Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro.

BACKGROUND: Chronic airway disease (CAD) is characterized by chronic airway inflammation and colonization of the lungs by pro-inflammatory pathogens. However, while various other bacterial species are present in the lower airways, it is not fully understood how they influence inflammation. We aimed to identify novel anti-inflammatory species present in lower airway samples of patients with CAD. METHODS: Paired sputum microbiome and inflammatory marker data of adults with CAD across three separate cohorts (Australian asthma and bronchiectasis, Scottish bronchiectasis) was analyzed using Linear discriminant analysis Effect Size (LEfSE) and Spearman correlation analysis to identify species associated with a low inflammatory profile in patients. RESULTS: We identified the genus Aggregatibacter as more abundant in patients with lower levels of airway inflammatory markers in two CAD cohorts (Australian asthma and bronchiectasis). In addition, the relative abundance of Aggregatibacter was inversely correlated with sputum IL-8 (Australian bronchiectasis) and IL-1ß levels (Australian asthma and bronchiectasis). Subsequent in vitro testing, using a physiologically relevant three-dimensional lung epithelial cell model, revealed that Aggregatibacter spp. (i.e. A. actinomycetemcomitans, A. aphrophilus) and their cell-free supernatant exerted anti-inflammatory activity without influencing host cell viability. CONCLUSIONS: These findings suggest that Aggregatibacter spp. might act to reduce airway inflammation in CAD patients.

Oral microbiota in autistic children: Diagnosis-related differences and associations with clinical characteristics.

Similar to the gut microbiome, oral microbiome compositions have been suggested to play an important role in the etiology of autism. However, empirical research on how variations in the oral microbiome relate to clinical-behavioral difficulties associated with autism remains sparse. Furthermore, it is largely unknown how potentially confounding lifestyle variables, such as oral health and nutrition, may impact these associations. To fill this gap, the current study examined diagnosis-related differences in oral microbiome composition between 80 school-aged autistic children (8-12 years; 64 boys, 16 girls) versus 40 age-matched typically developing peers (32 boys, 8 girls). In addition, associations with individual differences in social functioning (SRS-2), repetitive behavior (RBS-R) and anxiety (SCARED) were explored, as well as the impact of several lifestyle variables regarding nutrition and oral health. Results provide important indications that the bacterial genera Solobacterium, Stomatobaculum, Ruminococcaceae UCG.014, Tannerella and Campylobacter were significantly more abundant in autistic compared to non-autistic children. Furthermore, the former four bacteria that were significantly more abundant in the autistic children showed significant associations with parent-reported social difficulties, repetitive and restrictive behavior and with parent-reported anxiety-like behavior. Importantly, associations among oral microbiome and quantitative diagnostic characteristics were not significantly driven by differences in lifestyle variables. This exploratory study reveals significant differences in oral microbiome composition between autistic and non-autistic children, even while controlling for potential confounding lifestyle variables. Furthermore, the significant associations with clinical characteristics suggest that individual differences in microbiome composition might be involved in shaping the clinical phenotype of autism. However, these associations warrant further exploration of the oral microbiome's potential beyond the oral cavity and specifically with respect to neuropsychiatric conditions.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

A case study of the diet-microbiota-parasite interplay in bumble bees.

