Continuous-flow Culture Research Articles

426 Forages Containing Plant Secondary Compounds Can Improve Digestibility, Rumen Ammonia and Rumen Metabolites of Pasture: an in Vitro Continuous Culture Case Study

Abstract Forages containing plant secondary metabolites (PSM) have been investigated for use in reducing nitrogen waste in feedlot and dairy cattle diets but use as supplements to grazing beef cattle to capture the positive effects of PSM is unclear. Here we investigate potential improvement in rumen digestibility and metabolome, rumen ammonia production and flow from feed protein due to PSM bioactivity in forages used to supplement pasture grass using in vitro methods. Can PSM-containing forages offer a practical way to improve range nutrition and provide delivery of phytochemical benefits? Phytochemical- containing forages birdsfoot trefoil (BFT), sainfoin (SAN), small burnet (SMB) and cicer milkvetch (CMV) supplemented grass pasture diets (200 g/kg of DM). In vitro true dry matter digestibility was determined using 2-stage in vitro methods. Randomly assigned diets of PAS supplemented with one of the PSM-containing forage diets (PAS+X) or not supplemented (PAS) were randomly applied to in vitro dual flow continuous culture (CC) vessels inoculated with grazing beef cow rumen fluid as a metabolic host. For 2-stage digestibility, SMB digestibility exceeded that of CMV, followed by BFT and SAN. The grass diet (PAS) was the least digestible (Table 1). Saponin was detected in CMV samples, which is a new finding. For CC, compared with PAS, minimum culture pH for PAS+CMV was highest (P < 0.02) but no differences were observed in pH between PAS, and PAS+BFT and PAS+SAN. Compared with PAS, the molar proportion of the fatty acid propionate increased with PAS+CMV(P = 0.03) in contrast to the acetate- to- propionate ratio found highest for PAS and decreased for all diets with supplementation of PSM forages. Concentration of rumen NH3–N flow to rumen fluid per gram of protein intake was lower (P < 0.01) with PAS+CMV and PAS+SMB, compared with grass only. Feeding CMV and SMB to grazing cattle may improve nutrient utilization, metabolome, and environmental sustainability of beef cattle production by improving diet digestibility and reducing ammonia waste.

Algae Cultivation as Measure for the Sanitation of Organic Waste—A Case Study Based on the Alga Galdieria sulphuraria Grown on Food Waste Hydrolysate in a Continuous Flow Culture

Due to its growth under harsh acidic conditions, the microalga Galdieria sulphuraria may offer the opportunity to combine sanitation and the utilization of organic waste streams. To further deepen the knowledge of alternative waste treatment strategies that allow for holistic utilization, the control and removal of microbial contaminants via non-sterile heterotrophic G. sulphuraria on food waste hydrolysate were investigated in a continuous flow bioreactor culture. Furthermore, a substrate reservoir and harvested biomass were stored under non-sterile conditions over a period of 12 days. Despite the non-sterile conditions, the microbial load of the biomass could be kept under control. Neither the pathogen Salmonella sp. nor the coliform bacteria Escherichia coli could be found. Only nine counts per g of biomass were found for species belonging to Enterococcus spp., Enterobacteriacae, and moulds. Aerobic spore formers were counted with 2700 counts per g of biomass. Most of the aerobic mesophilic counts were formed by yeasts (1.5 × 106 vs. 1.3 × 106 counts per g biomass). The results revealed that, when using acidic growth conditions, contamination will not take over the culture; thus, the sterilization of waste materials can be skipped. It is assumed that such an approach can result in efficient processes for future waste-based bioeconomy strategies.

