Implications of past and present genetic connectivity for management of the saltwater crocodile (Crocodylus porosus).

Effective management of protected species requires information on appropriate evolutionary and geographic population boundaries and knowledge of how the physical environment and life-history traits combine to shape the population structure and connectivity. Saltwater crocodiles (Crocodylus porosus) are the largest and most widely distributed of living crocodilians, extending from Sri Lanka to Southeast Asia and down to northern Australia. Given the long-distance movement capabilities reported for C. porosus, management units are hypothesised to be highly connected by migration. However, the magnitude, scale, and consistency of connection across managed populations are not fully understood. Here we used an efficient genotyping method that combines DArTseq and sequence capture to survey ≈ 3000 high-quality genome-wide single nucleotide polymorphisms from 1176 C. porosus sampled across nearly the entire range of the species in Queensland, Australia. We investigated historical and present-day connectivity patterns using fixation and diversity indices coupled with clustering methods and the spatial distribution of kin pairs. We inferred kinship using forward simulation coupled with a kinship estimation method that is robust to unspecified population structure. The results demonstrated that the C. porosus population has substantial genetic structure with six broad populations correlated with geographical location. The rate of gene flow was highly correlated with spatial distance, with greater differentiation along the east coast compared to the west. Kinship analyses revealed evidence of reproductive philopatry and limited dispersal, with approximately 90% of reported first and second-degree relatives showing a pairwise distance of <50 km between sampling locations. Given the limited dispersal, lack of suitable habitat, low densities of crocodiles and the high proportion of immature animals in the population, future management and conservation interventions should be considered at regional and state-wide scales.

Penicillin-Binding Protein 1 (PBP1) of Staphylococcus aureus Has Multiple Essential Functions in Cell Division.

Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific ß-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate. IMPORTANCE Bacterial cell wall peptidoglycan is essential, and its synthesis is the target of clinically important antibiotics such as ß-lactams. ß-lactams target penicillin-binding proteins (PBPs) that assemble new peptidoglycan from its building blocks. The human pathogen Staphylococcus aureus only has two essential PBPs that can carry out all the functions necessary for growth and division. In the absence of the confounding antibiotic resistance-associated PBP PBP2A, PBP1 is required for cell division, and here, we have found that it has several essential functions, both as an enzyme and as a coordinator by binding to cell division proteins and to its peptidoglycan product, via its PASTA domains. This has led to a new model for cell division with PBP1 responsible for the synthesis of the characteristic architectural features of the septum.

SPaCE Swab: Point-of-Care Sensor for Simple and Rapid Detection of Acute Wound Infection.

Wound infection is commonly observed after surgery and trauma but is difficult to diagnose and poorly defined in terms of objective clinical parameters. The assumption that bacteria in a wound correlate with infection is false; all wounds contain microorganisms, but not all wounds are clinically infected. This makes it difficult for clinicians to determine true wound infection, especially in wounds with pathogenic biofilms. If an infection is not properly treated, pathogenic virulence factors, such as rhamnolipids from Pseudomonas aeruginosa, can modulate the host immune response and cause tissue breakdown. Life-threatening sepsis can result if the organisms penetrate deep into host tissue. This communication describes the sensor development for five important clinical microbial pathogens commonly found in wounds: Staphylococcus aureus, P. aeruginosa, Candida albicans/auris, and Enterococcus faecalis (the SPaCE pathogens). The sensor contains liposomes encapsulating a self-quenched fluorescent dye. Toxins, expressed by SPaCE infecting pathogens in early-stage infected wounds, break down the liposomes, triggering dye release, thus changing the sensor color from yellow to green, an indication of infection. Five clinical species of bacteria and fungi, up to 20 strains each (totaling 83), were grown as early-stage biofilms in ex vivo porcine burn wounds. The biofilms were then swabbed, and the swab placed in the liposome suspension. The population density of selected pathogens in a porcine wound biofilm was quantified and correlated with colorimetric response. Over 88% of swabs switched the sensor on (107-108 CFU/swab). A pilot clinical study demonstrated a good correlation between sensor switch-on and early-stage wound infection.

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope.

