Search

Abstract Imbalanced microbiota may contribute to the pathophysiology of irritable bowel syndrome (IBS), thus fecal microbiota transplantation (FMT) has been suggested as a potential treatment. Previous studies on the relationship between clinical improvement and microbiota after FMT have been inconclusive. In this study, we used 16S rRNA gene amplicon and shotgun metagenomics data from a randomized, placebo controlled FMT trial on 49 IBS patients to analyze changes after FMT in microbiota composition and its functional potential, and to identify connections between microbiota and patients’ clinical outcome. As a result, we found that the successful modulation of microbiota composition and functional profiles by FMT from a healthy donor was not associated with the resolution of symptoms in IBS patients. Notably, a donor derived strain of Prevotella copri dominated the microbiota in those patients in the FMT group who had a low relative abundance of P. copri pre-FMT. The results highlight the multifactorial nature of IBS and the role of recipient’s microbiota in the colonization of donor’s strains.

Background: Alterations in gut-brain axis communication pathways and the gut microbiota ecosystem caused by early life stress have been extensively described as critical players in the pathophysiology of stress-induced disorders. However, the extent to which stress-induced gut microbiota alterations manifest in early life and contribute to the sex-specific susceptibility to distinct gut-brain phenotypes in adulthood has yet to be defined. Methods: Male and female Sprague-Dawley rat offspring underwent maternal separation (3h/day from postnatal day 2–12). Faecal samples were collected before weaning for gut microbiota 16S rRNA sequencing and metabolomic analysis. Visceral pain sensitivity and negative valence behaviours were assessed in adulthood using colorectal distension and the forced swim test respectively. Behavioural data were processed in a two-step cluster analysis to identify groupings within the dataset. Multi-omics analysis was carried out to investigate if the microbial signatures following early life stress were already defined according to the membership of the adult behavioural phenotypes. Results: Maternal separation resulted in increased visceral hypersensitivity while showing a trend for a sex-dependent increase in negative valence behaviour in adulthood. The cluster analysis revealed four clusters within the dataset representing distinct pathophysiological domains reminiscent of the behavioural consequences of early-life stress: 1. resilient, 2. pain, 3. immobile and 4. comorbid. The early life gut microbiota of each of these clusters show distinct alterations in terms of diversity, genus level differential abundance, and functional modules. Multi-omic integrations points towards a role for different metabolic pathways underlying each cluster-specific phenotype. Conclusion: Our study is the first to identify distinct phenotypes defined by susceptibility or resilience to gut-brain dysfunction induced by early life stress. The gut microbiota in early life shows sex-dependent alterations in each cluster that precede specific behavioural phenotypes in adulthood. Future research is warranted to determine the causal relationship between early-life stress-induced changes in the gut microbiota and to understand the trajectory leading to the manifestation of different behavioural phenotypes in adulthood.

Abstract Background Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. Results Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and β-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. Conclusion Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract

Abstract Background Post weaning diarrhoea (PWD) causes piglet morbidity and mortality at weaning and is a major driver for antimicrobial use worldwide. New regulations in the EU limit the use of in-feed antibiotics (Ab) and therapeutic zinc oxide (ZnO) to prevent PWD. New approaches to control PWD are needed, and understanding the role of the microbiota in this context is key. In this study, shotgun metagenome sequencing was used to describe the taxonomic and functional evolution of the faecal microbiota of the piglet during the first two weeks post weaning within three experimental groups, Ab, ZnO and no medication, on commercial farms using antimicrobials regularly in the post weaning period. Results Diversity was affected by day post weaning (dpw), treatment used and diarrhoea but not by the farm. Microbiota composition evolved towards the dominance of groups of species such as Prevotella spp. at day 14dpw. ZnO inhibited E. coli overgrowth, promoted higher abundance of the family Bacteroidaceae and decreased Megasphaera spp. Animals treated with Ab exhibited inconsistent taxonomic changes across time points, with an overall increase of Limosilactobacillus reuteri and Megasphaera elsdenii. Samples from non-medicated pigs showed virulence-related functions at 7dpw, and specific ETEC-related virulence factors were detected in all samples presenting diarrhoea. Differential microbiota functions of pigs treated with ZnO were related to sulphur and DNA metabolism, as well as mechanisms of antimicrobial and heavy metal resistance, whereas Ab treated animals exhibited functions related to antimicrobial resistance and virulence. Conclusion Ab and particularly ZnO maintained a stable microbiota composition and functionality during the two weeks post weaning, by limiting E. coli overgrowth, and ultimately preventing microbiota dysbiosis. Future approaches to support piglet health should be able to reproduce this stable gut microbiota transition during the post weaning period, in order to maintain optimal gut physiological and productive conditions.

ABSTRACTMicroorganisms are ubiquitous in nature and are central to human, animal, environmental, and planetary health. They play a particularly important role in the food chain and the production of high-quality, safe, and health-promoting foods, especially fermented foods. This important role is not always apparent to members of the public. Here, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge, and practical skills relating to microbiology, introduced through the medium of producing fermented food, i.e., milk kefir or water kefir. During the course of Kefir4All, 123 citizen scientists, from second-level school and non-school settings, participated in a study to track changes in the microbial composition of kefirs, by performing and recording details of milk kefir or water kefir fermentations they performed in their homes or schools over the 21-week project. At the start of the study, the citizen scientists were provided with milk or water kefir grains to initiate the fermentations. Both types of kefir grain are semi-solid, gelatinous-like substances, composed of exopolysaccharides and proteins, containing a symbiotic community of bacteria and yeast. The experimental component of the project was complemented by a number of education and outreach events, including career talks and a site visit to our research center (Kefir Day). At the end of the study, a report was provided to each citizen scientist, in which individualized results of their fermenting activities were detailed. A number of approaches were taken to obtain feedback and other insights from the citizen scientists. Evaluations took place before and after the Kefir4All project to gauge the citizen scientist’s self-reported awareness, knowledge, and interest in microbiology and fermented foods. Further insights into the level of citizen science participation were gained through assessing the number of samples returned for analysis and the level of participation of the citizen scientists throughout the project. Notably, the survey results revealed a self-reported, increased interest in, and general knowledge of, science among the Kefir4All citizen scientists after undertaking the project and a willingness to take part in further citizen science projects. Ultimately, Kefir4All represents an example of the successful integration of citizen science into existing education and research systems.

