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The common presence of microbial communities (microbiotas) that form life-long associations with particular body sites of vertebrates are considered to represent symbiotic partnerships. These multi-organismal associations are different than ‘holobiont’ partnerships defined by Margulis which have restricted diversity and feature high fidelity, intergenerational transmission of symbionts. Comparison of the nutritional and physiological properties of germfree, defined microbiota, and conventional mice help to reveal the extent of the symbiosis between the vertebrate host and microbial communities of high diversity. The mouse-microbiota association is not obligate, but gut microbiota function enables additional energy harvest from the diet. This represents a major evolutionary innovation and therefore could be considered to be an example of symbiogenesis. However, this advantage is balanced by the expenditure of energy required to maintain structures and physiology that prevent invasion of murine tissues by the microbiota. Extrapolation of observations from gnotobiotes to humans helps to develop an appreciation of the human-gut microbiota relationship with emphasis on factors that may have assisted human survival. [ABSTRACT FROM AUTHOR]

Background: Host tp53 mutations are frequently found during the early stages of colitis-associated colorectal cancer (CAC), but whether such mutations induce gut microbiota dysbiosis and chronic intestinal inflammation that contributes to the development of CAC, remains unknown. Results: We found that zebrafish tp53 mutant larvae exhibited elevated intestinal inflammation, by monitoring the NFκB activity in the mid-distal intestines of zebrafish larvae using an NFκB:EGFP transgenic reporter line in vivo as well as neutrophil infiltration into the intestine. This inflammation was due to dysbiotic gut microbiota with reduced diversity, revealed using both 16S rRNA amplicon sequencing and a germfree larva model. In this dysbiosis, Aeromonas spp. were aberrantly enriched as major pathobionts and exhibited the capacity for aggressive colonization in tp53 mutants. Importantly, the ex-germfree experiments supported the causality of the host tp53 mutation for inducing the inflammation. Transcriptome and high-performance liquid chromatography analyses of the host gastrointestinal tracts identified dysregulated sialic acid (SA) metabolism concomitant with increased host Neu5Gc levels as the key determinant of aberrant inflammation, which was reversed by the sialidase inhibitors oseltamivir and Philippin A. Conclusions: These results demonstrate a crucial role for host tp53 in maintaining symbiosis and immune homeostasis via SA metabolism. Disturbed SA metabolism via a tp53 mutation may be exploited by specific elements of the gut microbiome, eliciting both dysbiosis and inflammation. Manipulating sialometabolism may therefore provide an efficacious therapeutic strategy for tp53 mutation-induced dysbiosis, inflammation, and ultimately, related cancers. -YW-NrG28r8zR7UJjY8FmQ Video Abstract [ABSTRACT FROM AUTHOR]

ABSTRACT Omnivorous animals, including humans, harbor diverse, species-rich gut communities that impact their growth, development, and homeostasis. Model invertebrates are broadly accessible experimental platforms that enable linking specific species or species groups to host phenotypes, yet often their specialized diets and distinct gut microbiota make them less comparable to human and other mammalian and gut communities. The omnivorous cockroach Periplaneta americana harbors ∼4 × 102 bacterial genera within its digestive tract and is enriched with taxa commonly found in omnivorous mammals (i.e., Proteobacteria, Bacteroidetes, and Firmicutes). These features make P. americana a valuable platform for identifying microbe-mediated host phenotypes with potential translations to mammals. Rearing P. americana insects under germfree conditions resulted in prolonging development time by ∼30% and an up to ∼8% reduction in body size along three dimensions. Germfree rearing resulted in downregulation of gene networks involved in growth, energy homeostasis, and nutrient availability. Reintroduction of a defined microbiota comprised of a subset of P. americana commensals to germfree insects did not recover normal growth and developmental phenotypes or transcriptional profiles observed in conventionally reared insects. These results are in contrast with specialist-feeding model insects (e.g., Drosophila), where introduction of a single endemic bacterial species to germfree condition-reared specimens recovered normal host phenotypes. These data suggest that understanding microbe-mediated host outcomes in animals with species-rich communities should include models that typically maintain similarly diverse microbiomes. The dramatic transcriptional, developmental, and morphological phenotypes linked to gut microbiome status in this study illustrates how microbes are key players in animal growth and evolution. IMPORTANCE Broadly accessible model organisms are essential for illustrating how microbes are engaged in the growth, development, and evolution of animals. We report that germfree rearing of omnivorous Periplaneta americana cockroaches resulted in growth defects and severely disrupted gene networks that regulate development, which highlights the importance of gut microbiota in these host processes. Absence of gut microbiota elicited a starvation-like transcriptional response in which growth and development were inhibited while nutrient scavenging was enhanced. Additionally, reintroduction of a subset of cockroach gut bacterial commensals did not broadly recover normal expression patterns, illustrating that a particular microbiome composition may be necessary for normal host development. Invertebrate microbiota model systems that enable disentangling complex, species-rich communities are essential for linking microbial taxa to specific host phenotypes.