Effect of chemostat turnover rate and select antibiotics on Salmonella Typhimurium in the presence of porcine gastrointestinal tract bacteria

The effect of select antibiotics on Salmonella Typhimurium DT104 persistence in a porcine cecal continuous flow culture was examined under two different washout rates. Porcine continuous flow cultures were conducted in the presence or absence of Gram-positive antibiotic carbadox. Carbadox eliminated chemostat anaerobes culturable on Brucella agar under 24 h turnover conditions, allowing Salmonella Typhimurium to persist for 15 days. Decreasing the culture dilution rate from a 24 to 48 h turnover time enabled Salmonella Typhimurium to maintain higher population levels more reflective of the theoretical washout rate when grown with than without added carbadox. This observation suggests that at the slower growth rate the presence of carbadox was antagonistic to the indigenous competitive exclusion potential of the mixed microbial population.

Effect of feeding frequency on growth performance, feed transit and digestive enzyme activity of Acipenser dabryanus juveniles

Dabry's sturgeon (Acipenser dabryanus) is critically endangered in China. However, the lack of research on the feeding strategy of this species is detrimental to its conservation management. The present study evaluated the effects of feeding frequency (1, 2, 3, 4 and 5 times per day) on the growth performance, feed transit and digestive enzyme activity of juvenile Dabry's sturgeons (initial weight 0.42 ± 0.09 g) in a continuous flow culture system at a water temperature of 18.2–18.5°C. Single-meal and normal-feeding (1, 2, 3, 4 and 5 times per day) experimental methods were adopted to assess the effect of different feeding frequencies on feed transit. The fish in each treatment of the normal-feeding experiment were fed normally according to the original feeding frequency (1, 2, 3, 4 and 5 times per day). The optimum feeding frequency for juvenile Dabry's sturgeons was determined to be 4 times per day based on the growth performance, feed conversion rate and digestive enzyme activity. The results of the single-meal experiment demonstrated that changes in daily feeding frequency modified residence time in the stomach (S1–S5: 27.2–10.4 h) and intestine (S2–S5: range from 14 to 29.85 h). However, the obtained gastric emptying time varied significantly depending on the experimental approach. The single-meal experiment approach resulted in a longer gastric emptying time of fish fed 1 and 2 times per day (S1: >35 h; S2: 35 h) than those in the normal-feeding experiment (N1: 16 h; N2: 24 h), demonstrating that the single-meal method might change the gastrointestinal emptying strategy of fish. Furthermore, the digestive enzyme activity had daily rhythmic changes and the average digestive enzyme activity was affected by feeding frequency. The average digestive enzyme activity of the fish in N3 or N4 was higher than those of fish in other groups (p < 0.05). In summary, this study provides much useful information for the healthy farming of juvenile Dabry's sturgeons and towards the understanding of digestive physiology, which is of great significance for the protection of Dabry's sturgeon.

MiniBioReactor Array (MBRA) in vitro gut model: a reliable system to study microbiota-dependent response to antibiotic treatment.

BackgroundAntimicrobial drugs are mostly studied for their impact on emergence of bacterial antibiotic resistance, but their impact on the gut microbiota is also of tremendous interest. In vitro gut models are important tools to study such complex drug–microbiota interactions in humans.MethodsThe MiniBioReactor Array (MBRA) in vitro microbiota system; a single-stage continuous flow culture model, hosted in an anaerobic chamber; was used to evaluate the impact of three concentrations of a third-generation cephalosporin (ceftriaxone) on faecal microbiota from two healthy donors (treatment versus control: three replicates per condition). We conducted 16S microbiome profiling and analysed microbial richness, diversity and taxonomic changes. β-Lactamase activities were evaluated and correlated with the effects observed in the MBRA in vitro system.ResultsThe MBRA preserved each donor’s specificities, and differences between the donors were maintained through time. Before treatment, all faecal cultures belonging to the same donor were comparable in composition, richness, and diversity. Treatment with ceftriaxone was associated with a decrease in α-diversity, and an increase in β-diversity index, in a concentration-dependent manner. The maximum effect on diversity was observed after 72 h of treatment. Importantly, one donor had a stronger microbiota β-lactamase activity that was associated with a reduced impact of ceftriaxone on microbiota composition.ConclusionsMBRA can reliably mimic the intestinal microbiota and its modifications under antibiotic selective pressure. The impact of the treatment was donor- and concentration-dependent. We hypothesize these results could be explained, at least in part, by the differences in β-lactamase activity of the microbiota itself. Our results support the relevance and promise of the MBRA system to study drug–microbiota interactions.

