Comparative accessory gene fingerprinting of surface water Escherichia coli reveals genetically diverse naturalized population.

AIMS: To utilize comparative accessory gene fingerprinting to discriminate between naturalized and faecal Escherichia coli, with particular emphasis on strains from phylogroup B1. METHODS AND RESULTS: Fourteen accessory genes that were potentially ecotype-specific were selected on the basis of comparative genomic DNA sequence analysis between faecal and environmental strains and also using a literature-based strategy. PCR assays were designed for each gene, and used to screen 107 faecal strains from various hosts and 106 environmental strains from surface water and sediment. While none of the 14 accessory genes were ecotype-specific, six of the genes were ecotype-enriched. Specifically, toxin-antitoxin system genes were more abundant among faecal strains, whereas genes involved in iron acquisition, complement resistance/surface exclusion, and biofilm formation were more abundant among environmental strains. These six genes were used to form composite fingerprints which revealed the presence of several ecotype-specific and -enriched fingerprints. Notably, some of the environmental strain-specific or -enriched fingerprints consisted of strains putatively belonging to clade ET-1, which has been previously recognized as a naturalized subpopulation. CONCLUSIONS: Unlike single genes which did not reliably distinguish between faecal and naturalized phylogroup B1 E. coli strains, composite fingerprints of ecotype-enriched accessory genes may offer a novel method for distinguishing between these two populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Accessory gene fingerprinting may have important practical implications for improving the specificity of methods that are widely used for quantifying and identifying the sources of faecal contamination in surface water.

Listeria monocytogenes batch culture growth response to metabolic inhibitors.

In certain environments nutrient and energy sources available to microorganisms can be limited. Foodborne pathogens must efficiently adapt in order to be successfully transmitted through the food chain to their hosts. For the intracellular foodborne pathogen Listeria monocytogenes, little is known regarding its response to nutrient/energy-limiting conditions. The alternative stress responsive sigma factor σ(B) has been reported to contribute to survival under specific stresses. Therefore, the effects of several metabolic inhibitors on growth of L. monocytogenes wild-type and a ΔsigB mutant were examined. In the absence of inhibitors, both strains reached stationary phase after 18 h at 23°C and 10 h at 37°C. All of the metabolic inhibitors slowed growth of either strain, with few differences observed among the different inhibitors.

Longitudinal shifts in bacterial diversity and fermentation pattern in the rumen of steers grazing wheat pasture.

Grazing steers on winter wheat forage is routinely practiced in the Southern Great Plains of the US. Here, we investigated the dynamics in bacterial populations of both solid and liquid ruminal fractions of steers grazing on maturing wheat forage of changing nutritive quality. The relationship between bacterial diversity and fermentation parameters in the liquid fraction was also investigated. During the first 28 days, the wheat was in a vegetative phase with a relatively high crude protein content (CP; 21%), which led to the incidence of mild cases of frothy bloat among steers. Rumen samples were collected on days 14, 28, 56 and 76, separated into solid and liquid fractions and analyzed for bacterial diversity using 16S pyrotag technology. The predominant phyla identified were Bacteroidetes (59-77%) and Firmicutes (20-33%) across both ruminal fractions. Very few differences were observed in the rumen bacterial communities within solid and liquid fractions on day 14. However, by day 28, the relatively high CP content complemented a distinct bacterial and chemical composition of the rumen fluid that was characterized by a higher ratio (4:1) of Bacteroidetes:Firmicutes and a corresponding lower acetate:propionate (3:1) ratio. Further, a greater accumulation of biofilm (mucopolysaccharide complex) on day 28 was strongly associated with the abundance of Firmicutes lineages such as Clostridium, Ruminococcus, Oscillospira and Moryella (P<0.05) in the fiber fraction. Such changes were diminished as the CP concentration declined over the course of the study. The abundance of Firmicutes was noticeable by 76 d in both fractions which signifies the development of a core microbiome associated with digestion of a more recalcitrant fiber in the mature wheat. This study demonstrates dynamics in the rumen microbiome and their association with fermentation activity in the rumen of steers during the vegetative (bloat-prone) and reproductive stages of wheat forage.

Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.