Gut microbiota significantly influence the plasma and liver lipidome. An interconnecting metabolite is acetate generated after degradation and fermentation of dietary fiber by the gut microbiota, which is metabolized in the liver into longer chain fatty acids and complex lipids reaching the circulation. Whether these systemic changes are accompanied by alternations of the intestinal lipidome is unclear. Therefore, we quantified glycerophospholipids, sphingolipids and sterols in ileum and colon, the two segments containing the highest densities of microbes in the gastrointestinal tract, of germfree and specific pathogen free mice using mass spectrometry-based lipidomics. We found that the presence of gut microbes lowers the free cholesterol content in colon while elevating phosphatidylcholine levels. Further, PUFA-containing phosphatidylcholine and -ethanolamine fractions are increased in ileum and colon of germfree compared to SPF mice. A total fatty acid analysis by GC–MS revealed higher levels of arachidonic and docosahexaenoic acid in the ileum of germfree mice indicating that the gut microbiota inhibits PUFA metabolism in the small intestine.

ABSTRACT Disrupted interactions between host and intestinal bacteria are implicated in colorectal cancer (CRC) development. However, activities derived from these bacteria and their interplay with the host are unclear. Here, we examine this interplay by performing mouse and microbiota RNA sequencing on colon tissues and 16S and small RNA sequencing on stools from germfree (GF) and gnotobiotic ApcMinΔ850/+;Il10−/− mice associated with microbes from biofilm-positive human CRC tumor (BF+T) and biofilm-negative healthy (BF-bx) tissues. The bacteria in BF+T mice differentially expressed (DE) >2,900 genes, including genes related to bacterial secretion, virulence, and biofilms but affected only 62 host genes. Small RNA sequencing of stools from these cohorts revealed eight significant DE host microRNAs (miRNAs) based on biofilm status and several miRNAs that correlated with bacterial taxon abundances. Additionally, computational predictions suggest that some miRNAs preferentially target bacterial genes while others primarily target mouse genes. 16S rRNA sequencing of mice that were reassociated with mucosa-associated communities from the initial association revealed a set of 13 bacterial genera associated with cancer that were maintained regardless of whether the reassociation inoculums were initially obtained from murine proximal or distal colon tissues. Our findings suggest that complex interactions within bacterial communities affect host-derived miRNA, bacterial composition, and CRC development. IMPORTANCE Bacteria and bacterial biofilms have been implicated in colorectal cancer (CRC), but it is still unclear what genes these microbial communities express and how they influence the host. MicroRNAs regulate host gene expression and have been explored as potential biomarkers for CRC. An emerging area of research is the ability of microRNAs to impact growth and gene expression of members of the intestinal microbiota. This study examined the bacteria and bacterial transcriptome associated with microbes derived from biofilm-positive human cancers that promoted tumorigenesis in a murine model of CRC. The murine response to different microbial communities (derived from CRC patients or healthy people) was evaluated through RNA and microRNA sequencing. We identified a complex interplay between biofilm-associated bacteria and the host during CRC in mice. These findings may lead to the development of new biomarkers and therapeutics for identifying and treating biofilm-associated CRCs.

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease.

