Microbiome composition and presence of cultivable commensal groups of Southern Tamanduas (Tamandua tetradactyla) varies with captive conditions.

Southern Tamanduas (Tamandua tetradactyla) belong to the specialized placental myrmecophages. There is not much information about their intestinal microbiome. Moreover, due to their food specialization, it is difficult to create an adequate diet under breeding conditions. Therefore, we used 16S rDNA amplicon sequencing to analyze the fecal microbiome of captive Southern Tamanduas from four locations in the Czech Republic and evaluated the impact of the incoming diet and facility conditions on microbiome composition. Together with the microbiome analysis, we also quantified and identified cultivable commensals. The anteater fecal microbiome was dominated by the phyla Bacillota and Bacteroidota, while Pseudomonadota, Spirochaetota, and Actinobacteriota were less abundant. At the taxonomic family level, Lachnospiraceae, Prevotellaceae, Bacteroidaceae, Oscillospiraceae, Erysipelotrichaceae, Spirochaetaceae, Ruminococcaceae, Leuconostocaceae, and Streptococcaceae were mainly represented in the fecal microbiome of animals from all locations. Interestingly, Lactobacillaceae dominated in the location with a zoo-made diet. These animals also had significantly lower diversity of gut microbiome in comparison with animals from other locations fed mainly with a complete commercial diet. Moreover, captive conditions of analyzed anteater included other factors such as the enrichment of the diet with insect-based products, probiotic interventions, the presence of other animals in the exposure, which can potentially affect the composition of the microbiome and cultivable microbes. In total, 63 bacterial species from beneficial commensal to opportunistic pathogen were isolated and identified using MALDI-TOF MS in the set of more than one thousand selected isolates. Half of the detected species were present in the fecal microbiota of most animals, the rest varied across animals and locations.

Commensal Bacteria Impact on Intestinal Toll-like Receptor Signaling in Salmonella-Challenged Gnotobiotic Piglets.

Gnotobiotic (GN) animals with simple and defined microbiota can help to elucidate host-pathogen interferences. Hysterectomy-derived germ-free (GF) minipigs were associated at 4 and 24 h post-hysterectomy with porcine commensal mucinolytic Bifidobacterium boum RP36 (RP36) strain or non-mucinolytic strain RP37 (RP37) or at 4 h post-hysterectomy with Lactobacillus amylovorus (LA). One-week-old GN minipigs were infected with Salmonella Typhimurium LT2 strain (LT2). We monitored histological changes in the ileum, mRNA expression of Toll-like receptors (TLRs) 2, 4, and 9 and their related molecules lipopolysaccharide-binding protein (LBP), coreceptors MD-2 and CD14, adaptor proteins MyD88 and TRIF, and receptor for advanced glycation end products (RAGE) in the ileum and colon. LT2 significantly induced expression of TLR2, TLR4, MyD88, LBP, MD-2, and CD14 in the ileum and TLR4, MyD88, TRIF, LBP, and CD14 in the colon. The LT2 infection also significantly increased plasmatic levels of inflammatory markers interleukin (IL)-6 and IL-12/23p40. The previous colonization with RP37 alleviated damage of the ileum caused by the Salmonella infection, and RP37 and LA downregulated plasmatic levels of IL-6. A defined oligo-microbiota composed of bacterial species with selected properties should probably be more effective in downregulating inflammatory response than single bacteria.

Culture-dependent screening of endospore-forming clostridia in infant feces.

