiotic intervention were analysed using quantitative real-time polymerase chain reaction (qPCR). Eight bacterial targets within the gastrointestinal microbiota with a putative IBS association were measured. RESULTS: A phylotype with 94% similarity to Ruminococcus torques remained abundant in the placebo group, but was decreased in the probiotic group during the intervention (P = 0.02 at 6 months). In addition, the clostridial phylotype, Clostridium thermosuccinogenes 85%, was stably elevated during the intervention (P = 0.00 and P = 0.02 at 3 and 6 months, respectively). The bacterial alterations detected were in accordance with previously discovered alleviation of symptoms. CONCLUSIONS: The probiotic supplement was thus shown to exert specific alterations in the IBS-associated microbiota towards the bacterial 16S rDNA phylotype quantities described previously for subjects free of IBS. These changes may have value as non-invasive biomarkers in probiotic intervention studies.

Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces.

In this study, a total of 94 lactic acid bacterial (LAB) isolates of porcine small intestinal and fecal origin were screened for their probiotic properties. The aim was to evaluate whether their isolation site and putative species identity play a role in these characteristics and whether either of these can be used as a predictive factor for the probiotic potential of bacterial isolates. The isolates were preliminarily identified by partial 16S rRNA gene sequencing and characterized in vitro for their pH and bile tolerance, adhesion capacity towards porcine enterocytes isolated from five intestinal sites and for antimicrobial activity towards five indicator pathogens. The interdependence of these characteristics was statistically evaluated. The isolates tolerated low pH and bile well. Adherence to the enterocytes of different origins did not correlate with the strain isolation site. In general, higher adherence was observed to colon cells in comparison to the small intestinal enterocytes. Culture filtrates of the isolates caused a decrease of up to three orders of magnitude in the intestinal pathogen cell numbers. The inhibition was mostly due to lactic and other organic acids. The predominating phylotypes identified were Lactobacillus reuteri and Lactobacillus salivarius, of which the former generally had the best adhesion capacity, whereas the latter appeared to be the best inhibitor. Based on the results, several strains of the pig Lactobacillus isolates tested may function as promising candidates for use in probiotic products. However, it was not possible to use the isolation site or the species identity of the isolates as reliable preliminary screening factors.

Acute phase response and clinical manifestation in outbreaks of interdigital phlegmon in dairy herds.

Several Finnish dairy herds have suffered from outbreaks of interdigital phlegmon (IP). In these new types of outbreaks, morbidity was high and clinical signs severe, resulting in substantial economic losses for affected farms. In our study, we visited 18 free stall dairy herds experiencing an outbreak of IP and 3 control herds without a similar outbreak. From a total of 203 sampled cows, 60 suffered from acute stage IP. We demonstrated that acute phase response of bovine IP was evident and therefore an appropriate analgesic should be administered in the treatment of affected animals. The response was most apparent in herds with high morbidity in IP and with a bacterial infection comprising Fusobacterium necrophorum and Dichelobacter nodosus, indicating that combination of these two bacterial species affect the severity of the disease.

Microbial community analysis reveals high level phylogenetic alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel syndrome sufferers.

BACKGROUND: A growing amount of scientific evidence suggests that microbes are involved in the aetiology of irritable bowel syndrome (IBS), and the gastrointestinal (GI) microbiota of individuals suffering from diarrhoea-predominant IBS (IBS-D) is distinguishable from other IBS-subtypes. In our study, the GI microbiota of IBS-D patients was evaluated and compared with healthy controls (HC) by using a high-resolution sequencing method. The method allowed microbial community analysis on all levels of microbial genomic guanine plus cytosine (G+C) content, including high G+C bacteria. METHODS: The collective faecal microbiota composition of ten IBS-D patients was analysed by examining sequences obtained using percent G+C (%G+C) -based profiling and fractioning combined with 16S rRNA gene clone library sequencing of 3267 clones. The IBS-D library was compared with an analogous healthy-control library of 23 subjects. Real-time PCR analysis was used to identify phylotypes belonging to the class Gammaproteobacteria and the order Coriobacteriales. RESULTS: Significant differences were found between clone libraries of IBS-D patients and controls. The microbial communities of IBS-D patients were enriched in Proteobacteria and Firmicutes, but reduced in the number of Actinobacteria and Bacteroidetes compared to control. In particular, 16S rDNA sequences belonging to the family Lachnospiraceae within the phylum Firmicutes were in greater abundance in the IBS-D clone library. CONCLUSIONS: In the microbiota of IBS-D sufferers, notable differences were detected among the prominent bacterial phyla (Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria) localized within the GI tract.

Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide.

In order to gain insight into the effects of human breast milk on the development of the intestinal bifidobacteria and associated health effects, the transcriptome of Bifidobacterium longum LMG 13197 grown in breast milk and formula milk containing galactooligosaccharides (GOS) and long-chain fructooligosaccharides was compared to that obtained in a semisynthetic medium with glucose. Total RNA was isolated from exponentially growing cells and hybridized to a clone library-based microarray. Inserts of clones with significant hybridization signals were sequenced and identified. The B. longum transcriptomes obtained during growth on human and formula milk were more similar to each other than to that obtained from growth in semisynthetic medium with glucose. Remarkably, there were only a few genes implicated in carbohydrate metabolism that were similarly upregulated during growth in both human and formula milk although oligosaccharides were added to the formula. Common highly upregulated genes notably included putative genes for cell surface type 2 glycoprotein-binding fimbriae that are implicated in attachment and colonization in the intestine. Genes involved in carbohydrate metabolism formed the dominant group specifically upregulated in breast milk and included putative genes for N-acetylglucosamine degradation and for metabolism of mucin and human milk oligosaccharides via the galactose/lacto-N-biose gene cluster. This supports the notion that the bifidogenic effect of human milk is to a great extent based on its oligosaccharides. The transcriptional effect of semisynthetic medium containing GOS, which, like human milk, contains a large amount of lactose and galactose, on the B. longum transcriptome was also studied and revealed substantial similarity with carbohydrate-utilization genes upregulated during growth in human milk. This knowledge provides leads to optimizing formula milk to better simulate the observed bifidogenic effects of human breast milk.

Survey of interdigital phlegmon outbreaks and their risk factors in free stall dairy herds in Finland.

BACKGROUND: Severe outbreaks of interdigital phlegmon (IP) associated with a high morbidity and major economic losses have occurred in Finland in the past decade. A survey was performed to indicate the current occurrence of infectious hoof diseases and to identify herd level risk factors predisposing to an outbreak of IP. RESULTS: Responses to a questionnaire revealed that an outbreak of IP defined as morbidity ≥5% within the 1st month of the outbreak, had occurred in 18.0% of the respondent study farms. Risk factors for an outbreak included animal transport between herds, i.e. either animal purchase or contract heifer rearing, enlargement or renovation of the barn, and if the fields of the farm had been organically cultivated. Having any kind of mechanical ventilation in comparison to natural ventilation seemed to lower the risk of IP. Additionally, the farms that had experienced an outbreak of IP often had other infectious hoof diseases. However, it was unclear which disease appeared first. CONCLUSIONS: More attention is needed before and during enlargement or renovation of the barn and substantial planning is crucial for every part of the enlargement process in dairy farms.

PCR-ELISA I: Application to simultaneous analysis of mixed bacterial samples composed of intestinal species.

Sixteen oligonucleotide identification probes, designed in this study or adapted from literature, were tested for a PCR-ELISA application to simultaneously detect under standardised conditions selected intestinal bacteria, lactobacilli and bifidobacteria. The level of specificity obtained with most of the probes fulfilled the set criteria. The lack of efficiency of PCR performed with the primers, proposed to be specific for the entire eubacteria domain, and compromises made in hybridisation conditions due to simultaneous usage of multiple probes reduced the sensitivity of the PCR-ELISA test. The method was, however, found to be suitable for detecting predominant members of the intestinal flora. Applicability of the PCR-ELISA test could be further widened using primers with a more restricted specificity in the PCR step, as was demonstrated for the detection of Bifidobacterium with genus-specific primers. Advantages of the PCR-ELISA method include convenient performance and the possibility to test rapidly large amounts of samples with a multitude of probes.

PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation.

A PCR-ELISA method was extended for detection of most common Bifidobacterium species in humans and applied to a feeding trial including administration of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide (GOS)-containing syrup as probiotic and prebiotic preparations, respectively. For PCR-ELISA, oligonucleotide probes based on 16S rDNA sequences were designed and tested for specificity and sensitivity with nine different bifidobacterial species followed by analysis of faecal samples. Bifidobacteria were monitored for their fluctuations during and after the feeding trial. Bifidobacterium longum was the most common species found in the faecal samples, followed by B. adolescentis and B. bifidum. During ingestion of the probiotic B. lactis Bb-12, the strain appeared in the faeces but was absent again one week after finishing of the trial. The species that were observed in the faecal samples taken prior to the feeding experiments persisted also in samples derived from the pre-feeding and feeding periods. The most consistent change observed was the decrease in the relative amount of B. longum in the test group ingesting either B. lactis Bb-12 alone or in combination with GOS-syrup. Since the amounts of B. longum increased again in the post-feeding sample with these subjects, it may suggest that to some extent B. lactis Bb-12 is able to transiently replace B. longum.

