Exhaled nitric oxide is associated with inflammatory biomarkers and risk of acute respiratory exacerbations in children with HIV-associated chronic lung disease.

OBJECTIVES: Chronic lung disease is a recognized complication in children with HIV. Acute respiratory exacerbations (ARE) are common among this group and cause significant morbidity. Exhaled nitric oxide (eNO) is a known marker of local airway inflammation. We investigated the association between eNO and ARE, biomarkers of systemic inflammation, and the effect of azithromycin on eNO levels. METHODS: Individuals aged 6-19 years with HIV-associated chronic lung disease in Harare, Zimbabwe, were enrolled in a placebo-controlled randomized trial investigating the effect of 48-week azithromycin treatment on lung function and ARE. eNO levels and biomarkers were measured at inclusion and after treatment in a consecutively enrolled subset of participants. Linear regression and generalized linear models were used to study associations between eNO and ARE, biomarkers, and the effect of azithromycin on eNO levels. RESULTS: In total, 172 participants were included in this sub-study, 86 from the placebo group and 86 from the azithromycin group. Participants experiencing at least one ARE during follow-up had significantly higher eNO levels at baseline than participants who did not (geometric mean ratio 1.13, 95% confidence interval [CI] 1.03-1.24, p = 0.015), adjusted for trial arm, age, sex and history of tuberculosis. Matrix metalloproteinase (MMP)-3, -7, and -10 were significantly associated with higher baseline eNO levels. At 48 weeks, azithromycin treatment did not affect eNO levels (geometric mean ratio 0.86, 95% CI 0.72-1.03, p = 0.103). CONCLUSION: Higher baseline eNO levels were a risk factor for ARE. eNO was associated with proinflammatory biomarkers previously found to contribute to the development of chronic lung disease. The potential use of eNO as a marker of inflammation and risk factor for ARE in HIV-associated chronic lung disease needs further investigation.

Flexible and Biocompatible Antifouling Polyurethane Surfaces Incorporating Tethered Antimicrobial Peptides through Click Reactions.

Efficient, simple antibacterial materials to combat implant-associated infections are much in demand. Herein, the development of polyurethanes, both cross-linked thermoset and flexible and versatile thermoplastic, suitable for "click on demand" attachment of antibacterial compounds enabled via incorporation of an alkyne-containing diol monomer in the polymer backbone, is described. By employing different polyolic polytetrahydrofurans, isocyanates, and chain extenders, a robust and flexible material comparable to commercial thermoplastic polyurethane is prepared. A series of short synthetic antimicrobial peptides are designed, synthesized, and covalently attached in a single coupling step to generate a homogenous coating. The lead material is shown to be biocompatible and does not display any toxicity against either mouse fibroblasts or reconstructed human epidermis according to ISO and OECD guidelines. The repelling performance of the peptide-coated materials is illustrated against colonization and biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis on coated plastic films and finally, on coated commercial central venous catheters employing LIVE/DEAD staining, confocal laser scanning microscopy, and bacterial counts. This study presents the successful development of a versatile and scalable polyurethane with the potential for use in the medical field to reduce the impact of bacterial biofilms.

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

The novel bacteriocin romsacin from Staphylococcus haemolyticus inhibits Gram-positive WHO priority pathogens.

IMPORTANCE: Bacteria produce bacteriocins to inhibit growth of other bacterial species. We have studied the antimicrobial activity of a new bacteriocin produced by the skin bacterium S. haemolyticus. The bacteriocin is effective against several types of Gram-positive bacteria, including highly virulent and antibiotic-resistant strains such as Staphylococcus aureus and Enterococcus faecium. Effective antimicrobials are important for the treatment of infections and the success of major surgery and chemotherapy. Bacteriocins can be part of the solution to the global concern of antimicrobial resistance.

Development of early life gut resistome and mobilome across gestational ages and microbiota-modifying treatments.

