Gnotobiotic Experiments Research Articles

Roles of microbiota in autoimmunity in Arabidopsis leaves

Over the past three decades, researchers have isolated plant mutants that show constitutively activated defence responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. Here, from a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes an S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high-humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, showing lesions, and having a high basal level of defence gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several ‘lesion mimic’-type autoimmune mutants in Arabidopsis. It is worth noting that autoimmune phenotypes caused by mutations in two Nucleotide-Binding, Leucine-Rich Repeat (NLR) genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of at least two classes of autoimmunity (microbiota-dependent versus microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom. These parallels highlight the intricate relationship between host immunity and microbial communities across various biological systems.

A guide to germ-free and gnotobiotic mouse technology to study health and disease.

The intestinal microbiota has major influence on human physiology and modulates health and disease. Complex host-microbe interactions regulate various homeostatic processes, including metabolism and immune function, while disturbances in microbiota composition (dysbiosis) are associated with a plethora of human diseases and are believed to modulate disease initiation, progression and therapy response. The vast complexity of the human microbiota and its metabolic output represents a great challenge in unraveling the molecular basis of host-microbe interactions in specific physiological contexts. To increase our understanding of these interactions, functional microbiota research using animal models in a reductionistic setting are essential. In the dynamic landscape of gut microbiota research, the use of germ-free and gnotobiotic mouse technology, in which causal disease-driving mechanisms can be dissected, represents a pivotal investigative tool for functional microbiota research in health and disease, in which causal disease-driving mechanisms can be dissected. A better understanding of the health-modulating functions of the microbiota opens perspectives for improved therapies in many diseases. In this review, we discuss practical considerations for the design and execution of germ-free and gnotobiotic experiments, including considerations around germ-free rederivation and housing conditions, route and timing of microbial administration, and dosing protocols. This comprehensive overview aims to provide researchers with valuable insights for improved experimental design in the field of functional microbiota research.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Monitoring and contamination incidence of gnotobiotic experiments performed in microisolator cages

With the increased interest in the microbiome research, gnotobiotic animals and techniques emerged again as valuable tools to investigate functional effects of host-microbe and microbe-microbe interactions. The increased demand for gnotobiotic experiments has resulted in the greater need for housing systems for short-term maintenance of gnotobiotic animals. During the last six years, the gnotobiotic facility of the Hannover Medical School has worked intensively with different housing systems for gnotobiotic animals. Here, we report our experience in handling, contamination incidence, and monitoring strategies that we apply for controlling gnotobiotic experiments. From our experience, the risk of introducing contaminants to animals housed in microisolator cages is higher than in isolators. However, with strict operating protocols, the contamination rate in these systems can be minimized. In addition to spore-forming bacteria and fungi from the environment, spore-forming bacteria from defined bacterial communities used in experiments represent the major risk for contamination of gnotobiotic experiments performed in microisolator cages. The presence/absence of contaminants in germ-free animals can be easily monitored by preparation of wet mounts and Gram staining of fecal samples. Contaminants in animals colonized with specific microorganisms need to be tracked with methods such as next-generation sequencing. However, when using PCR-based methods it is important to consider that relatively small amounts of bacterial DNA detected likely originates from food, bedding, or reagents and is not to be interpreted as true contamination.

Microbial Colonization Coordinates the Pathogenesis of a Klebsiella pneumoniae Infant Isolate

Enterobacteriaceae are among the first colonizers of neonate intestine. Members of this family, such as Escherichia and Klebsiella, are considered pathobionts and as such are capable of inducing local and systemic disease under specific colonization circumstances. Interplay between developing microbiota and pathogenic function of pathobionts are poorly understood. In this study, we investigate the functional interaction between various colonization patterns on an early colonizer, K. pneumoniae. K. pneumoniae 51-5 was isolated from stool of a healthy, premature infant, and found to contain the genotoxin island pks associated with development of colorectal cancer. Using intestinal epithelial cells, macrophages, and primary splenocytes, we demonstrate K. pneumoniae 51-5 upregulates expression of proinflammatory genes in vitro. Gnotobiotic experiments in Il10−/− mice demonstrate the neonate isolate induces intestinal inflammation in vivo, with increased expression of proinflammatory genes. Regulation of microbiota assembly revealed K. pneumoniae 51-5 accelerates onset of inflammation in Il10−/− mice, most significantly when microbiota is naturally acquired. Furthermore, K. pneumoniae 51-5 induces DNA damage and cell cycle arrest. Interestingly, K. pneumoniae 51-5 induced tumors in ApcMin/+; Il10−/− mice was not significantly affected by absence of colibactin activating enzyme, ClbP. These findings demonstrate pathogenicity of infant K. pneumoniae isolate is sensitive to microbial colonization status.

