Prevalence of persistent SARS-CoV-2 in a large community surveillance study.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

Acquisition and Development of the Extremely Preterm Infant Microbiota Across Multiple Anatomical Sites.

OBJECTIVES: Microbial communities influencing health and disease are being increasingly studied in preterm neonates. There exists little data, however, detailing longitudinal microbial acquisition, especially in the most extremely preterm (<26 weeks' gestation). This study aims to characterize the development of the microbiota in this previously under-represented cohort. METHODS: Seven extremely preterm infant-mother dyads (mean gestation 23.6 weeks) were recruited from a single neonatal intensive care unit. Oral and endotracheal secretions, stool, and breast milk (n = 157 total), were collected over the first 60 days of life. Targeted 16S rRNA gene sequencing identified bacterial communities present. RESULTS: Microbiota of all body sites were most similar immediately following birth and diverged longitudinally. Throughout the sampling period Escherichia, Enterococcus, Staphylococcus, and an Enterobacteriaceae were dominant and well dispersed across all sites. Temporal divergence of the stool from other microbiota was driven by decreasing diversity and significantly greater proportional abundance of Bifidobacteriaceae compared to other sites. CONCLUSIONS: Four taxa dominated all anatomical sampling sites. Rare taxa promoted dissimilarity. Cross-seeding between upstream communities and the stool was demonstrated, possibly relating to buccal colostrum/breast milk exposure and indwelling tubes. Given the importance of dysbiosis in health and disease of extremely preterm infants, better understanding of microbial acquisition within this context may be of clinical benefit.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

Polymicrobial airway bacterial communities in adult bronchiectasis patients.

BACKGROUND: Chronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation. RESULTS: The lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung. CONCLUSIONS: We have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Profiling the Enterobacterales Community Isolated from Retail Foods in England.

Enterobacterales include foodborne pathogens of importance to public health and are often targeted in food surveillance programs as both safety and hygiene indicators. Furthermore, Enterobacterales are important in the context of antimicrobial resistance dissemination, also impacting infection treatment efficacy. In this study, the prevalence and characteristics of Enterobacterales in UK retail foods were examined. From 110 retail food samples, 253 Enterobacterales were recovered, with 16S rRNA sequencing revealing a diverse species community, including enteropathogens; the most common were Proteus mirabilis and Escherichia coli (18% each). Antimicrobial resistance was common, with 160/253 (63%) isolates being resistant to at least 1 antimicrobial. Resistance to all tested antimicrobials was observed. Thirteen percent of isolates were multidrug resistant, including 2 isolates each resistant to 8 or 9 of 9 antimicrobials tested. Klebsiella isolates possessed relatively higher levels of antimicrobial resistance to other species. Hafnia, Kluyvera, and Proteus isolates produced significantly higher biofilm biomass than Klebsiella (p = 0.038, 0.028, and 0.042, respectively) or Escherichia (p = 0.001, 0.008, and 0.001, respectively). Simultaneous curli fimbriae and cellulose production was noted in 7% of isolates at 37 °C, but not at 15 °C. This research demonstrates a high diversity of Enterobacterales within UK retail foods, alongside notable antimicrobial resistance phenotypes in enteropathogenic species, highlighting the need for effective surveillance and interventions.

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence.

Bacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognise and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognises cognate motifs has not been fully understood. In this study we characterise BREX from pathogenic Salmonella and present X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that both methyltransferase activity and defence require larger BREX complexes. Through rational engineering of PglX we broaden both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is used to recognise specific DNA sequences for BREX activity, contributing to motif recognition for both phage defence and host methylation.

Built environment microbiomes transition from outdoor to human-associated communities after construction and commissioning.

