Stephen Lloyd Michell

The tier 1 bioterrorism agent Burkholderia pseudomallei causes melioidosis, a tropical disease with fatality rates that can exceed 40% despite antibiotic therapy. Antibiotic failure is likely to be, at least in part, due to biofilm-dwelling B. pseudomallei, and therefore, an improved understanding of how this pathogen regulates biofilm formation could reveal new opportunities for clinical intervention. The antimicrobial radical nitric oxide (NO) plays a key role in host immune defenses against bacteria, and the ability of B. pseudomallei to sense and mitigate NO toxicity is vital for establishing infection. NO-sensing proteins (NosPs), which have recently emerged as key regulators of biofilm formation in many bacterial species, use a FIST domain to sense NO via a bound heme. We hypothesized that the NosP homolog in B. pseudomallei would regulate biofilm formation and mediate NO-protective responses. We used [γ- ³² P]ATP autophosphorylation assays to show that NosP of B. pseudomallei controls the autophosphorylation rate of an associated histidine kinase protein (NosK) in an NO-dependent manner. NosK was found to phosphorylate a response regulator protein (NosR) with an HD-GYP output domain, which is associated with c-di-GMP signaling, therefore implicating NosP in modulating c-di-GMP-regulated phenotypes. Unmarked, in-frame deletion of either nosP or nosK caused significant changes in B. pseudomallei biofilm formation and increased sensitivity to nitrosative stress, in addition to affecting other virulence traits such as growth and swimming motility. These results indicate that NosP and NosK signaling control a range of infection-relevant phenotypes and may serve as targets for novel therapeutic intervention. IMPORTANCE: Melioidosis is an emerging, potentially life-threatening infection caused by the bacterium Burkholderia pseudomallei , killing ~89,000 people per year globally. Antibiotic therapy fails in ~10%–40% of cases, and hence, an improved understanding of the molecular mechanisms that control B. pseudomallei virulence could reveal new approaches for improving melioidosis treatment. Biofilm formation and resistance to the antimicrobial radical NO are virulence traits that help bacteria establish infections. Here, we show that two proteins in B. pseudomallei , NosP and NosK, work together to detect NO and regulate a suite of virulence traits, including NO resistance, biofilm formation, growth, and swimming motility. This work, therefore, improves our understanding of the molecular mechanisms that control infection-related phenotypes in B. pseudomallei .

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Antimicrobial resistance poses one of the greatest threats to global health and there is an urgent need for new therapeutic options. Phages are viruses that infect and kill bacteria and phage therapy could provide a valuable tool for the treatment of multidrug-resistant infections. In this study, water samples collected by citizen scientists as part of the Citizen Phage Library (CPL) project, and wastewater samples from the Environment Agency yielded phages with activity against clinical strains Klebsiella pneumoniae BPRG1484 and Enterobacter cloacae BPRG1482. A total of 169 and 163 phages were found for K. pneumoniae and E. cloacae, respectively, within four days of receiving the strains. A third strain (Escherichia coli BPRG1486) demonstrated cross-reactivity with 42 E. coli phages already held in the CPL collection. Seed lots were prepared for four K. pneumoniae phages and a cocktail combining these phages was found to reduce melanisation in a Galleria mellonella infection model. The resources and protocols utilised by the Citizen Phage Library enabled the rapid isolation and characterisation of phages targeted against multiple strains. In the future, within a clearly defined regulatory framework, phage therapy could be made available on a named-patient basis within the UK.