Plant-Growth Promotion and Biocontrol Properties of Three Streptomyces spp. Isolates to Control Bacterial Rice Pathogens.

Bacterial Panicle Blight caused by Burkholderia glumae is a major disease of rice, which has dramatically affected rice production around the world in the last years. In this study we describe the assessment of three Streptomyces isolates as biocontrol agents for B. glumae. Additionally, the presence of other plant-growth promoting abilities and their possible beneficial effects upon their inoculation on rice plants was evaluated as an ecological analysis for their future inoculation in rice crops. Two isolates (A20 and 5.1) inhibited growth of virulent B. glumae strains, as well as a wide range of bacterial and fungal species, while a third strain (7.1) showed only antifungal activity. In vitro tests demonstrated the ability of these strains to produce siderophores, Indoleacetic acid (IAA), extracellular enzymes and solubilizing phosphate. Greenhouse experiments with two rice cultivars indicated that Streptomyces A20 is able to colonize rice plants and promote plant growth in both cultivars. Furthermore, an egfp tagged mutant was generated and colonization experiments were performed, indicating that Streptomyces A20 –GFP was strongly associated with root hairs, which may be related to the plant growth promotion observed in the gnotobiotic experiments. In order to characterize the antimicrobial compounds produced by strain A20 bacteria, mass spectrometry analyses were performed. This technique indicated that A20 produced several antimicrobial compounds with sizes below 3 kDa and three of these molecules were identified as Streptotricins D, E and F. These findings indicate the potential of Streptomyces A20 as a biocontrol inoculant to protect rice plants against bacterial diseases.

Role of Gut Microbiota in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, caused by both genetic and environmental factors. Recently, investigators have focused on the gut microbiota, which is thought to be an environmental agent affecting the development of RA. Here we review the evidence from animal and human studies that supports the role of the gut microbiota in RA. We and others have demonstrated that the abundance of Prevotella copri is increased in some early RA. We have also used gnotobiotic experiments to show that dysbiosis in RA patients contributed to the development of Th17 cell-dependent arthritis in intestinal microbiota-humanized SKG mice. On the other hand, Prevotella histicola from human gut microbiota suppressed the development of arthritis. In summary, Prevotella species are involved in the pathogenesis of arthritis.

SAT0079 The analysis of fecal microbiota in rheumatoid arthritis patients compared to healthy volunteers using bacterial RRNA-targeted reverse transcription-quantitative PCR

BackgroundGut microbiota have long been thought to contribute to inflammatory disease, and some have considered the bacterial flora as one of the environmental factors causing rhumatoid arthritis (RA). Recently, two...

Abstract ED06-04: The microbiota and development of colitis-associated colorectal cancer