The microbiota of the built environment is linked to usage, materials and, perhaps most importantly, human health. Many studies have attempted to identify ways of modulating microbial communities within built environments to promote health. None have explored how these complex communities assemble initially, following construction of new built environments. This study used high-throughput targeted sequencing approaches to explore bacterial community acquisition and development throughout the construction of a new build. Microbial sampling spanned from site identification, through the construction process to commissioning and use. Following commissioning of the building, bacterial richness and diversity were significantly reduced (P < 0.001) and community structure was altered (R2 = 0.14; P = 0.001). Greater longitudinal community stability was observed in outdoor environments than indoor environments. Community flux in indoor environments was associated with human interventions driving environmental selection, which increased 10.4% in indoor environments following commissioning. Increased environmental selection coincided with a 12% reduction in outdoor community influence on indoor microbiomes (P = 2.00 × 10-15). Indoor communities became significantly enriched with human associated genera including Escherichia, Pseudomonas, and Klebsiella spp. These data represent the first to characterize the initial assembly of bacterial communities in built environments and will inform future studies aiming to modulate built environment microbiota.

Bacteriophage communities are a reservoir of unexplored microbial diversity in neonatal health and disease.

Acquisition and development of the gut microbiome are vital for immune education in neonates, especially those born preterm. As such, microbial communities have been extensively studied in the context of postnatal health and disease. Bacterial communities have been the focus of research in this area due to the relative ease of targeted bacterial sequencing and the availability of databases to align and validate sequencing data. Recent increases in high-throughput metagenomic sequencing accessibility have facilitated research to investigate bacteriophages within the context of neonatal gut microbial communities. Focusing on unexplored viral diversity, has identified novel bacteriophage species and previously uncharacterised viral diversity. In doing so, studies have highlighted links between bacteriophages and bacterial community structure in the context of health and disease. However, much remains unknown about the complex relationships between bacteriophages, the bacteria they infect and their human host. With a particular focus on preterm infants, this review highlights opportunities to explore the influence of bacteriophages on developing microbial communities and the tripartite relationships between bacteriophages, bacteria and the neonatal human host. We suggest a focus on expanding collections of isolated bacteriophages that will further our understanding of the growing numbers of bacteriophages identified in metagenomes.

Vanillin Cross-Linked Chitosan Film with Controlled Release of Green Tea Polyphenols for Active Food Packaging.

We report a novel cross-linked chitosan composite film containing vanillin, glycerol, and green tea extract. The effects of vanillin-mediated cross-linking and the incorporation of antimicrobial green tea polyphenols were investigated. The cross-linking effect, confirmed by Fourier transform infrared (FTIR) analysis, increased the tensile strength of the biopolymer film to 20.9 ± 3 MPa. The release kinetics of polyphenols from the chitosan-vanillin matrix was studied, and we reported an initial burst release (8 h) followed by controlled release (8 to 400 h). It was found that both vanillin and green tea polyphenols were successful inhibitors of foodborne bacteria, with a minimum inhibitory concentration of the tea polyphenols determined as 0.15 mg/mL (Staphylococcus aureus). These active components also displayed strong antioxidant capacities, with polyphenols quenching >80% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals at all concentrations tested. Degradation results revealed that there was a significant (>85%) mass loss of all samples after being buried in compost for 12 weeks. The biopolymeric films, prepared by solvent casting methods, adhere to green chemistry and waste valorization principles. The one-pot recipe reported may also be applied to other cross-linkers and active compounds with similar chemical functionalities. Based on the obtained results, the presented material provides a promising starting point for the development of a degradable active packaging material.

The core phageome and its interrelationship with preterm human milk lipids.

Phages and lipids in human milk (HM) may benefit preterm infant health by preventing gastrointestinal pathobiont overgrowth and microbiome modulation. Lipid association may promote vertical transmission of phages to the infant. Despite this, interrelationships between lipids and phages are poorly characterized in preterm HM. Shotgun metagenomics and untargeted lipidomics of phage and lipid profiles from 99 preterm HM samples reveals that phages are abundant and prevalent from the first week and throughout the first 100 days of lactation. Phage-host richness of preterm HM increases longitudinally. Core phage communities characterized by Staphylococcus- and Propionibacterium-infecting phages are significantly correlated with long-chain fatty acid abundances over lactational age. We report here a phage-lipid interaction in preterm HM, highlighting the potential importance of phage carriage in preterm HM. These results reveal possible strategies for phage carriage in HM and their importance in early-life microbiota development.

Comparative genomics of Shiga toxin encoding bacteriophages.