AIMS: Diets and parasites influence the gut bacterial symbionts of bumble bees, but potential interactive effects remain overlooked. The main objective of this study was to assess the isolated and interactive effects of sunflower pollen, its phenolamides, and the widespread trypanosomatid Crithidia sp. on the gut bacterial symbionts of Bombus terrestris males. METHODS AND RESULTS: Bumble bee males emerged in microcolonies fed on either (i) willow pollen (control), (ii) sunflower pollen, or (iii) willow pollen spiked with phenolamide extracts from sunflower pollen. These microcolonies were infected by Crithidia sp. or were pathogen-free. Using 16S rRNA amplicon sequencing (V3-V4 region), we observed a significant alteration of the beta diversity but not of the alpha diversity in the gut microbial communities of males fed on sunflower pollen compared to males fed on control pollen. Similarly, infection by the gut parasite Crithidia sp. altered the beta diversity but not the alpha diversity in the gut microbial communities of males, irrespective of the diet. By contrast, we did not observe any significant alteration of the beta or alpha diversity in the gut microbial communities of males fed on phenolamide-enriched pollen compared to males fed on control pollen. Changes in the beta diversity indicate significant dissimilarities of the bacterial taxa between the treatment groups, while the lack of difference in alpha diversity demonstrates no significant changes within each treatment group. CONCLUSIONS: Bumble bees harbour consistent gut microbiota worldwide, but our results suggest that the gut bacterial communities of bumble bees are somewhat shaped by their diets and gut parasites as well as by the interaction of these two factors. This study confirms that bumble bees are suitable biological surrogates to assess the effect of diet and parasite infections on gut microbial communities.

The wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta microbiota are host specific and dominated by endosymbionts and environmental microorganisms.

We characterized the microbial communities of the crop, midgut, hindgut, and ovaries of the wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta through 16S rRNA gene and ITS2 amplicon sequencing and a large-scale isolation campaign. The bacterial communities of these bees were dominated by endosymbionts of the genera Wolbachia and Spiroplasma. Bacterial and yeast genera representing the remaining predominant taxa were linked to an environmental origin. While only a single sampling site was examined for Andrena vaga, Anthophora plumipes, and Colletes cunicularius, and two sampling sites for Osmia cornuta, the microbiota appeared to be host specific: bacterial, but not fungal, communities generally differed between the analyzed bee species, gut compartments and ovaries. This may suggest a selective process determined by floral and host traits. Many of the gut symbionts identified in the present study are characterized by metabolic versatility. Whether they exert similar functionalities within the bee gut and thus functional redundancy remains to be elucidated.

Microbiota and pathogens in an invasive bee: Megachile sculpturalis from native and invaded regions.

The present study aimed to characterise the bacterial, fungal and parasite gut community of the invasive bee Megachile sculpturalis sampled from native (Japan) and invaded (USA and France) regions via 16S rRNA and ITS2 amplicon sequencing and PCR detection of bee microparasites. The bacterial and fungal gut microbiota communities in bees from invaded regions were highly similar and differed strongly from those obtained in Japan. Core amplicon sequence variants (ASVs) within each population represented environmental micro-organisms commonly present in bee-associated niches that likely provide beneficial functions to their host. Although the overall bacterial and fungal communities of the invasive M. sculpturalis in France and the co-foraging native bees Anthidium florentinum and Halictus scabiosae, were significantly different, five out of eight core ASVs were shared suggesting common environmental sources and potential transmission. None of the 46 M. sculpturalis bees analysed harboured known bee pathogens, while microparasite infections were common in A. florentinum, and rare in H. scabiosae. A common shift in the gut microbiota of M. sculpturalis in invaded regions as a response to changed environmental conditions, or a founder effect coupled to population re-establishment in the invaded regions may explain the observed microbial community profiles and the absence of parasites. While the role of pathogen pressure in shaping biological invasions is still debated, the absence of natural enemies may contribute to the invasion success of M. sculpturalis.

DNA extraction protocol impacts ocular surface microbiome profile.

Purpose: The aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed. Methods: Both eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants. Results: Samples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis. Conclusion: High-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology.