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease. [ABSTRACT FROM AUTHOR]

ABSTRACT Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr−/−) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr−/− mice and CONV-R Ldlr−/− mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr−/− mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr−/− mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr−/− mice on HFD were diminished compared to CONV-R Ldlr−/− controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr−/− mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr−/− mice relative to CONV-R Ldlr−/− mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr−/− mice on type III collagen. IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr−/− mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Microbiome alterations have been shown to affect stroke outcome. However, to what extent the presence of a gut microbiome per se is affecting post-stroke neuroinflammation has not been tested. By comparing germfree mice with recolonized (Ex-GF) and conventional SPF mice, we were able to demonstrate that bacterial colonization reduces stroke volumes. Bacterial colonization increased cerebral expression of cytokines as well as microglia/macrophage cell counts in contrast to improved stroke outcome. Interestingly, the microbiome-mediated brain protection was absent in lymphocyte-deficient mice. These findings support the concept of lymphocyte-driven protective neuroinflammation after stroke under control of the microbiome. [ABSTRACT FROM AUTHOR]

Objective Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. Methods GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. Results GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17β-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order Clostridiales (phylum Firmicutes ) as a characteristic feature of normal SPF mice fed lard. Conclusions In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism. [ABSTRACT FROM AUTHOR]

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study. [ABSTRACT FROM AUTHOR]

The enteric nervous system (ENS), considered a separate branch of the autonomic nervous system, is located throughout the length of the gastrointestinal (GI) tract as a series of interconnected ganglionated plexi. Given the proximity of the intestinal microbiota to the ENS, it is perhaps not surprising that the gut microbiota can influence its development and function. However, these interactions are complex and may be either direct or indirect, often involving signalling initiated by microbe-derived components, metabolites or host-derived intermediaries which subsequently affect enteric nerve excitability and GI function. Individual microbes and strains can differentially influence ENS activity and neurochemistry. In this review we will briefly summarise the role of the microbiota on ENS development, and, in some more detail, explore the mechanisms by which the microbiota can influence ENS activity and function. [ABSTRACT FROM AUTHOR]

Background: Bacteroides sp. are dominant constituents of the human and animal intestinal microbiota require porphyrins (i.e., protoporphyrin IX or iron-charged heme) for normal growth. The highly stimulatory effect of porphyrins on Bacteroides growth lead us to propose their use as a potential determinant of bacterial colonization. However, showing a role for porphryins would require sensitive detection methods that work in complex samples such as feces. Results: We devised a highly sensitive semi-quantitative porphyrin detection method (detection limit 1-4 ng heme or PPIX) that can be used to assay pure or complex biological samples, based on Bacteroides growth stimulation. The test revealed that healthy colonized or non-colonized murine and human hosts provide porphyrins in feces, which stimulate Bacteroides growth. In addition, a common microbiota constituent, Escherichia coli, is shown to be a porphyrin donor, suggesting a novel basis for intestinal bacterial interactions. Conclusions: A highly sensitive method to detect porphyrins based on bacterial growth is devised and is functional in complex biological samples. Host feces, independently of their microbiota, and E. coli, which are present in the intestine, are shown to be porphryin donors. The role of porphyrins as key bioactive molecules can now be assessed for their impact on Bacteroides and other bacterial populations in the gut. [ABSTRACT FROM AUTHOR]

Background & Aims Regulation of bile acid homeostasis in mammals is a complex process regulated via extensive cross-talk between liver, intestine and intestinal microbiota. Here we studied the effects of gut microbiota on bile acid homeostasis in mice. Methods Bile acid homeostasis was assessed in four mouse models. Germfree mice, conventionally-raised mice, Asbt -KO mice and intestinal-specific Gata4 -iKO mice were treated with antibiotics (bacitracin, neomycin and vancomycin; 100 mg/kg) for five days and subsequently compared with untreated mice. Results Attenuation of the bacterial flora by antibiotics strongly reduced fecal excretion and synthesis of bile acids, but increased the expression of the bile acid synthesis enzyme CYP7A1 . Similar effects were seen in germfree mice. Intestinal bile acid absorption was increased and accompanied by increases in plasma bile acid levels, biliary bile acid secretion and enterohepatic cycling of bile acids. In the absence of microbiota, the expression of the intestinal bile salt transporter Asbt was strongly increased in the ileum and was also expressed in more proximal parts of the small intestine. Most of the effects of antibiotic treatment on bile acid homeostasis could be prevented by genetic inactivation of either Asbt or the transcription factor Gata4. Conclusions Attenuation of gut microbiota alters Gata4-controlled expression of Asbt, increasing absorption and decreasing synthesis of bile acids. Our data support the concept that under physiological conditions microbiota stimulate Gata4, which suppresses Asbt expression, limiting the expression of this transporter to the terminal ileum. Our studies expand current knowledge on the bacterial control of bile acid homeostasis. [ABSTRACT FROM AUTHOR]