BACKGROUND: Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. RESULTS: The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. CONCLUSIONS: The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.

Optimisation and Application of a Novel Method to Identify Bacteriophages in Maternal Milk and Infant Stool Identifies Host-Phage Communities Within Preterm Infant Gut.

Human milk oligosaccharides, proteins, such as lactoferrin, and bacteria represent just some of the bioactive components of mother's breast milk (BM). Bacteriophages (viruses that infect bacteria) are an often-overlooked component of BM that can cause major changes in microbial composition and metabolism. BM bacteriophage composition has been explored in term and healthy infants, suggesting vertical transmission of bacteriophages occurs between mothers and their infants. Several important differences between term and very preterm infants (<30 weeks gestational age) may limit this phenomenon in the latter. To better understand the link between BM bacteriophages and gut microbiomes of very preterm infants in health and disease, standardised protocols are required for isolation and characterisation from BM. In this study, we use isolated nucleic acid content, bacteriophage richness and Shannon diversity to validate several parameters applicable during bacteriophage isolation from precious BM samples. Parameters validated include sample volume required; centrifugal sedimentation of microbes; hydrolysis of milk samples with digestive enzymes; induction of temperate bacteriophages and concentration/purification of isolated bacteriophage particles in donor milk (DM). Our optimised method enables characterisation of bacteriophages from as little as 0.1 mL BM. We identify viral families that were exclusively identified with the inclusion of induction of temperate bacteriophages (Inoviridae) and hydrolysis of milk lipid processes (Iridoviridae and Baculoviridae). Once applied to a small clinical cohort we demonstrate the vertical transmission of bacteriophages from mothers BM to the gut of very preterm infants at the species level. This optimised method will enable future research characterising the bacteriophage composition of BM in very preterm infants to determine their clinical relevance in the development of a healthy preterm infant gut microbiome.

Gut Microbial Stability is Associated with Greater Endurance Performance in Athletes Undertaking Dietary Periodization.

Dietary manipulation with high-protein or high-carbohydrate content are frequently employed during elite athletic training, aiming to enhance athletic performance. Such interventions are likely to impact upon gut microbial content. This study explored the impact of acute high-protein or high-carbohydrate diets on measured endurance performance and associated gut microbial community changes. In a cohort of well-matched, highly trained endurance runners, we measured performance outcomes, as well as gut bacterial, viral (FVP), and bacteriophage (IV) communities in a double-blind, repeated-measures design randomized control trial (RCT) to explore the impact of dietary intervention with either high-protein or high-carbohydrate content. High-dietary carbohydrate improved time-trial performance by +6.5% (P < 0.03) and was associated with expansion of Ruminococcus and Collinsella bacterial spp. Conversely, high dietary protein led to a reduction in performance by -23.3% (P = 0.001). This impact was accompanied by significantly reduced diversity (IV: P = 0.04) and altered composition (IV and FVP: P = 0.02) of the gut phageome as well as enrichment of both free and inducible Sk1virus and Leuconostoc bacterial populations. Greatest performance during dietary modification was observed in participants with less substantial shifts in community composition. Gut microbial stability during acute dietary periodization was associated with greater athletic performance in this highly trained, well-matched cohort. Athletes, and those supporting them, should be mindful of the potential consequences of dietary manipulation on gut flora and implications for performance, and periodize appropriately. IMPORTANCE Dietary periodization is employed to improve endurance exercise performance but may impact on gut microbial communities. Bacteriophage are implicated in bacterial cell homeostasis and have been identified as biomarkers of disequilibrium in the gut ecosystem possibly brought about through dietary periodization. We find high-carbohydrate and high-protein diets to have opposing impacts on endurance performance in highly trained athlete populations. Reduced performance is linked with disturbance of microbial stasis in the gut. We demonstrate bacteriophage communities are the most sensitive component of the gut microbiota to increased gut stress following dietary manipulation. Athletes undertaking dietary periodization should be aware of potential negative impacts of drastic changes to dietary composition on gut microbial stasis and, in turn, endurance performance.

The role of viral genomics in understanding COVID-19 outbreaks in long-term care facilities.