Introduction: While modulation of the human adult gut microbiota is a trending strategy to improve health, the underlying mechanisms are poorly understood. Methods: This study aimed to assess the predictive value of the ex vivo, reactor-based, high-throughput SIFR® (Systemic Intestinal Fermentation Research) technology for clinical findings using three structurally different prebiotics [inulin (IN), resistant dextrin (RD) and 2'-fucosyllactose (2'FL)]. Results: The key finding was that data obtained within 1-2 days were predictive for clinical findings upon repeated prebiotic intake over weeks: among hundreds of microbes, IN stimulated Bifidobacteriaceae, RD boosted Parabacteroides distasonis, while 2'FL specifically increased Bifidobacterium adolescentis and Anaerobutyricum hallii. In line with metabolic capabilities of these taxa, specific SCFA (short-chain fatty acids) were produced thus providing insights that cannot be obtained in vivo where such metabolites are rapidly absorbed. Further, in contrast to using single or pooled fecal microbiota (approaches used to circumvent low throughput of conventional models), working with 6 individual fecal microbiota enabled correlations that support mechanistic insights. Moreover, quantitative sequencing removed the noise caused by markedly increased cell densities upon prebiotic treatment, thus allowing to even rectify conclusions of previous clinical trials related to the tentative selectivity by which prebiotics modulate the gut microbiota. Counterintuitively, not the high but rather the low selectivity of IN caused only a limited number of taxa to be significantly affected. Finally, while a mucosal microbiota (enriched with Lachnospiraceae) can be integrated, other technical aspects of the SIFR® technology are a high technical reproducibility, and most importantly, a sustained similarity between the ex vivo and original in vivo microbiota. Discussion: By accurately predicting in vivo results within days, the SIFR® technology can help bridge the so-called "Valley of Death" between preclinical and clinical research. Facilitating development of test products with better understanding of their mode of action could dramatically increase success rate of microbiome modulating clinical trials.Graphical Abstract.

Convivina is a specialised core gut symbiont of the invasive hornet Vespa velutina.

We provide a culturomics analysis of the cultivable bacterial communities of the crop, midgut and hindgut compartments, as well as the ovaries, of the invasive insect Vespa velutina, along with a cultivation-independent analysis of samples of the same nest through 16S rRNA amplicon sequencing. The Vespa velutina bacterial symbiont community was dominated by the genera Convivina, Fructobacillus, Lactiplantibacillus, Lactococcus, Sphingomonas and Spiroplasma. Lactococcus lactis and Lactiplantibacillus plantarum represented generalist core lactic acid bacteria (LAB) symbionts, while Convivina species and Fructobacillus fructosus represented highly specialised core LAB symbionts with strongly reduced genome sizes. Sphingomonas and Spiroplasma were the only non-LAB core symbionts but were not isolated. Convivina bacteria were particularly enriched in the hornet crop and included Convivina intestini, a species adapted towards amino acid metabolism, and Convivina praedatoris sp. nov. which was adapted towards carbohydrate metabolism.

The Resemblance between Bacterial Gut Colonization in Pigs and Humans.

Thorough understanding of the initial colonization process of human intestines is important to optimize the prevention of microbiota-associated diseases, and also to further improve the current microbial therapies. In recent years, therefore, colonization of the human gut has gained renewed interest. However, due to a lack of standardization of life events that might influence this early colonization process in humans, many generally accepted insights are based on deduction and assumption. In our review, we compare knowledge on colonization in humans with research in piglets, because the intestinal tract of pigs is remarkably similar to that of humans and the early-life events are more standardized. We assess potential similarities and challenge some concepts that have been widely accepted in human microbiota research. Bacterial colonization of the human gut is characterized by successive waves in a progressive process, to a complex gut microbiota community. After re-analyzing available data from piglets, we found that the bacterial colonization process is very similar in terms of the wave sequence and functionality of each wave. Moreover, based on the piglet data, we found that, in addition to external factors such as suckling and nutrition, the bacterial community itself appears to have a major influence on the colonization success of additional bacteria in the intestine. Thus, the colonization process in piglets might rely, at least in part, on niche dependency, an ecological principle to be considered in the intestinal colonization process in humans.

Microbial diversity and antimicrobial susceptibility in endotracheal tube biofilms recovered from mechanically ventilated COVID-19 patients.