The new field of microbiome research studies the microbes within multicellular hosts and the many effects of these microbes on the host's health and well-being. We now know that microbes influence metabolism, immunity and even behavior. Essential questions, which are just starting to be answered, are what are the mechanisms by which these bacteria affect specific host characteristics. One important but understudied mechanism appears to involve hormones. Although the precise pathways of microbiota-hormonal signaling have not yet been deciphered, specific changes in hormone levels correlate with the presence of the gut microbiota. The microbiota produces and secretes hormones, responds to host hormones and regulates expression levels of host hormones. Here, we summarize the links between the endocrine system and the gut microbiota. We categorize these interactions by the different functions of the hormones, including those affecting behavior, sexual attraction, appetite and metabolism, gender and immunity. Future research in this area will reveal additional connections, and elucidate the pathways and consequences of bacterial interactions with the host endocrine system. [ABSTRACT FROM AUTHOR]

Infection of Swiss/NIH mice with Leishmania major was compared with infection in isogenic resistant C57BL/6 and susceptible BALB/c mice. Swiss/NIH mice showed self-controlled lesions in the injected foot pad. The production of high levels of interferon-g (IFN-g) and low levels of interleukin-4 (IL-4) by cells from these animals suggests that they mount a Th1-type immune response. The importance of the indigenous microbiota on the development of murine leishmaniasis was investigated by infecting germfree Swiss/NIH in the hind footpad with L. major and conventionalizing after 3 weeks of infection. Lesions from conventionalized Swiss/NIH mice were significantly larger than conventional mice. Histopathological analysis of lesions from conventionalized animals showed abscesses of variable shapes and sizes and high numbers of parasitized macrophages. In the lesions from conventional mice, besides the absence of abscess formation, parasites were rarely observed. On the other hand, cells from conventional and conventionalized mice produced similar Th1-type response characterized by high levels of IFN-g and low levels of IL-4. In this study, we demonstrated that Swiss/NIH mice are resistant to L. major infection and that the absence of the normal microbiota at the beginning of infection significantly influenced the lesion size and the inflammatory response at the site of infection.

Abstract: Background and aims: Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety. Methods: Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not. Results: GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats. Conclusions: In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior. [Copyright &y& Elsevier]

Background There is increasing evidence suggesting the existence of an interaction between commensal microbiota, the gut and the brain. The aim of this study was to examine the influence of commensal microbiota on the host behaviors in a contamination-free environment, which was verified by culture-based methods. Methods Open-field and marble-burying tests were used to analyze anxiety-like behaviors and locomotor activity in gnotobiotic BALB/c mice with a common genetic background in a sterile isolator. The monoamine levels in several regions of the brain were measured in germfree ( GF) mice and commensal fecal microbiota-associated mice ( EX- GF). Key Results A 24-h exposure to the environment outside the sterile isolators rendered GF mice less anxious than those not contaminated, while there was no change in the locomotion. EX- GF mice, the gnotobiotic mice with normal specific pathogen-free microbiota, were less anxious and active than GF mice using open-field and marble-burying tests. The norepinephrine, dopamine, and serotonin turnover rates were higher in the EX- GF mice than in the GF mice in most regions of the brain, suggesting that monoaminergic neurotransmission might increase in the EX- GF mice comparing the GF mice. Monoassociation with Brautia coccoides reduced the anxiety level, but it did not affect the locomotor activity. In contrast, colonization with Bifidobacterium infantis decreased the locomotor activity, while having little effect on the anxiety level. Conclusions & Inferences These results strongly support the current view that gut microorganisms modulate brain development and behavior. [ABSTRACT FROM AUTHOR]

There is increasing interest in the bidirectional communication between the mammalian host and prokaryotic cells. Catecholamines (CA), candidate molecules for such communication, are presumed to play an important role in the gut lumen; however, available evidence is limited because of the lack of actual data about luminal CA. This study evaluated luminal CA levels in the gastrointestinal tract and elucidated the involvement of gut microbiota in the generation of luminal CA by comparing the findings among specific pathogen-free mice (SPF-M), germ-free mice (GF-M), and gnotobiotic mice. Substantial levels of free dopamine and norepinephrine were identified in the gut lumen of SPF-M. The free CA levels in the gut lumen were lower in GF-M than in SPF-M. The majority of CA was a biologically active, free form in SPF-M, whereas it was a biologically inactive, conjugated form in GF-M. The association of GF-M with either Clostridium species or SPF fecal flora, both of which have abundant β-glucuronidase activity, resulted in the drastic elevation of free CA. The inoculation of E. coli strain into GF-M induced a substantial amount of free CA, but the inoculation of its mutant strain deficient in the β-glucuronidase gene did not. The intraluminal administration of DA increased colonic water absorption in an in vivo ligated loop model of SPF-M, thus suggesting that luminal DA plays a role as a proabsorptive modulator of water transport in the colon. These results indicate that gut microbiota play a critical role in the generation of free CA in the gut lumen. [ABSTRACT FROM AUTHOR]