BACKGROUND: Only a few studies dealt with the occurrence of endospore-forming clostridia in the microbiota of infants without obvious health complications. METHODS: A methodology pipeline was developed to determine the occurrence of endospore formers in infant feces. Twenty-four fecal samples (FS) were collected from one infant in monthly intervals and were subjected to variable chemical and heat treatment in combination with culture-dependent analysis. Isolates were identified by MALDI-TOF mass spectrometry, 16S rRNA gene sequencing, and characterized with biochemical assays. RESULTS: More than 800 isolates were obtained, and a total of 21 Eubacteriales taxa belonging to the Clostridiaceae, Lachnospiraceae, Oscillospiraceae, and Peptostreptococcaceae families were detected. Clostridium perfringens, C. paraputrificum, C. tertium, C. symbiosum, C. butyricum, and C. ramosum were the most frequently identified species compared to the rarely detected Enterocloster bolteae, C. baratii, and C. jeddahense. Furthermore, the methodology enabled the subsequent cultivation of less frequently detectable gut taxa such as Flavonifractor plautii, Intestinibacter bartlettii, Eisenbergiella tayi, and Eubacterium tenue. The isolates showed phenotypic variability regarding enzymatic activity, fermentation profiles, and butyrate production. CONCLUSIONS: Taken together, this approach suggests and challenges a cultivation-based pipeline that allows the investigation of the population of endospore formers in complex ecosystems such as the human gastrointestinal tract.

Breastfeeding and the major fermentation metabolite lactate determine occurrence of Peptostreptococcaceae in infant feces.

Previous studies indicated an intrinsic relationship between infant diet, intestinal microbiota composition and fermentation activity with a strong focus on the role of breastfeeding on microbiota composition. Yet, microbially formed short-chain fatty acids acetate, propionate and butyrate and other fermentation metabolites such as lactate not only act as substrate for bacterial cross-feeding and as mediators in microbe-host interactions but also confer antimicrobial activity, which has received considerably less attention in the past research. It was the aim of this study to investigate the nutritional-microbial interactions that contribute to the development of infant gut microbiota with a focus on human milk oligosaccharide (HMO) fermentation. Infant fecal microbiota composition, fermentation metabolites and milk composition were analyzed from 69 mother-infant pairs of the Swiss birth cohort Childhood AlleRgy nutrition and Environment (CARE) at three time points depending on breastfeeding status defined at the age of 4 months, using quantitative microbiota profiling, HPLC-RI and 1H-NMR. We conducted in vitro fermentations in the presence of HMO fermentation metabolites and determined the antimicrobial activity of lactate and acetate against major Clostridiaceae and Peptostreptococcaceae representatives. Our data show that fucosyllactose represented 90% of the HMOs present in breast milk at 1- and 3-months post-partum with fecal accumulation of fucose, 1,2-propanediol and lactate indicating fermentation of HMOs that is likely driven by Bifidobacterium. Concurrently, there was a significantly lower absolute abundance of Peptostreptococcaceae in feces of exclusively breastfed infants at 3 months. In vitro, lactate inhibited strains of Peptostreptococcaceae. Taken together, this study not only identified breastfeeding dependent fecal microbiota and metabolite profiles but suggests that HMO-derived fermentation metabolites might exert an inhibitory effect against selected gut microbes.

Species and Strain Variability among Sarcina Isolates from Diverse Mammalian Hosts.

Sarcina spp. has been isolated from the gastrointestinal tracts of diverse mammalian hosts. Their presence is often associated with host health complications, as is evident from many previously published medical case reports. However, only a handful of studies have made proper identification. Most other identifications were solely based on typical Sarcina-like morphology without genotyping. Therefore, the aim of this work was culture detection and the taxonomic classification of Sarcina isolates originating from different mammalian hosts. Sarcina-like colonies were isolated and collected during cultivation analyses of animal fecal samples (n = 197) from primates, dogs, calves of domestic cattle, elephants, and rhinoceroses. The study was carried out on apparently healthy animals kept in zoos or by breeders in the Czech Republic and Slovakia. Selected isolates were identified and compared using 16S rRNA gene sequencing and multi-locus sequence analysis (MLSA; Iles, pheT, pyrG, rplB, rplC, and rpsC). The results indicate the taxonomic variability of Sarcina isolates. S. ventriculi appears to be a common gut microorganism in various captive primates. In contrast, a random occurrence was also recorded in dogs. However, dog isolate N13/4e could represent the next potential novel Sarcina taxonomic unit. Also, a potentially novel Sarcina species was found in elephants, with occurrences in all tested hosts. S. maxima isolates were detected rarely, only in rhinoceroses. Although Sarcina bacteria are often linked to lethal diseases, our results indicate that Sarcina spp. appear to be a common member of the gut microbiota and seem to be an opportunistic pathogen. Further characterization and pathogenic analyses are required.

