Evaluation of soluble corn fiber on chemical composition and nitrogen-corrected true metabolizable energy and its effects on in vitro fermentation and in vivo responses in dogs.

Dietary fermentable fiber is known to benefit intestinal health of companion animals. Soluble corn fiber (SCF) was evaluated for its chemical composition, nitrogen-corrected true ME (TMEn) content, in vitro digestion and fermentation characteristics, and in vivo effects on nutrient digestibility, fecal fermentation end products, and modulation of the fecal microbiome of dogs. Soluble corn fiber contained 78% total dietary fiber, all present as soluble dietary fiber; 56% was low molecular weight soluble fiber (did not precipitate in 95% ethanol). The SCF also contained 26% starch and 8% resistant starch and had a TMEn value of 2.6 kcal/g. Soluble corn fiber was first subjected to in vitro hydrolytic-enzymatic digestion to determine extent of digestibility and then fermented using dog fecal inoculum, with fermentative outcomes measured at 0, 3, 6, 9, and 12 h. Hydrolytic-enzymatic digestion of SCF was only 7%. In vitro fermentation showed increased (P < 0.05) concentrations of short-chain fatty acids through 12 h, with acetate, propionate, and butyrate reaching peak concentrations of 1,803, 926, and 112 µmol/g DM, respectively. Fermentability of SCF was higher (P < 0.05) than for cellulose but lower (P < 0.05) than for pectin. In the in vivo experiment, 10 female dogs (6.4 ± 0.2 yr and 22 ± 2.1 kg) received 5 diets with graded concentrations of SCF (0, 0.5, 0.75, 1.0, or 1.25% [as-is basis]) replacing cellulose in a replicated 5 × 5 Latin square design. Dogs were first acclimated to the experimental diets for 10 d followed by 4 d of total fecal collection. Fresh fecal samples were collected to measure fecal pH and fermentation end products and permit a microbiome analysis. For microbiome analysis, extraction of DNA was followed by amplification of the V4 to V6 variable region of the 16S rRNA gene using barcoded primers. Sequences were classified into taxonomic levels using a nucleotide basic local alignment search tool (BLASTn) against a curated GreenGenes database. Few changes in nutrient digestibility or fecal fermentation end products or stool consistency were observed, and no appreciable modulation of the fecal microbiome occurred. In conclusion, SCF was fermentable in vitro, but higher dietary concentrations may be necessary to elicit potential in vivo responses.

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.

Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models.

Faecal microbiota of domestic cats fed raw whole chicks v. an extruded chicken-based diet.

Extruded cat foods differ greatly in macronutrient distribution compared with wild-type diets (i.e. small mammals, reptiles, birds and insects). Based on the literature, this variability likely impacts faecal microbial populations. A completely randomised design was utilised to test the impacts of two dietary treatments on faecal microbial populations: (1) chicken-based extruded diet (EXT; n 3 cats) and (2) raw 1-3-d-old chicks (CHI; n 5 cats). Cats were adapted to diets for 10 d. Bacterial DNA was isolated from faecal samples and amplicons of the 16S rRNA V4-V6 region were generated and analysed by 454 pyrosequencing. Faeces of cats fed CHI had greater (P < 0·05) proportions of the following bacterial genera: unidentified Lachnospiraceae (15 v. 5 %), Peptococcus (9 v. 3 %) and Pseudobutyrivibrio (4 v. 1 %). Faeces of cats fed EXT had greater (P < 0·05) proportions of Faecalibacterium (1·0 v. 0·2 %) and Succinivibrio (1·2 v. < 0·1 %). Five genera, including Lactobacillus and Bifidobacterium, were present in a majority of samples (two to three out of three) from cats fed EXT, but were not detected in the samples (zero of five) for cats fed CHI. These shifts in faecal bacterial populations compared with feeding a whole-prey diet may impact the functional capacities of the microbiota and its interaction with the host. Further research is warranted to determine the impacts of these shifts on long-term health of domestic cats.

Salmonellosis impacts the proportions of faecal microbial populations in domestic cats fed 1-3-d-old chicks.

There has been a recent increase in the feeding of unconventional diets, including whole-prey diets, to domestic pet cats. Our objective was to characterise faecal microbial populations of domestic cats fed whole and ground (6·35 mm grind) raw 1-3-d-old chicks (Rodent Pro). Faecal samples were collected from neutered male domestic cats (mean age = 5·7 years) fed these diet items in a crossover design. Bacterial DNA was isolated from faecal samples and amplicons of the 16S rRNA V4-V6 region were generated and analysed by 454 pyrosequencing. Faecal microbial populations of cats fed whole v. ground chicks did not differ. During the study, three cats presented with symptoms of infection (anorexia or diarrhoea) and tested clinically positive for Salmonella using a standard PCR method. The remaining cats tested negative. Data were analysed post hoc to test for differences in microbial populations due to clinical status. The predominant genera were Clostridium (9-30 %), unidentified Lachnospiraceae (10-28 %), Blautia (4-19 %), Peptococcus (2-19 %) and Fusobacterium (2-14 %). Faeces of cats testing clinically positive for Salmonella had higher (P ≤ 0·05) proportions of the genera Coprococcus (5·6 v. 0·4 %) and Escherichia (subgenera Shigella; 1·1 v. 0·3 %). Salmonella was not detected in faecal samples utilising the pyrosequencing method; however, there was a shift in microbial populations due to clinical status. The clinical symptoms reported herein may be not only due to the Salmonella itself, but also shifts in other gut microbial populations.

Alteration of digestive tract microbiome in neonatal Holstein bull calves by bacitracin methylene disalicylate treatment and scours.