We reviewed all genomic epidemiology studies on COVID-19 in long-term care facilities (LTCFs) that had been published to date. We found that staff and residents were usually infected with identical, or near identical, SARS-CoV-2 genomes. Outbreaks usually involved one predominant cluster, and the same lineages persisted in LTCFs despite infection control measures. Outbreaks were most commonly due to single or few introductions followed by a spread rather than a series of seeding events from the community into LTCFs. The sequencing of samples taken consecutively from the same individuals at the same facilities showed the persistence of the same genome sequence, indicating that the sequencing technique was robust over time. When combined with local epidemiology, genomics allowed probable transmission sources to be better characterised. The transmission between LTCFs was detected in multiple studies. The mortality rate among residents was high in all facilities, regardless of the lineage. Bioinformatics methods were inadequate in a third of the studies reviewed, and reproducing the analyses was difficult because sequencing data were not available in many facilities.

Raltegravir is a substrate for SLC22A6: a putative mechanism for the interaction between raltegravir and tenofovir.

The identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEM(VBL100), and Caco-2 cells. Transport by uptake transporters was assessed by using a Xenopus laevis oocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEM(VBL100), and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. The K(m) and V(max) for SLC22A6 transport were 150 µM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 µM tenofovir with a 50% inhibitory concentration (IC(50)) of 14.0 µM, and tenofovir inhibited 1 µM raltegravir with an IC(50) of 27.3 µM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.

The clinical and microbiological utility of inhaled aztreonam lysine for the treatment of acute pulmonary exacerbations of cystic fibrosis: An open-label randomised crossover study (AZTEC-CF).

BACKGROUND: The objective of this study was to explore the clinical and microbiological outcomes associated with substituting inhaled aztreonam lysine for an intravenous antibiotic in the treatment of acute pulmonary exacerbations of CF. METHODS: An open-label randomised crossover pilot trial was conducted at a UK CF centre among 16 adults with CF and P. aeruginosa infection. Median [IQR] age was 29.5 [24.5-32.5], mean ± SD forced expiratory volume in 1 second (FEV1) was 52.4 ± 14.7 % predicted. Over the course of two exacerbations, participants were randomised to sequentially receive 14 days of inhaled aztreonam lysine plus IV colistimethate (AZLI+IV), or dual IV antibiotics (IV+IV). Primary outcome was absolute change in % predicted FEV1. Other outcomes evaluated changes in quality of life, bacterial load and the lung microbiota. RESULTS: The difference between mean change in lung function at day 14 between AZLI+IV and IV+IV was +4.6% (95% CI 2.1-7.2, p=0.002). The minimum clinically important difference of the Cystic Fibrosis Revised Questionnaire (CFQ-R) was achieved more frequently with AZLI+IV (10/12, 83.3%) than IV+IV (7/16, 43.8%), p=0.05. No differences were observed for modulation of serum white cell count, C-reactive protein or sputum bacterial load. Microbiome compositional changes were observed with IV+IV (Bray-Curtis r2=0.14, p=0.02), but not AZLI+IV (r2=0.03, p=0.64). CONCLUSION: In adults with CF and P. aeruginosa infection experiencing an acute pulmonary exacerbation, AZLI+IV improved lung function and quality of life compared to the current standard treatment. These findings support the need for larger definitive trials of inhaled antibiotics in the acute setting. CLINICAL TRIAL REGISTRATION: EudraCT 2016-002832-34 ClinicalTrials.org NCT02894684.

The Impact of NOD2 Genetic Variants on the Gut Mycobiota in Crohn's Disease Patients in Remission and in Individuals Without Gastrointestinal Inflammation.