Abstract The human intestinal microbiota is a community of 1013-1014 microorganisms that reside in the intestine and normally participate in a symbiotic relationship with their eukaryotic host. The microbiome contains a wealth of information, encompassing 150 fold as many genes as the human genome. Recent studies have hinted that a core human microbiome exists at the gene level, with a large number of microbial genes and pathways shared among individuals. Deviations from this core microbiome could potentially affect human health and promote disease state. Thus characterizing the vast intestinal microbial community present in healthy as well as pathologic conditions will likely revolutionize our conception of bacteria/host interactions and identify new physiological processes influenced by the microbiota. Already the microbiota has been linked to cardiac development, angiogenesis, innate and adaptive immunity, metabolism, nutrient acquisition, and gastrointestinal development and homeostasis. Furthermore, alterations in the microbial community are associated with multiple diseases, including obesity, fatty liver disease, type 1 and type 2 diabetes, kidney disease, arthritis and inflammatory bowel diseases (IBD). Eukaryotes are equipped with an elegant repertoire of innate receptors each recognizing specific conserved microbial patterns present on various microorganisms, such as components of bacterial cell walls, locomotion system or nucleic acids. These microbial sensors are termed pattern recognition receptors (PRR) and include retinoic acid inducible gene-I like RNA helicases (RLH), C-type lectin receptors (CLR), Nucleotide-binding domain leucine-rich repeat proteins (NLR; also known as Nod-like receptors), and Toll-like receptors (TLR). Although the etiology of colitis-associated colorectal cancer remains elusive, innate host sensors have been shown to modulate the pathogenesis. Indeed, using mice exposed to the colon-specific carcinogen azoxymethane (AOM) compound and to the mucosal-damaging agent dextran sulfate sodium (DSS), numerous studies have revealed the differential contribution of innate sensors to CAC. For example, the NLR protein NOD1, a close relative of the IBD susceptible gene NOD2 protects against the development of colitis and colorectal cancer. Similarly, the adaptor protein MyD88 frequently utilized by TLR family members is protective in the context of AOM/DSS-induced colorectal cancer. Furthermore, the intracellular sensor NLRP3 also appears protective against colitis and CAC. In contrast, TLR4 signaling promotes the development of colorectal cancer in the AOM/DSS model. Although informative, these studies only capture the impact of various innate host responses on the development of colorectal cancer. The true impact of the microbiota could only be investigated using germ-free mice and gnotobiotic technology. To avoid the use of a chronic injury model (DSS), our laboratory has substituted the spontaneous IL-10−/− mouse model of intestinal inflammation for the AOM/DSS model. We have focused our attention on the role of microbial community on the development of colitis-associated colorectal cancer. Using this AOM/Il10−/− model of colorectal cancer in combination with gnotobiotic techniques, we observed a clear role for the microbiota in the development and progression of the pathology. Using metagenomic approaches, we observed a shift in bacterial community composition in mice bearing tumors compared to healthy controls. Interestingly, introduction of the probiotic VSL#3 changed microbial community structure and promoted the development of colorectal cancer in Il10−/− mice. Gnotobiotic experiments showed that bacteria-induced intestinal inflammation is not sufficient to promote colorectal cancer. Indeed, although the severity of colitis at the clinical and molecular levels is similar between Escherichia coli NC101 (E. coli) and Enterococcus faecalis (E. faecalis) monoassociated Il10−/− mice, tumor numbers were enhanced by more than 40 fold by the former commensal bacterium. We will present evidence that microbial composition and its associated genome/transcriptome plays a key role in the development of CAC. These findings would suggest that although chronic inflammation is a part of the pathology, microbial community and associated microbial genome is at the forefront of CAC susceptibility. Altering the composition of the microbiota may represent a novel means by which to modulate development of colorectal cancer in IBD patients. Citation Information: Cancer Prev Res 2010;3(12 Suppl):ED06-04.

Rotifer ( Brachionus “Cayman”) culture performance improvement through l-carnitine addition is not related to fatty acid metabolism

The purpose of this paper was to investigate the nature of the effect of l-carnitine on the cultures of the marine rotifer Brachionus “Cayman” and its microflora. Xenic and gnotobiotic experiments were set up with rotifers, fed axenic wild-type yeast ( Saccharomyces cerevisiae) or xenic microalgae ( Tetraselmis suecica) and incubated with l-carnitine concentrations of 0.1, 1, 10, 60, 100 and 1000 mg L −1. Axenic neonates were obtained by separating amictic eggs from the rotifers by blending, prior to disinfection with glutaraldehyde and hatching. The gnotobiotic cultures had a significantly lower growth rate and egg ratio (%), compared to both xenic trials. The xenic cultures fed algae performed significantly better than those fed baker's yeast, when comparing population density, growth rate and egg ratio. A significant effect of l-carnitine addition was only found in yeast-fed cultures. Microbial assays, conducted with similar doses of autoclaved l-carnitine, revealed that several bacterial species present in the community of conventional Brachionus “Cayman” cultures, could possibly utilize l-carnitine for growth. These results suggest that the improvement of population density, growth rate and egg ratio in xenic rotifer cultures supplemented with l-carnitine, is most likely due to the stimulation by this compound of certain species of (beneficial) microorganisms, which are in turn valorized by the rotifers, yielding improved culture performance. Because of the absence of a positive effect under gnotobiotic conditions, a direct effect of l-carnitine on fatty acid metabolism, as suggested in literature, is unlikely.

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants

Thiosulfate oxidizing bacteria isolated from crop plants were tested for their traits related to plant growth promotion and their ability to solubilize Morocco rock phosphate (RP) through oxidation of thiosulfate to sulfuric acid. All the tested strains grew in Nfb medium (except Dyella ginsengisoli) and possessed beta-1,3 glucanase activity (except Burkholderia kururiensis). Of the fourteen tested strains, 2 were found positive for siderophore production, 3 each for phytohormones (IAA), and salicylic acid production. Based on qualitative and quantitative assays, 5 strains were found to efficiently solubilize tri-calcium phosphate in Pikovskaya's medium. Nine strains exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity. In gnotobiotic experiments, Pandoraea sputorum ATSB28 which possessed the lowest ACC deaminase (0.44 nM of alpha-Keto butyrate formed min(-1) mg of protein(-1)) activity increased the primary root length of canola by 166%. Inoculation of Pandoraea sp. strain ATSB30 in mixture containing RP and thiosulfate significantly enhanced the water extractable-P (1147 microg P g RP(-1)) and bicarbonate extractable-P (1144 microg P g RP(-1)) on day 45. Glucose amendment resulted in increased RP solubilization as compared to glucose unamended treatments. Thiosulfate oxidizing bacteria tested in this study possessed at least one or more plant growth promoting traits apart from thiosulfate oxidation and solubilized the RP.