In patients with acute respiratory failure, mechanical ventilation through an endotracheal tube (ET) may be required to correct hypoxemia and hypercarbia. However, biofilm formation on these ETs is a risk factor for infections in intubated patients, as the ET can act as a reservoir of microorganisms that can cause infections in the lungs. As severely ill COVID-19 patients often need to be intubated, a better knowledge of the composition of ET biofilms in this population is important. In Spring 2020, during the first wave of the COVID-19 pandemic in Europe, 31 ETs were obtained from COVID-19 patients at Ghent University Hospital (Ghent, Belgium). Biofilms were collected from the ET and the biofilm composition was determined using culture-dependent (MALDI-TOF mass spectrometry and biochemical tests) and culture-independent (16S and ITS1 rRNA amplicon sequencing) approaches. In addition, antimicrobial resistance was assessed for isolates collected via the culture-dependent approach using disc diffusion for 11 antimicrobials commonly used to treat lower respiratory tract infections. The most common microorganisms identified by the culture-dependent approach were those typically found during lung infections and included both presumed commensal and potentially pathogenic microorganisms like Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans. More unusual organisms, such as Paracoccus yeei, were also identified, but each only in a few patients. The culture-independent approach revealed a wide variety of microbes present in the ET biofilms and showed large variation in biofilm composition between patients. Some biofilms contained a diverse set of bacteria of which many are generally considered as non-pathogenic commensals, whereas others were dominated by a single or a few pathogens. Antimicrobial resistance was widespread in the isolates, e.g. 68% and 53% of all isolates tested were resistant against meropenem and gentamicin, respectively. Different isolates from the same species recovered from the same ET biofilm often showed differences in antibiotic susceptibility. Our data suggest that ET biofilms are a potential risk factor for secondary infections in intubated COVID-19 patients, as is the case in mechanically-ventilated non-COVID-19 patients.

The Effect of Topical Anesthetics on 16S Ribosomal Ribonucleic Acid Amplicon Sequencing Results in Ocular Surface Microbiome Research.

PURPOSE: To clarify the short-term effect of topical anesthetics on 16S ribosomal ribonucleic acid amplicon sequencing results in ocular surface microbiome research. METHODS: Both eyes of 24 eligible volunteers undergoing general anesthesia were sampled. Before sampling, a drop of artificial tears or a drop of topical anesthetic was applied in a randomized way. By using artificial tears as a control, we assured blinding of the executer and took a potential diluting effect into account. Bacterial DNA was extracted using the QIAGEN RNeasy PowerMicrobiome Kit with specific adaptations. Amplified DNA was sequenced with the Illumina MiSeq sequencing platform. RESULTS: Four sample pairs were excluded due to low yield of bacterial DNA. In the remaining 20 sample pairs, no differences were observed with topical anesthetics at the levels of amplicon sequence variants (ASVs), phylum, genera, or alpha and beta diversity. Weighted UniFrac distance confirmed that the intraindividual distance between the right and left eye was smaller than the effect of the topical anesthetic. Interestingly, however, we identified Cutibacterium as a potential discriminative biomarker for topical anesthetic use. Overall, a significantly higher number of observed reads were assigned to genera with Gram-positive characteristics. CONCLUSIONS: Based on our targeted, double-blinded, within-subject study, topical anesthetics do not affect the overall sequencing results but display a specific effect on Cutibacterium. When comparing research results, the impact of topical anesthetics on prevalence and abundance of Cutibacterium should be considered. TRANSLATIONAL RELEVANCE: Understanding and standardization of sampling techniques are indispensable to properly execute clinical microbiome research.

Executioner caspases 3 and 7 are dispensable for intestinal epithelium turnover and homeostasis at steady state.

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.

Gut Microbiome Profiling Uncovers a Lower Abundance of Butyricicoccus in Advanced Stages of Chronic Kidney Disease.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins which exert deleterious effects on various organ systems. Several of these uremic toxins originate from the bacterial metabolization of aromatic amino acids in the colon. This study assessed whether the gut microbial composition varies among patients in different stages of CKD. Uremic metabolites were quantified by UPLC/fluorescence detection and microbial profiling by 16S rRNA amplicon sequencing. Gut microbial profiles of CKD patients were compared among stages 1-2, stage 3 and stages 4-5. Although a substantial inter-individual difference in abundance of the top 15 genera was observed, no significant difference was observed between groups. Bristol stool scale (BSS) correlated negatively with p-cresyl sulfate and hippuric acid levels, irrespective of the intake of laxatives. Butyricicoccus, a genus with butyrate-generating properties, was decreased in abundance in advanced stages of CKD compared to the earlier stages (p = 0.043). In conclusion, in this cross-sectional study no gradual differences in the gut microbial profile over the different stages of CKD were observed. However, the decrease in the abundance of Butyricicoccus genus with loss of kidney function stresses the need for more in-depth functional exploration of the gut microbiome in CKD patients not on dialysis.

