The presence of biofilm structures in atherosclerotic plaques of arteries from legs amputated as a complication of diabetic foot ulcers.

OBJECTIVE: Atherosclerosis, rather than microcirculatory impairment caused by endothelial cell dysfunction, is the main driver of circulatory compromise in patients with diabetic limbs. The presence of atherosclerotic plaque at the trifurcation is a significant contributor to amputation of diabetic legs. The presence of bacteria and other microorganisms in atherosclerotic plaque has long been known, however, the cause of chronic inflammation and the role of bacteria/viruses in atherosclerosis have not been studied in detail. The objective of this study was to clarify the cause of the chronic inflammation within atherosclerotic plaques, and determine if any bacteria and/or viruses are involved in the inflammatory pathway. METHOD: This study uses fluorescence microscopy and fluorescence in-situ hybridisation (FISH) to identify components of biofilm in atherosclerotic arteries. These tools are also used to identify individual bacteria, and determine the architectural spatial location within the atherosclerotic plaque where the bacteria can be found. RESULTS: The results indicate that the presence of biofilms in grossly involved arteries may be an important factor in chronic inflammatory pathways of atherosclerotic progression, in the amputated limbs of patients with diabetic foot ulcers and vascular disease. CONCLUSION: While the presence of bacterial biofilm structures in atherosclerotic plaque does not prove that biofilm is the proximate cause of atherosclerosis, it could contribute to the persistent inflammation associated with it. Second, the synergistic relationship between the atherosclerotic infection and the diabetic foot ulcer may ultimately contribute to higher amputation rates in diabetics. DECLARATION OF INTEREST: RAW and RDW have equity interest in PathoGenius, a clinical laboratory using DNA to identify microbes.

Prevention of antibiotic-associated metabolic syndrome in mice by intestinal alkaline phosphatase.

AIMS: To examine whether co-administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice. METHODS: A total of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7-day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high-fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition. RESULTS: Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co-administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides). CONCLUSIONS: Co-administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.

Intestinal aganglionosis is associated with early and sustained disruption of the colonic microbiome.

BACKGROUND: Congenital aganglionosis (Hirschsprung's disease) results in colonic dysmotility and a risk for Hirschsprung's-associated enterocolitis (HAEC), whose cause is unknown. We hypothesized that aganglionosis leads to microbiome changes that may contribute to HAEC risk. METHODS: Colon and fecal samples were collected from endothelin receptor B-null (Ednrb(-/-) ) mice, an established model of colorectal aganglionosis, at postnatal day 7 (P7), P20, and P24. We determined microbiome composition by 16S ribosomal RNA gene pyrosequencing and fecal metabolite profile by nuclear magnetic resonance spectroscopy. KEY RESULTS: Wild-type (WT) mice exhibited increasing species diversity with age, with mutant mice possessing even greater diversity. WT and mutant microbiomes, both fecal and colonic, significantly segregated by principal coordinates analysis based on species composition at all ages examined. Importantly, mutant mice contained more Bacteroidetes and less Firmicutes than WT, with additional genus- and species-level differences observed. Notably, mutant P7 colon was dominated by coagulase-negative Staphylococcus species, which were rare in WT. Mutant fecal metabolite profiles also differed, particularly in the abundance of formate, a short-chain fatty acid product of microbial fermentation. CONCLUSIONS & INFERENCES: Colorectal aganglionosis is associated with early and sustained disruption of the normal colonic and fecal microbiome, supporting the enteric nervous system as a determinant of microbiome composition. Furthermore, the differences observed suggest a potential contributory role for the microbiome in the etiology of HAEC. These findings provide a basis for further studies to determine the causative role of specific bacterial communities in HAEC and the potential to restore the normal microbiome in Hirschsprung's disease.

Molecular diagnostics and personalised medicine in wound care: assessment of outcomes.