BACKGROUND: Gestational age (GA) and associated level of gastrointestinal tract maturation are major factors driving the initial gut microbiota composition in preterm infants. Besides, compared to term infants, premature infants often receive antibiotics to treat infections and probiotics to restore optimal gut microbiota. How GA, antibiotics, and probiotics modulate the microbiota's core characteristics, gut resistome and mobilome, remains nascent. METHODS: We analysed metagenomic data from a longitudinal observational study in six Norwegian neonatal intensive care units to describe the bacterial microbiota of infants of varying GA and receiving different treatments. The cohort consisted of probiotic-supplemented and antibiotic-exposed extremely preterm infants (n = 29), antibiotic-exposed very preterm (n = 25), antibiotic-unexposed very preterm (n = 8), and antibiotic-unexposed full-term (n = 10) infants. The stool samples were collected on days of life 7, 28, 120, and 365, and DNA extraction was followed by shotgun metagenome sequencing and bioinformatical analysis. FINDINGS: The top predictors of microbiota maturation were hospitalisation length and GA. Probiotic administration rendered the gut microbiota and resistome of extremely preterm infants more alike to term infants on day 7 and ameliorated GA-driven loss of microbiota interconnectivity and stability. GA, hospitalisation, and both microbiota-modifying treatments (antibiotics and probiotics) contributed to an elevated carriage of mobile genetic elements in preterm infants compared to term controls. Finally, Escherichia coli was associated with the highest number of antibiotic-resistance genes, followed by Klebsiella pneumoniae and Klebsiella aerogenes. INTERPRETATION: Prolonged hospitalisation, antibiotics, and probiotic intervention contribute to dynamic alterations in resistome and mobilome, gut microbiota characteristics relevant to infection risk. FUNDING: Odd-Berg Group, Northern Norway Regional Health Authority.

Dual inhibition of complement C5 and CD14 attenuates inflammation in a cord blood model.

BACKGROUND: Escherichia coli and Group B streptococci (GBS) are the main causes of neonatal early-onset sepsis (EOS). Despite antibiotic therapy, EOS is associated with high morbidity and mortality. Dual inhibition of complement C5 and the Toll-like receptor co-factor CD14 has in animal studies been a promising novel therapy for sepsis. METHODS: Whole blood was collected from the umbilical cord after caesarean section (n = 30). Blood was anti-coagulated with lepirudin. C5 inhibitor (eculizumab) and anti-CD14 was added 8 min prior to, or 15 and 30 min after adding E. coli or GBS. Total bacterial incubation time was 120 min (n = 16) and 240 min (n = 14). Cytokines and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. RESULTS: Dual inhibition significantly attenuated TCC formation by 25-79% when adding inhibitors with up to 30 min delay in both E. coli- and GBS-induced inflammation. TNF, IL-6 and IL-8 plasma concentration were significantly reduced by 28-87% in E. coli-induced inflammation when adding inhibitors with up to 30 min delay. The dual inhibition did not significantly reduce TNF, IL-6 and IL-8 plasma concentration in GBS-induced inflammation. CONCLUSION: Dual inhibition of C5 and CD14 holds promise as a potential future treatment for severe neonatal EOS. IMPACT: Neonatal sepsis can cause severe host inflammation with high morbidity and mortality, but there are still no effective adjunctive immunologic interventions available. Adding CD14 and complement C5 inhibitors up to 30 min after incubation of E. coli or Group B streptococci in a human umbilical cord blood model significantly reduced complement activation and cytokine release. Dual inhibition of C5 and CD14 is a potential future therapy to modulate systemic inflammation in severe cases of neonatal sepsis.

Lactoferricin-inspired peptide AMC-109 augments the effect of ciprofloxacin against Pseudomonas aeruginosa biofilm in chronic murine wounds.