Enterocyte proliferation and apoptosis in the caudal small intestine is influenced by the composition of colonizing commensal bacteria in the neonatal gnotobiotic pig

We previously reported marked differences in small intestinal morphology, including changes in crypt depth and villous height, after inoculation of germ-free pigs with different bacterial species. In an attempt to identify the mechanisms governing changes in villous morphology associated with bacterial colonization, 2 gnotobiotic experiments were performed. In each experiment, 16 piglets were allocated to 4 treatment groups including germ-free (GF), monoassociation with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized with sow feces (SF). Piglets were reared under gnotobiotic conditions until 14 d of age, at which time whole intestinal tissue and enterocytes were collected for histological, gene expression, and protein analysis. Proliferating cell nuclear antigen, tumor necrosis factor alpha (TNFalpha), Fas ligand (FasL), CD3epsilon, caspase 3 (casp3), and toll-like receptors (TLR)2, 4, and 9 expression were measured by quantitative PCR. Activated casp3 was measured by Western blot. Increased abundance of activated casp3 and transcripts encoding proliferating cell nuclear antigen, TNFalpha, CD3epsilon, and FasL was observed in SF and EC treatment groups compared with GF and LF. Expression of TLR2 was increased (P < 0.05) in the SF treatment and tended to be greater (P < 0.08) in EC relative to LF and GF. Results indicate that conventional bacteria and E. coli but not L. fermentum increase overall cell turnover by stimulating increased apoptosis through the expression of FasL and TNFalpha and by increasing cell proliferation. The differential regulation of TLR expression indicates that microbially induced changes may be mediated in part by these receptors. Induction of inflammatory responses and activation of apoptosis through death receptors appears to play a significant role in enterocyte turnover mediated by commensal bacteria.

Survival in the soil of the ectomycorrhizal fungus Laccaria bicolor and the effects of a mycorrhiza helper Pseudomonas fluorescens

In disinfected forest nursery soils, inoculating Douglas fir ( Pseudotsuga menziesii) seedlings with the ectomycorrhizal fungal strain Laccaria bicolor S238N significantly increases tree growth after outplantating. However, the success of the inoculation depends on survival of the fungal inoculum in the soil during the pre-symbiotic life of the fungus. We followed the survival of L. bicolor S238N in autoclaved nursery soil in the glasshouse, and under gnotobiotic conditions in autoclaved or γ-irradiated nursery soil. We also studied the effect of the mycorrhiza helper bacterium Pseudomonas fluorescens BBc6R8, which promotes the Douglas fir- L. bicolor S238N symbiosis, on fungal viability. In the glasshouse, fungal viability was assessed by trapping with Douglas fir seedlings. We showed that the fungus retained its viability in a pre-symbiotic state in the soil at least for 23 weeks, which is much longer than that reported in the literature for other ectomycorrhizal fungi. The bacterium did not significantly modify the survival of the fungus. In the gnotobiotic experiments, ergosterol, a specific fungal membrane component, was used to quantify fungal biomass. Fungal behaviour differed with the disinfection technique used, which modified the chemical characteristics of the initial soil. There was no fungal growth in the autoclaved soil but there was a rapid increase of fungal biomass in the irradiated soil. The effect of the bacterium on fungal biomass also varied with a significant stimulation in the autoclaved soil vs. a significant inhibition in the irradiated soil. Our results show that the beneficial effect of the bacterium on the fungus depends on the condition of the fungus, i.e. the greatest benefit occurs when the fungus is growing under unfavourable conditions.

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N2 fixation (BNF) using an agar growth medium containing 5 mg N l–1of 15N-labelled (NH4)2SO4. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with 15N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N2-fixing bacteria and plants.

Saprophytic soil microflora in relation to yield reductions in soil repeatedly cropped with barley (Hordeum vulgare L.)