This study investigated the effect of phytonutrients (PN) supplied postruminally on nutrient utilization, gut microbial ecology, immune response, and productivity of lactating dairy cows. Eight ruminally cannulated Holstein cows were used in a replicated 4×4 Latin square. Experimental periods lasted 23 d, including 14-d washout and 9-d treatment periods. Treatments were control (no PN) and daily doses of 2g/cow of either curcuma oleoresin (curcumin), garlic extract (garlic), or capsicum oleoresin (capsicum). Phytonutrients were pulse-dosed into the abomasum of the cows, through the rumen cannula, 2 h after feeding during the last 9 d of each experimental period. Dry matter intake was not affected by PN, although it tended to be lower for the garlic treatment compared with the control. Milk yield was decreased (2.2 kg/d) by capsicum treatment compared with the control. Feed efficiency, milk composition, milk fat and protein yields, milk N efficiency, and 4.0% fat-corrected milk yield were not affected by treatment. Rumen fermentation variables, apparent total-tract digestibility of nutrients, N excretion with feces and urine, and diversity of fecal bacteria were also not affected by treatment. Phytonutrients had no effect on blood chemistry, but the relative proportion of lymphocytes was increased by the capsicum treatment compared with the control. All PN increased the proportion of total CD4(+) cells and total CD4(+) cells that co-expressed the activation status signal and CD25 in blood. The percentage of peripheral blood mononuclear cells (PBMC) that proliferated in response to concanavalin A and viability of PBMC were not affected by treatment. Cytokine production by PBMC was not different between control and PN. Expression of mRNA in liver for key enzymes in gluconeogenesis, fatty acid oxidation, and response to reactive oxygen species were not affected by treatment. No difference was observed due to treatment in the oxygen radical absorbance capacity of blood plasma but, compared with the control, garlic treatment increased 8-isoprostane levels. Overall, the PN used in this study had subtle or no effects on blood cells and blood chemistry, nutrient digestibility, and fecal bacterial diversity, but appeared to have an immune-stimulatory effect by activating and inducing the expansion of CD4 cells in dairy cows. Capsicum treatment decreased milk yield, but this and other effects observed in this study should be interpreted with caution because of the short duration of treatment.

Equine stomachs harbor an abundant and diverse mucosal microbiota.

Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 were Firmicutes (>83% reads/sample), mainly Streptococcus spp., Lactobacillus spp. and, Sarcina spp. Cluster 2 was more diverse, with predominantly Proteobacteria, Bacteroidetes, and Firmicutes, consisting of Actinobacillus spp. Moraxella spp., Prevotella spp., and Porphyromonas spp. Helicobacter sp. sequences were not identified in any of 53,920 reads. FISH (n = 9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual.

Pyrosequencing reveals the complex polymicrobial nature of invasive pyogenic infections: microbial constituents of empyema, liver abscess, and intracerebral abscess.

The polymicrobial nature of invasive pyogenic infections may be underestimated by routine culture practices, due to the fastidious nature of many organisms and the loss of viability during transport or from prior antibacterials. Pyrosequencing was performed on brain and liver abscesses and pleural fluid and compared to routine culture data. Forty-seven invasive pyogenic infection samples from 44 patients [6 intracerebral abscess (ICA), 21 pyogenic liver abscess (PLA), and 18 pleural fluid (PF) samples] were assayed. Pyrosequencing identified an etiologic microorganism in 100 % of samples versus 45 % by culture, p <0.01. Pyrosequencing was also more likely than traditional cultures to classify infections as polymicrobial, 91 % versus 17 %, p <0.001. The median number of genera identified by pyrosequencing compared to culture was 1 [interquartile range (IQR) 1-3] versus 0 (IQR 0-1) for ICA, 7 (IQR 1-15) versus 1 (IQR 0-1) for PLA, and 15 (IQR 9-19) versus 0 (IQR 0-1) for PF. Where organisms were cultured, they typically represented the numerically dominant species identified by pyrosequencing. Complex microbial communities are involved in invasive pyogenic infection of the lung, liver, and brain. Defining the polymicrobial nature of invasive pyogenic infections is the first step towards appreciating the clinical and diagnostic implications of these complex communities.

Development of colonic microflora as assessed by pyrosequencing in dairy calves fed waste milk.