Release of HMGB1 and Toll-like Receptors 2, 4, and 9 Signaling Are Modulated by Bifidobacterium animalis subsp. lactis BB-12 and Salmonella Typhimurium in a Gnotobiotic Piglet Model of Preterm Infants.

Gnotobiotic (GN) animals with defined microbiota allow us to study host-microbiota and microbiota-microbiota interferences. Preterm germ-free (GF) piglets were mono-associated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to ameliorate/prevent the consequences of infection with the Salmonella Typhimurium strain LT2 (LT2). Goblet cell density; expression of Toll-like receptors (TLRs) 2, 4, and 9; high mobility group box 1 (HMGB1); interleukin (IL)-6; and IL-12/23p40 were analyzed to evaluate the possible modulatory effect of BB12. BB12 prevented an LT2-induced decrease of goblet cell density in the colon. TLRs signaling modified by LT2 was not influenced by the previous association with BB12. The expression of HMGB1, IL-6, and IL12/23p40 in the jejunum, ileum, and colon and their levels in plasma were all decreased by BB12, but these changes were not statistically significant. In the colon, differences in HMGB1 distribution between the GF and LT2 piglet groups were observed. In conclusion, the mono-association of GF piglets with BB12 prior to LT2 infection partially ameliorated the inflammatory response to LT2 infection.

Feed Insects as a Reservoir of Granadaene-Producing Lactococci.

Insects are a component of the diet of different animal species and have been suggested as the major source of human dietary protein for the future. However, insects are also carriers of potentially pathogenic microbes that constitute a risk to food and feed safety. In this study, we reported the occurrence of a hemolytic orange pigmented producing phenotype of Lactococcus garvieae/petauri/formosensis in the fecal microbiota of golden lion tamarins (Leontopithecus rosalia) and feed larvae (Zophobas atratus). Feed insects were identified as a regular source of L. garvieae/petauri/formosensis based on a reanalysis of available 16S rRNA gene libraries. Pan-genome analysis suggested the existence of four clusters within the L. garvieae/petauri/formosensis group. The presence of cyl cluster indicated that some strains of the L. garvieae/petauri/formosensis group produced a pigment similar to granadaene, an orange cytotoxic lipid produced by group B streptococci, including Streptococcus agalactiae. Pigment production by L. garvieae/petauri/formosensis strains was dependent on the presence of the fermentable sugars, with no pigment being observed at pH <4.7. The addition of buffering compounds or arginine, which can be metabolized to ammonium, restored pigment formation. In addition, pigment formation might be related to the source of peptone. These data suggest that edible insects are a possible source of granadaene-producing lactococci, which can be considered a pathogenic risk with zoonotic potential.

The bifidobacterial distribution in the microbiome of captive primates reflects parvorder and feed specialization of the host.

Bifidobacteria, which commonly inhabit the primate gut, are beneficial contributors to host wellbeing. Anatomical differences and natural habitat allow an arrangement of primates into two main parvorders; New World monkeys (NWM) and Old World monkeys (OWM). The number of newly described bifidobacterial species is clearly elevated in NWM. This corresponds to our finding that bifidobacteria were the dominant group of cultivated gut anaerobes in NWM, while their numbers halved in OWM and were often replaced by Clostridiaceae with sarcina morphology. We examined an extended MALDI-TOF MS database as a potential identification tool for rapid screening of bifidobacterial distribution in captive primates. Bifidobacterial isolates of NWM were assigned mainly to species of primate origin, while OWM possessed typically multi-host bifidobacteria. Moreover, bifidobacterial counts reflected the feed specialization of captive primates decreasing from frugivore-insectivores, gummivore-insectivores, frugivore-folivores to frugivore-omnivores. Amplicon sequencing analysis supported this trend with regards to the inverse ratio of Actinobacteria and Firmicutes. In addition, a significantly higher diversity of the bacterial population in OWM was found. The evolution specialization of primates seems to be responsible for Bifidobacterium abundance and species occurrence. Balanced microbiota of captive primates could be supported by optimized prebiotic and probiotic stimulation based on the primate host.