Abstract: Thousands of Artemia larvae (1000 to 4800 ind bottles−1) were reared axenically with live and cooked cell wall deficient Saccharomyces cerevisiae (SC) strains as microbial diet to examine the protective effects against vibriosis and the participation of phenoloxidase. Firstly, six different cell wall deficient SC strains (fks1, knr4, kre6, chs3, gas1 and mnn9) were compared for best larval growth and phenoloxidase-like (PO-L) responses after infection with Vibrio proteolyticus. Live SC-mnn9 and SC-gas1 provided the best (P <0.05) dietary support for Artemia growth and induced higher PO-L (P <0.05). Secondly, SC-mnn9, SC-gas1 and the SC-wild type (WT) were prepared as cooked microbial diet and were offered to germfree (GF) larvae. Growth, PO-L responses and survival after infection with Vibrio campbelii were higher (P <0.05) in larvae fed with cooked SC-mnn9 and SC-gas1 compared to SC-WT. Cooked SC-WT was a poor diet for GF Artemia, nevertheless, a rapid diet swap to cooked SC-gas1 or SC mnn9 improved the PO-L and survival against V. campbelii which suggested that the protective effect of SC-mnn9 and SC-gas1 could also be effective in larvae that have been undernourished. Artemia''s PO-L seemed to vary in accordance to growth status, health or disease caused by vibriosis. This apparent capacity of Artemia to express PO-L under health and disease offers an interesting tool to explore its participation in the innate defense system of crustacean larval stages. [Copyright &y& Elsevier]

Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut-homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM-1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM-1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3+β7+ cells were present in similar numbers in CV and GF intestine at birth. They were CD62L– in CV mice and were accompanied by further CD3+β7+CD62L– T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus-derived T cells seed the intestinal lamina propria before birth via β–MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool. [ABSTRACT FROM AUTHOR]

Abstract: Interstitial nephritis occurs spontaneously in kd/kd mice, but the mechanisms leading to this disease have not been fully elucidated. The earliest manifestation of a phenotype is the appearance of ultrastructural defects in the mitochondria of mice as young as 42days of age. To examine the influence of the environment on the phenotype, homozygous B6.kd/kd mice were transferred from specific pathogen-free (SPF) conditions to a germfree (GF) environment, and the development of the disease was observed. The GF state resulted in a highly significant reduction in the frequency of tubulointerstitial nephritis. In addition, GF conditions markedly reduced the appearance of the mitochondrial phenotype, with no sign of mitochondrial abnormalities in GF mice of up to 155days of age. These results suggest that environmental factors are involved in the progression of all known manifestations of this disease phenotype. [Copyright &y& Elsevier]

Three fermented, ropy, non-dairy, oat-based products were evaluated for their effect on serum lipids, faecal cholesterol and faecal short chain fatty acids in germfree and conventional rats. Three different exopolysaccharide (EPS) producing lactic acid bacteria strains were used to ferment the non-dairy oat-base (Adavena® G40) (Ceba Foods AB, Lund, Sweden). Two commercial non-dairy products based on oats (Mill Milk™) (Ceba Foods AB, Lund, Sweden) and rice (Rice Dream®) (Imagine Foods, London, UK) were used as non-ropy and unfermented controls. All the standardized feeds were sterilized before being fed to the animals. Adult, germfree and conventional AGUS rats, were fed the above sterile diets ad libitum for 21 days. Blood samples and faecal samples were collected and the animals’ weight gain was monitored throughout the study. No significant change in serum lipids or faecal excretion of cholesterol was observed between the groups on the different diets. A difference in faecal SCFA pattern was observed in conventional rats fed on the oat-based diets in comparison to the group fed on the rice-based diet. More evidence is needed to support the effect of fermented, ropy, oat-based products and their potential effect on serum lipids, faecal cholesterol/coprostanol levels and amounts of short chain fatty acids. [Copyright &y& Elsevier]