Probiotic and milk technological properties of Lactobacillus brevis.

Two Lactobacillus brevis strains ATCC 8287 and ATCC 14869(T), were evaluated for their applicability as putative probiotics in dairy products. The strains expressed good in vitro adherence to human Caco-2 and Intestine 407 cells and tolerated well low pH, bile acids and pancreatic fluid under in vitro conditions. In antimicrobial activity assays, strain ATCC 8287 showed inhibitory properties toward selected potential harmful microorganisms, particularly against Bacillus cereus. Both L. brevis strains were resistant to vancomycin, which is typical for the genus Lactobacillus. The L. brevis strains were not able to acidify milk to yoghurt but were suitable as supplement strains in yoghurts. This was shown by producing a set of yoghurt products and analysing their rheological and sensory properties during a cold storage period of 28 days. Survival of the strains through human intestine was examined in 1-week feeding trials. Despite its human origin, L. brevis ATCC 14869(T) could not survive through the gastrointestinal (GI) tract, whereas L. brevis ATCC 8287 was detected in the faecal samples taken during and immediately after ingestion of the strain. In conclusion, L. brevis ATCC 8287 is a promising candidate as a probiotic supplement in dairy products.

Association of symptoms with gastrointestinal microbiota in irritable bowel syndrome.

AIM: To investigate the correlations between self-reported symptoms of irritable bowel syndrome (IBS) and the gastrointestinal (GI) microbiota composition. METHODS: Fecal samples were collected from a total of 44 subjects diagnosed with IBS. Their symptoms were monitored with a validated inflammatory bowel disease questionnaire adjusted for IBS patients. Thirteen quantitative real-time polymerase chain reaction assays were applied to evaluate the GI microbiota composition. Eubacteria and GI bacterial genera (Bifidobacterium, Lactobacillus and Veillonella), groups (Clostridium coccoides/Eubacterium rectale, Desulfovibrio desulfuricans) and distinct bacterial phylotypes [closest 16S rDNA sequence resemblance to species Bifidobacterium catenulatum, Clostridium cocleatum, Collinsella aerofaciens (C. aerofaciens), Coprococcus eutactus (C. eutactus), Ruminococcus torques and Streptococcus bovis] with a suspected association with IBS were quantified. Correlations between quantities or presence/absence data of selected bacterial groups or phylotypes and various IBS-related symptoms were investigated. RESULTS: Associations were observed between subjects' self-reported symptoms and the presence or quantities of certain GI bacteria. A Ruminococcus torques (R. torques)-like (94% similarity in 16S rRNA gene sequence) phylotype was associated with severity of bowel symptoms. Furthermore, among IBS subjects with R. torques 94% detected, the amounts of C. cocleatum 88%, C. aerofaciens-like and C. eutactus 97% phylotypes were significantly reduced. Interesting observations were also made concerning the effect of a subject's weight on GI microbiota with regard to C. aerofaciens-like phylotype, Bifidobacterium spp. and Lactobacillus spp. CONCLUSION: Bacteria seemingly affecting the symptom scores are unlikely to be the underlying cause or cure of IBS, but they may serve as biomarkers of the condition.

Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification.

AIM: To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS). METHODS: The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three time-points with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS: Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 +/- 0.90 vs -3.33 +/- 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 +/- 0.90 vs -3.08 +/- 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients' intestinal microbiota than in that of control subjects (-2.43 +/- 1.49 vs -4.02 +/- 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 +/- 0.53 vs -2.92 +/- 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 +/- 1.83 vs -3.69 +/- 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION: Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.

The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is a significant gastrointestinal disorder with unknown etiology. The symptoms can greatly weaken patients' quality of life and account for notable economical costs for society. Contribution of the gastrointestinal microbiota in IBS has been suggested. Our objective was to characterize putative differences in gastrointestinal microbiota between patients with IBS and control subjects. These differences could potentially have a causal relationship with the syndrome. METHODS: Microbial genomes from fecal samples of 24 patients with IBS and 23 controls were collected, pooled in a groupwise manner, and fractionated according to their guanine cytosine content. Selected fractions were analyzed by extensive high-throughput 16S ribosomal RNA gene cloning and sequencing of 3753 clones. Some of the revealed phylogenetic differences were further confirmed by quantitative polymerase chain reaction assays on individual samples. RESULTS: The coverage of the clone libraries of IBS subtypes and control subjects differed significantly (P < .0253). The samples were also distinguishable by the Bayesian analysis of bacterial population structure. Moreover, significant (P < .05) differences between the clone libraries were found in several bacterial genera, which could be verified by quantitative polymerase chain reaction assays of phylotypes belonging to the genera Coprococcus, Collinsella, and Coprobacillus. CONCLUSIONS: The study showed that fecal microbiota is significantly altered in IBS. Further studies on molecular mechanisms underlying these alterations are needed to elucidate the exact role of intestinal bacteria in IBS.