Abstract Background Antimicrobials and heavy metals such as zinc oxide (ZnO) have been commonly used on Irish commercial pig farms for a 2-week period post-weaning to help prevent infection. In 2022, the prophylactic use of antimicrobials and ZnO was banned within the European Union due to concerns associated with the emergence of antimicrobial resistance (AMR) and contamination of the environment with heavy metals. In this study, faecal and environmental samples were taken from piglets during the weaning period from ten commercial farms, of which five farms used antimicrobial or ZnO prophylaxis (AB-ZnO farms) and five which had not used antimicrobials or ZnO for the previous 3 years (AB-ZnO free farms). A total of 50 samples were compared using a metagenomic approach. Results The results of this study showed some significant differences between AB-ZnO and AB-ZnO free farms and suggested positive selection for AMR under antimicrobial and ZnO treatment. Moreover, strong differences between environmental and faecal samples on farms were observed, suggesting that the microbiome and its associated mobile genetic elements may play a key role in the composition of the resistome. Additionally, the age of piglets affected the resistome composition, potentially associated with changes in the microbiome post-weaning. Conclusions Overall, our study showed few differences in the resistome of the pig and its environment when comparing AB-ZnO farms with AB-ZnO free farms. These results suggest that although 3 years of removal of in-feed antimicrobial and ZnO may allow a reduction of AMR prevalence on AB-ZnO farms, more time, repeated sampling and a greater understanding of factors impacting AMR prevalence will be required to ensure significant and persistent change in on-farm AMR.

Abstract It has been established that the human gut microbiota is central to health, and, consequently, there has been a growing desire to positively modulate its composition and/or function through, for example, the use of fermented foods, prebiotics or probiotics. Here, we compare the relative impact of the daily consumption of an inulin-enriched diet (n = 10), a commercial probiotic-containing fermented milk product (FMP) (n = 10), or a traditional kefir FMP (n = 9), over a 28-day period on the gut microbiome and urine metabolome of healthy human adults. None of the treatments resulted in significant changes to clinical parameters or biomarkers tested. However, shotgun metagenomic analysis revealed that kefir consumption resulted in a significant change in taxonomy, in the form of an increased abundance of the sub-dominant FMP-associated species Lactococcus raffinolactis, which further corresponded to shifts in the urine metabolome. Overall, our results indicated that daily consumption of a single portion of kefir alone resulted in detectable changes to the gut microbiota and metabolome of consumers.

IntroductionPost-weaning diarrhoea (PWD) is a multifactorial disease that affects piglets after weaning, contributing to productive and economic losses. Its control includes the use of in-feed prophylactic antibiotics and therapeutic zinc oxide (ZnO), treatments that, since 2022, are no longer permitted in the European Union due to spread of antimicrobial resistance genes and pollution of soil with heavy metals. A dysbiosis in the microbiota has been suggested as a potential risk factor of PWD onset. Understanding pig’s microbiota development around weaning and its changes in response to ZnO and antibiotics is crucial to develop feasible alternatives to prophylactic and metaphylactic antimicrobial use.MethodsThis study used shotgun metagenomic sequencing to investigate the environmental and faecal microbiota on 10 farms using (Treated) or not using (ZnO-free) in-feed antibiotics and ZnO during the first 14 days post-weaning (dpw). Environmental samples from clean pens were collected at weaning day (0dpw), and faecal samples at 0, 7 and 14dpw. Diarrhoeic faecal samples were collected at 7dpw when available.ResultsThe analysis of data revealed that the faecal microbiota composition and its functionality was impacted by the sampling time point (microbiota maturation after weaning) but not by the farm environment. Treatment with antibiotics and ZnO showed no effects on diversity indices while the analyses of microbiota taxonomic and functional profiles revealed increased abundance of taxa and metabolic functions associated with Phascolarctobacterium succinatutens or different species of Prevotella spp. on the Treated farms, and with Megasphaera elsdenii and Escherichia coli on the ZnO-free farms. The analysis of diarrhoea samples revealed that the treatment favoured the microbiota transition or maturation from 0dpw to 14dpw in Treated farms, resembling the composition of healthy animals, when compared to diarrhoea from ZnO-free farms, which were linked in composition to 0dpw samples.DiscussionThe results provide a comprehensive overview of the beneficial effects of ZnO and antibiotics in PWD in the microbiota transition after weaning, preventing the overgrowth of pathogens such as pathogenic E. coli and revealing the key aspects in microbiota maturation that antibiotics or ZnO alternatives should fulfil.

Abstract This study examined the effects of maternal and/or post-weaning Bacillus altitudinis supplementation on the microbiota in sow colostrum and faeces, and offspring digesta and faeces. Sows (n = 12/group) were assigned to: (1) standard diet (CON), or (2) CON supplemented with probiotic B. altitudinis spores (PRO) from day (d)100 of gestation to weaning (d26 of lactation). At weaning, offspring were assigned to CON or PRO for 28d, resulting in: (1) CON/CON, (2) CON/PRO, (3) PRO/CON, and (4) PRO/PRO, after which all received CON. Samples were collected from sows and selected offspring (n = 10/group) for 16S rRNA gene sequencing. Rothia was more abundant in PRO sow colostrum. Sow faeces were not impacted but differences were identified in offspring faeces and digesta. Most were in the ileal digesta between PRO/CON and CON/CON on d8 post-weaning; i.e. Bacteroidota, Alloprevotella, Prevotella, Prevotellaceae, Turicibacter, Catenibacterium and Blautia were more abundant in PRO/CON, with Firmicutes and Blautia more abundant in PRO/PRO compared with CON/CON. Lactobacillus was more abundant in PRO/CON faeces on d118 post-weaning. This increased abundance of polysaccharide-fermenters (Prevotella, Alloprevotella, Prevotellaceae), butyrate-producers (Blautia) and Lactobacillus likely contributed to previously reported improvements in growth performance. Overall, maternal, rather than post-weaning, probiotic supplementation had the greatest impact on intestinal microbiota.