3-Hydroxypropionic acid contributes to the antibacterial activity of glycerol metabolism by the food microbe Limosilactobacillus reuteri.

Strains of Limosilactobacillus reuteri are used as starter and bioprotective cultures and contribute to the preservation of food through the production of fermentation metabolites lactic and acetic acid, and of the antimicrobial reuterin. Reuterin consists of acrolein and 3-hydroxypropionaldehyde (3-HPA), which can be further metabolized to 1,3-propanediol and 3-hydroxypropionic acid (3-HP). While reuterin has been the focus of many investigations, the contribution of 3-HP to the antimicrobial activity of food related reuterin-producers is unknown. We show that the antibacterial activity of 3-HP was stronger at pH 4.8 compared to pH 5.5 and 6.6. Gram-positive bacteria were in general more resistant against 3-HP and propionic acid than Gram-negative indicator strains including common food pathogens, while spoilage yeast and molds were not inhibited by ≤ 640 mM 3-HP. The presence of acrolein decreased the minimal inhibitory activity of 3-HP against E. coli indicating synergistic antibacterial activity. 3-HP was formed during the growth of the reuterin-producers, and by resting cells of L. reuteri DSM 20016. Taken together, this study shows that food-related reuterin producers strains synthesize a second antibacterial compound, which might be of relevance when strains are added as starter or bioprotective cultures to food products.

Antibiotic susceptibility screening of primate-associated Clostridium ventriculi.

Clostridium ventriculi (syn. Sarcina ventriculi) is a Gram-positive opportunistic pathogen with sarcina morphology. In the case of gastrointestinal disorders, the treatment is often empirical. Due to the common occurrence in primates and the potential risk of dysbiosis; the antibiotic susceptibility screening of C. ventriculi strains isolated from guenon monkeys and crested gibbons to 58 antibiotics was performed to reduce potentially ineffective antibiotic use in case of disease. Isolates were found to be susceptible to the majority of the tested antibiotics, mainly to (fluoro)quinolones, macrolides, penicillins, and tetracyclines. The susceptibility profiles were similar despite the hosts. Tested strains showed also natural resistance to a few antibiotics on the genus level. Detected in vitro antibiotic efficiency is consistent with documented human treatment cases.

Five novel bifidobacterial species isolated from faeces of primates in two Czech zoos: Bifidobacterium erythrocebi sp. nov., Bifidobacterium moraviense sp. nov., Bifidobacterium oedipodis sp. nov., Bifidobacterium olomucense sp. nov. and Bifidobacterium panos sp. nov.

