Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms.

OBJECTIVE: Chlorhexidine mouthrinses are marketed in different formulations. This study aimed at investigating qualitative and quantitative changes in in-vitro multispecies oral biofilms, induced by different chlorhexidine-containing mouthrinses. BACKGROUND DATA: Earlier studies comparing chlorhexidine mouthrinses are either clinical studies or in-vitro studies assessing the antimicrobial efficacy of the mouthrinses. However, no clear investigations are available regarding ecological impact of different chlorhexidine formulations on in-vitro multispecies oral biofilms after rinsing with different chlorhexidine formulations. METHODS: Nine commercially available chlorhexidine mouthrinses were selected. Multispecies oral communities (14 species) were grown for 48 h in a Biostat-B Twin bioreactor. After that, they were used to develop biofilms on the surface of hydroxyapatite disks in 24-well pates for 48 h. Biofilms were then rinsed once or multiple times with the corresponding mouthrinse. Biofilms were collected before starting the rinsing experiment and every 24 h for 3 days and vitality quantitative PCR was performed. The experiment was repeated 3 independent times on 3 different days and the results were analyzed using a linear mixed model. RESULTS: The mouthrinses provoked different effects in terms of change in total viable bacterial load (VBL), ecology, and community structure of the multispecies biofilms. There was no relation between chlorhexidine concentrations, presence, or absence of cetylpyridinium chloride and/or alcohol, and the observed effects. Some tested chlorhexidine mouthrinses (MC, HG, HH, and HI) strongly lowered the total VBL (≈1007 Geq/ml), but disrupted biofilm symbiosis (≥40% of the biofilms communities are pathobionts). On the other hand, other tested chlorhexidine mouthrinses (MD, ME, and HF) had limited impact on total VBL (≥1010 Geq/ml), but improved the biofilm ecology and community structure (≤10% of the biofilms communities are pathobionts). CONCLUSION: Not all chlorhexidine mouthrinses have the same effect on oral biofilms. Their effect seems to be strongly product dependent and vary according to their compositions and formulations.

Continuous H2/CO2 fermentation for acetic acid production under transient and continuous sulfide inhibition.

Waste gas fermentation powered by renewable H2 is reaching kiloton scale. The presence of sulfide, inherent to many waste gases, can cause inhibition, requiring additional gas treatment. In this work, acetogenesis and methanogenesis inhibition by sulfide were studied in a 10-L mixed-culture fermenter, supplied with CO2 and connected with a water electrolysis unit for electricity-powered H2 supply. Three cycles of inhibition (1.3 mM total dissolved sulfide (TDS)) and recovery were applied, then the fermenter was operated at 0.5 mM TDS for 35 days. During operation at 0.5 mM TDS the acetate production rate reached 7.1 ± 1.5 mmol C L-1 d-1. Furthermore, 43.7 ± 15.6% of the electrons, provided as H2, were distributed to acetate and 7.7 ± 4.1% to butyrate, the second most abundant fermentation product. Selectivity of sulfide as inhibitor was demonstrated by a 7 days lag-phase of methanogenesis recovery, compared to 48 h for acetogenesis and by the less than 1% electrons distribution to CH4, under 0.5 mM TDS. The microbial community was dominated by Eubacterium, Proteiniphilum and an unclassified member of the Eggerthellaceae family. The taxonomic diversity of the community decreased and conversely the phenotypic diversity increased, during operation. This work illustrated the scale-up potential of waste gas fermentations, by elucidating the effect of sulfide as a common gas impurity, and by demonstrating continuous, potentially renewable supply of electrons.

A Viability Quantitative PCR Dilemma: Are Longer Amplicons Better?

The development of viability quantitative PCR (v-qPCR) has allowed for a more accurate assessment of the viability of a microbial sample by limiting the amplification of DNA from dead cells. Although valuable, v-qPCR is not infallible. One of the most limiting factors for accurate live/dead distinction is the length of the qPCR amplicon used. However, no consensus or guidelines exist for selecting and designing amplicon lengths for optimal results. In this study, a wide range of incrementally increasing amplicon lengths (68 to 906 base pairs [bp]) was used on live and killed cells of nine bacterial species treated with a viability dye (propidium monoazide [PMA]). Increasing amplicon lengths up to approximately 200 bp resulted in increasing quantification cycle (Cq) differences between live and killed cells while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased the Cq difference but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. IMPORTANCE Viability quantitative PCR (v-qPCR) has evolved into a valuable, mainstream technique for determining the number of viable microorganisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently, there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live/dead distinction.