The objective of the current study was to examine the effect of pasteurization of waste milk, used to feed dairy calves, on the bacterial diversity of their lower gut. Using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing, fecal samples from dairy calves, ages 1 wk to 6 mo old and fed either pasteurized or nonpasteurized waste milk, were analyzed for bacterial diversity. Calves were maintained on 2 separate farms and, aside from how the waste milk was treated, were housed and cared for similarly. Fifteen calves were sampled from each age group (1, 2, and 4 wk, and 2, 4, and 6 mo of age; n=90 samples per milk treatment, 180 total samples) on each farm via rectal palpation and the samples shipped and frozen before analysis. In general, bacterial diversity, as represented by the total number of different species, was greater for the calves fed pasteurized waste milk at all ages (except 1 wk of age) and increased with increasing age in both treatments. Proteobacteria, Bacteroidetes, and Firmicutes were the predominant phyla. Differences in phyla and class were observed among treatments and age of calf but with no consistent trends. Salmonella were detected in 9 out of 14 (64%) of the 1-wk-old calves fed nonpasteurized milk. Treponema, an important beneficial bacterium in cattle rumen, was more prevalent in the pasteurized waste milk-fed animals and became higher in the older animals from this group. Escherichia-Shigella were detected among treatments at all ages, and highest at 1 wk of age, averaging approximately 21 and 20% of all bacteria for calves fed pasteurized and nonpasteurized waste milk, respectively, and decreasing as calves aged (2.6 and 1.3%). The consistent detection of Salmonella in the younger animals fed nonpasteurized milk and its absence in all other groups is an important finding related to this feeding practice.

Effect of sublethal heat stress on Salmonella Typhimurium virulence.

AIMS: To determine the virulence gene expression of Salmonella Typhimurium in response to sublethal heat stress and determine the adhesion and invasion pattern of heat-stressed Salmonella in Caco-2 intestinal epithelial cells. METHODS AND RESULTS: Transcriptional profiling was employed to capture the virulence gene response of Salm. Typhimurium at 42°C sublethal heat stress. Data indicated an induction of SPI-2 and SPI-5 genes and a repression of SPI-1-encoded genes due to heat stress. Gene expression pattern also showed induced transcription of fimbriae genes and genes present within the stress-associated Rpo regulon. Changes in adhesion and invasion pattern of heat-stressed Salm. Typhimurium were tested in Caco-2 cells. Heat-stressed Salm. Typhimurium showed greater adhesion to Caco-2 cells compared with nonstressed control cells. CONCLUSIONS: Salmonella Typhimurium exposed to sublethal heat stress responds by altered virulence gene expression, which further enhances the adhesion of bacterial cells to intestinal Caco-2 cells. Results indicate a role of physiological stress in Salm. Typhimurium in promoting microbial virulence and host cell vulnerability to infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Studying the Salmonella virulence genes expression in response to sublethal heat stress is crucial for the understanding of the virulence status of Salmonella in temperature-abused foods. Results of this study provide information about the gene response and virulence status of Salmonella pathogenicity factors in response to sublethal heat stress towards host cells.

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.

Survey of fungi and yeast in polymicrobial infections in chronic wounds.

OBJECTIVE: To assess the incidence, abundance and species diversity of fungi in chronic wounds, as well as to describe the associations of major fungi populations. METHOD: Comprehensive molecular diagnostic reports were evaluated from a total of 915 chronic wounds in a retrospective study. RESULTS: Of the 915 clinical specimens, 208 (23%) were positive for fungal species. These samples were further compared in a compiled dataset, and sub-classified among the four major chronic wound types (decubitus ulcer, diabetic foot ulcer, non-healing surgical wound, and venous leg ulcer). The most abundant fungi were yeasts in the genus Candida; however, Curvularia, Malessezia, Aureobasidium, Cladosporium, Ulocladium, Engodontium and Trichtophyton were also found to be prevalent components of these polymicrobial infections. A notable bacterial/fungal negative correlation was found to be apparent between Staphylococcus and Candida. There were also significant relationships between both bacterial and fungal genera and patient metadata including gender, diabetes status and cardiovascular comorbidities. CONCLUSION: This microbial survey shows that fungi are more important wound pathogens and opportunistic pathogens than previously reported, exemplifying the impact of these under-reported pathogens. With the application of modern cost-effective and comprehensive molecular diagnostics, clinicians can now identify and address this significant component of chronic wound bioburden with targeted therapies, thereby improving healing trajectories.