OBJECTIVES: Chronic wounds are characterised by prolonged inflammation, low mitogenic activity, high protease/low inhibitor activity, microbiota changes and biofilm formation, combined with the aetiology of the original insult. One strategy to promote healing is to terminate the parasitism-like relationship between the biofilm-growing pathogen and host response. Antimicrobial peptide AMC-109 is a potential treatment with low resistance potential and broad-spectrum coverage with rapid bactericidal effect. We aimed to investigate whether adjunctive AMC-109 could augment the ciprofloxacin effect in a chronic Pseudomonas aeruginosa wound model. METHODS: Third-degree burns were inflicted on 33 BALB/c mice. Pseudomonas aeruginosa embedded in seaweed alginate was injected sub-eschar to mimic biofilm. Mice were randomised to receive AMC-109, combined AMC-109 and ciprofloxacin, ciprofloxacin, or placebo for 5 days followed by sample collection. RESULTS: A lower bacterial load was seen in the double-treated group compared with either monotherapy group (AMC-109, p = 0.0076; ciprofloxacin, p = 0.0266). To evaluate the innate host response, cytokines and growth factors were quantified. The pro-inflammatory response was dampened in the double-treated mice compared with the mono-ciprofloxacin-treated group (p = 0.0009). Lower mobilisation of neutrophils from the bone marrow was indicated by reduced G-CSF in all treatment groups compared with placebo. Improved tissue remodelling was indicated by the highest level of tissue inhibitor of metalloproteases and low metalloprotease level in the double-treated group. CONCLUSION: AMC-109 showed adjunctive antipseudomonal abilities augmenting the antimicrobial effect of ciprofloxacin in this wound model. The study indicates a potential role for AMC-109 in treating chronic wounds with complicating biofilm infections.

In vitro and in vivo antibacterial properties of peptide AMC-109 impregnated wound dressings and gels.

Synthetic mimics of antimicrobial peptides (AMPs) is a promising class of molecules for a variety of antimicrobial applications. Several hurdles must be passed before effective systemic infection therapies with AMPs can be achieved, but the path to effective topical treatment of skin, nail, and soft tissue infections appears less challenging to navigate. Skin and soft tissue infection is closely coupled to the emergence of antibiotic resistance and represents a major burden to the healthcare system. The present study evaluates the promising synthetic cationic AMP mimic, AMC-109, for treatment of skin infections in vivo. The compound is evaluated both in impregnated cotton wound dressings and in a gel formulation against skin infections caused by Staphylococcus aureus and methicillin resistant S. aureus. Both the ability to prevent colonization and formation of an infection, as well as eradicate an ongoing infection in vivo with a high bacterial load, were evaluated. The present work demonstrates that AMC-109 displays a significantly higher antibacterial activity with up to a seven-log reduction in bacterial loads compared to current clinical standard therapy; Altargo cream (1% retapamulin) and Fucidin cream (2% fusidic acid) in the in vivo wound models. It is thus concluded that AMC-109 represents a promising entry in the development of new and effective remedies for various skin infections.

Staphylococcus borealis sp. nov., isolated from human skin and blood.

When analysing a large cohort of Staphylococcus haemolyticus, using whole-genome sequencing, five human isolates (four from the skin and one from a blood culture) with aberrant phenotypic and genotypic traits were identified. They were phenotypically similar with yellow colonies, nearly identical 16S rRNA gene sequences and initially speciated as S. haemolyticus based on 16S rRNA gene sequence and MALDI-TOF MS. However, compared to S. haemolyticus, these five strains demonstrate: (i) considerable phylogenetic distance with an average nucleotide identity <95 % and inferred DNA-DNA hybridization <70  %; (ii) a pigmented phenotype; (iii) urease production; and (iv) different fatty acid composition. Based on the phenotypic and genotypic results, we conclude that these strains represent a novel species, for which the name Staphylococcus borealis sp. nov. is proposed. The novel species belong to the genus Staphylococcus and is coagulase- and oxidase-negative and catalase-positive. The type strain, 51-48T, is deposited in the Culture Collection University of Gothenburg (CCUG 73747T) and in the Spanish Type Culture Collection (CECT 30011T).

Effects of fecal microbiota transplantation in subjects with irritable bowel syndrome are mirrored by changes in gut microbiome.