The yield-depressing effect due to repeated cropping (monoculture) of barley reported from long-term field experiments was observed as a reduction in plant growth in short-term pot experiments. The nature of the monoculture effect was investigated by mixing field soils with different cropping histories in different proportions in the greenhouse, while the influence of rhizosphere microflora from the monocultured and crop rotation soils on barley growth was studied in gnotobiotic experiments. Indigenous bacterial populations and the pH of the test soils were also measured. Significantly more bacteria were found in the crop rotation soil compared to the monocultured soil, but the two soils did not differ in pH. Greenhouse experiments showed that in the monocultured soil, seed germination was delayed and plant dry weight reduced, and that these effects had a biological origin. Attempts were also made to induce the monoculture effect in the crop rotation soil by inoculation with known harmful bacteria. The results from the experiments with mixed soils and with soil inoculation indicated that where crop rotation was practised the soil was more sensitive to bacterial effects than the monocultured soil. The rhizosphere microflora from the monocultured soil did not affect plant weights in short-term gnotobiotic experiments, but it significantly stimulated the number of lateral roots compared with the crop rotation microflora. This stimulation could not be related to differences in bacterial counts, pH, or ion concentrations in the plant-growing medium.

Migration of materials and microorganisms in the dental pulp of dogs and rats

Data from a wide range of studies on pulp response to materials in rats and dogs and a series of gnotobiotic experiments designed to investigate pulp response to caries have been reviewed for evidence of migration of materials and bacteria within the pulp. Migration of materials after direct pulp capping was unusual in the rat molar pulp. In the dog, by contrast, migration of materials from direct pulp capping sites was frequently observed. When material particles were contained within phagocytic cells, there was little evidence of any pulp response; free material within the tissues was frequently associated with inflammation or necrosis. In the gnotobiotic studies, phagocytosis of strains of Streptococcus mutans by the pulp in carious teeth was seen.

Influence of dietary lactose on the gut flora of chicks.

1. The gut microflora of chicks fed on a purified diet containing 300 g lactose plus 300 g starch/kg was compared with that of control birds receiving a diet containing 600 g starch/kg. 2. In 14-d-old conventional chicks, lactose in the diet decreased the incidence of lactobacilli and clostridia in the caecal contents, although when presenting lactose-fed chicks the counts of lactobacilli exceeded those of control chicks. 3. High counts of Proteus sp. were present in the caeca of control birds but they were completely suppressed in conventional birds fed on the lactose diet. In vitro tests showed that this inhibition was partially due to Escherichia coli and Streptococcus faecalis. 4. The growth of Lactobacillus acidophilus was inhibited by lactose when gnotobiotic chicks were monoassociated but not when polyassociated. The protective effect was shown in vitro to be due to L. salivarius. 5. The pH was markedly lowered in the caecum of conventional and polyassociated chicks receiving dietary lactose. Of the strains used in gnotobiotic experiments E. coli, S. faecalis and L. salivarius produced the lowest pH values in the caeca.

6] Germfree rats

Evidence is accumulating that suggests that the bacterial flora has a pervasive influence on the physiology, immune status, and metabolism and nutrition of the animal host. The effect of the flora can best be studied by making observations on animals whose flora has either been simplified or eliminated entirely. Such studies require gnotobiotic animals. This chapter describes the methods that can be employed by laboratories primarily engaged in fields, such as bacteriology, pharmacology, toxicology, or nutrition, where gnotobiotic experiments may occasionally be useful. Only a few hundred square feet of space are required for the use of these methods and the space can readily be converted to other purposes when an isolator is not in use. The description in the chapter is limited to a method of performing gnotobiotic experiments that are found particularly suitable for studying the metabolism of xenobiotic compounds and for certain nutritional experiments. The gnotobiotic techniques are used to establish whether a metabolic reaction occurring in a conventional rat can be attributed to its flora.

Fermentation of L-sorbose by microorganisms of the human dental plaque.

Microorganisms with the ability to ferment L-sorbose were recovered from human dental plaque. According to their characterization these bacteria could be identified as Lactobacillus casei. Their fermentation capacity was tested in vitro with L-sorbose as sole carbohydrate source. Sorbose consumption and acid production were both analyzed by high pressure liquid chromatography. The amount of fermentation end product – lactic acid – thus detected is slightly higher than that produced under comparable conditions by Streptococcus mutans with sucrose as carbohydrate source. The cariogenic potential of the combination L. casei – L-sorbose was investigated by gnotobiotic animal experiments. The monoassociation of L. casei with rats fed an L-sorbose diet led to higher caries scores than S. mutans in gnotobiotic rats with a sucrose test diet. However, in conventional animal experiments the cariogenicity of L-sorbose was significantly lower than that of sucrose, but still higher than, e. g., that of xylitol.