This chapter will summarise the role of some functionally active intestinal microbes acting on man and animal on some anatomical, physiological and biochemical functions. The influences on immunological functions will be presented separately. The baselines for these functions refer to studies performed in material from germfree animals and healthy newborns, and values obtained are compared to functions in a macro-organism which have been in affected by a micro-flora. In principle, these functions are similar in all the organisms so far investigated, only the levels vary, due to age, and different dietary components and environmental factors. Alterations of the normal pattern of these functions mainly reflects altered ecological conditions in the organism and the reasons for these are sometimes known-sometimes unknown. The consequences after alterations of these functions have to be investigated in several ways, i.e., both with regard to local and distal effects on the host organism. [ABSTRACT FROM AUTHOR]

The intestinal flora may act on cholesterol metabolism either, directly by converting the molecule to coprostanol, or indirectly, by influencing upon intestinal bile acid metabolism, mainly by deconjugation. In the present study, sixteen probiotic strains belonging to the genera: Bifidobacterium, Enterococcus, Lactobacillus and Streptococcus were investigated for possible direct action on cholesterol metabolism in vitro and in vivo in ex-germfree mice monoassociated with each probiotic. Additionally, deconjugation of taurodeoxycholic acid (TDC) and of glycodeoxycholic acid (GDC) was investigated in vitro as an indirect effect upon cholesterol metabolism, using the same strains. Gas-liquid chromatography and thin-layer chromatography were used. None of the probiotic strains, was able to transform cholesterol to coprostanol in vitro or in vivo. Of the total probiotics analyzed, B. bifidum B12, L. acidophilus La5, L. acidophilus ATCC4356, L. fermentum ATCC14931, L. plantarum 299v, L. rhamnosus ATCC7469 and E. faecium were able to deconjugate both TDC and GDC in vitro. We conclude that the formation of coprostanol does not account for the supposed cholesterol-lowering effect of the probiotics tested. As some of the probiotics were able to deconjugate TDC and/or GDC in vitro, this microbial function should be studied more extensively in vivo. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the cell kinetics and epithelial structure in the intestinal tract in cohorts of young and old germfree (GF) and conventional AGUS rats of both sexes. The young rats were 35 days old and the old rats were 190960 days old. All rats were fed the same diet and water ad libitum. At the beginning of the experiments, the rats were starved for 2.5 h. Thereafter, all animals were given vincristine 1 mg/kg intraperitoneally (i.p.) 4 h before they were sacrificed. Specimens from eight parts of the intestine were investigated. The cell kinetic parameters determined in the small intestine were percentage of cells in mitosis, growth fraction (GrF), total number of cells in the crypts and villi, depth of the crypts, height of villi and the crypt/villus (C/V) ratio. In the colon, the percentage of cells in mitosis, GrF and the total number of cells in the crypts, as well as depth of the crypts, were determined. Taken together, the effect of gender was most pronounced in the upper part of the small intestine, the effect of age was most pronounced in the lower small intestine, whereas the effect of microbial status was relatively more pronounced in the duodenum and in the large intestine. Thus, these factors are acting in a compartmentalized manner in the intestinal tract. In conclusion, gender, age and microbial status have to be taken. [ABSTRACT FROM AUTHOR]

Studies in mice using germfree animals as controls for microbial colonization have shown that the gut microbiome mediates diet-induced obesity. Such studies use diets rich in saturated fat, however, Western diets in the United States America are enriched in soybean oil, composed of unsaturated fatty acids, either linoleic or oleic acid. Here, we addressed whether the microbiome is a variable in fat metabolism in mice on a soybean oil diet. We used conventionally-raised, low-germ, and germfree mice fed for 10 weeks diets either high or low in high-linoleic-acid soybean oil as the sole source of fat. Conventional and germfree mice gained relative fat weight and all mice consumed more calories on the high fat vs. low fat soybean oil diet. Plasma fatty acid levels were generally dependent on diet, with microbial colonization status affecting iso-C18:0, C20:3n-6, C14:0, and C15:0 levels. Colonization status, but not diet, impacted levels of liver sphingolipids including ceramides, sphingomyelins, and sphinganine. Our results confirm that absorbed fatty acids are mainly a reflection of the diet and that microbial colonization influences liver sphingolipid pools regardless of diet. [ABSTRACT FROM AUTHOR]