Irritable bowel syndrome (IBS) is a common disorder of the lower gastrointestinal tract. The pathophysiology is far from settled, but a gut microbial dysbiosis is hypothesized to be a contributing factor. We earlier published a randomized double-blind placebo-controlled clinical trial on fecal microbiota transplantation (FMT) for IBS - the REFIT trial. The present data set describes the engraftment and includes participants from the study who received active FMT; 14 participants with effect of FMT (Effect) and 8 without (No effect). Samples were collected at baseline, after 6 and 12 months. Samples from the transplants (Donor) served as a comparator. In total 66 recipient samples and 17 donor samples were subjected to deep metagenomic sequencing, and taxonomic and functional analyses were performed. Alpha diversity measures showed a significantly increased diversity and evenness in the IBS groups compared to the donors. Taxonomic profiles showed higher relative abundance of phylum Firmicutes, and lower relative abundance of phylum Bacteroidetes, compared to donors at baseline. This profile was shifted toward the donor profile following FMT. Imputed growth rates showed that the resulting growth pattern was a conglomerate of donor and recipient activity. Thirty-four functional subclasses showed distinct differences between baseline samples and donors, most of which were shifted toward a donor-like profile after FMT. All of these changes were less pronounced in the No effect group. We conclude that FMT induces long-term changes in gut microbiota, and these changes mirror the clinical effect of the treatment. The study was registered in ClinicalTrials.gov (NCT02154867).

Ribosome-Mediated Attenuation of vga(A) Expression Is Shaped by the Antibiotic Resistance Specificity of Vga(A) Protein Variants.

Vga(A) protein variants confer different levels of resistance to lincosamides, streptogramin A, and pleuromutilins (LSAP) by displacing antibiotics from the ribosome. Here, we show that expression of vga(A) variants from Staphylococcus haemolyticus is regulated by cis-regulatory RNA in response to the LSAP antibiotics by the mechanism of ribosome-mediated attenuation. The specificity of induction depends on Vga(A)-mediated resistance rather than on the sequence of the riboregulator. Fine tuning between Vga(A) activity and its expression in response to the antibiotics may contribute to the selection of more potent Vga(A) variants because newly acquired mutation can be immediately phenotypically manifested.

Identification of surface proteins in a clinical Staphylococcus haemolyticus isolate by bacterial surface shaving.

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.

Comparative Genomic Analysis of Staphylococcus haemolyticus Reveals Key to Hospital Adaptation and Pathogenicity.

Staphylococcus haemolyticus is a skin commensal gaining increased attention as an emerging pathogen of nosocomial infections. However, knowledge about the transition from a commensal to an invasive lifestyle remains sparse and there is a paucity of studies comparing pathogenicity traits between commensal and clinical isolates. In this study, we used a pan-genomic approach to identify factors important for infection and hospital adaptation by exploring the genomic variability of 123 clinical isolates and 46 commensal S. haemolyticus isolates. Phylogenetic reconstruction grouped the 169 isolates into six clades with a distinct distribution of clinical and commensal isolates in the different clades. Phenotypically, multi-drug antibiotic resistance was detected in 108/123 (88%) of the clinical isolates and 5/46 (11%) of the commensal isolates (p < 0.05). In the clinical isolates, we commonly identified a homolog of the serine-rich repeat glycoproteins sraP. Additionally, three novel capsular polysaccharide operons were detected, with a potential role in S. haemolyticus virulence. Clinical S. haemolyticus isolates showed specific signatures associated with successful hospital adaption. Biofilm forming S. haemolyticus isolates that are resistant to oxacillin (mecA) and aminoglycosides (aacA-aphD) are most likely invasive isolates whereas absence of these traits strongly indicates a commensal isolate. We conclude that our data show a clear segregation of isolates of commensal origin, and specific genetic signatures distinguishing the clinical isolates from the commensal isolates. The widespread use of antimicrobial agents has probably promoted the development of successful hospital adapted clones of S. haemolyticus clones through acquisition of mobile genetic elements or beneficial point mutations and rearrangements in surface associated genes.

The synthetic antimicrobial peptide LTX21 induces inflammatory responses in a human whole blood model and a murine peritoneum model.