Bacteria are surrounded by a complex cell envelope made up of one or two membranes supplemented with a layer of peptidoglycan (PG). The envelope is responsible for the protection of bacteria against lysis in their oft-unpredictable environments and it contributes to cell integrity, morphology, signaling, nutrient/small-molecule transport, and, in the case of pathogenic bacteria, host-pathogen interactions and virulence. The cell envelope requires considerable remodeling during cell division in order to produce genetically identical progeny. Several proteinaceous machines are responsible for the homeostasis of the cell envelope and their activities must be kept coordinated in order to ensure the remodeling of the envelope is temporally and spatially regulated correctly during multiple cycles of cell division and growth. This review aims to highlight the complexity of the components of the cell envelope, but focusses specifically on the molecular apparatuses involved in the synthesis of the PG wall, and the degree of cross talk necessary between the cell division and the cell wall remodeling machineries to coordinate PG remodeling during division. The current understanding of many of the proteins discussed here has relied on structural studies, and this review concentrates particularly on this structural work.

Protocol for the development of a core indicator set for reporting burn wound infection in trials: ICon-B study.

INTRODUCTION: Systematic reviews of high-quality randomised controlled trials are necessary to identify effective interventions to impact burn wound infection (BWI) outcomes. Evidence synthesis requires that BWI is reported in a consistent manner. Cochrane reviews investigating interventions for burns report that the indicators used to diagnose BWI are variable or not described, indicating a need to standardise reporting. BWI is complex and diagnosed by clinician judgement, informed by patient-reported symptoms, clinical signs, serum markers of inflammation and bacteria in the wound. Indicators for reporting BWI should be important for diagnosis, frequently observed in patients with BWI and assessed as part of routine healthcare. A minimum (core) set of indicators of BWI, reported consistently, will facilitate evidence synthesis and support clinical decision-making. AIMS: The Infection Consensus in Burns study aims to identify a core indicator set for reporting the diagnosis of BWI in research studies. METHODS: (1) Evidence review: a systematic review of indicators used in trials and observational studies reporting BWI outcomes to identify a long list of candidate indicators; (2) refinement of the long list into a smaller set of survey questions with an expert steering group; (3) a two-round Delphi survey with 100 multidisciplinary expert stakeholders, to achieve consensus on a short list of indicators; (4) a consensus meeting with expert stakeholders to agree on the BWI core indicator set. ETHICS AND DISSEMINATION: Participants will be recruited through professional bodies, such that ethical approval from the National Health Service (NHS) Health Research Authority (HRA) is not needed. The core indicator set will be disseminated through peer-reviewed publication, co-production with journal editors, research funders and professional bodies, and presentation at national conferences. PROSPERO REGISTRATION NUMBER: CRD42018096647.

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the d-galacturonic acid-α-1,2-l-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second d-galacturonic acid side chain (linked ß-1,3 to l-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/ß)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting d-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.

Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.

The SILKIE (Skin graftIng Low friKtIon Environment) study: a non-randomised proof-of-concept and feasibility study on the impact of low-friction nursing environment on skin grafting success rates in adult and paediatric burns.

OBJECTIVES: To evaluate the impact of low-friction (LF) bedding on graft loss in an acute burn care setting, and to examine the feasibility and costs of using LF bedding compared with standard care. DESIGN: Proof of concept before and after study with feasibility of delivering the intervention. SETTING: Three burns services within two UK hospital trusts. PARTICIPANTS: Inclusion criteria were patients older than 4 weeks, who received a skin graft after burn injury and were admitted overnight. The comparator cohort were eligible patients admitted in a 12-month period before the intervention. INTERVENTION: Introduction of LF sheets and pillowcases during a 15-month period. OUTCOME MEASURES: For proof of concept, the LF and comparator cohorts were compared in terms of number of regrafting operations (primary), percentage graft loss, hospital length of stay (LoS) and LoS cost (secondary). Feasibility outcomes were practicality and safety of using LF bedding. RESULTS: 131 patients were eligible for the LF cohort and 90 patients for the comparator cohort. Although the primary outcome of the proportion needing regrafting was halved in the LF cohort, the confidence interval (CI) crossed 1 (OR (95% CI): 0.56 (0.16 to 1.88)). Partial graft loss (any loss) was significantly reduced in the LF cohort (OR (95% CI): 0.27 (0.14, 0.51)). Inpatient LoS was no different between the two cohorts (difference in median days (95% CI): 0 (-2 to 1)), and the estimated difference in LoS cost was £-1139 (-4829 to 2551). Practical issues were easily resolved, and no safety incidents occurred while patients were nursed on LF bedding. CONCLUSIONS: LF bedding is safe to use in burned patients with skin grafts and we have shown proof of concept for the intervention. Further economic modelling is required to see if an appropriately powered randomised control trial would be worthwhile or if roll out across the National Health Service is justified. TRIAL REGISTRATION NUMBER: ISRCTN82599687.