The effects of bacitracin methylene disalicylate (BMD) and scours on the fecal microbiome, animal performance, and health were studied in Holstein bull calves. Holstein bull calves (n = 150) were obtained from a single source at 12 to 24 h of age. Bull calves were randomly assigned to 1 of 2 treatments including CON (no BMD; n = 75 calves) and BMD (n = 75 calves). Starting 3 d after arrival, BMD was added into milk replacer (0.5 g/feeding; twice daily) and fed to the calves for 10 consecutive d. No differences (P > 0.10) were observed in ADG for d 0 to 28 and d 0 to 56, DMI for d 0 to 28, d 29 to 56, and d 0 to 56, or G:F for d 0 to 28, d 29 to 56, and d 0 to 56; ADG for d 29 to 56 tended to increase (P < 0.10) for BMD-treated calves compared with controls. Fecal samples were collected from 15 scouring calves and 10 cohorts (nonscouring calves received on the same day and administered the same treatment as the scouring calves). Animal morbidity and fecal score did not vary between the 2 treatments. Mortality was not influenced by the treatments in the BMD administration period or throughout the experiment. Fecal samples were subjected to pyrotagged 454 FLX pyrosequencing of 16S rRNA gene amplicon to examine compositional dynamics of fecal microbes. Escherichia, Enterococcus, and Shigella had greater (P < 0.05) populations in the BMD group whereas Dorea, Roseburia, Fecalibacterium, Papillibacter, Collinsella, Eubacterium, Peptostreptococcus, and Prevotella were decreased (P < 0.05) by BMD treatment. Genus populations were also compared between scouring and nonscouring calves. Streptococcus was the only genus that had notable increase (P < 0.05) in fecal samples from scouring calves whereas populations of Bacteroides, Roseburia, and Eubacterium were markedly (P < 0.05) greater in nonscouring calves. These results show that BMD has the ability to alter the composition of the fecal microbiome but failed to improve performance in Holstein bull calves. Discrepancy of microorganism profiles between scouring and nonscouring calves might be associated with the occurrence of scours and bacterial genera identified might be potential target of treating diarrhea.

Rumen bacterial, archaeal, and fungal diversity of dairy cows in response to ingestion of lauric or myristic acid.

The objective of this experiment, part of a larger study, was to investigate changes in rumen bacterial, archaeal, and fungal diversity in cows fed medium-chain saturated fatty acids. In the main study, 6 lactating dairy cows were dosed intraruminally with 240 g/(cow · d) of stearic (SA, control), lauric (LA), or myristic (MA) acid in a replicated 3 × 3 Latin square design trial. Experimental periods were 28 d, and cows were transfaunated between periods. Lauric acid decreased protozoal counts in the rumen by 96% compared with SA and MA (compared with SA, MA had no effect on ruminal protozoa). Whole ruminal contents samples were collected 2, 4, 6, 8, 10, 14, 18, and 24 h after the morning feeding on d 23 of each experimental period, stored frozen, and later composited by cow and period for microbial profile analyses, which involved tag-encoded flexible (FLX) amplicon pyrosequencing to provide diversity analyses of gastrointestinal bacterial, archaeal, and fungal populations of the cattle. The LA treatment, either directly or through its effect on protozoa, had a profound effect on the microbial ecology of the rumen. Ruminal populations of Prevotella, Bacteroides, and Enterorhabdus were decreased (P = 0.04 to P < 0.001) by more than 2-fold in LA treatments compared with SA, and Clostridium populations were decreased (P = 0.01) in LA- compared with MA-treated cows. The proportion of Ruminococcus was not affected by treatment, although the LA treatment had the least proportion of Ruminococcus. Proportions of Eubacterium, Butyrivibrio, Olsenella, and Lactobacillus genera were increased (P = 0.03 to 0.01) by LA compared with MA or SA. The LA treatment, possibly through its effect on protozoa physically associated with archaea, resulted in an increase (P = 0.01) in the archaeal methanogenic genus Methanosphaera and a decrease (P = 0.01) in Methanobrevibacter. Few changes in fungal populations caused by treatment were detected. Collectively, results indicate that LA, either through antiprotozoal or direct antimicrobial effects, altered bacterial and archaeal populations in the rumen of dairy cows, but effects on fungal populations were not clear.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of bacterial diversity in the rumen and feces of cattle fed different levels of dried distillers grains plus solubles using bacterial tag-encoded FLX amplicon pyrosequencing.

Dietary components and changes cause shifts in the gastrointestinal microbial ecology that can play a role in animal health and productivity. However, most information about the microbial populations in the gut of livestock species has not been quantitative. In the present study, we utilized a new molecular method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) that can perform diversity analyses of gastrointestinal bacterial populations. In the present study, cattle (n = 6) were fed a basal feedlot diet and were subsequently randomly assigned to 1 of 3 diets (n = 2 cows per diet). In each diet, 0, 25, or 50% of the concentrate portion of the ration was replaced with dried distillers grain (DDGS). Ruminal and fecal bacterial populations were different when animals were fed DDGS compared with controls; ruminal and fecal Firmicute:Bacteroidetes ratios were smaller (P = 0.07) in the 25 and 50% DDG diets compared with controls. Ruminal pH was decreased (P < 0.05) in ruminal fluid from cattle fed diets containing 50% compared with 0% DDGS. Using bTEFAP, the normal microbiota of cattle were examined using modern molecular methods to understand how diets affect gastrointestinal ecology and the gastrointestinal contribution of the microbiome to animal health and production.

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.