Five Bifidobacterium strains, VB23T, VB24T, VB25T, VB26T and VB31T, were isolated from chimpanzee (Pan troglodytes), cotton-top tamarin (Saguinus oedipus), Goeldi's marmoset (Callimico goeldii), moustached tamarin (Saguinus mystax) and patas monkey (Erythrocebus patas), respectively, which were kept in two Czech zoos. These strains were isolated from faecal samples and were Gram-positive, non-motile, non-sporulating, anaerobic and fructose-6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA revealed close relatedness between VB23T and Bifidobacterium angulatum LMG 11039T (96.0 %), VB24T and Bifidobacterium pullorum subsp. pullorum DSM 20433T (96.1 %), VB25T and Bifidobacterium goeldii LMG 30939T (96.5 %), VB26T and Bifidobacterium imperatoris LMG 30297T (98.1 %), and VB31T and B. angulatum LMG 11039T (99.40 %). Internal transcribed spacer profiling revealed that VB23T, VB24T, VB25T, VB26T and VB31T had highest similarity to Bifidobacterium breve LMG 13208T (77.2 %), Bifidobacterium longum subsp. infantis ATCC 15697T (85.8 %), Bifidobacterium biavatii DSM 23969T (76.9 %), B. breve LMG 13208T (81.2 %) and B. angulatum LMG 11039T (88.2 %), respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses with their closest neighbours supported the independent phylogenetic positions of the strains with values between 86.3 and 94.3 % for ANI and 25.8 and 54.9 % for dDDH. These genomic and phylogenetic analyses suggested that the evaluated strains were novel Bifidobacterium species named Bifidobacterium erythrocebi sp. nov. (VB31T=DSM 109960T=CCUG 73843T), Bifidobacterium moraviense sp. nov. (VB25T=DSM 109958T=CCUG 73842T), Bifidobacterium oedipodis sp. nov. (VB24T=DSM 109957T=CCUG 73932T), Bifidobacterium olomucense sp. nov. (VB26T=DSM 109959T=CCUG 73845T) and Bifidobacterium panos sp. nov. (VB23T=DSM 109963T=CCUG 73840T).

Decoding the Genomic Variability among Members of the Bifidobacterium dentium Species.

Members of the Bifidobacterium dentium species are usually identified in the oral cavity of humans and associated with the development of plaque and dental caries. Nevertheless, they have also been detected from fecal samples, highlighting a widespread distribution among mammals. To explore the genetic variability of this species, we isolated and sequenced the genomes of 18 different B. dentium strains collected from fecal samples of several primate species and an Ursus arctos. Thus, we investigated the genomic variability and metabolic abilities of the new B. dentium isolates together with 20 public genome sequences. Comparative genomic analyses provided insights into the vast metabolic repertoire of the species, highlighting 19 glycosyl hydrolases families shared between each analyzed strain. Phylogenetic analysis of the B. dentium taxon, involving 1140 conserved genes, revealed a very close phylogenetic relatedness among members of this species. Furthermore, low genomic variability between strains was also confirmed by an average nucleotide identity analysis showing values higher than 98.2%. Investigating the genetic features of each strain, few putative functional mobile elements were identified. Besides, a consistent occurrence of defense mechanisms such as CRISPR-Cas and restriction-modification systems may be responsible for the high genome synteny identified among members of this taxon.

Bifidobacterium canis sp. nov., a novel member of the Bifidobacterium pseudolongum phylogenetic group isolated from faeces of a dog (Canis lupus f. familiaris).

A fructose-6-phosphate phosphoketolase-positive strain (GSD1FST) was isolated from a faecal sample of a 3 weeks old German Shepherd dog. The closest related taxa to isolate GSD1FST based on results from the EZBioCloud database were Bifidobacterium animalis subsp. animalis ATCC 25527T, Bifidobacterium animalis subsp. lactis DSM 10140T and Bifidobacterium anseris LMG 30189T, belonging to the Bifidobacterium pseudolongum phylogenetic group. The resulting 16S rRNA gene identities (compared length of 1454 nucleotides) towards these taxa were 97.30, 97.23 and 97.09 %, respectively. The pairwise similarities of strain GSD1FST using argS, atpA, fusA, hsp60, pyrG, rpsC, thrS and xfp gene fragments to all valid representatives of the B. pseudolongum phylogenetic group were in the concatenated range of 83.08-88.34 %. Phylogenomic analysis based on whole-genome methods such as average nucleotide identity revealed that bifidobacterial strain GSD1FST exhibits close phylogenetic relatedness (88.17 %) to Bifidobacetrium cuniculi LMG 10738T. Genotypic characteristics and phylogenetic analyses based on nine molecular markers, as well as genomic and comparative phenotypic analyses, clearly proved that the evaluated strain should be considered as representing a novel species within the B. pseudolongum phylogenetic group named as Bifidobacterium canis sp. nov. (GSD1FST=DSM 105923T=LMG 30345T=CCM 8806T).