BACKGROUND AND AIMS: Historical and emerging data implicate fungi in Crohn's disease [CD] pathogenesis. However, a causal link between mycobiota, dysregulated immunity, and any impact of NOD2 variants remains elusive. This study aims to evaluate associations between NOD2 variants and faecal mycobiota in CD patients and non-CD subjects. METHODS: Faecal samples were obtained from 34 CD patients [18 NOD2 mutant, 16 NOD2 wild-type] identified from the UK IBD Genetics Consortium. To avoid confounding influence of mucosal inflammation, CD patients were in clinical remission and had a faecal calprotectin <250 µg/g; 47 non-CD subjects were included as comparator groups, including 22 matched household [four NOD2 mutant] and 25 non-household subjects with known NOD2 genotype [14 NOD2 mutant] identified by the NIHR BioResource Cambridge. Faecal mycobiota composition was determined using internal transcribed spacer 1 [ITS1] sequencing and was compared with 16S rRNA gene sequences and volatile organic compounds. RESULTS: CD was associated with higher numbers of fungal observed taxonomic units [OTUs] [p = 0.033]. Principal coordinates analysis using Jaccard index [p = 0.018] and weighted Bray-Curtis dissimilarities [p = 0.01] showed Candida spp. clustered closer to CD patients whereas Cryptococcus spp. clustered closer to non-CD. In CD, we found higher relative abundance of Ascomycota [p = 0.001] and lower relative abundance Basidiomycota [p = 0.019] phyla. An inverse relationship was found between bacterial and fungal Shannon diversity in NOD2 wild-type which was independent of CD [r = -0.349; p = 0.029]. CONCLUSIONS: This study confirms compositional changes in the gut mycobiota in CD and provides evidence that fungi may play a role in CD pathogenesis. No NOD2 genotype-specific differences were observed in the faecal mycobiota.

High-throughput method for rapid induction of prophages from lysogens and its application in the study of Shiga Toxin-encoding Escherichia coli strains.

A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains and demonstrated the ability to detect both lambdoid and stx-carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the Q gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.

Temperate Bacteriophages from Chronic Pseudomonas aeruginosa Lung Infections Show Disease-Specific Changes in Host Range and Modulate Antimicrobial Susceptibility.

Temperate bacteriophages are a common feature of Pseudomonas aeruginosa genomes, but their role in chronic lung infections is poorly understood. This study was designed to identify the diverse communities of mobile P. aeruginosa phages by employing novel metagenomic methods, to determine cross infectivity, and to demonstrate the influence of phage infection on antimicrobial susceptibility. Mixed temperate phage populations were chemically mobilized from individual P. aeruginosa, isolated from patients with cystic fibrosis (CF) or bronchiectasis (BR). The infectivity phenotype of each temperate phage lysate was evaluated by performing a cross-infection screen against all bacterial isolates and tested for associations with clinical variables. We utilized metagenomic sequencing data generated for each phage lysate and developed a novel bioinformatic approach allowing resolution of individual temperate phage genomes. Finally, we used a subset of the temperate phages to infect P. aeruginosa PAO1 and tested the resulting lysogens for their susceptibility to antibiotics. Here, we resolved 105 temperate phage genomes from 94 lysates that phylogenetically clustered into 8 groups. We observed disease-specific phage infectivity profiles and found that phages induced from bacteria isolated from more advanced disease infected broader ranges of P. aeruginosa isolates. Importantly, when infecting PAO1 in vitro with 20 different phages, 8 influenced antimicrobial susceptibility. This study shows that P. aeruginosa isolated from CF and BR patients harbors diverse communities of inducible phages, with hierarchical infectivity profiles that relate to the progression of the disease. Temperate phage infection altered the antimicrobial susceptibility of PAO1 at subinhibitory concentrations of antibiotics, suggesting they may be precursory to antimicrobial resistance.IMPORTANCE Pseudomonas aeruginosa is a key opportunistic respiratory pathogen in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. The genomes of these pathogens are enriched with mobile genetic elements including diverse temperate phages. While the temperate phages of the Liverpool epidemic strain have been shown to be active in the human lung and enhance fitness in a rat lung infection model, little is known about their mobilization more broadly across P. aeruginosa in chronic respiratory infection. Using a novel metagenomic approach, we identified eight groups of temperate phages that were mobilized from 94 clinical P. aeruginosa isolates. Temperate phages from P. aeruginosa isolated from more advanced disease showed high infectivity rates across a wide range of P. aeruginosa genotypes. Furthermore, we showed that multiple phages altered the susceptibility of PAO1 to antibiotics at subinhibitory concentrations.