Wounds can commonly become infected with polymicrobial biofilms containing bacterial and fungal microorganisms. Microbial colonization of the wound can interfere with sufficient healing and repair, leading to high rates of chronicity in certain individuals, which can have a huge socioeconomic burden worldwide. One route for alleviating biofilm formation in chronic wounds is sufficient treatment of the infected area with topical wound washes and ointments. Thus, the primary aim here was to create a complex in vitro biofilm model containing a range of microorganisms commonly isolated from the infected wound milieu. These polymicrobial biofilms were treated with three conventional anti-biofilm wound washes, chlorhexidine (CHX), povidone-iodine (PVP-I), and hydrogen peroxide (H2O2), and efficacy against the microorganisms assessed using live/dead qPCR. All treatments reduced the viability of the biofilms, although H2O2 was found to be the most effective treatment modality. These biofilms were then co-cultured with 3D skin epidermis to assess the inflammatory profile within the tissue. A detailed transcriptional and proteomic profile of the epidermis was gathered following biofilm stimulation. At the transcriptional level, all treatments reduced the expression of inflammatory markers back to baseline (untreated tissue controls). Olink technology revealed a unique proteomic response in the tissue following stimulation with untreated and CHX-treated biofilms. This highlights treatment choice for clinicians could be dictated by how the tissue responds to such biofilm treatment, and not merely how effective the treatment is in killing the biofilm.

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: the isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

The investigation of the microbial populations of the human body, known as the microbiome, has led to a revolutionary field of science, and understanding of its impacts on human development and health. The majority of microbiome research to date has focussed on bacteria and other kingdoms of life, such as fungi. Trailing behind these is the interrogation of the gut viruses, specifically the phageome. Bacteriophages, viruses that infect bacterial hosts, are known to dictate the dynamics and diversity of bacterial populations in a number of ecosystems. However, the phageome of the human gut, while of apparent importance, remains an area of many unknowns. In this paper we discuss the role of bacteriophages within the human gut microbiome. We examine the methods used to study bacteriophage populations, how this evolved over time and what we now understand about the phageome. We review the phageome development in infancy, and factors that may influence phage populations in adult life. The role and action of the phageome is then discussed at both a biological-level, and in the broader context of human health and disease.

Klebsiella infections, including catheter associated urinary tract infections, are a considerable burden on health care systems. This is due to their difficulty to treat, caused by antimicrobial resistance and their ability to form biofilms. In this study, we investigated the use of a Klebsiella phage cocktail to reduce biofilm viability. We used two methodologies to investigate this, a standard 96-well plate assay and a more complicated Foley catheter-based model. The phage cocktail was used alone and in combination with clinically relevant antibiotic treatments. Viability was measured by both a resazurin based stain and colony forming unit counts, of cells sloughed off from the biofilm. We showed that phage infection dynamics and host survival vary significantly in different standard laboratory media, presumably due to the expression of different surface receptors and capsule composition by the bacteria effecting phage binding. This underscores the importance of a realistic model for developing phage therapy. We demonstrate that bacteriophage-based treatments are a viable option for preventing Klebsiella colonisation and biofilm formation on urinary catheters. Phage cocktails were able to significantly reduce the amount of biofilm that formed when they were present during early biofilm formation. The phages used in this study were unable to significantly reduce a pre-formed mature biofilm, despite encoding depolymerases. Phages applied together with antimicrobial treatments, showed synergistic interactions, in some cases the combined treatment was much more effective than antimicrobial treatments alone. We show that phage cocktails have the potential to prevent Klebsiella biofilms in catheters, if used early or as a preventative treatment and will work well alongside standard antibiotics in the treatment of catheter-associated urinary tract infections (CAUTI).

