Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and well-being1,2. Intestinal colonisation begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla2. Culture independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases1. However, due to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown3. Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole genome sequencing, phylogenetic analysis and computational modeling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. Applying this approach to healthy individuals, we isolated 137 bacterial species from characterised and candidate novel families, genera and species that were archived as pure cultures. Whole genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50-60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialised for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a significant proportion of oxygen-sensitive intestinal bacteria can transmit between individuals, impacting microbiota heritability.

Understanding gut microbiome functions requires cultivated bacteria for experimental validation and reference bacterial genome sequences to interpret metagenome datasets and guide functional analyses. We present the Human Gastrointestinal Bacteria Culture Collection (HBC), a comprehensive set of 737 whole-genome-sequenced bacterial isolates, representing 273 species (105 novel species) from 31 families found in the human gastrointestinal microbiota. The HBC increases the number of bacterial genomes derived from human gastrointestinal microbiota by 37%. The resulting global Human Gastrointestinal Bacteria Genome Collection (HGG) classifies 83% of genera by abundance across 13,490 shotgun-sequenced metagenomic samples, improves taxonomic classification by 61% compared to the Human Microbiome Project (HMP) genome collection and achieves subspecies-level classification for almost 50% of sequences. The improved resource of gastrointestinal bacterial reference sequences circumvents dependence on de novo assembly of metagenomes and enables accurate and cost-effective shotgun metagenomic analyses of human gastrointestinal microbiota.

Interactions of anaerobic gut bacteria, such as Clostridium difficile , with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei , and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei . Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host–anaerobe and pathogen–commensal interaction studies in vitro .

Novel antimicrobials are urgently needed to combat drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Studying plants and other natural materials used in historical infection remedies may enable further discoveries to help fill the antibiotic discovery gap. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, known as 'Bald's eyesalve', and showed it had promising antibacterial activity. In this current paper, we have found this bactericidal activity extends to a range of Gram-negative and Grampositive wound pathogens in planktonic culture and, crucially, that this activity is maintained against Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes in a soft-tissue wound biofilm model. While the presence of garlic in the mixture can explain the activity against planktonic cultures, garlic has no activity against biofilms. We have found the potent anti-biofilm activity of Bald's eyesalve cannot be attributed to a single ingredient and requires the combination of all ingredients to achieve full activity. Our work highlights the need to explore not only single compounds but also mixtures of natural products for treating biofilm infections and underlines the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline. Widespread multidrug resistance of once-susceptible pathogens, combined with a lack of success in developing novel antimicrobials, has resulted in a looming crisis. Antimicrobial resistance leads to problematic infections, threatens the success of routine surgery and cancer treatments 1 , and is estimated to kill 10 million people per year by 2050 2,3. One particularly troublesome area is biofilm-associated infection. Biofilm infections are estimated to cost the UK's National Health Service over a billion pounds every year, with this cost only set to increase 4,5. Biofilms are communities of bacteria that produce a protective extracellular matrix and are especially persistent 6. Biofilm eradication often requires 100-1,000 times higher antibiotic concentrations to achieve clearance than the same bacteria growing planktonically (as individual free-floating cells) 7. In vivo, biofilms may essentially be completely impervious to antibiotic treatment 8. The path to overcoming biofilm infections requires a multifaceted response, including the discovery and clinical deployment of novel antimicrobials. This search for novel candidates must be directed towards those pathogenic bacteria with the highest health and economic impact (e.g. ESKAPE group 9,10) and will be expedited if early discovery work on leads takes into account the inherent difficulties of treating biofilm-associated infections 6,11 .

22The rise in antimicrobial resistance has prompted the development of alternatives, 23 such as plant-derived compounds, to combat bacterial infections. Bald's eyesalve, a 24 remedy used in the Early Medieval period, has previously been shown to have efficacy 25 against Staphylococcus aureus grown in an in vitro model of soft tissue infection. This 26 remedy also had bactericidal activity against methicillin-resistant S. aureus (MRSA) in 27 a chronic mouse wound. However, the safety profile of Bald's eyesalve has not yet 28 been demonstrated, and this is vital before testing in humans. Here, we determined 29 the safety potential of Bald's eyesalve using in vitro, ex vivo, and in vivo models 30 representative of skin or eye infections. We also confirmed that Bald's eyesalve is 31 active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of 32 cytotoxicity were observed in eyesalve-treated cell lines representative of skin and 33 immune cells. Results from a bovine corneal opacity and permeability test 34 demonstrated slight irritation to the cornea that resolved within 10 minutes. The slug 35 mucosal irritation assay revealed that a low level of mucus was secreted by slugs 36 exposed to eyesalve, indicating mild mucosal irritation. We obtained promising results 37 from mouse wound closure experiments; no visible signs of irritation or inflammation 38 were observed. Our results suggest that Bald's eyesalve could be tested further on 39 human volunteers to assess safety for topical application against bacterial infections. 40 41 Importance: Alternative treatment for bacterial infections are needed to combat the 42 ever increasing repertoire of bacteria resistant to antibiotics. A medieval plant-based 43 remedy, Bald's eyesalve, shows promise as a substitute for the treatment of these 44infections. For any substance to be effective in the treatment of bacterial infections in 45 humans, it is important to consider the safety profile. This is a key consideration in 46 3 order to have the necessary regulatory approval. We demonstrate the safety profile of 47 Bald's eyesalve using a variety of models, including whole-organ and whole-animal 48 models. Our results show that Bald's eyesalve is mildly toxic to cultured human cells, 49 but potentially suitable for patch tests on healthy human volunteers to assess safety 50 for later clinical trials. Our work has the potential to transform the management of 51 diseases caused by bacterial infections, such as diabetic foot ulcers, through topical 52 application of a natural product cocktail based on Bald's eyesalve. 53 54

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The rise in antimicrobial resistance has prompted the development of alternatives to combat bacterial infections. Bald’s eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus in in vitro and in vivo models of chronic wounds. However, the safety profile of Bald’s eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald’s eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald’s eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 min. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs indicating moderate mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald’s eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.

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