A Microbiota-Dependent Response to Anticancer Treatment in an In Vitro Human Microbiota Model: A Pilot Study With Hydroxycarbamide and Daunorubicin.

BackgroundAnticancer drug efficacy is linked to the gut microbiota’s composition, and there is a dire need to better understand these interactions for personalized medicine. In vitro microbiota models are promising tools for studies requiring controlled and repeatable conditions. We evaluated the impact of two anticancer drugs on human feces in the MiniBioReactor Array (MBRA) in vitro microbiota system.MethodsThe MBRA is a single-stage continuous-flow culture model, hosted in an anaerobic chamber. We evaluated the effect of a 5-day treatment with hydroxycarbamide or daunorubicine on the fecal bacterial communities of two healthy donors. 16S microbiome profiling allowed analysis of microbial richness, diversity, and taxonomic changes.ResultsIn this host-free setting, anticancer drugs diversely affect gut microbiota composition. Daunorubicin was associated with significant changes in alpha- and beta-diversity as well as in the ratio of Firmicutes/Bacteroidetes in a donor-dependent manner. The impact of hydroxycarbamide on microbiota composition was not significant.ConclusionWe demonstrated, for the first time, the impact of anticancer drugs on human microbiota composition, in a donor- and molecule-dependent manner in an in vitro human microbiota model. We confirm the importance of personalized studies to better predict drug-associated-dysbiosis in vivo, linked to the host’s response to treatment.