BACKGROUND: Tuberculosis remains a global health challenge, with early diagnosis key to its reduction. Face-mask sampling detects exhaled Mycobacterium tuberculosis. We aimed to investigate bacillary output from patients with pulmonary tuberculosis and to assess the potential of face-mask sampling as a diagnostic method in active case-finding. METHODS: We did a 24-h longitudinal study in patients from three hospitals in Pretoria, South Africa, with microbiologically confirmed pulmonary tuberculosis. Patients underwent 1 h of face-mask sampling eight times over a 24-h period, with contemporaneous sputum sampling. M tuberculosis was detected by quantitative PCR. We also did an active case-finding pilot study in inhabitants of an informal settlement near Pretoria. We enrolled individuals with symptoms of tuberculosis on the WHO screening questionnaire. Participants provided sputum and face-mask samples that were tested with the molecular assay Xpert MTB/RIF Ultra. Sputum-negative and face-mask-positive individuals were followed up prospectively for 20 weeks by bronchoscopy, PET-CT, and further sputum analysis to validate the diagnosis. FINDINGS: Between Sept 22, 2015, and Dec 3, 2015, 78 patients with pulmonary tuberculosis were screened for the longitudinal study, of whom 24 completed the study (20 had HIV co-infection). M tuberculosis was detected in 166 (86%) of 192 face-mask samples and 38 (21%) of 184 assessable sputum samples obtained over a 24-h period. Exhaled M tuberculosis output showed no diurnal pattern and did not associate with cough frequency, sputum bacillary content, or chest radiographic disease severity. On May 16, 2018, 45 individuals were screened for the prospective active case-finding pilot study, of whom 20 had tuberculosis symptoms and were willing to take part. Eight participants were diagnosed prospectively with pulmonary tuberculosis, of whom six were exclusively face-mask positive at screening. Four of these participants (three of whom were HIV-positive) had normal findings on chest radiography but had treatment-responsive early tuberculosis-compatible lesions on PET-CT scans, with Xpert-positive sputum samples after 6 weeks. INTERPRETATION: Face-mask sampling offers a highly efficient and non-invasive method for detecting exhaled M tuberculosis, informing the presence of active infection both with greater consistency and at an earlier disease stage than with sputum samples. The approach shows potential for diagnosis and screening, particularly in difficult-to-reach communities. FUNDING: Wellcome Trust, CARA (Council for At-Risk Academics), University of Leicester, the UK Medical Research Council, and the National Institute for Health Research. VIDEO ABSTRACT.

The rumen contains a multi-kingdom, commensal microbiome, including protozoa, bacteria, archaea, fungi and viruses, which enables ruminant herbivores to ferment and utilize plant feedstuffs that would be otherwise indigestible. Within the rumen, virus populations are diverse and highly abundant, often out-numbering the microbial populations that they both predate on and co-exist with. To date the research effort devoted to understanding rumen-associated viral populations has been considerably less than that given to the other microbial populations, yet their contribution to maintaining microbial population balance, intra-ruminal microbial lysis, fiber breakdown, nutrient cycling and genetic transfer may be highly significant. This review follows the technological advances which have contributed to our current understanding of rumen viruses and drawing on knowledge from other environmental and animal-associated microbiomes, describes the known and potential roles and impacts viruses have on rumen function and speculates on the future directions of rumen viral research.

The emerging pathogenic multidrug-resistant yeast Candida auris is an important source of healthcare-associated infections and of growing global clinical concern. The ability of this organism to survive on surfaces and withstand environmental stressors creates a challenge for eradicating it from hospitals. A panel of C. auris clinical isolates was evaluated on different surface environments against the standard disinfectant sodium hypochlorite and high-level disinfectant peracetic acid. C. auris was shown to selectively tolerate clinically relevant concentrations of sodium hypochlorite and peracetic acid in a surface-dependent manner, which may explain its ability to successfully persist within the hospital environment.

Candida auris has emerged as a significant clinical entity as it can cause outbreaks within the healthcare setting. A key feature of its nosocomial properties is that it can transfer between patients, yet little is known about the mechanisms behind this. A panel of C. auris clinical isolates were screened for their planktonic and sessile susceptibilities to skin disinfection challenge using povidone iodine, chlorhexidine and hydrogen peroxide. C. auris biofilms displayed increased tolerance to these strategies compared with planktonic cells. Additionally, analysis using a complex biofilm model demonstrated reduced susceptibility against clinically-relevant concentrations of chlorhexidine and hydrogen peroxide, with eradication achieved only using povidone iodine. Principal component analysis (PCA) also revealed distinct clustering of C. auris biofilms compared with C. albicans and C. glabrata biofilms, and directionality with respect to different treatments. These findings indicate differential responses of different Candida species with respect to antiseptic challenge against biofilms, with C. auris appearing to be more resilient as a complex community.