OBJECTIVE: This large, level A, retrospective cohort study set out to compare healing outcomes in three large cohorts of wound patients managed universally for bioburden: standard of care group, who were prescribed systemic antibiotics on the basis of empiric and traditional culture-based methodologies; treatment group 1, who were prescribed an improved selection of systemic antibiotics based on the results of molecular diagnostics; treatment group 2 who received personalised topical therapeutics (including antibiotics) based on the results of molecular diagnostics. METHOD: Apart from the differences in diagnostic methods and antibiotic treatments described above, all three cohorts were subjected to the same biofilm-based wound care protocol, which included evaluation of the host and bioburden, frequent sharp debridement, use of wound dressings and comprehensive standard care (reperfusion therapy, nutritional support, offloading, compression and management of comorbidities). RESULTS: In all, 1378 patients were recruited into the study. In the standard of care group 48.5% of patients (244/503) healed completely during the 7-month study period. This increased to 62.4% (298/479) in treatment group 1 and 90.4% (358/396) in treatment group 2. Cox proportional hazards analysis revealed the time to complete closure decreased by 26% in treatment group 1 (p<0.001) and 45.9% in treatment group 2 (p<0.001) compared with the standard of care group. Patients in treatment group 2 had >200% better odds of healing at any given time point compared with the other cohorts. CONCLUSION: Implementation of personalised topical therapeutics guided by molecular diagnosis resulted in statistically and clinically significant improvements in outcome. The integration of molecular diagnostics and personalised medicine provides a directed and targeted approach to wound care. CONFLICT OF INTEREST: SED and RDW are owners of PathoGenius Laboratories, a clinical diagnostic laboratory. SED and RDW are owners of Research and Testing Laboratory, which develops molecular diagnostics. CJ and JK are clinical advisors for PathoGenius. CJ and JK are owners of Southeastern Medical Compounding, Savannah, GA and Southeastern Medical Technologies, Savannah, GA.

Healing and healing rates of chronic wounds in the age of molecular pathogen diagnostics.

OBJECTIVE: To compare healing outcomes at a wound healing centre both before and after the introduction of molecular pathogen diagnostics. METHOD: An IT consultant was recruited to analyse the medical records of patients at a wound healing centre, comparing patient groups from 2007 and 2009 - before and after the introduction of comprehensive molecular pathogen diagnostic methods. RESULTS: Before the implementation of molecular diagnostics, 244/503 patients (48.5%) healed completely, while after implementation 298/479 patients (62.4%) healed. Furthermore, based on survival analysis and after controlling for potential confounding factors, time to healing was significantly shorter in 2009 than 2007 (p<0.05). Specifically, biofilm-based wound care, along with the implementation of comprehensive molecular diagnostics for venous leg ulcers, pressure ulcers and diabetic foot ulcers and all wounds combined showed, respectively, 21%, 23%, 25% and 22% reductions in the time to healing. In addition, after implementing molecular diagnostics, the use of expensive fi rst-line antibiotics also declined in 2009, while a broader range of targeted antibiotics was used. CONCLUSION: The results of modern molecular pathogen diagnostic applications allow comprehensive evaluation of the microbial bioburden in chronic wounds. This comprehensive diagnostic in turn has led to a more precise and targeted therapeutic approach to wound care. With the comprehensive nature of molecular diagnostics future advances in topical patient specific therapeutics are now possible.

Molecular and functional assessment of bacterial community convergence in metal-amended soils.

Species diversity and the structure of microbial communities in soils are thought to be a function of the cumulative selective pressures within the local environment. Shifts in microbial community structure, as a result of metal stress, may have lasting negative effects on soil ecosystem dynamics if critical microbial community functions are compromised. Three soils in the vicinity of a copper smelter, previously contaminated with background, low and high levels of aerially deposited metals, were amended with metal-salts to determine the potential for metal contamination to shape the structural and functional diversity of microbial communities in soils. We hypothesized that the microbial communities native to the three soils would initially be unique to each site, but would converge on a microbial community with similar structure and function, as a result of metal stress. Initially, the three different sites supported microbial communities with unique structural and functional diversity, and the nonimpacted site supported inherently higher levels of microbial activity and biomass, relative to the metal-contaminated sites. Amendment of the soils with metal-salts resulted in a decrease in microbial activity and biomass, as well as shifts in microbial community structure and function at each site. Soil microbial communities from each site were also observed to be sensitive to changes in soil pH as a result of metal-salt amendment; however, the magnitude of these pH-associated effects varied between soils. Microbial communities from each site did not converge on a structurally or functionally similar community following metal-salt amendment, indicating that other factors may be equally important in shaping microbial communities in soils. Among these factors, soil physiochemical parameters like organic matter and soil pH, which can both influence the bioavailability and toxicity of metals in soils, may be critical.

Evidence that miRNAs are different from other RNAs.

An examination of 513 known pre-miRNAs and 237 other RNAs (tRNA, rRNA, and mRNA) revealed that miRNAs were significantly different from other RNAs (p < 0.001). miRNA genes were less conserved than other RNA genes, although their mature miRNA sequences were highly conserved. The A+U content of pre-miRNAs was higher than non-coding RNA (p < 0.001), but lower than mRNAs. The nucleotides in pre-miRNAs formed more hydrogen bonds and base pairs than in other RNAs. miRNAs had higher negative adjusted minimal folding free energies than other RNAs except tRNAs (p < 0.001). The MFE index (MFEI) was a sufficient criterion to distinguish miRNAs from all coding and non-coding RNAs (p < 0.001). The MFEI for miRNAs was 0.97, significantly higher than tRNAs (0.64), rRNAs (0.59), or mRNAs (0.65). Our findings should facilitate the prediction and identification of new miRNAs using computational and experimental strategies.