Treponema peruense sp. nov., a commensal spirochaete isolated from human faeces.

A Gram-stain-negative, obligatory anaerobic spirochaete (RCC2812T) was isolated from a faecal sample obtained from an individual residing in a remote Amazonian community in Peru. The bacterium showed highest 16S rRNA gene sequence similarity to the pig intestinal spirochete Treponema succinifaciens (89.48 %). Average nucleotide identity values between strain RCC2812T and all available Treponema genomes from validated type strains were all <73 %, thus clearly lower than the species delineation threshold. The DNA G+C content of RCC2812T was 41.24 mol%. Phenotypic characterization using the API-ZYM and API 20A systems confirmed the divergent position of this bacterium within the genus Treponema. Strain RCC2812T could be differentiated from the phylogenetically most closely related T. succinifaciens by the presence of alkaline phosphatase and α -glucosidase activities. Unlike T. succinifaciens, strain RCC2812T grew equally well with or without serum. Strain RCC2812T is the first commensal Treponema isolated from the human faecal microbiota of remote populations, and based on the collected data represents a novel Treponema species for which the name Treponema peruense sp. nov. is proposed. The type strain is RCC2812T (=LMG 31794T=CIP 111910T).

The bacterial diversity of raw Moroccon camel milk.

Dromedary camel milk is generally considered a valuable and marketable commodity but its production suffers from poor hygienic conditions that result in low microbiological quality and the presence of various pathogens. The objective of the present study was to provide a detailed report of the bacterial species level composition of Moroccan raw camel milk samples that can serve as a starting point for the selection of starter cultures to facilitate a change in manufacturing practices to an improved and safer production system. The composition of the bacterial community in four freshly collected raw camel milk samples was analyzed by performing a large-scale isolation campaign combined with 16S rRNA gene amplicon sequencing. A total of 806 isolates were obtained from four raw camel milk samples using ten combinations of growth media and incubation conditions. Subsequent isolate dereplication using MALDI-TOF mass spectrometry and identification of representative isolates through sequence analysis of protein encoding and 16S rRNA genes revealed the presence of established and novel dairy lactic acid bacteria, as well as bacteria that are considered indicators of poor hygienic conditions and psychrotrophic spoilage organisms. The large numbers of Lactococcus and Enterococcus isolates obtained present an interesting resource for starter culture selection.

Isolation, characterization and antibiotic resistance of Proteus mirabilis from Belgian broiler carcasses at retail and human stool.

Proteus mirabilis is an important pathogen involved in human urinary tract infections, and also more isolated from stools of patients with diarrheal disease than from healthy patients. The role of food, especially poultry products as source for human infection and multi-resistant strains remains unclear. As a resident in broilers' intestines, P. mirabilis can contaminate broiler carcasses due to slaughter practices, and be a risk for human infection. The present study evaluated the performance of five isolation media, and subsequently examined the presence of P. mirabilis on broiler carcasses at retail. Additionally, isolates were characterized by the Dienes' test, repetitive element PCR fingerprinting and pulsed-field gel electrophoresis, and their antibiotic resistance profile determined. Using a combined isolation protocol on blood agar, xylose lysine deoxycholate agar and violet red bile glucose agar, P. mirabilis was isolated from 29 out of 80 broiler carcasses (36.25%) with a mean contamination level of 2.25 ± 0.50 log10 CFU/g. A high strain heterogeneity was present in isolates from broilers and human stool. The same strains were not shared, but the antibiotic resistance profiling was similar. A role of poultry products as source for human infection should be taken into account.