A retrospective study: Can we differentiate between repeat self-inflicted burn patients and those who commit a self-inflicted burn as an individual occurrence?

Self-inflicted burns (SIBs) are a significant cause of burns morbidity worldwide. A sub-group of SIB patients demonstrate recurrent SIB behaviour causing repeated morbidity and an increasing strain on hospital resources. The ability to predict which patients are likely to demonstrate repeat behaviour will allow for more targeted interventions in this group. This study aimed to identify the factors that differentiate patients who repeat SIB from those who commit SIB as an individual occurrence. A three-step approach was adopted: (1) initial data collection through the locally held records of the International Burns Injury Database (iBID); (2) follow-up data of SIB patient information were extracted from patient notes and (3) statistical data analysis. Seventy-five records remained for analysis. Seventeen patients were identified as going on to commit SIB more than once and so classified as 'repeat SIB' patients (22.7%). Repeat SIB patients appeared to be more commonly female and Caucasian with a mean total body surface area of less than half the individual occurrence group. The repeat SIB group was also more likely to commit burns to their limbs and demonstrate previous non-burn deliberate self-harm behaviour. 'Cold' burns were also committed more commonly in the repeat SIB group. This paper describes the largest sample of repeat SIB patients in the literature. It appears that repeat SIB patients have a set of differentiating factors that, when combined, allow for some element of prediction of these behaviours.

Comparing outcomes of sheet grafting with 1:1 mesh grafting in patients with thermal burns: a randomized trial.

BACKGROUND: In many units, the standard mesh ratio is 1.5:1, but in our unit we have a 1:1 mesher, which does not expand the skin but provides regular fenestrations. There is some evidence that the unexpanded 1.5:1 meshed graft compares favourably with sheet grafts from a cosmetic perspective whilst reducing the risk of graft failure secondary to a subgraft haematoma, but none comparing the 1:1 meshed graft with the sheet graft. We conducted a randomized trial to compare surgical outcomes in unfenestrated sheet grafts with 1:1 meshed grafts. METHODS: All patients aged ≥16 years undergoing skin grafts with either a sheet or a 1:1 mesh for burn reconstruction were included. Patients on steroids, those with conditions that impair healing, and burns >20% were excluded. Patients were randomized into the sheet grafting or mesh graft using a computer-generated allocation system. The mean percentage of graft loss was assessed by a Visitrak overlay system. At 3-4 months, 7-8 months and at 1 year, photos were taken for scar assessment using the Vancouver Scar Score (VSS). RESULTS: Out of 72 patients, 48 patients (24 sheet vs. 24 mesh) completed the trial at 12 months. The mean age was 58 years (range 21-90). There was no total loss of graft in either group. The mean percentage of graft loss due to haematoma formation was higher in the sheet graft group (10%) compared to the 1:1 mesh group (6%) (P<0.062). The VSS score was 5 in both groups at 12 months. There was no significant difference in scar quality between the treatment groups. CONCLUSION: These results show that the 1:1 mesh graft is superior to the sheet graft with regard to graft loss, although this result is not statistically significant. There are comparable findings in terms of cosmetic perspective at 12 months post-operatively in both arms of the trial.

Progression of wound pH during the course of healing in burns.

The aim of this study was to measure the pH on the wound surface of 30 burn patients and test the hypothesis that wound surface pH is correlated to healing time and burn depth. Inclusion criteria were any adult outpatient with burn injury. Patient age was 17 to 75 years (mean, 44), burn depth ranged from superficial to full thickness with a TBSA of 0.4 to 4%. Cause of burn included scalds, flame burn, and contact burns. On admission, and at each dressing change, the pH on the wound surface was measured. The pH in both healing and nonhealing wounds was found to decrease with each dressing change. At the second dressing change, wounds that went on to heal were found to have a significantly lower pH of 7.32 in comparison with pH 7.73 in wounds that failed to heal and therefore required subsequent grafting (P = .004). Wound pH was also correlated to depth at the second dressing change (superficial = pH 6.05, full thickness = pH 8.0). The correlation between pH and wound outcome could be used as an additional diagnostic tool to predict poor healing in wounds. Early identification of a nonhealing wound may allow a more aggressive treatment regimen, including skin grafting, to bring about rapid wound healing.