Summary: A comprehensive metagenomics-based investigation of the microorganisms present within milk kefir communities from across the globe was carried out with a view to defining the milk kefir pan-metagenome, including details relating to core and non-core components. Milk kefir samples, generated by inoculating full fat, pasteurized cow’s milk with 64 kefir grains sourced from 25 different countries, were analyzed. We identified core features, including a consistent pattern of domination by representatives from the species Lactobacillus helveticus or the sub-species Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactococcus lactis subsp. lactis or Lla. cremoris subsp. cremoris in each kefir. Notably, even in kefirs where the lactococci did not dominate, they and 51 associated metabolic pathways were identified across all metagenomes. These insights can contribute to future efforts to create tailored kefir-based microbial communities for different applications and assist regulators and producers to ensure that kefir products have a microbial composition that reflects the artisanal beverage.

Abstract The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals.

Summary Porcine reproductive and respiratory syndrome (PRRS) is a viral disease defined by reproductive problems, respiratory distress and a negative impact on growth rate and general condition. Virulent PRRS virus (PRRSV) strains have emerged in the last years with evident knowledge gaps in their impact on the host immune response. Thus, the present study examines the impact of acute PRRS virus (PRRSV) infection, with two strains of different virulence, on selected immune parameters and on the gut microbiota composition of infected pigs using 16S rRNA compositional sequencing. Pigs were infected with a low virulent (PRRS_3249) or a virulent (Lena) PRRSV‐1 strain and euthanized at 1, 3, 6, 8 or 13 days post‐inoculation (dpi). Faeces were collected from each animal at the necropsy time‐point. Alpha and beta diversity analyses demonstrated that infection, particularly with the Lena strain, impacted the microbiome composition from 6 dpi onwards. Taxonomic differences revealed that infected pigs had higher abundance of Treponema and Methanobrevibacter (FDR

Abstract The objective was to evaluate the effect of dietary Bacillus altitudinis spore supplementation during day (D)0–28 post-weaning (PW) and/or D29–56 PW compared with antibiotic and zinc oxide (AB + ZnO) supplementation on pig growth and gut microbiota. Eighty piglets were selected at weaning and randomly assigned to one of five dietary treatments: (1) negative control (Con/Con); (2) probiotic spores from D29–56 PW (Con/Pro); (3) probiotic spores from D0–28 PW (Pro/Con); (4) probiotic spores from D0–56 PW (Pro/Pro) and (5) AB + ZnO from D0–28 PW. Overall, compared with the AB + ZnO group, the Pro/Con group had lower body weight, average daily gain and feed intake and the Pro/Pro group tended to have lower daily gain and feed intake. However, none of these parameters differed between any of the probiotic-treated groups and the Con/Con group. Overall, AB + ZnO-supplemented pigs had higher Bacteroidaceae and Prevotellaceae and lower Lactobacillaceae and Spirochaetaceae abundance compared to the Con/Con group, which may help to explain improvements in growth between D15–28 PW. The butyrate-producing genera Agathobacter, Faecalibacterium and Roseburia were more abundant in the Pro/Con group compared with the Con/Con group on D35 PW. Thus, whilst supplementation with B. altitudinis did not enhance pig growth performance, it did have a subtle, albeit potentially beneficial, impact on the intestinal microbiota.

Abstract Background The microorganisms that inhabit food processing environments (FPE) can strongly influence the associated food quality and safety. In particular, the possibility that FPE may act as a reservoir of antibiotic-resistant microorganisms, and a hotspot for the transmission of antibiotic resistance genes (ARGs) is a concern in meat processing plants. Here, we monitor microbial succession and resistome dynamics relating to FPE through a detailed analysis of a newly opened pork cutting plant over 1.5 years of activity. Results We identified a relatively restricted principal microbiota dominated by Pseudomonas during the first 2 months, while a higher taxonomic diversity, an increased representation of other taxa (e.g., Acinetobacter, Psychrobacter), and a certain degree of microbiome specialization on different surfaces was recorded later on. An increase in total abundance, alpha diversity, and β-dispersion of ARGs, which were predominantly assigned to Acinetobacter and associated with resistance to certain antimicrobials frequently used on pig farms of the region, was detected over time. Moreover, a sharp increase in the occurrence of extended-spectrum β-lactamase-producing Enterobacteriaceae and vancomycin-resistant Enterococcaceae was observed when cutting activities started. ARGs associated with resistance to β-lactams, tetracyclines, aminoglycosides, and sulphonamides frequently co-occurred, and mobile genetic elements (i.e., plasmids, integrons) and lateral gene transfer events were mainly detected at the later sampling times in drains. Conclusions The observations made suggest that pig carcasses were a source of resistant bacteria that then colonized FPE and that drains, together with some food-contact surfaces, such as equipment and table surfaces, represented a reservoir for the spread of ARGs in the meat processing facility. Video Abstract

In healthy hosts the gut microbiota is restricted to gut tissues by several barriers some of which require MyD88-dependent innate immune sensor pathways. Nevertheless, some gut taxa have been reported to disseminate to systemic tissues. However, the extent to which this normally occurs during homeostasis in healthy organisms is still unknown. In this study, we recovered viable gut bacteria from systemic tissues of healthy wild type (WT) and MyD88−/− mice. Shotgun metagenomic-sequencing revealed a marked increase in the relative abundance of L. johnsonii in intestinal tissues of MyD88−/− mice compared to WT mice. Lactobacillus johnsonii was detected most frequently from multiple systemic tissues and at higher levels in MyD88−/− mice compared to WT mice. Viable L. johnsonii strains were recovered from different cell types sorted from intestinal and systemic tissues of WT and MyD88−/− mice. L. johnsonii could persist in dendritic cells and may represent murine immunomodulatory endosymbionts.

Abstract Efficient and accurate identification of microorganisms throughout the food chain can potentially allow the identification of sources of contamination and the timely implementation of control measures. High throughput DNA sequencing represents a potential means through which microbial monitoring can be enhanced. While Illumina sequencing platforms are most typically used, newer portable platforms, such as the Oxford Nanopore Technologies (ONT) MinION, offer the potential for rapid analysis of food chain microbiomes. Initial assessment of the ability of rapid MinION-based sequencing to identify microbes within a simple mock metagenomic mixture is performed. Subsequently, we compare the performance of both ONT and Illumina sequencing for environmental monitoring of an active food processing facility. Overall, ONT MinION sequencing provides accurate classification to species level, comparable to Illumina-derived outputs. However, while the MinION-based approach provides a means of easy library preparations and portability, the high concentrations of DNA needed is a limiting factor.

