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.

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.

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.

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.

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.

Propagation of anaerobic bacteria within an aerobic multi-species chronic wound biofilm model.

OBJECTIVE: Most chronic wound biofilms have been shown to have significant populations of anaerobes. In order to better screen antimicrobial and antibiofilm therapeutics, we evaluated the ability of key anaerobes to incorporate and propagate within our aerobic chronic wound biofilm. METHOD: We had previously developed a rapid model to simulate polymicrobial chronic wound biofilms. This model incorporated meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and Pseudomonas aeruginosa. The model was used along with a variety of anaerobes to determine whether this biofilm model would support propagation of anaerobes similar to that we have identified in chronic wounds. RESULTS: Using our previously defined Lubbock Chronic Wound Biofilm (LCWB) model combined with quantitative PCR, anaerobic bacteria were shown to proliferate through integration into the biofilm under aerobic conditions. Using electron microscopy we show close association between aerobes and anaerobes within the biofilm suggesting a synergistic relationship. CONCLUSION: We have expanded the utility of the LCBW to show the ability of clinically significant anaerobic bacteria to thrive in aerobic conditions. The expansion of this model can further simulate the functional characteristics of chronic wound pathogenic biofilms and the species that dwell within them allowing improved ability to evaluate therapeutics that target anaerobes.

Identification of yeast in chronic wounds using new pathogen-detection technologies.

OBJECTIVE: To evaluate the ability of two new diagnostic methods to detect and accurately identify yeast associated with chronic wound infections. METHOD: Fungal tag-encoded FLX amplicon pyrosequencing (fTEFAP), a universal fungal identification method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, and a new quantitative polymerase chain reaction (qPCR) wound pathogen panel were used to evaluate three chronic wounds suspected to contain yeast. RESULTS: Forty wound samples were analysed in addition to the three samples suspected of containing yeast. The qPCR panel, which targets Candida albicans, detected this yeast in two of the three wound samples. In contrast, fTEFAP detected yeast in each of the three samples: two showed Candida albicans and the third Candida parapsilosis. fTEFAP also identified a lower level of Candida tropicalis in one of the wounds that was positive for Candida albicans. The qPCR wound panel results were returned within two hours, while the fTEFAP results were returned within 24 hours. CONCLUSION: Two new molecular methods have been developed to aid wound pathogen diagnostics. The quantitative PCR wound panel is rapid but is limited to major wound-associated bacteria and yeasts. The universal fTEFAP and bTEFAP methods take 24 hours to return results but are able to detect the relative contribution of any bacteria of yeast in a chronic wound diagnostic sample. DECLARATION OF INTEREST: Southwest Regional Wound Care Center is a clinical wound-care provider seeking to improve the ability of wound care practitioners to help patients. The Research and Testing Laboratory develops molecular methods including fTEFAP, bTEFAP and the quantitative PCR wound panel.

Bacterial diversity in surgical site infections: not just aerobic cocci any more.

OBJECTIVE: To evaluate the microbial diversity in chronic surgical site infections (SSIs). METHOD: Bacterial populations in 23 chronic SSIs were identified using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP),which is an universal bacterial identification method.These results were then validated using quantitative polymerase chain reaction (qPCR). RESULTS: bTEFAP identified two previously uncharacterised Bacteroidales in all of the SSIs and showed that it was the predominant population in the majority of these chronic wounds. Other bacteria identified included Corynebacterium spp., Peptoniphilus spp., Staphylococcus spp., Staphylococcus aureus, Serratia marcescens, Prevotella spp. and Pseudomonas aeruginosa. Rarefaction analysis of the data indicated that, on average, six genera occurred in any given SSI, suggesting that such infections are multispecies. On average, over 60% of the bacteria evaluated in the SSIs were anaerobic bacilli. The previous literature indicates that aerobic cocci predominate in such wounds. CONCLUSION: This modern molecular survey indicates that our previous understanding of which bacteria cause SSIs may be faulty. The high prevalence of anaerobic bacilli and the overwhelming predominance of two previously uncharacterised Bacteroidales suggest that such bacteria may be a leading contributor to such infections. Further research on the identification and treatment of such bacteria are warranted.

Effects of biofilm treatments on the multi-species Lubbock chronic wound biofilm model.

OBJECTIVE: To evaluate the efficacy of several biofilm effectors in inhibiting biofilm formation in an in vitro multi-species chronic wound biofilm model. METHOD: The Lubbock Chronic Wound Biofilm (LCWB) model has been described in detail elsewhere. Pathogens used in the model are Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus. These are three of the most important species associated with biofilms. Here, the model was exposed to the following biofilm effectors: xylitol, salicylic acid, farnesol, erythritol and two proprietary, semi-solid, wound-dressing formulations currently under development (Sanguitec gels). RESULTS: Biofilm formation was completely inhibited in the LCWB model following treatment with 20% xylitol, 10% erythritol, 1,000 microg/ml farnesol, 20mM salicylic acid or 0.1% of either of the two Sanguitec gel formulations. Salicylic acid specifically inhibited S. aureus (p<0.01) at 10mM and 20mM, consequently increasing the ratios of P. aeruginosa and E. faecalis within the biofilm. Xylitol had an increasing inhibitory effect on P. aeruginosa (p<0.01) at all concentrations evaluated. Erythritol had an inhibitory effect on P. aeruginosa and S. aureus growth (p<0.01) at over 5% concentrations. The inhibitory effect of both Sanguitec gel formulations was more broadly effective, with an increasingly inhibitory effect on all LCWB species (p<0.01). CONCLUSION: The LCWB model provides a multi-species format with which to evaluate the effect of biofilm effectors on wound flora in a biofilm phenotype. These results suggest that different treatments can target specific populations within a biofilm. Salicylic acid preferentially targeted S. aureus, xylitol preferentially targeted P. aeruginosa, while erythritol preferentially targeted both P. aeruginosa and S. aureus. In contrast, the two Sanguitec gel formulations provided a broad, less preferential, inhibition of biofilm development. DECLARATION OF INTEREST: Research and Testing Laboratory is a for-profit enterprise that develops molecular methods and performs service research work on biofilms. Sanguitec gel was developed by JPK and CEJ.

Evaluation of the bacterial diversity in cecal contents of laying hens fed various molting diets by using bacterial tag-encoded FLX amplicon pyrosequencing.

Laying hens are typically induced to molt to begin a new egg-laying cycle by withdrawing feed for up to 12 to 14 d. Fasted hens are more susceptible to colonization and tissue invasion by Salmonella enterica serovar Enteritidis. Much of this increased incidence in fasted hens is thought to be due to changes in the native intestinal microflora. An alternative to feed withdrawal involves feeding alfalfa meal crumble to hens, which is indigestible by poultry but provides fermentable substrate to the intestinal microbial population and reduces Salmonella colonization of hens compared with feed withdrawal. The present study was designed to quantify differences in the cecal microbial population of hens (n=12) fed a typical layer ration, undergoing feed withdrawal, or being fed alfalfa crumble by using a novel tag bacterial diversity amplification method. Bacteroides, Prevotella, and Clostridium were the most common genera isolated from all treatment groups. Only the ceca of hens undergoing feed withdrawal (n=4) contained Salmonella. The number of genera present was greatest in the alfalfa crumble-fed group and least in the feed withdrawal group (78 vs. 54 genera, respectively). Overall, the microbial diversity was least and Lactobacillius populations were not found in the hens undergoing feed withdrawal, which could explain much of these hens' sensitivity to colonization by Salmonella.