Initial butyrate producers during infant gut microbiota development are endospore formers.

The acquisition of the infant gut microbiota is key to establishing a host-microbiota symbiosis. Microbially produced metabolites tightly interact with the immune system, and the fermentation-derived short-chain fatty acid butyrate is considered an important mediator linked to chronic diseases later in life. The intestinal butyrate-forming bacterial population is taxonomically and functionally diverse and includes endospore formers with high transmission potential. Succession, and contribution of butyrate-producing taxa during infant gut microbiota development have been little investigated. We determined the abundance of major butyrate-forming groups and fermentation metabolites in faeces, isolated, cultivated and characterized the heat-resistant cell population, which included endospores, and compared butyrate formation efficiency of representative taxa in batch cultures. The endospore community contributed about 0.001% to total cells, and was mainly composed of the pioneer butyrate-producing Clostridium sensu stricto. We observed an increase in abundance of Faecalibacterium prausnitzii, butyrate-producing Lachnospiraceae and faecal butyrate levels with age that is likely explained by higher butyrate production capacity of contributing taxa compared with Clostridium sensu stricto. Our data suggest that a successional arrangement and an overall increase in abundance of butyrate forming populations occur during the first year of life, which is associated with an increase of intestinal butyrate formation capacity.

Bifidobacterium ß-Glucosidase Activity and Fermentation of Dietary Plant Glucosides Is Species and Strain Specific.

Dietary plant glucosides are phytochemicals whose bioactivity and bioavailability can be modified by glucoside hydrolase activity of intestinal microbiota through the release of acylglycones. Bifidobacteria are gut commensals whose genomic potential indicates host-adaption as they possess a diverse set of glycosyl hydrolases giving access to a variety of dietary glycans. We hypothesized bifidobacteria with ß-glucosidase activity could use plant glucosides as fermentation substrate and tested 115 strains assigned to eight different species and from different hosts for their potential to express ß-glucosidases and ability to grow in the presence of esculin, amygdalin, and arbutin. Concurrently, the antibacterial activity of arbutin and its acylglycone hydroquinone was investigated. Beta-glucosidase activity of bifidobacteria was species specific and most prevalent in species occurring in human adults and animal hosts. Utilization and fermentation profiles of plant glucosides differed between strains and might provide a competitive benefit enabling the intestinal use of dietary plant glucosides as energy sources. Bifidobacterial ß-glucosidase activity can increase the bioactivity of plant glucosides through the release of acylglycone.

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium.

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

Colonization of Germ-Free Piglets with Commensal Lactobacillus amylovorus, Lactobacillus mucosae, and Probiotic E. coli Nissle 1917 and Their Interference with Salmonella Typhimurium.

Non-typhoid Salmonellae are worldwide spread food-borne pathogens that cause diarrhea in humans and animals. Their multi-drug resistances require alternative ways to combat this enteric pathogen. Mono-colonization of a gnotobiotic piglet gastrointestinal tract with commensal lactobacilli Lactobacillus amylovorus and Lactobacillus mucosae and with probiotic E. coli Nissle 1917 and their interference with S. Typhimurium infection was compared. The impact of bacteria and possible protection against infection with Salmonella were evaluated by clinical signs, bacterial translocation, intestinal histology, mRNA expression of villin, claudin-1, claudin-2, and occludin in the ileum and colon, and local intestinal and systemic levels of inflammatory cytokines IL-8, TNF-α, and IL-10. Both lactobacilli colonized the gastrointestinal tract in approximately 100× lower density compare to E. coli Nissle and S. Typhimurium. Neither L. amylovorus nor L. mucosae suppressed the inflammatory reaction caused by the 24 h infection with S. Typhimurium. In contrast, probiotic E. coli Nissle 1917 was able to suppress clinical signs, histopathological changes, the transcriptions of the proteins, and the inductions of the inflammatory cytokines. Future studies are needed to determine whether prebiotic support of the growth of lactobacilli and multistrain lactobacilli inoculum could show higher protective effects.