Abstract Background The global threat of antimicrobial resistance (AMR) is a One Health problem impacted by antimicrobial use (AMU) for human and livestock applications. Extensive Iberian swine production is based on a more sustainable and eco-friendly management system, providing an excellent opportunity to evaluate how sustained differences in AMU impact the resistome, not only in the animals but also on the farm environment. Here, we evaluate the resistome footprint of an extensive pig farming system, maintained for decades, as compared to that of industrialized intensive pig farming by analyzing 105 fecal, environmental and slurry metagenomes from 38 farms. Results Our results evidence a significantly higher abundance of antimicrobial resistance genes (ARGs) on intensive farms and a link between AMU and AMR to certain antimicrobial classes. We observed differences in the resistome across sample types, with a higher richness and dispersion of ARGs within environmental samples than on those from feces or slurry. Indeed, a deeper analysis revealed that differences among the three sample types were defined by taxa-ARGs associations. Interestingly, mobilome analyses revealed that the observed AMR differences between intensive and extensive farms could be linked to differences in the abundance of mobile genetic elements (MGEs). Thus, while there were no differences in the abundance of chromosomal-associated ARGs between intensive and extensive herds, a significantly higher abundance of integrons in the environment and plasmids, regardless of the sample type, was detected on intensive farms. Conclusions Overall, this study shows how AMU, production system, and sample type influence, mainly through MGEs, the profile and dispersion of ARGs in pig production. Video Abstract

Abstract Background Mounting evidence suggests a role for the gut microbiota in modulating brain physiology and behaviour, through bi-directional communication, along the gut-brain axis. As such, the gut microbiota represents a potential therapeutic target for influencing centrally mediated events and host behaviour. It is thus notable that the fermented milk beverage kefir has recently been shown to modulate the composition of the gut microbiota in mice. It is unclear whether kefirs have differential effects on microbiota-gut-brain axis and whether they can modulate host behaviour per se. Methods To address this, two distinct kefirs (Fr1 and UK4), or unfermented milk control, were administered to mice that underwent a battery of tests to characterise their behavioural phenotype. In addition, shotgun metagenomic sequencing of ileal, caecal and faecal matter was performed, as was faecal metabolome analysis. Finally, systemic immunity measures and gut serotonin levels were assessed. Statistical analyses were performed by ANOVA followed by Dunnett's post hoc test or Kruskal-Wallis test followed by Mann-Whitney U test. Results Fr1 ameliorated the stress-induced decrease in serotonergic signalling in the colon and reward-seeking behaviour in the saccharin preference test. On the other hand, UK4 decreased repetitive behaviour and ameliorated stress-induced deficits in reward-seeking behaviour. Furthermore, UK4 increased fear-dependent contextual memory, yet decreased milk gavage-induced improvements in long-term spatial learning. In the peripheral immune system, UK4 increased the prevalence of Treg cells and interleukin 10 levels, whereas Fr1 ameliorated the milk gavage stress-induced elevation in neutrophil levels and CXCL1 levels. Analysis of the gut microbiota revealed that both kefirs significantly changed the composition and functional capacity of the host microbiota, where specific bacterial species were changed in a kefir-dependent manner. Furthermore, both kefirs increased the capacity of the gut microbiota to produce GABA, which was linked to an increased prevalence in Lactobacillus reuteri. Conclusions Altogether, these data show that kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour. In addition, different kefirs may direct the microbiota toward distinct immunological and behavioural modulatory effects. These results indicate that kefir can positively modulate specific aspects of the microbiota-gut-brain axis and support the broadening of the definition of psychobiotic to include kefir fermented foods. Video abstract.

The effect of fermentation on the antithrombotic properties of polar lipids in ovine milk has been assessed through the production of yoghurts. The total lipids (TL), total neutral lipids (TNL), and total polar lipids (TPL) were extracted. The fatty acid profiles of all yoghurt polar lipids were analysed by GC-MS. The levels of MUFA increased in the fatty acids of the polar lipids, but there was a reduction in PUFA as milk was fermented to yoghurt. The bioactivity of each lipid extract was assessed against platelet-activating factor (PAF) induced platelet aggregation. All yoghurt polar lipids exhibited potent antithrombotic activities with IC50 values ranging from 45 to 77 µg. Shotgun metagenomics determined the species-level microbial composition and functional potential of the yoghurts. Yoghurts containing L. acidophilus seem to correlate with greater bioactivity. Several phospholipid biosynthetic genes have been identified in the most antithrombotic yoghurts. This study has demonstrated that fermentation enhances the antithrombotic properties of yoghurt polar lipids against PAF.

The use of probiotics such as Lactobacillus and Bifidobacterium spp. as a therapeutic against inflammatory bowel disease (IBD) is of significant interest. Lactobacillus salivarus strain UCC118TM is a commensal that has been shown to possess probiotic properties in vitro and anti-infective properties in vivo. However, the usefulness of UCC118 TM as a therapeutic against colitis remains unclear. This study investigates the probiotic potential of Lactobacillus salivarius, UCC118™ in a mouse model of colitis. DSS-induced colitis was coupled with pre-treatment or post-treatment with UCC118TM by daily oral gavage. In the pre-treatment model of colitis, UCC118TM reduced the severity of the disease in the early stages. Improvement in disease severity was coupled with an upregulation of tissue IL-10 levels and increased expression of macrophage M2 markers. This anti-inflammatory activity of UCC118TM was further confirmed in vitro, using a model of LPS-treated bone marrow-derived macrophages. Taken together, these results suggest that UCC118TM may promote the resolution of inflammation. This was supported in a mouse model of established DSS-induced colitis whereby UCC118TM treatment accelerated recovery, as evidenced by weight, stool, histological markers and the recovery of microbiome-associated dysbiosis with an increased abundance of beneficial commensal species. These results demonstrate the potential of Lactobacillus salivarius UCC118TM as a probiotic-based therapeutic strategy to promote health through the upregulation of anti-inflammatory IL-10 and protect against dysbiosis during IBD.