Introduction. Diabetic foot infection (DFI) is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria. Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro. Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd., UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs. Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth. Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram-positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis , Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced. Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

Summary: With the rapid increase of aquaculture contributing to sustainable food security, comes the need to better understand seafood associated diseases. One of the major aquatic bacterial genera responsible for human infections from seafood is Vibrio , especially from oysters. Currently, in vivo study of bacterial interactions within oysters is limited by the inability to promote high‐level uptake of bacteria by oysters. This study has therefore evolved current natural marine snow protocols to generate ‘artificial’ marine snow, into which bacteria can be incorporated to facilitate extensive uptake by oysters. This presents an adaptable model for bacterial study within filter‐feeding shellfish. Using this model, we demonstrate for the first time the antibacterial activity of Vibrio vulnificus Type 6 secretion systems in vivo , revealing an important role for the T6SS in V . vulnificus ecology.

Vibrio vulnificus is a bacterium responsible for severe gastroenteritis, sepsis and wound infections. Gastroenteritis and sepsis are commonly associated with the consumption of raw oysters, whereas wound infection is often associated with the handling of contaminated fish. Although classical virulence factors of this emerging pathogen are well characterised, there remains a paucity of knowledge regarding the general biology of this species. To investigate the presence of previously unreported virulence factors, we applied whole genome sequencing to a panel of ten V. vulnificus strains with varying virulence potentials. This identified two novel type 6 secretion systems (T6SSs), systems that are known to have a role in bacterial virulence and population dynamics. By utilising a range of molecular techniques and assays we have demonstrated the functionality of one of these T6SSs. Furthermore, we have shown that this system is subject to thermoregulation and is negatively regulated by increasing salinity concentrations. This secretion system was also shown to be involved in the killing of V. vulnificus strains that did not possess this system and a model is proposed as to how this interaction may contribute to population dynamics within V. vulnificus strains. In addition to this intra-species killing, this system also contributes to the killing of inter bacterial species and may have a role in the general composition of Vibrio species in the environment.

Background: Clostridium difficile infection poses a significant healthcare burden. However, the derivation of a simple, evidence based prediction rule to assist patient management has not yet been described. This study aimed to identify such a prediction rule to stratify hospital inpatients according to risk of all-cause mortality, at initial diagnosis of infection. Method: Univariate, multivariate and decision tree procedures were used to deduce a prediction rule from over 186 variables; retrospectively collated from clinical data for 213 patients. The resulting prediction rule was validated on independent data from a cohort of 158 patients described by Bhangu et al. (Colorectal Disease, 12(3):241-246, 2010). Results: Serum albumin levels (g/L) (P = 0.001), respiratory rate (resps /min) (P = 0.002), C-reactive protein (mg/L) (P = 0.034) and white cell count (mcL) (P = 0.049) were predictors of all-cause mortality. Threshold levels of serum albumin ≤ 24.5 g/L, C- reactive protein >228 mg/L, respiratory rate >17 resps/min and white cell count >12 × 10³ mcL were associated with an increased risk of all-cause mortality. A simple four variable prediction rule was devised based on these threshold levels and when tested on the initial data, yield an area under the curve score of 0.754 (P < 0.001) using receiver operating characteristics. The prediction rule was then evaluated using independent data, and yield an area under the curve score of 0.653 (P = 0.001). Conclusions: Four easily measurable clinical variables can be used to assess the risk of mortality of patients with Clostridium difficile infection and remains robust with respect to independent data.

Bacterial lipoproteins are a set of membrane proteins with many different functions. Due to this broad-ranging functionality, these proteins have a considerable significance in many phenomena, from cellular physiology through cell division and virulence. Here we give a general overview of lipoprotein biogenesis and highlight examples of the roles of lipoproteins in bacterial disease caused by a selection of medically relevant Gram-negative and Gram-positive pathogens:Mycobacterium tuberculosis,Streptococcus pneumoniae,Borrelia burgdorferi, andNeisseria meningitidis. Lipoproteins have been shown to play key roles in adhesion to host cells, modulation of inflammatory processes, and translocation of virulence factors into host cells. As such, a number of lipoproteins have been shown to be potential vaccines. This review provides a summary of some of the reported roles of lipoproteins and of how this knowledge has been exploited in some cases for the generation of novel countermeasures to bacterial diseases.