Neuronal units linked to microvascular modules in cerebral cortex: response elements for imaging the brain.

How neuronal activity changes cerebral blood flow is of biological and practical importance. The rodent whisker-barrel system has special merits as a model for studies of changes in local cerebral blood flow (LCBF). Stimulus-evoked changes in neural firing and 'intrinsic signals' recorded through a cranial window were used to define regions of interest for repeated flow measurements. Whisker-activated changes in flow were measured with intravascular markers at the pia. LCBF changes were always prompt and localized over the appropriate barrel. Stimulus-related changes in parenchymal flow monitored continuously with H2 electrodes recorded short latency flow changes initiated in middle cortical layers. Activation that increased flow to particular barrels often led to reduced flow to adjacent cortex. Dye was injected into single penetrating arterioles from the pia of the fixed brain and injected into arterioles in slices of cortex where barrels were evident without stains. Arteriolar and venular domains at the surface were not directly related to underlying barrels. Capillary tufts in layer IV were mainly coincident with barrels. The matching between a capillary plexus (a vascular module) and a barrel (a functional neuronal unit) is a spatial organization of neurons and blood vessels that optimizes local interactions between the two. The paths of communication probably include: neurons to neurons, neurons to glia, neurons to vessels, glia to vessels, vessels to vessels and vessels to brain. Matching a functional grouping of neurons with a vascular module is an elegant means of reducing the risk of embarrassment for energy-expensive neuronal activity (ion pumping) while minimizing energy spent for delivery of the energy (cardiac output). For imaging studies this organization sets biological limits to spatial, temporal and magnitude resolution. Reduced flow to nearby inactive cortex enhances local differences.

Localized dynamic changes in cortical blood flow with whisker stimulation corresponds to matched vascular and neuronal architecture of rat barrels.

The hypothesis that functional groups of neurons in whisker barrels are linked to a modular organization of cortical vessels was tested. Endovascular casts demonstrated cortical capillary networks resembling the whisker barrel pattern that were fed from the middle cerebral artery. In histological sections, dense capillaries apparently were confined to single barrels and were supplied by one or a few penetrating arterioles. The barrel field in cortical layer IV was localized in relation to surface arteriovenous patterns. Living vessels were imaged through a closed cranial window under anesthesia with a fluorescence microscope and SIT or ICCD cameras. After intracarotid injections of fluorescein isothiocyanatedextrans, saline, or 3 microns latex beads, changes in arteriolar diameter, arteriovenous transit times (AVTTs), and bead velocities were measured. When row C whiskers were stroked at 4-5 Hz for 1 min, blood flow increased in arterioles that supplied contralateral row C barrels as demonstrated by postmortem histology. AVTTs slowed significantly in vessels supplying adjacent cortex. We hypothesize that cerebral vascular units supply individual whisker barrels and are functionally linked to them for precise focal regulation of cerebral blood flow.

The effect of pancreatic polypeptide on glucose disposal after surgical alterations of the pancreas.

Surgical alterations of the pancreas result in anatomic changes that can affect postoperative glucose metabolism. Pancreas transplantation results in reduction of beta-cell mass, systemic release of insulin, and denervation. The authors hypothesized that such alterations affect peripheral glucose disposal to induce an "insensitivity" to endogenously (systemically) released insulin. Additionally, they hypothesized that surgically induced deficiency of the postprandial hormone, pancreatic polypeptide, might contribute to altered glucose disposal. The authors studied two surgical models in dogs known to be devoid of pancreatic polypeptide--70% proximal pancreatectomy (PPx) and PPx plus distal pancreas autotransplantation (PAT/B). Oral glucose challenge and euglycemic hyperinsulinemic clamp studies were performed before and after a 16-day "pulsed" infusion of pancreatic polypeptide. Both surgical procedures resulted in elevations in the integrated glucose response after oral glucose, which was not affected by pancreatic polypeptide infusion. Euglycemic clamp studies showed decreased hepatic glucose output (Ra) and overall glucose disposal (Rd) in the fasted state for both surgical groups. The transplant animals demonstrated significant decreases in Rd during the hyperinsulinemic challenge (3.2 +/- 0.01 versus 5.7 +/- 0.01 mg/kg/minute at 60 to 120 minutes for PAT/B versus control). After 16 days of pancreatic polypeptide infusion, however, basal Ra, as well as basal and 60- to 120-minute Rd values, were returned to control values in the transplant group. The authors conclude that pancreas transplantation results in altered glucose disposal, possibly due to an altered effectiveness of systemically released insulin. They conclude that pancreatic polypeptide is an important modulator of peripheral insulin action. Therefore, the role of pancreatic polypeptide must be taken into account when evaluating postoperative glucose metabolism in canine models of pancreas transplantation.