Obesity is endemic in human populations in the western society, and with mounting evidence that the intestinal ecological environment plays a major role in its pathogenesis, identification of therapies based on intestinal microbiota modulation are gaining attention. Obesity in companion animals is also a common clinical problem. We set out using a multidimensional approach, to determine the effectiveness and safety of a weight loss program for horses incorporating diet restriction and exercise. In addition, we aimed to investigate the effect of this program on the overall intestinal health of overweight sedentary horses. The investigation comprised of a randomized, controlled, 6-week study of 14 overweight sedentary horses and ponies who were blocked for age, gender, and breed (controls n = 7, treatment n = 7). The treatment group were fed a restricted diet (1.4% of body weight dry matter intake) and the control group a maintenance diet (2% of body weight as dry matter intake) over the study period. The treatment group were subjected to a prescribed exercise regime, while the control group were exercised to mimic foraging conditions. Several clinical measurements were taken at the start and end of the study, including morphological parameters, ultrasound measurements of subcutaneous fat, and blood pressure. Fecal microbiota analysis was performed using 16S rRNA gene sequence analysis, and fecal metabolome was analyzed using NMR spectroscopy, on samples taken at weeks 1, 3, and 6 of the study. All horses completed the study period successfully. However, two of the treatment group had to have modified exercise regimes. The treatment group showed significant weight loss (p < 0.00001) and an associated decrease in waste circumference (p < 0.0001) when compared with the control group. The alpha-diversity of the fecal microbiota in the treatment group showed a significant increase from the start to the end of the study period (p < 0.05); however, there was no significant difference between groups at any sampling point. There were significant changes (p < 0.05) in the metabolome in both groups between the start and end of the study, but not between groups at any sampling point. Finally, the resting blood pressure of all horses was significantly lower by the end of the study.

This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses.

Sex steroids, derived mainly from gonads, can shape microbiota composition; however, the impact of gonadectomy and sex on steroid production in the gut (i.e., gut steroids), and its interaction with microbiota composition, needs to be clarified. In this study, steroid environment and gut steroidogenesis were analysed by liquid chromatography tandem mass spectrometry and expression analyses. Gut microbiota composition as branched- and short-chain fatty acids were determined by 16S rRNA gene sequence analysis and gas chromatography flame ionisation detection, respectively. Here, we first demonstrated that levels of pregnenolone (PREG), progesterone (PROG), and isoallopregnanolone (ISOALLO) were higher in the female rat colon, whereas the level of testosterone (T) was higher in males. Sexual dimorphism on gut steroidogenesis is also reported after gonadectomy. Sex, and more significantly, gonadectomy, affects microbiota composition. We noted that a number of taxa and inferred metabolic pathways were associated with gut steroids, such as positive associations between Blautia with T, dihydroprogesterone (DHP), and allopregnanolone (ALLO), whereas negative associations were noted between Roseburia and T, ALLO, PREG, ISOALLO, DHP, and PROG. In conclusion, this study highlights the novel sex-specific association between microbiota and gut steroids with possible relevance for the gut-brain axis.

ABSTRACT Fermented foods have been the focus of ever greater interest as a consequence of purported health benefits. Indeed, it has been suggested that consumption of these foods helps to address the negative consequences of “industrialization” of the human gut microbiota in Western society. However, as the mechanisms via which the microbes in fermented foods improve health are not understood, it is necessary to develop an understanding of the composition and functionality of the fermented-food microbiota to better harness desirable traits. Here, we considerably expand the understanding of fermented-food microbiomes by employing shotgun metagenomic sequencing to provide a comprehensive insight into the microbial composition, diversity, and functional potential (including antimicrobial resistance and carbohydrate-degrading and health-associated gene content) of a diverse range of 58 fermented foods from artisanal producers from a number of countries. Food type, i.e., dairy-, sugar-, or brine-type fermented foods, was the primary driver of microbial composition, with dairy foods found to have the lowest microbial diversity. From the combined data set, 127 high-quality metagenome-assembled genomes (MAGs), including 10 MAGs representing putatively novel species of Acetobacter, Acidisphaera, Gluconobacter, Companilactobacillus, Leuconostoc, and Rouxiella, were generated. Potential health promoting attributes were more common in fermented foods than nonfermented equivalents, with water kefirs, sauerkrauts, and kvasses containing the greatest numbers of potentially health-associated gene clusters. Ultimately, this study provides the most comprehensive insight into the microbiomes of fermented foods to date and yields novel information regarding their relative health-promoting potential. IMPORTANCE Fermented foods are regaining popularity worldwide due in part to a greater appreciation of the health benefits of these foods and the associated microorganisms. Here, we use state-of-the-art approaches to explore the microbiomes of 58 of these foods, identifying the factors that drive the microbial composition of these foods and potential functional benefits associated with these populations. Food type, i.e., dairy-, sugar-, or brine-type fermented foods, was the primary driver of microbial composition, with dairy foods found to have the lowest microbial diversity and, notably, potential health promoting attributes were more common in fermented foods than nonfermented equivalents. The information provided here will provide significant opportunities for the further optimization of fermented-food production and the harnessing of their health-promoting potential.

Background: Evidence is accruing to suggest that microbiota-gut-brain signalling plays a regulatory role in cardiorespiratory physiology. Chronic intermittent hypoxia (CIH), modelling human sleep apnoea, affects gut microbiota composition and elicits cardiorespiratory morbidity. We investigated if treatment with prebiotics ameliorates cardiorespiratory dysfunction in CIH-exposed rats. Methods: Adult male rats were exposed to CIH (96 cycles/day, 6.0% O2 at nadir) for 14 consecutive days with and without prebiotic supplementation (fructo- and galacto-oligosaccharides) beginning two weeks prior to gas exposures. Findings: CIH increased apnoea index and caused hypertension. CIH exposure had modest effects on the gut microbiota, decreasing the relative abundance of Lactobacilli species, but had no effect on microbial functional characteristics. Faecal short-chain fatty acid (SCFA) concentrations, plasma and brainstem pro-inflammatory cytokine concentrations and brainstem neurochemistry were unaffected by exposure to CIH. Prebiotic administration modulated gut microbiota composition and diversity, altering gut-metabolic (GMMs) and gut-brain (GBMs) modules and increased faecal acetic and propionic acid concentrations, but did not prevent adverse CIH-induced cardiorespiratory phenotypes. Interpretation: CIH-induced cardiorespiratory dysfunction is not dependant upon changes in microbial functional characteristics and decreased faecal SCFA concentrations. Prebiotic-related modulation of microbial function and resultant increases in faecal SCFAs were not sufficient to prevent CIH-induced apnoea and hypertension in our model. Our results do not exclude the potential for microbiota-gut-brain axis involvement in OSA-related cardiorespiratory morbidity, but they demonstrate that in a relatively mild model of CIH, sufficient to evoke classic cardiorespiratory dysfunction, such changes are not obligatory for the development of morbidity, but may become relevant in the elaboration and maintenance of cardiorespiratory morbidity with progressive disease. Funding: Department of Physiology and APC Microbiome Ireland, University College Cork, Ireland. APC Microbiome Ireland is funded by Science Foundation Ireland, through the Government's National Development Plan.