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections.

Polymicrobial inter-kingdom biofilm infections represent a clinical management conundrum. The presence of co-isolation of bacteria and fungi complicates the ability to routinely administer single antimicrobial regimens, and synergy between the microorganisms influences infection severity. We therefore investigated the nosocomial pathogens Staphylococcus aureus and Candida albicans with respect to antimicrobial intervention. We characterized the interaction using biofilm assays and evaluated the effect of miconazole treatment using in vitro and in vivo assays. Finally, we assessed the impact of biofilm extracellular matrix (ECM) on these interactions. Data indicated that the C. albicans mycofilms supported adhesion and colonization by S. aureus through close interactions with hyphal elements, significantly increasing S. aureus biofilm formation throughout biofilm maturation. Miconazole sensitivity was shown to be reduced in both mono- and dual-species biofilms compared to planktonic cells. Within a three-dimensional biofilm model sensitivity was also hindered. Galleria mellonella survival analysis showed both enhanced pathogenicity of the dual-species infection, which was concomitantly desensitized to miconazole treatment. Analysis of the ECM revealed the importance of extracellular DNA, which supported the adhesion of S. aureus and the development of the dual-species biofilm structures. Collectively, these data highlight the clinical importance of dual-species inter-kingdom biofilm infections, though also provides translational opportunities to manage them more effectively.

Diabetic foot ulcer treatment currently focuses on targeting bacterial biofilms, while dismissing fungi. To investigate this, we used an in vitro biofilm model containing bacteria and fungi, reflective of the wound environment, to test the impact of antimicrobials. Here we showed that while monotreatment approaches influenced biofilm composition, this had no discernible effect on overall quantity. Only by combining bacterium- and fungus-specific antibiotics were we able to decrease the biofilm bioburden, irrespective of composition.

Chronic diabetic foot ulcers are frequently colonised and infected by polymicrobial biofilms that ultimately prevent healing. This study aimed to create a novel in vitro inter-kingdom wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical wound treatments. Inter-kingdom triadic biofilms composed of Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be quantitatively greater in this model compared to a simple substratum when assessed by conventional culture, metabolic dye and live dead qPCR. These biofilms were both structurally complex and compositionally dynamic in response to topical therapy, so when treated with either chlorhexidine or povidone iodine, principal component analysis revealed that the 3-D cellulose model was minimally impacted compared to the simple substratum model. This study highlights the importance of biofilm substratum and inclusion of relevant polymicrobial and inter-kingdom components, as these impact penetration and efficacy of topical antiseptics.

BACKGROUND: the aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform. RESULTS: Conventional laboratory culture showed positive growth from only 55 % of the patients, whereas 16S AS was positive for 75 % of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU's). S. aureus was isolated in 72 % of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp. Anaerococcus spp. and Corynebacterium spp. with the addition of Staphylococcus spp. in new ulcers. The majority of OTU's residing in both new and recurrent ulcers (over 67 %) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers). CONCLUSIONS: the abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer "starts afresh" and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer.

It has been reported that targeted disruption of ampD I or mrcA causes β-lactamase hyperproduction in Stenotrophomonas maltophilia. We show here that β-lactamase-hyperproducing laboratory selected mutants and clinical isolates can have wild-type ampD I and mrcA genes, implicating mutation of at least one additional gene in this phenotype. The involvement of mutations at multiple loci in the activation of β-lactamase production in S. maltophilia reveals that there are significant deviations from the enterobacterial paradigm of AmpR-mediated control of β-lactamase induction. We do show, however, that S. maltophilia ampD I can complement a mutation in Escherichia coli ampD. This suggests that an anhydromuropeptide degradation product of peptidoglycan is used to activate AmpR in S. maltophilia, as is also the case in enteric bacteria.