FUNGAL PERITONITIS IN THE SETTING OF NECROTIZING PANCREATITIS

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Spontaneous fungal peritonitis (SFP) is a fungal infection of ascitic fluid, defined as over 250 polymorphic neutrophils (PMNs) and fungal growth on culture. It has a high mortality rate and often is recognized late in clinical courses. SFP is commonly associated with cirrhosis or advanced end stage liver disease. This is due to perturbation of mucosal blood flow and bacterial growth in the intestines, and changes in the permeability of the mucosal wall of the gastrointestinal tract (1-3). However, amongst patients without cirrhosis, there is limited data describing patients with SFP. Herein we describe a patient with no history of cirrhosis presenting with necrotizing pancreatitis and ileus who developed SFP. CASE PRESENTATION: A 60-year-old woman with a past medical history of hypertension presented to our hospital unresponsive with septic shock. She was intubated, resuscitated, placed on vasopressors and treated with broad spectrum antibiotics. Further evaluation with laboratory data demonstrated a lipase > 4000 u/L. Computed tomography imaging was consistent with necrotizing pancreatitis, ileus and small bowel obstruction. Her course was complicated by development of a large phlegmon requiring cystogastrostomy for drainage, in addition to total parenteral nutrition (TPN). Despite drainage and antibiotics she continued to have worsening abdominal distension and vasopressor requirement. An abdominal ultrasound demonstrated loculated ascites with septations (a). An abdominal drain was placed with subsequent drainage of 2.2 liters of moss-colored ascitic fluid (b), with over 2000 PMNs and growth of Candida albicans, Enterococcus faecalis and Stenotrophomonas maltophilia. Her antibiotics were adjusted to fluconazole, oxacillin and minocycline. With subsequent drainage and appropriate antibiotics, her vasopressor requirement resolved, she was liberated from the ventilator and her mental status improved. DISCUSSION: We describe a patient who develops SFP without its commonly known risk factors. We suspect the use of broad-spectrum antibiotics during the patient's hospital course, in conjunction with mucosal breakdown of the enteral tract in the setting of ileus and severe necrotizing pancreatitis created a favorable environment for fungal overgrowth. TPN is not known to cause SFP specifically, but with fungal infections in general. Antibiotics in the setting of TPN use may have caused further alterations of microbial balance, increased fungal dominance (4-6) and resulted in microbial translocation and polymicrobial peritonitis. CONCLUSIONS: It is critical to recognize SFP in patients without liver disease. Other less common risk factors such as antibiotics, perturbation of the enteral tract with diseases such as pancreatitis, ileus and TPN can increase one's risk of SFP. Early recognition and treatment can improve patient outcomes in patients presenting with septic shock. REFERENCE #1: Patel D. et al. Spontaneous fungal peritonitis as a rare complication of ascites secondary to cardiac cirrhosis: a case report. Am J Case Report. 2019; 20:1526-1529. REFERENCE #2: Shizuma T. Spontaneous bacterial and fungal peritonitis in patients with liver cirrhosis: a literature review. World J Hepatol. 2018 Feb 27; 10(2): 254-266. REFERENCE #3: Lahmer T., Brandi A., Rasch S., Schmid RM., Huber W. Fungal peritonitis: underestimated disease in critically ill patients with liver cirrhosis and spontaneous peritonitis. PLoS ONE. 2016 July 8; 11(7).4. Van Leeuwen PA, et al. Clinical significance of translocation. Gut. Jan 1994; 31(1 Suppl):S28-34). Doi: 10.1136/gut.35.1_suppl.s28.5. Bernhardt H, Zimmermann K, Knoke M. The continuous flow culture as an in vitro model in experimental mycology. Mycoses. 1999;42 Suppl 2:29-32. PMID: 10865900.6. Yang S. et al. A risk factor analysis of healthcare-associated fungal infections in an intensive are unit: a retrospective cohort study. BMC Infectious Diseases. 2013; 13(10) DISCLOSURES: No relevant relationships by Andrew Deitchman, source=Web Response No relevant relationships by Austin Hager, source=Web Response No relevant relationships by Diane Wang, source=Web Response

In vitro Digestibility, In situ Degradability, Rumen Fermentation and N Metabolism of Camelina Co-Products for Beef Cattle Studied with A Dual Flow Continuous Culture System of Camelina Co-Products for Beef Cattle.

Simple SummaryCurrently, vegetable protein sources such as soybean meal and rapeseed meal are expensive and with volatile prices. These economic circumstances are driving the research of potential new protein resources for beef cattle diets that can reduce the ration cost without compromising animal productive yields. As possible candidates, camelina meal and camelina expeller have been studied; they are co-products with a high protein percentage, obtained after oil extraction from the oil seeds of Camelina sativa. The objectives of this study were to characterize these camelina co-products and ascertain if they could be useful ingredients for beef cattle diets. The results indicate that the diets formulated with camelina meal and camelina expeller do not show differences in the efficiency of microbial protein synthesis compared to the current reference proteins, camelina meal diet being the most similar to soybean meal and rapeseed meal diets, and camelina expeller the diet with the highest fermentation potential. The results of soybean meal as an individual ingredient reveal more differences with camelina co-products. In vivo studies are necessary to draw conclusions, but in vitro results obtained suggest that camelina meal and camelina expeller are potential substitutes for rapeseed meal in beef cattle diets.Camelina meal (CM) and camelina expeller (CE) were compared with soybean meal (SM) and rapeseed meal (RM). Trial 1 consisted of a modified Tilley and Terry in vitro technique. Trial 2 was an in situ technique performed by incubating nylon bags within cannulated cows. Trial 3 consisted in dual-flow continuous culture fermenters. In Trial 1, CM, CE and RM showed similar DM digestibility and OM digestibility, and SM was the most digestible ingredient (p < 0.05). Trial 2 showed that CE had the numerically highest DM degradability, but CP degradability was similar to RM. Camelina meal had a DM degradability similar to SM and RM and had an intermediate coefficient of CP degradability. In Trial 3, CE diet tended to present a higher true OM digestibility than SM diet (p = 0.06). Total volatile fatty acids (VFA) was higher in CE and CM diets than in SM diet (p = 0.009). Crude protein degradation tended to be higher (p = 0.07), and dietary nitrogen flow tended to be lower (p = 0.06) in CE diet than in CM diet. The efficiency of microbial protein synthesis was not affected by treatment (p > 0.05). In conclusion, CE and CM as protein sources differ in CP coefficient of degradability but their results were similar to RM. More differences were detected with regard to SM.