Postsurgical adhesions occur commonly after surgical procedures and are the source of substantial postoperative morbidity. No preventive or prophylactic regimen against adhesions has proven successful in all circumstances. The reasons for this are not clear. The basic mechanisms causing adhesion formation have not been elucidated fully, and furthermore, lack of accurate methods of measuring adhesions may be a contributing factor. Postoperative adhesions may occur in all kinds of surgery but are especially prominent in the abdomen, where the bowel flora may be a compromising factor. This study was undertaken to study the influence of the gastrointestinal microflora on adhesion formation. Germfree and ex-germfree DA rats were subjected to a cecal crush model, and adhesions were evaluated after 7 days using 3 different scoring scales. Germfree rats formed significantly fewer adhesions than their ex-germfree (conventionalized) counterparts. The differences were so great that all three scoring scales achieved significance ( p < .005). This study corroborates that the endogenous bowel flora per se is involved in adhesion formation without causing frank infection. [ABSTRACT FROM AUTHOR]

The cecum of germfree rats, as studied by light microscopy and scanning and transmission electron microscopy, differs in many respects from the cecum of conventional rats. Epithelial cells in germfree rats are taller and have larger nuclei and longer microvilli than similar cells in conventional rats. The cecal mucosa of germfree rats shows a larger variability in the arrangement of the crypts of Lieberkühn than does the mucosa of conventional rats. Some crypts are funnel-shaped and connected close to the mucosal surface with adjacent similar crypts to form long valleys. Less wide crypts open on elevated regions of the mucosal surface. The lamina propria of germfree animals is devoid of plasma cells but rich in mast cells. Germfree animals show hypertrophy of the tunica muscularis externa. In conventional rats the cecal lumen contains a large variety of morphologically different bacteria. However, the lumen of the crypts of Lieberkühn contains only one type of elongated bacteria, which are present in large amounts. This finding suggests that symbiotic relations may be of particular importance in the crypts of Lieberkühn in the cecum. [ABSTRACT FROM AUTHOR]

Gut microbiota significantly influence the plasma and liver lipidome. An interconnecting metabolite is acetate generated after degradation and fermentation of dietary fiber by the gut microbiota, which is metabolized in the liver into longer chain fatty acids and complex lipids reaching the circulation. Whether these systemic changes are accompanied by alternations of the intestinal lipidome is unclear. Therefore, we quantified glycerophospholipids, sphingolipids and sterols in ileum and colon, the two segments containing the highest densities of microbes in the gastrointestinal tract, of germfree and specific pathogen free mice using mass spectrometry-based lipidomics. We found that the presence of gut microbes lowers the free cholesterol content in colon while elevating phosphatidylcholine levels. Further, PUFA-containing phosphatidylcholine and -ethanolamine fractions are increased in ileum and colon of germfree compared to SPF mice. A total fatty acid analysis by GC–MS revealed higher levels of arachidonic and docosahexaenoic acid in the ileum of germfree mice indicating that the gut microbiota inhibits PUFA metabolism in the small intestine. [ABSTRACT FROM AUTHOR]

Omnivorous animals, including humans, harbor diverse, species-rich gut communities that impact their growth, development, and homeostasis. Model invertebrates are broadly accessible experimental platforms that enable linking specific species or species groups to host phenotypes, yet often their specialized diets and distinct gut microbiota make them less comparable to human and other mammalian and gut communities. The omnivorous cockroach Periplaneta americana harbors ∼4 × 10 2 bacterial genera within its digestive tract and is enriched with taxa commonly found in omnivorous mammals (i.e., Proteobacteria, Bacteroidetes , and Firmicutes ). These features make P. americana a valuable platform for identifying microbe-mediated host phenotypes with potential translations to mammals. Rearing P. americana insects under germfree conditions resulted in prolonging development time by ∼30% and an up to ∼8% reduction in body size along three dimensions. Germfree rearing resulted in downregulation of gene networks involved in growth, energy homeostasis, and nutrient availability. Reintroduction of a defined microbiota comprised of a subset of P. americana commensals to germfree insects did not recover normal growth and developmental phenotypes or transcriptional profiles observed in conventionally reared insects. These results are in contrast with specialist-feeding model insects (e.g., Drosophila ), where introduction of a single endemic bacterial species to germfree condition-reared specimens recovered normal host phenotypes. These data suggest that understanding microbe-mediated host outcomes in animals with species-rich communities should include models that typically maintain similarly diverse microbiomes. The dramatic transcriptional, developmental, and morphological phenotypes linked to gut microbiome status in this study illustrates how microbes are key players in animal growth and evolution. IMPORTANCE Broadly accessible model organisms are essential for illustrating how microbes are engaged in the growth, development, and evolution of animals. We report that germfree rearing of omnivorous Periplaneta americana cockroaches resulted in growth defects and severely disrupted gene networks that regulate development, which highlights the importance of gut microbiota in these host processes. Absence of gut microbiota elicited a starvation-like transcriptional response in which growth and development were inhibited while nutrient scavenging was enhanced. Additionally, reintroduction of a subset of cockroach gut bacterial commensals did not broadly recover normal expression patterns, illustrating that a particular microbiome composition may be necessary for normal host development. Invertebrate microbiota model systems that enable disentangling complex, species-rich communities are essential for linking microbial taxa to specific host phenotypes.