Background: Multiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. One behavioral trait where individuals assign incentive salience to food stimuli (“sign-trackers”, ST) are more impulsive compared to those that do not (“goal-trackers”, GT), as well as more sensitive to drugs and drug stimuli. Furthermore, this GT/ST phenotype predicts differences in other behavioral measures. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors determined by the GT/ST phenotype. Methods: Outbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA gene amplicon sequencing. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype. Results: Overall microbiome composition was not associated to the GT/ST phenotype. However, microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. Female STs had notable correlations between microbiome and attentional deficient. In both males and females, many measures were correlated with the bacterial families Ruminocococcaceae and Lachnospiraceae. Conclusions: These data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex.

ABSTRACT Here, the role of the dairy-processing chain as a reservoir of antimicrobial resistance (AR) determinants and a source of novel biocontrol quorum-sensing inhibitors is assessed through a functional metagenomics approach. A metagenomic library comprising ∼22,000 recombinant clones was built from DNA isolated from raw milk, raw milk cheeses, and cheese-processing environment swab samples. The high-throughput sequencing of 9,216 recombinant clones showed that lactic acid bacteria (LAB) dominated the microbial communities of raw milk cheese, while Gram-negative microorganisms of animal or soil origin dominated the microbiota of raw milk and cheese-processing environments. Although functional screening of the metagenomic library did not recover potential quorum-sensing inhibitors, in silico analysis using an in-house database built specifically for this study identified homologues to several genes encoding proteins with predicted quorum-quenching activity, among which, the QsdH hydrolase was the most abundant. In silico screening of the library identified LAB, and especially Lactococcus lactis, as a relevant reservoir of AR determinants in cheese. Functional screening of the library allowed the isolation of 13 recombinant clones showing an increased resistance toward ampicillin, which in all cases was accompanied by a reduced susceptibility to a wide range of β-lactam antibiotics. This study shows that the dairy-processing environment is a rich reservoir of AR determinants, which vary by sample source, and suggests that combining next-generation sequencing with functional metagenomics can be of use in overcoming the limitations of both approaches. IMPORTANCE The study shows the potential of functional metagenomics analyses to uncover the diversity of functions in microbial communities prevailing in dairy products and their processing environments, evidencing that lactic acid bacteria (LAB) dominate the cheese microbiota, whereas Gram-negative microorganisms of animal or soil origin dominate the microbiota of milk and cheese-processing environments. The functional and in silico screening of the library allowed the identification of LAB, and especially Lactococcus lactis, as a relevant reservoir of antimicrobial resistance (AR) determinants in cheese. Quorum-quenching (QQ) determinants were not recovered through the execution of wet-lab function-based screenings but were detected through in silico sequencing-based analyses.

Abstract Background The use of shotgun metagenomics to analyse low-complexity microbial communities in foods has the potential to be of considerable fundamental and applied value. However, there is currently no consensus with respect to choice of species classification tool, platform, or sequencing depth. Here, we benchmarked the performances of three high-throughput short-read sequencing platforms, the Illumina MiSeq, NextSeq 500, and Ion Proton, for shotgun metagenomics of food microbiota. Briefly, we sequenced six kefir DNA samples and a mock community DNA sample, the latter constructed by evenly mixing genomic DNA from 13 food-related bacterial species. A variety of bioinformatic tools were used to analyse the data generated, and the effects of sequencing depth on these analyses were tested by randomly subsampling reads. Results Compositional analysis results were consistent between the platforms at divergent sequencing depths. However, we observed pronounced differences in the predictions from species classification tools. Indeed, PERMANOVA indicated that there was no significant differences between the compositional results generated by the different sequencers (p = 0.693, R 2 = 0.011), but there was a significant difference between the results predicted by the species classifiers (p = 0.01, R 2 = 0.127). The relative abundances predicted by the classifiers, apart from MetaPhlAn2, were apparently biased by reference genome sizes. Additionally, we observed varying false-positive rates among the classifiers. MetaPhlAn2 had the lowest false-positive rate, whereas SLIMM had the greatest false-positive rate. Strain-level analysis results were also similar across platforms. Each platform correctly identified the strains present in the mock community, but accuracy was improved slightly with greater sequencing depth. Notably, PanPhlAn detected the dominant strains in each kefir sample above 500,000 reads per sample. Again, the outputs from functional profiling analysis using SUPER-FOCUS were generally accordant between the platforms at different sequencing depths. Finally, and expectedly, metagenome assembly completeness was significantly lower on the MiSeq than either on the NextSeq (p = 0.03) or the Proton (p = 0.011), and it improved with increased sequencing depth. Conclusions Our results demonstrate a remarkable similarity in the results generated by the three sequencing platforms at different sequencing depths, and, in fact, the choice of bioinformatics methodology had a more evident impact on results than the choice of sequencer did.

The effect of fermentation on the antithrombotic properties of polar lipids in raw and pasteurised caprine milk was assessed through the production of various cheeses using the same starter culture. The total lipids (TL), total neutral lipids (TNL), and total polar lipids (TPL) were extracted from each milk and cheese and the TPL fatty acid profiles were analysed by GC–MS. It was determined that fermentation influenced the polar lipid fatty acid composition. The milk and cheese polar lipids exhibited potent antithrombotic activities with IC50 values ranging from 79 to 226 µg against platelet-activating factor (PAF) induced platelet aggregation. Finally, shotgun metagenomics determined the species-level microbial composition and functional potential of each milk and cheese. Several microbe-encoded phospholipid biosynthetic genes were identified in the most antithrombotic cheeses. Lactococcus lactis and other microbial species may play a significant role in determining the antithrombotic properties and fatty acid composition of caprine cheese polar lipids.