Nutrients digestibility, metabolic parameters and milk production in postpartum Holstein cows fed pomegranate (Punica granatum L.) by-products silage under heat stress condition

Two experiments were conducted to determine the best level of pomegranate by-products silage (PBS) in dairy cows ration and then its effect on transition dairy cows under heat stress condition was studied. In the first experiment, corn silage (CS) was replaced with the incremental levels of PBS [0, 60, 120 and 180 g PBS/kg dry matter (DM)] in total mixed ration (TMR) and then the nutrient digestibility and rumen fermentation parameters were studied using a dual flow continuous culture system. In the second experiment, effects of feeding PBS (120 g PBS addition per kg DM of TMR) on in vivo nutrient digestibility, ruminal fermentation, blood metabolites and milk yield were investigated in 20 postpartum transition dairy cows under heat stress condition [average of temperature-humidity index (THI); 74.3 ± 2.58]. Fresh pomegranate by-products (PB) including peel and seed were ensiled in a bunker silo with addition 20 g urea/kg DM. Results of the first experiment showed that PBS had about 49 g phenolic compounds/kg DM. Replacing of CS in TMR with 60 g and 120 g PBS improved neutral detergent fiber (aNDF) and acid detergent fiber (ADF) digestibility. TMR supplemented with 120 g PBS produced greatest total volatile fatty acids (VFA) and molar proportion of propionate after fermentation, but molar proportion of acetate and acetate:propionate ratio were lowest. Results of second experiment showed that replacing of CS with 120 g PBS increased total milk production and energy corrected milk (ECM), but it decreased milk protein and lactose content. Feeding fresh cows with 120 g PBS increased their lying behavior compared to the control group (712.1 vs. 572.6 min. d). Cows fed 120 g PBS had higher ADF digestibility and lower crude protein (CP) digestibility than that the control group. Cows fed 120 g PBS numerically had greater serum glucose. It was concluded that replacing of CS with 120 g PBS had potential to increase milk yield in Holstein dairy cows under heat stress condition without any adverse effect on their blood metabolites or health.

Role of Dilution Rate and Nutrient Availability in the Formation of Microbial Biofilms.

We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates (0.09, 0.28, and 0.81 h-1). The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21× larger and its estimated growth rate 2.4× higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.

Effects of live yeast (Saccharomyces cerevisiae) and type of cereal on rumen microbial fermentation in a dual flow continuous culture fermentation system.

Live yeast additives may help optimize ruminal fermentation of high-grain diets, especially when the starch of the ration is highly fermentable. Eight dual flow continuous culture fermenters were used in a 2×2 factorial design in two replicated periods of 9days (six for adaptation and three for sampling) to determine the effect of live yeast and type of cereal on rumen microbial fermentation and nutrient digestibility. Main factors were the addition of live yeast: no yeast (NY) vs. 2×107 CFU of yeast/g of diet (LY); and type of cereal in the diet: corn (CO) vs. barley (BA). All fermenters were fed 80g dry matter/day of a 10-90 forage to concentrate diet and pH was allowed to fluctuate with an upper (6.6) and lower (5.5) limit. Treatment BA increased OM digestion, valerate proportion, peptides and ammonia N fractions, ammonia N flow, crude protein degradation and target copies of Megasphaera elsdenii; and decreased NDF digestion, propionate proportion, branched-chain VFA (BCVFA) concentration, AA-N fraction and nonammonia N flow. Treatment LY increased BCVFA and decreased ammonia N fraction and flow, and the target copies of Streptococcus bovis. Treatment LY decreased the slope of pH drop, the area under pH 6.0 and the gas production. These results suggest potential benefits of LY in stabilizing the fermentation of BA-based diets.