The global spread of antimicrobial resistance and the increasing number of immune-compromised patients are major challenges in modern medicine. Targeting bacterial virulence or the human host immune system to increase host defence are important strategies in the search for novel antimicrobial drugs. We investigated the inflammatory response of the synthetic short antimicrobial peptide LTX21 in two model systems: a human whole blood ex vivo model and a murine in vivo peritoneum model - both reflecting early innate immune response. In the whole blood model, LTX21 increased the secretion of a range of different cytokines, decreased the level of tumour necrosis factor (TNF) and activated the complement system. In a haemolysis assay, we found 2.5% haemolysis at a LTX21 concentration of 500 mg/L. In the murine model, increased influx of white blood cells (WBCs) and polymorphonuclear neutrophils (PMNs) in the murine peritoneal cavity was observed after treatment with LTX21. In addition, LTX21 increased monocyte chemoattractant protein-1 (MCP-1). In conclusion, LTX21 affected the inflammatory response; the increase in cytokine secretion, complement activation and WBC influx indicates an activated inflammatory response. The present results indicate the impact of LTX21 on the host-pathogen interplay. Whether this will also affect the course of infection has to be investigated.

Proteome profiling of secreted and membrane vesicle associated proteins of an invasive and a commensal Staphylococcus haemolyticus isolate.

Bacterial membrane vesicles (MVs) mediate bacterial virulence by enabling secretion and long distance delivery of bacterial effector molecules. Staphylococcus haemolyticus has now been demonstrated to produce membrane vesicles (MVs). The protein content of S. haemolyticus MVs was identified by Mass spectrometry and compared to proteins identified in the total secretome. This information is presented in this data article. Further background and interpretation of the data can be found in the article: Comparative exoproteome profiling of an invasive and a commensal S. haemolyticus isolate (Cavanagh et al., in press). Data are available via Proteome Xchange with identifier PXD010389.

Comparative exoproteome profiling of an invasive and a commensal Staphylococcus haemolyticus isolate.

Staphylococcus haemolyticus is a skin commensal emerging as an opportunistic pathogen. Nosocomial isolates of S. haemolyticus are the most antibiotic resistant members of the coagulase negative staphylococci (CoNS), but information about other S. haemolyticus virulence factors is scarce. Bacterial membrane vesicles (MVs) are one mediator of virulence by enabling secretion and long distance delivery of bacterial effector molecules while protecting the cargo from proteolytic degradation from the environment. We wanted to determine if the MV protein cargo of S. haemolyticus is strain specific and enriched in certain MV associated proteins compared to the totalsecretome. The present study shows that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. The MV cargo of both strains was enriched in proteins involved in adhesion and acquisition of iron. The MV cargo of the clinical strain was further enriched in antimicrobial resistance proteins. Data are available via ProteomeXchange with identifier PXD010389. BIOLOGICAL SIGNIFICANCE: Clinical isolates of Staphylococcus haemolyticus are usually multidrug resistant, their main virulence factor is formation of biofilms, both factors leading to infections that are difficult to treat. We show that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. Identification of staphylococcal membrane vesicles can potentially be used in novel approaches to combat staphylococcal infections, such as development of vaccines.

Fluorescence assay to predict activity of the glycopeptide antibiotics.

Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.

Effects of Probiotic Supplementation on the Gut Microbiota and Antibiotic Resistome Development in Preterm Infants.