ABSTRACT Optimal feed efficiency (FE) in pigs is important for economic and environmental reasons. Previous research identified FE-associated bacterial taxa within the intestinal microbiota of growing pigs. This study investigated whether FE-associated bacteria and selected FE-associated physiological traits were consistent across geographic locations (Republic of Ireland [ROI] [two batches of pigs, ROI1 and ROI2], Northern Ireland [NI], and Austria [AT]), where differences in genetic, dietary, and management factors were minimized. Pigs (n = 369) were ranked, within litter, on divergence in residual feed intake (RFI), and 100 extremes were selected (50 with high RFI and 50 with low RFI) across geographic locations for intestinal microbiota analysis using 16S rRNA amplicon sequencing and examination of FE-associated physiological parameters. Microbial diversity varied by geographic location and intestinal sampling site but not by RFI rank, except in ROI2, where more-feed-efficient pigs had greater ileal and cecal diversity. Although none of the 188 RFI-associated taxonomic differences found were common to all locations/batches, Lentisphaerae, Ruminococcaceae, RF16, Mucispirillum, Methanobrevibacter, and two uncultured genera were more abundant within the fecal or cecal microbiota of low-RFI pigs in two geographic locations and/or in both ROI batches. These are major contributors to carbohydrate metabolism, which was reflected in functional predictions. Fecal volatile fatty acids and salivary cortisol were the only physiological parameters that differed between RFI ranks. Despite controlling genetics, diet specification, dietary phases, and management practices in each rearing environment, the rearing environment, encompassing maternal influence, herd health status, as well as other factors, appears to impact intestinal microbiota more than FE. IMPORTANCE Interest in the role of intestinal microbiota in determining FE in pigs has increased in recent years. However, it is not known if the same FE-associated bacteria are found across different rearing environments. In this study, geographic location and intestinal sampling site had a greater influence on the pig gut microbiome than FE. This presents challenges when aiming to identify consistent reliable microbial biomarkers for FE. Nonetheless, seven FE-associated microbial taxa were common across two geographic locations and/or two batches within one location, and these indicated a potentially “healthier” and metabolically more capable microbiota in more-feed-efficient pigs. These taxa could potentially be employed as biomarkers for FE, although bacterial consortia, rather than individual taxa, may be more likely to predict FE. They may also merit consideration for use as probiotics or could be targeted by dietary means as a strategy for improving FE in pigs in the future.

ABSTRACT Salmonella colonization and infection in production animals such as pigs are a cause for concern from a public health perspective. Variations in susceptibility to natural infection may be influenced by the intestinal microbiota. Using 16S rRNA compositional sequencing, we characterized the fecal microbiome of 15 weaned pigs naturally infected with Salmonella at 18, 33, and 45 days postweaning. Dissimilarities in microbiota composition were analyzed in relation to Salmonella infection status (infected, not infected), serological status, and shedding pattern (nonshedders, single-point shedders, intermittent-persistent shedders). Global microbiota composition was associated with the infection outcome based on serological analysis. Greater richness within the microbiota postweaning was linked to pigs being seronegative at the end of the study at 11 weeks of age. Members of the Clostridia, such as Blautia, Roseburia, and Anaerovibrio, were more abundant and part of the core microbiome in nonshedder pigs. Cellulolytic microbiota (Ruminococcus and Prevotella) were also more abundant in noninfected pigs during the weaning and growing stages. Microbial profiling also revealed that infected pigs had a higher abundance of Lactobacillus and Oscillospira, the latter also being part of the core microbiome of intermittent-persistent shedders. These findings suggest that a lack of microbiome maturation and greater proportions of microorganisms associated with suckling increase susceptibility to infection. In addition, the persistence of Salmonella shedding may be associated with an enrichment of pathobionts such as Anaerobiospirillum. Overall, these results suggest that there may be merit in manipulating certain taxa within the porcine intestinal microbial community to increase disease resistance against Salmonella in pigs. IMPORTANCE Salmonella is a global threat for public health, and pork is one of the main sources of human salmonellosis. However, the complex epidemiology of the infection limits current control strategies aimed at reducing the prevalence of this infection in pigs. The present study analyzes for the first time the impact of the gut microbiota in Salmonella infection in pigs and its shedding pattern in naturally infected growing pigs. Microbiome (16S rRNA amplicon) analysis reveals that maturation of the gut microbiome could be a key consideration with respect to limiting the infection and shedding of Salmonella in pigs. Indeed, seronegative animals had higher richness of the gut microbiota early after weaning, and uninfected pigs had higher abundance of strict anaerobes from the class Clostridia, results which demonstrate that a fast transition from the suckling microbiota to a postweaning microbiota could be crucial with respect to protecting the animals.

The cystic fibrosis (CF) lung harbours a diverse microbiome and reduced diversity in the CF lung has been associated with advancing age, increased inflammation and poorer lung function. Data suggest that the window for intervention is early in CF, yet there is a paucity of studies on the lung microbiome in children with CF. The objective of this study was to thoroughly characterise the lower airway microbiome in pre-school children with CF. Bronchoalveolar lavage (BAL) samples were collected annually from children attending the three clinical centres. Clinical and demographic data were collated on all subjects alongside BAL inflammatory markers. 16S rRNA gene sequencing was performed on the Illumina MiSeq platform. Bioinformatics and data analysis were performed using Qiime and R project software. Data on 292 sequenced BALs from 101 children with CF and 51 without CF show the CF lung microbiome, while broadly similar to that in non-CF children, is distinct. Alpha diversity between the two cohorts was indistinguishable at this early age. The CF diagnosis explained only 1.1% of the variation between the cohort microbiomes. However, several key genera were significantly differentially abundant between the groups. While the non-CF lung microbiome diversity increased with age, diversity reduced in CF with age. Pseudomonas and Staphylococcus were more abundant with age, while genera such as Streptococcus, Porphyromonas and Veillonella were less abundant with age. There was a negative correlation between alpha diversity and interleukin-8 and neutrophil elastase in the CF population. Neither current flucloxacillin or azithromycin prophylaxis, nor previous oral or IV antibiotic exposure, was correlated with microbiome diversity. Consecutive annual BAL samples over 5 years from a subgroup of children demonstrated diverse patterns of development in the first years of life.