Temperature controlled dual hypoxic chamber design for in vitro ischemia experiments

In vitro ischemia models are designed to study various aspects of hypo-perfusion, focusing on the consequences of acute events under body temperature. Cold ischemia is less investigated even though the beneficial effects of cooling is expected. The aim of the present work was to develop a device modeling cold and warm ischemia in vitro. Oxygen-glucose deprivation was applied with continuous nitrogen flow and glucose-free cell culture media to mimic ischemia. The temperature in both chambers were independently set between 4 and 37°C. Samples were placed inside for the ischemic period, followed by a reperfusion stage under standard cell culture conditions. We tested rat calvaria bone pieces undergoing 1, 7, 12 and 24h of ischemia at 4 and 37°C. After 24h of reperfusion, cell number was measured with a tetrazolium cell viability assay. One hour of warm ischemia paradoxically increased the post-reperfusion cell count, while cold-ischemia had an opposite effect. After 7h of warm ischemia the cells were already unable to recover, while under cold ischemia 60% of the cells were still functioning. After 12h of cold ischemia 50% of the cells were still be able to recover, while at 24h even the low temperature was unable to keep the cells alive. The markedly different effect of warm and cold ischemia suggests that this newly designed system is capable of reliable and reproducible modeling of ischemic conditions. Moreover, it also enables deeper investigations in the pathophysiology of cold ischemia at cellular and tissue level.

Utilization of food waste in continuous flow cultures of the heterotrophic microalga Chlorella pyrenoidosa for saturated and unsaturated fatty acids production

Algal cells are known for improved accumulation of long chain fatty acids when grown in continuous flow cultures under conditions where nutrients, such as nitrogen and/or phosphorous, are limited. Food waste has been shown to be an appropriate source of carbon, nitrogen and phosphorous compounds to be used as nutrients in heterotrophic microalgal biomass production. Food waste, however, has unpredictable contents of the three mentioned compounds, which makes an application in continuous flow cultures where defined concentrations of nutrients are required challenging. Therefore, this study aimed on developing continuous flow cultures of the heterotrophic microalga Chlorella pyrenoidosa solely based on mixed restaurant food waste for improved fatty acid production. Food waste was enzymatically digested using amylolytic or proteolytic enzymes, and the obtained hydrolysate was used for the cultivation of C. pyrenoidosa in continuous flow cultures under nitrogen and/or phosphate limitation. In a nutrient sufficient batch culture, C. pyrenoidosa contained 103.8 mg g−1 lipids, while up to three times more lipids was found in biomass cultured under nutrient limited conditions. C. pyrenoidosa also accumulated up to 619.0 mg g−1carbohydrates. With decreasing dilution rate and increasing nutrient stress, the carbohydrate content could be decreased to 390.9 mg g−1. The outcomes of this study extended our understanding on how to utilize complex media in continuous flow cultures of algal cells which are carried out under specific nutrient limitation for improved fatty acid accumulation.