Characterization of a mobile clpL gene from Lactobacillus rhamnosus.

Two genes encoding ClpL ATPase proteins were identified in a probiotic Lactobacillus rhamnosus strain, E-97800. Sequence analyses revealed that the genes, designated clpL1 and clpL2, share 80% identity. The clpL2 gene showed the highest degree of identity (98.5%) to a clpL gene from Lactobacillus plantarum WCFSI, while it was not detected in three other L. rhamnosus strains studied. According to Northern analyses, the expression of clpL1 and the clpL2 were induced during heat shock by > 20- and 3-fold, respectively. The functional promoter regions were determined by primer extension analyses, and the clpL1 promoter was found to be overlapped by an inverted repeat structure identical to the conserved CIRCE element, indicating that clpL1 belongs to the HrcA regulon in L. rhamnosus. No consensus binding sites for HrcA or CtsR could be identified in the clpL2 promoter region. Interestingly, the clpL2 gene was found to be surrounded by truncated transposase genes and flanked by inverted repeat structures nearly identical to the terminal repeats of the ISLpl1 from L. plantarum HN38. Furthermore, clpL2 was shown to be mobilized during prolonged cultivation at elevated temperature. The presence of a gene almost identical to clpL2 in L. plantarum and its absence in other L. rhamnosus strains suggest that the L. rhamnosus E-97800 has acquired the clpL2 gene via horizontal transfer. No change in the stress tolerance of the ClpL2-deficient derivative of E-97800 compared to the parental strain was observed.

Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with real-time PCR.

OBJECTIVE: The gut microbiota may contribute to the onset and maintenance of irritable bowel syndrome (IBS). In this study, the microbiotas of patients suffering from IBS were compared with a control group devoid of gastrointestinal (GI) symptoms. METHODS: Fecal microbiota of patients (n = 27) fulfilling the Rome II criteria for IBS was compared with age- and gender-matched control subjects (n = 22). Fecal samples were obtained at 3 months intervals. Total bacterial DNA was analyzed by 20 quantitative real-time PCR assays covering approximately 300 bacterial species. RESULTS: Extensive individual variation was observed in the GI microbiota among both the IBS- and control groups. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. Average results from three fecal samples suggested differences in the Clostridium coccoides subgroup and Bifidobacterium catenulatum group between IBS patients (n = 21) and controls (n = 15). Of the intestinal pathogens earlier associated with IBS, no indications of Helicobacter spp. or Clostridium difficile were found whereas one case of Campylobacter jejuni was identified by sequencing. CONCLUSIONS: With these real-time PCR assays, quantitative alterations in the GI microbiota of IBS patients were found. Increasing microbial DNA sequence information will further allow designing of new real-time PCR assays for a more extensive analysis of intestinal microbes in IBS.

Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria.

PCR primers and hybridization probes were designed for the 16S rRNA genes of six bacterial species or groups typically present in human faeces or used in the dairy industry. The primers and probes were applied for quantification of the target bacterial genomes added in artificial DNA mixtures or faecal DNA preparations, using dot-blot hybridization and real-time PCR with SYBR Green I and TaqMan chemistries. Dot-blot hybridization with (33)P-labelled oligonucleotide probes was shown to detect a 10 % target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targeted oligonucleotide probes without pre-enrichment of the 16S gene pool by PCR was thus limited to the detection of the predominant microbial groups. Real-time PCR was performed using a 96-well format and was therefore feasible for straightforward analysis of large sample amounts. Both chemistries tested could detect and quantify a subpopulation of 0.01 % from the estimated number of total bacterial genomes present in a population sample. The linear range of amplification varied between three and five orders of magnitude for the specific target genome while the efficiency of amplification for the individual PCR assays was between 88.3 and 104 %. Use of a thermally activated polymerase was required with the SYBR Green I chemistry to obtain a similar sensitivity level to the TaqMan chemistry. In comparison to dot-blot hybridization, real-time PCR was easier and faster to perform and also proved to have a superior sensitivity. The results suggest that real-time PCR has a great potential for analysis of the faecal microflora.