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

ABSTRACT Previous studies suggest a link between intestinal microbiota and porcine feed efficiency (FE). Therefore, we investigated whether fecal microbiota transplantation (FMT) in sows and/or neonatal offspring, using inocula derived from highly feed-efficient pigs, could improve offspring FE. Pregnant sows were assigned to control or FMT treatments and the subsequent offspring to control treatment, FMT once (at birth), or FMT four times (between birth and weaning). FMT altered sow fecal and colostrum microbiota compositions and resulted in lighter offspring body weight at 70 and 155 days of age when administered to sows and/or offspring. This was accompanied by FMT-associated changes within the offspring’s intestinal microbiota, mostly in the ileum. These included transiently higher fecal bacterial diversity and load and numerous compositional differences at the phylum and genus levels (e.g., Spirochaetes and Bacteroidetes at high relative abundances and mostly members of Clostridia, respectively), as well as differences in the abundances of predicted bacterial pathways. In addition, intestinal morphology was negatively impacted, duodenal gene expression altered, and serum protein and cholesterol concentrations reduced due to FMT in sows and/or offspring. Taken together, the results suggest poorer absorptive capacity and intestinal health, most likely explaining the reduced body weight. An additive effect of FMT in sows and offspring also occurred for some parameters. Although these findings have negative implications for the practical use of the FMT regime used here for improving FE in pigs, they nonetheless demonstrate the enormous impact of early-life intestinal microbiota on the host phenotype. IMPORTANCE Here, for the first time, we investigate FMT as a novel strategy to modulate the porcine intestinal microbiota in an attempt to improve FE in pigs. However, reprogramming the maternal and/or offspring microbiome by using fecal transplants derived from highly feed-efficient pigs did not recapitulate the highly efficient phenotype in the offspring and, in fact, had detrimental effects on lifetime growth. Although these findings may not be wholly attributable to microbiota transplantation, as antibiotic and purgative were also part of the regime in sows, similar effects were also seen in offspring, in which these interventions were not used. Nonetheless, additional work is needed to unravel the effects of each component of the FMT regime and to provide additional mechanistic insights. This may lead to the development of an FMT procedure with practical applications for the improvement of FE in pigs, which could in turn improve the profitability of pig production.

There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.

ABSTRACT Kefir is a putatively health-promoting dairy beverage that is produced when a kefir grain, consisting of a consortium of microorganisms, is added to milk to initiate a natural fermentation. Here, a detailed analysis was carried out to determine how the microbial population, gene content, and flavor of three kefirs from distinct geographic locations change over the course of 24-h fermentations. Metagenomic sequencing revealed that Lactobacillus kefiranofaciens was the dominant bacterial species in kefir during early stages of fermentations but that Leuconostoc mesenteroides became more prevalent in later stages. This pattern is consistent with an observation that genes involved in aromatic amino acid biosynthesis were absent from L. kefiranofaciens but were present in L. mesenteroides. Additionally, these shifts in the microbial community structure, and associated pathways, corresponded to changes in the levels of volatile compounds. Specifically, Acetobacter spp. correlated with acetic acid; Lactobacillus spp. correlated with carboxylic acids, esters and ketones; Leuconostoc spp. correlated with acetic acid and 2,3-butanedione; and Saccharomyces spp. correlated with esters. The correlation data suggest a causal relationship between microbial taxa and flavor that is supported by observations that addition of L. kefiranofaciens NCFB 2797 increased the levels of esters and ketones whereas addition of L. mesenteroides 213M0 increased the levels of acetic acid and 2,3-butanedione. Finally, we detected genes associated with probiotic functionalities in the kefir microbiome. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein and can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. IMPORTANCE Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. Author Video: An author video summary of this article is available.

This study examines the potential of next-generation sequencing based ‘genotyping-by-sequencing’ (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

BackgroundEarly life stress is a risk factor for many psychiatric disorders ranging from depression to anxiety. Stress, especially during early life, can induce dysbiosis in the gut microbiota, the key modulators of the bidirectional signalling pathways in the gut-brain axis that underline several neurodevelopmental and psychiatric disorders. Despite their critical role in the development and function of the central nervous system, the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on the regulation of gut-microbiota in early-life stress has not been explored.Methods and resultsHere, we show that long-term supplementation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) (80% EPA, 20% DHA) n-3 PUFAs mixture could restore the disturbed gut-microbiota composition of maternally separated (MS) female rats. Sprague-Dawley female rats were subjected to an early-life stress, maternal separation procedure from postnatal days 2 to 12. Non-separated (NS) and MS rats were administered saline, EPA/DHA 0.4 g/kg/day or EPA/DHA 1 g/kg/day, respectively. Analysis of the gut microbiota in adult rats revealed that EPA/DHA changes composition in the MS, and to a lesser extent the NS rats, and was associated with attenuation of the corticosterone response to acute stress.ConclusionsIn conclusion, EPA/DHA intervention alters the gut microbiota composition of both neurodevelopmentally normal and early-life stressed animals. This study offers insights into the interaction between n-3 PUFAs and gut microbes, which may play an important role in advancing our understanding of disorders of mood and cognitive functioning, such as anxiety and depression.

ObjectiveTo assess the effects of feeding Bt MON810 maize to pigs for 110 days on the intestinal microbiota.Methodology/principal findingsForty male pigs (∼40 days old) were blocked by weight and litter ancestry and assigned to one of four treatments; 1) Isogenic maize-based diet for 110 days (Isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by a Bt maize-based diet for 80 days (Isogenic/Bt); 4) Bt maize-based diet for 30 days followed by an isogenic maize-based diet for 80 days (Bt/Isogenic). Enterobacteriaceae, Lactobacillus and total anaerobes were enumerated in the feces using culture-based methods on days 0, 30, 60 and 100 of the study and in ileal and cecal digesta on day 110. No differences were found between treatments for any of these counts at any time point. The relative abundance of cecal bacteria was also determined using high-throughput 16 S rRNA gene sequencing. No differences were observed in any bacterial taxa between treatments, with the exception of the genus Holdemania which was more abundant in the cecum of pigs fed the isogenic/Bt treatment compared to pigs fed the Bt treatment (0.012 vs 0.003%; P≤0.05).Conclusions/significanceFeeding pigs a Bt maize-based diet for 110 days did not affect counts of any of the culturable bacteria enumerated in the feces, ileum or cecum. Neither did it influence the composition of the cecal microbiota, with the exception of a minor increase in the genus Holdemania. As the role of Holdemania in the intestine is still under investigation and no health abnormalities were observed, this change is not likely to be of clinical significance. These results indicate that feeding Bt maize to pigs in the context of its influence on the porcine intestinal microbiota is safe.