Engineering bio-inspired surface microstructures to control bacterial adhesion and biofilm growth

Event Abstract Back to Event Engineering bio-inspired surface microstructures to control bacterial adhesion and biofilm growth Dalal Asker1, 2, Nicolas Lavielle1 and Benjamin Hatton1 1 University of Toronto, Materials Science and Engineering, Canada 2 Alexandria University, Food Science & Technology Department, Egypt Introduction: Bacteria are highly effective at populating living or inanimate surfaces, which causes significant problems in the healthcare industry, such as hospital-acquired infections, and persistent biofilm infection of medical devices. There is much to learn on the roles of surface topology at nano and micro scales, wettability and local adhesion forces on bacterial attachment and growth. This talk will present recent results on non-wetting materials engineered to prevent bacterial attachment, based on superhydrophobic surface microtopographies[1]. Further, we have designed a novel class of ultra low-adhesion surfaces that incorporate a micron-scale thick lubricant layer immobilized at a surface, inspired by the Nepenthes pitcher plant[2],[3]. Materials and Methods: Herein we demonstrate the role for superhydrophobic non-wetting surfaces to control (short-term) cell attachment, and the role of surface feature size on cellular attachment. Micropost arrays were molded in polyurethane (PU) with increasing diameter, from 300 nm to 20 µm, from Si masters etched by photolithography. Samples were exposed to P. aeruginosa and E. coli for 5 min contact time, and compared to flat PU and commercial medical gloves. Silicone-based ‘slips’ samples were exposed to continuous flow culture of P. aeruginosa for up to 30 days. Results and Discussion: We have modelled bacterial cell attachment as particles acting under a balance of local surface tension forces[1]. Superhydrophobic surfaces, also bio-inspired based on non-wetting natural surfaces, were found to be highly effective to prevent bacterial cell attachment (reduced from 104 to 100 cells/mm2), when the micropost diameter was smaller than the cell size (1.5 µm or less), which points to a new paradigm of antimicrobial surface design. Silicone-based ‘slips’ surfaces were also found to effectively prevent bacterial cell adhesion (3 log to 4 log reduction in CFU), due to the inert, stabilized surface liquid interface (Fig 1)[4]. Conclusions: Engineering non-wetting microstructured surfaces can effectively prevent bacterial attachment by physical mechanisms, and can be effective for a broad range of cell types. As a result, these are not biologically-specific and are not susceptible to resistance. Figure 1. Suppression of bacterial cells attachment and biofilm formation by SLIPS under continuous flow conditions for up to 30 days. (A) Fluorescence imaging of P. aeruginosa overproducing Psl biofilm attached to the micro-structured PDMS control (a-e), and micro-structured PDMS SLIPS (f-l) surfaces, respectively.

Mitigation of in vitro hydrogen sulfide production using bismuth subsalicylate with and without monensin in beef feedlot diets.

The objective of this study was to determine if a sulfur binder, bismuth subsalicylate (BSS), alone or combined with monensin (MON) could decrease the production of HS by rumen microbes. In Exp. 1, two 24-h batch culture incubations were conducted using a substrate consisting of 50% corn, 40% distillers grains, 9.75% hay, and 0.25% mineral premix, on a DM basis. Five treatments including BSS concentrations of 0% (control), 0.5%, 1%, 2%, and 4% of DM were assigned in 5 replicates to 120-mL serum bottles containing rumen fluid, buffer, and 0.5 g of dietary substrate. Addition of 2% and 4% BSS decreased ( < 0.05) gas production, whereas all concentrations of BSS reduced ( < 0.05) HS production by 18%, 24%, 82%, and 99% for 0.5%, 1%, 2%, and 4% BSS, respectively. Final pH increased ( < 0.05) with 2% and 4% BSS treatments. At 4% of DM, BSS decreased ( < 0.05) total VFA concentration (m) and propionate (mol/100 mol) but increased acetate (mol/100 mol) and acetate to propionate ratio. Concentration of branched-chain VFA increased ( < 0.05) with the addition of 0.5% BSS, compared with the control. On the basis of these results, addition of BSS (1% of DM) and MON (5 mg/kg) were used to assess their effects on metabolism and HS release by rumen microbes in 8 dual flow continuous culture fermenters during two 10-d periods (Exp. 2). Treatments were arranged in a 2 × 2 factorial design. Substrate similar to that used in Exp. 1 was provided at 75 g DM/fermenter daily. Headspace HS concentration was reduced ( < 0.05) by 99% with BSS treatment but was not affected ( = 0.21) by MON. An overall increase ( < 0.05) in fermentation pH was found following addition of BSS. Addition of BSS increased ( < 0.05) digestion of NDF and ADF but decreased ( < 0.05) nonfiber carbohydrate digestion and total VFA concentration. Acetate and propionate (mol/100 mol) increased ( < 0.05) with BSS, whereas butyrate (mol/100 mol) and branched-chain VFA (m) decreased ( < 0.05). Addition of BSS increased ( < 0.05) NH-N concentration and NH-N outflow but decreased ( < 0.05) microbial N outflow. Results from this study showed no response to monensin addition, but BSS markedly reduced HS production and altered microbial fermentation during in vitro rumen fluid incubations.

Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs).

BackgroundContinuous-flow culture models are one tool for studying complex interactions between members of human fecal microbiotas because they allow studies to be completed during an extended period of time under conditions where pH, nutrient availability, and washout of waste products and dead cells can be controlled. Because many of the existing well-validated continuous-flow models are large and complex, we were interested in developing a simpler continuous-flow system that would allow microbial community dynamics to be examined in higher throughput while still maintaining complex microbial communities. To this end, we developed minibioreactor arrays (MBRAs), small volume bioreactors (15 ml) that allow simultaneous cultivation of up to 48 microbial communities in a single anaerobic chamber.ResultsWe used MBRA to characterize the microbial community dynamics of replicate reactors inoculated from three different human fecal donors and reactors seeded with feces pooled from these three donors. We found that MBRA could be used to efficiently cultivate complex microbial communities that were a subset of the initial fecal inoculum (15–25 % of fecal OTUs initially observed). After an initial acclimation period of approximately 1 week, communities in each reactor stabilized and exhibited day-to-day variation similar to that observed in stable mouse fecal communities. Replicate reactors were predominately populated by shared core microbial communities; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units (OTUs). Consistent with differences between fecal donors, MBRA communities present in reactors seeded with different fecal samples had distinct composition and structure.ConclusionsFrom these analyses, we conclude that MBRAs can be used to cultivate communities that recapitulate key features of human fecal communities and are a useful tool to facilitate higher-throughput studies of the dynamics of these communities.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0106-5) contains supplementary material, which is available to authorized users.

Evaluation of Surface Microtopography Engineered by Direct Laser Interference for Bacterial Anti-Biofouling

Modification of the biomaterial surface topography is a promising strategy to prevent bacterial adhesion and biofilm formation. In this study, we use direct laser interference patterning (DLIP) to modify polystyrene surface topography at sub-micrometer scale. The results revealed that three-dimensional micrometer structures have a profound impact on bacterial adhesion. Thus, line- and pillar-like patterns enhanced S. aureus adhesion, whereas complex lamella microtopography reduced S. aureus adhesion in static and continuous flow culture conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus adhesion both when the surface is coated with human serum proteins and when the material is implanted subcutaneously in a foreign-body associated infection model.

Enrichment of denitrifying methane-oxidizing microorganisms using up-flow continuous reactors and batch cultures.

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L−1·day−1 was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89–91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.

Characterization of Nontypable Haemophilus influenzae Isolates Recovered from Adult Patients with Underlying Chronic Lung Disease Reveals Genotypic and Phenotypic Traits Associated with Persistent Infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues1,2, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here, we developed a continuous flow culture apparatus (Nutrostat) for maintaining proliferating cells in low nutrient media for long periods of time and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNAi screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the upregulation of OXPHOS normally caused by glucose limitation as a result of either mtDNA mutations in Complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, anti-diabetic drugs that inhibit OXPHOS3,4, when cancer cells are grown in low glucose or as tumour xenografts. Remarkably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of Complex I function5. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.