Assessment of the adequacy of counselling regarding reproductive-related issues in women of childbearing age on anti-epileptic drugs.

BACKGROUND: The use of anti-epileptic drugs (AEDs) in women of childbearing age (WCBA) necessitates careful counselling regarding reproductive-related issues. AIM: (i) To compare documentation of appropriate counselling regarding reproductive-related issues in WCBA prescribed AEDs for non-epilepsy vs. epilepsy indications, and (ii) to examine whether the frequency of counselling improved after introduction of 'standardized typed advice'. DESIGN: Retrospective audit and quality assessment and improvement programme. METHODS: We analysed medical records of all WCBA prescribed gabapentin, pregabalin, topiramate, valproate or carbamazepine by a general neurology clinical service before (Study period A) and after (Study period B) introduction of standardized typed passages regarding potential teratogenicity ± interactions with hormonal contraception at a university teaching hospital. The χ2 test or the Fisher's exact test was employed, as appropriate. RESULTS: In WCBA prescribed AEDs for non-epilepsy indications, documentation of appropriate counselling regarding potential teratogenicity improved from 49% (17/35 patients) in Period A to 79% (27/34 patients) in Period B (P = 0.008). The frequency of counselling regarding teratogenicity was higher in patients prescribed AEDs for epilepsy compared with non-epilepsy indications in Study period A (100% vs. 49%, P = 0.002), but was no longer significantly different in Study period B (86% vs. 79%, P = 0.64). Documentation of counselling regarding potential interaction of enzyme-inducing AEDs with hormonal contraception did not significantly change between study periods. CONCLUSION: Significant improvements in documentation regarding potential teratogenicity of AEDs prescribed for non-epilepsy indications can be achieved by introducing standardized, typed passages copied to patients. Such a practice change is practical and widely applicable to neurological and non-neurological practice worldwide.

Evaluation of in vitro gas production and rumen bacterial populations fermenting corn milling (co)products.

The objective of this study was to evaluate the fermentation dynamics of 2 commonly fed corn (co)products in their intact and defatted forms, using the in vitro gas production (IVGP) technique, and to investigate the shifts of the predominant rumen bacterial populations using the 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) technique. The bTEFAP technique was used to determine the bacterial profile of each fermentation time at 24 and 48 h. Bacterial populations were identified at the species level. Species were grouped by substrate affinities (guilds) for cellulose, hemicellulose, pectin, starch, sugars, protein, lipids, and lactate. The 2 (co)products were a dried distillers grain (DDG) plus solubles produced from a low-heat drying process (BPX) and a high-protein DDG without solubles (HP). Chemical analysis revealed that BPX contained about 11.4% ether extract, whereas HP contained only 3.88%. Previous studies have indicated that processing methods, as well as fat content, of corn (co)products directly affect fermentation rate and substrate availability, but little information is available regarding changes in rumen bacterial populations. Fermentation profiles of intact and defatted BPX and HP were compared with alfalfa hay as a standard profile. Defatting before incubation had no effect on total gas production in BPX or HP, but reduced lag time and the fractional rate of fermentation of BPX by at least half, whereas there was no effect for HP. The HP feed supported a greater percentage of fibrolytic and proteolytic bacteria than did BPX. Defatting both DDG increased the fibrolytic (26.8 to 38.7%) and proteolytic (26.1 to 37.2%) bacterial guild populations and decreased the lactate-utilizing bacterial guild (3.06 to 1.44%). Information regarding the fermentation kinetics and bacterial population shifts when feeding corn (co)products may lead to more innovative processing methods that improve feed quality (e.g., deoiling) and consequently allow greater inclusion rates in dairy cow rations.

Molecular diagnosis of Raoultella planticola infection of a surgical site.

Raoultella planticola has been rarely diagnosed in clinical specimens. A case of a polymicrobial surgical site infection primarily caused by R. planticola in a 66-year-old Caucasian male with a fractured left tibia after an open reduction internal fixation of his left ankle is described and confirms this organism to be an opportunistic human pathogen. This pathogen was diagnosed with rapid clinical molecular pathogen diagnostic methods, which allowed an appropriate therapy to be implemented, thereby improving prognosis.

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.

Chronic wounds and the medical biofilm paradigm.

There is a growing recognition that biofilms are the principal cause of wound chronicity. The development of treatments for wound biofilms raises the prospect that chronic wounds can be treated, potentially saving many patients' lives.

Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window.

OBJECTIVE: To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment. METHOD: Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study. RESULTS: All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/ burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance increased in each of the models by 48 hours and reached original resistance levels by 72 hours. CONCLUSION: These data suggest the principles of biofilm-based wound care, along with the use of serial debridement to continually remove mature biofilm, followed by biofilm wound management strategies, including topical antibiotics while the bioburden is still immature and more susceptible, are valid.

Culture-independent characterization of bacteria and fungi in a poultry bioaerosol using pyrosequencing: a new approach.

Work in animal production facilities often results in exposure to organic dusts. Previous studies have documented decreases in pulmonary function and lung inflammation among workers exposed to organic dust in the poultry industry. Bacteria and fungi have been reported as components of the organic dust produced in poultry facilities. To date, little is known about the diversity and concentration of bacteria and fungi inside poultry buildings. All previous investigations have utilized culture-based methods for analysis that identify only biota cultured on selected media. The bacterial tag-encoded flexible (FLX) amplicon pyrosequencing (bTEFAP) and fungal tag-encoded flexible (FLX) amplicon pyrosequencing (fTEFAP) are modern and comprehensive approaches for determining biodiversity of microorganisms and have not previously been used to provide characterization of exposure to microorganisms in an occupational environment. This article illustrates the potential application of this novel technique in occupational exposure assessment as well as other settings. An 8-hr area sample was collected using an Institute of Medicine inhalable sampler attached to a mannequin in a poultry confinement building. The sample was analyzed using bTEFAP and fTEFAP. Of the bacteria and fungi detected, 116 and 39 genera were identified, respectively. Among bacteria, Staphylococcus cohnii was present in the highest proportion (23%). The total inhalable bacteria concentration was estimated to be 7503 cells/m³. Among the fungi identified, Sagenomella sclerotialis was present in the highest proportion (37%). Aspergillus ochraceus and Penicillium janthinellum were also present in high proportions. The total inhalable fungi concentration was estimated to be 1810 cells/m³. These estimates are lower than what has been reported by others using standard epifluorescence microscope methods. However, no study has used non-culture-based techniques, such as bTEFAP and fTEFAP, to evaluate bacteria and fungi in the inhalable fraction of a bioaerosol in a broiler production environment. Furthermore, the impact of this bTEFAP and fTEFAP technology has yet to be realized by the scientific community dedicated to evaluating occupational and environmental bioaerosol exposure.

Crustacean intestinal detergent promotes sterol solubilization.

Although crustacean tissue cholesterol content is high, Crustacea, like other arthropods; are incapable of cholesterol synthesis, and presumably are dependent for maintaining tissue cholesterol stores on the intestinal absorption of ingested sterol. A detergent, N-(N-dodecanoylsarcosyl)taurine, representative of a set of detergents synthesized by the crustacean hepatopancreas and secreted into the intestine, is capable of efficient cholesterol solubilization, and thus of promoting sterol absorption.

A database of expressed genes from Cochliomyia hominivorax (Diptera: Calliphoridae).

We used an expressed sequence tag and 454 pyrosequencing approach to initiate a study of the genome of the screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Two normalized cDNA libraries were constructed from RNA isolated from embryos and second instar larvae from the Panama 95 strain. Approximately 5,400 clones from each library were sequenced from both the 5' and 3' directions using the Sanger method. In addition, double-stranded cDNA was prepared from random-primed polyA RNA purified from embryos, second-instar larvae, adult males, and adult females. These four cDNA samples were used for 454 pyrosequencing that produced approximately 300,000 independent sequences. Sequences were assembled into a database of assembled contigs and singletons and used to search public protein databases and annotate the sequences. The full database consists of 6,076 contigs and 58,221 singletons assembled from both the traditional expressed sequence tag (EST) and 454 sequences. Annotation of the data led to the identification of several gene coding regions with possible roles in sex determination in the screwworm. This database will facilitate the design of microarray and other experiments to study screwworm gene expression on a larger scale than previously possible.

Regular debridement is the main tool for maintaining a healthy wound bed in most chronic wounds.

Sharp debridement is the most clinically and cost-effective way of physically removing and suppressing a biofilm. Continued debridement, as part of a multifaceted treatment strategy, will keep the biofilm in a weakened state.