The association of vertebrate hosts with the indigenous microbiota and its effect on the response to infections has long been a subject of scientific curiosity. From the first theory supported by Louis Pasteur that life would be impossible in the absence of associated microorganisms to the development of germfree mammals for research, a lot was learned about how the normal microbiota influences the environment in which pathogens may find themselves. In the present review, we attempt to summarize the more recent results from our group and others on the influence of the normal microbiota on the outcome of parasitic infections. Our results and those of others point to a complex relationship between the mammalian system and its indigenous microbiota, leading to greater resistance to some infections and enhanced susceptibility to others

Disrupted interactions between host and intestinal bacteria are implicated in colorectal cancer (CRC) development. However, activities derived from these bacteria and their interplay with the host are unclear. Here, we examine this interplay by performing mouse and microbiota RNA sequencing on colon tissues and 16S and small RNA sequencing on stools from germfree (GF) and gnotobiotic Apc Min Δ 850/+ ; Il10 -/- mice associated with microbes from biofilm-positive human CRC tumor (BF+T) and biofilm-negative healthy (BF-bx) tissues. The bacteria in BF+T mice differentially expressed (DE) >2,900 genes, including genes related to bacterial secretion, virulence, and biofilms but affected only 62 host genes. Small RNA sequencing of stools from these cohorts revealed eight significant DE host microRNAs (miRNAs) based on biofilm status and several miRNAs that correlated with bacterial taxon abundances. Additionally, computational predictions suggest that some miRNAs preferentially target bacterial genes while others primarily target mouse genes. 16S rRNA sequencing of mice that were reassociated with mucosa-associated communities from the initial association revealed a set of 13 bacterial genera associated with cancer that were maintained regardless of whether the reassociation inoculums were initially obtained from murine proximal or distal colon tissues. Our findings suggest that complex interactions within bacterial communities affect host-derived miRNA, bacterial composition, and CRC development. IMPORTANCE Bacteria and bacterial biofilms have been implicated in colorectal cancer (CRC), but it is still unclear what genes these microbial communities express and how they influence the host. MicroRNAs regulate host gene expression and have been explored as potential biomarkers for CRC. An emerging area of research is the ability of microRNAs to impact growth and gene expression of members of the intestinal microbiota. This study examined the bacteria and bacterial transcriptome associated with microbes derived from biofilm-positive human cancers that promoted tumorigenesis in a murine model of CRC. The murine response to different microbial communities (derived from CRC patients or healthy people) was evaluated through RNA and microRNA sequencing. We identified a complex interplay between biofilm-associated bacteria and the host during CRC in mice. These findings may lead to the development of new biomarkers and therapeutics for identifying and treating biofilm-associated CRCs., (Copyright © 2020 Tomkovich et al.)

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-14C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO2 was collected throughout the period. The conventional rats released 50% more 14C as 14CO2 than the germfree animals. The total amount of the label recovered as 14CO2 during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of 14CO2 accounted for approximately 75% of the 14C recovered in forms other than the original cholesterol-26-14C; 15–20% was found incorporated in water-soluble and fat-soluble fractions other than 3Β-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals.The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of 14CO2 from cholesterol-26-14C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr -/- mice and CONV-R Ldlr -/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr -/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr -/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr -/- mice on HFD were diminished compared to CONV-R Ldlr -/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr -/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr -/- mice relative to CONV-R Ldlr -/- mice. Ex vivo , this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr -/- mice on type III collagen. IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr -/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions., (Copyright © 2019 Kiouptsi et al.)