Objectives: In 2014 probiotic supplementation (Lactobacillus acidophilus and Bifidobacterium longum subspecies infantis; InfloranⓇ) was introduced as standard of care to prevent necrotizing enterocolitis (NEC) in extremely preterm infants in Norway. We aimed to evaluate the influence of probiotics and antibiotic therapy on the developing gut microbiota and antibiotic resistome in extremely preterm infants, and to compare with very preterm infants and term infants not given probiotics. Study design: A prospective, observational multicenter study in six tertiary-care neonatal units. We enrolled 76 infants; 31 probiotic-supplemented extremely preterm infants <28 weeks gestation, 35 very preterm infants 28-31 weeks gestation not given probiotics and 10 healthy full-term control infants. Taxonomic composition and collection of antibiotic resistance genes (resistome) in fecal samples, collected at 7 and 28 days and 4 months age, were analyzed using shotgun-metagenome sequencing. Results: Median (IQR) birth weight was 835 (680-945) g and 1,290 (1,150-1,445) g in preterm infants exposed and not exposed to probiotics, respectively. Two extremely preterm infants receiving probiotic developed NEC requiring surgery. At 7 days of age we found higher median relative abundance of Bifidobacterium in probiotic supplemented infants (64.7%) compared to non-supplemented preterm infants (0.0%) and term control infants (43.9%). Lactobacillus was only detected in small amounts in all groups, but the relative abundance increased up to 4 months. Extremely preterm infants receiving probiotics had also much higher antibiotic exposure, still overall microbial diversity and resistome was not different than in more mature infants at 4 weeks and 4 months. Conclusion: Probiotic supplementation may induce colonization resistance and alleviate harmful effects of antibiotics on the gut microbiota and antibiotic resistome. Clinical Trial Registration: Clinicaltrials.gov: NCT02197468. https://clinicaltrials.gov/ct2/show/NCT02197468.

Faecal microbiota transplantation versus placebo for moderate-to-severe irritable bowel syndrome: a double-blind, randomised, placebo-controlled, parallel-group, single-centre trial.

BACKGROUND: Irritable bowel syndrome (IBS) is a common condition characterised by abdominal pain, bloating, and poor quality of life. IBS might be caused by a gut dysbiosis. We aimed to compare faecal microbiota transplantation (FMT) with placebo in patients with IBS. METHODS: In this double-blind, randomised, placebo-controlled, parallel-group, single-centre study, we enrolled patients with IBS with diarrhoea or with diarrhoea and constipation (excluding dominating constipation) defined by the ROME III criteria, scored as moderate to severe according to the IBS severity scoring system (IBS-SSS; a score of ≥175). Eligible participants were aged 18-75 years and were recruited locally by general practitioners in northern Norway. We randomly assigned participants (2:1) in blocks of six to active or placebo FMT. Personnel not involved in the clinical performance of the trial generated the randomisation sequence using a randomisation website. Non-study personnel performed the final allocation and standardised the active and placebo transplants to make them identical in appearance and temperature. The faeces were freshly processed, and were used the same day (fresh transplant) or were stored in a freezer for later use (frozen transplant); participants' own faeces served as placebo. A dose of 8 mg loperamide was administered orally 2 h before endoscopy to retain the transplant. The transplant (50-80 g of faeces mixed with 200 mL of isotonic saline and 50 mL of 85% glycerol) was administered by a colonoscope to the caecum. The primary endpoint was symptom relief of more than 75 points assessed by IBS-SSS, 3 months after FMT. The primary analysis was done in the modified intention-to-treat population, excluding participants who did not undergo treatment or who were diagnosed with any other disease by pinch biopsies obtained during the treatment procedure. For the safety analysis, only participants who did not undergo treatment were excluded. The study is registered with ClinicalTrials.gov, number NCT02154867. The trial has been extended with an open-labelled study treating the placebo group with frozen FMT for further exploratory studies. FINDINGS: Between Jan 1, and Oct 30, 2015, we recruited 90 participants and randomly assigned them to active treatment (n=60) or placebo (n=30). Three participants did not undergo FMT and four were excluded after diagnosis of microscopic colitis, leaving 83 for final modified intention-to-treat analysis (55 in the active treatment group and 28 in the placebo group). 36 (65%) of 55 participants receiving active treatment versus 12 (43%) of 28 receiving the placebo showed response at 3 months (p=0·049). One participant had transient nausea and vertigo (active group) and was observed at the hospital for a few hours after the procedure. Two participants had soiling of transplant on their way home from treatment (one in each group) and three experienced self-limiting intermittent abdominal pain (one in the active group and two in the placebo group). No serious adverse events could be attributed to FMT. INTERPRETATION: FMT induced significant symptom relief in patients with IBS. However, larger multicentre studies are needed to confirm the results. FUNDING: HelseNord and the Norwegian Centre of Rural Medicine, University of Tromsø.