Mainstream partial nitritation/anammox with integrated fixed-film activated sludge: Combined aeration and floc retention time control strategies limit nitrate production.

Implementation of mainstream partial nitritation/anammox (PN/A) can lead to more sustainable and cost-effective sewage treatment. For mainstream PN/A reactor, an integrated fixed-film activated sludge (IFAS) was operated (26 °C). The effects of floccular aerobic sludge retention time (AerSRTfloc), a novel aeration strategy, and N-loading rate were tested to optimize the operational strategy. The best performance was observed with a low, but sufficient AerSRTfloc (~7d) and continuous aeration with two alternating dissolved oxygen setpoints: 10 min at 0.07-0.13 mg O2 L-1 and 5 min at 0.27-0.43 mg O2 L-1. Nitrogen removal rates were 122 ± 23 mg N L-1 d-1, and removal efficiencies 73 ± 13%. These conditions enabled flocs to act as nitrite sources while the carriers were nitrite sinks, with low abundance of nitrite oxidizing bacteria. The operational strategies in the source-sink framework can serve as a guideline for successful operation of mainstream PN/A reactors.

Microbial enrichment, functional characterization and isolation from a cold seep yield piezotolerant obligate hydrocarbon degraders.

Deep-sea environments can become contaminated with petroleum hydrocarbons. The effects of hydrostatic pressure (HP) in the deep sea on microbial oil degradation are poorly understood. Here, we performed long-term enrichments (100 days) from a natural cold seep while providing optimal conditions to sustain high hydrocarbon degradation rates. Through enrichments performed at increased HP and ambient pressure (AP) and by using control enrichments with marine broth, we demonstrated that both pressure and carbon source can have a big impact on the community structure. In contrast to previous studies, hydrocarbonoclastic operational taxonomic units (OTUs) remained dominant at both AP and increased HP, suggesting piezotolerance of these OTUs over the tested pressure range. Twenty-three isolates were obtained after isolation and dereplication. After recultivation at increased HP, an Alcanivorax sp. showed promising piezotolerance in axenic culture. Furthermore, preliminary co-cultivation tests indicated synergistic growth between some isolates, which shows promise for future synthetic community construction. Overall, more insights into the effect of increased HP on oil-degrading communities were obtained as well as several interesting isolates, e.g. a piezotolerant hydrocarbonoclastic bacterium for future deep-sea bioaugmentation investigation.

Effect of water temperature on cellular stress responses in meagre (Argyrosomus regius).

Μeagre (Argyrosomus regius) is a newly introduced species in the aquaculture sector characterized by high growth rates and good nutritional value. However, the optimum conditions such as water temperature for the rearing of this species are still under research. The aim of the present study was to investigate several biochemical responses in the heart, muscle, liver, and intestine of meagre reared under different water temperatures (17, 23, and 26 °C) in comparison to the initial acclimation temperature at 20 °C. To assess its cellular stress response, the molecular markers of Hsp70 and Hsp90 induced levels, p38 MAPK and p44/42 MAPK phosphorylated levels, ubiquitin conjugates, and Bcl-2 levels were determined. Moreover, to investigate its antioxidant defense, the activities of superoxide dismutase, catalase, and glutathione reductase were measured. The obtained results in meagre showed a distinct tissue-specific response regarding the variations observed in the studied parameters under different acclimation temperatures. Specifically, most examined tissues presented a decrease in the levels of the examined markers compared to 20 °C, while in the intestine, an increase of Hsp70 and p38 MAPK levels was observed at 23 and 26 °C. However, data on the final weight and length of the fish lead to the conclusion that high water temperatures such as 26 °C might be the best for the rearing of this species. Therefore, it is assumed that the observed differential molecular responses may act in a cell-protective way at all temperatures, irrespectively of the effect of temperature on growth parameters.

Homoacetogenesis and microbial community composition are shaped by pH and total sulfide concentration.

Biological CO2 sequestration through acetogenesis with H2 as electron donor is a promising technology to reduce greenhouse gas emissions. Today, a major issue is the presence of impurities such as hydrogen sulfide (H2 S) in CO2 containing gases, as they are known to inhibit acetogenesis in CO2 -based fermentations. However, exact values of toxicity and inhibition are not well-defined. To tackle this uncertainty, a series of toxicity experiments were conducted, with a mixed homoacetogenic culture, total dissolved sulfide concentrations ([TDS]) varied between 0 and 5 mM and pH between 5 and 7. The extent of inhibition was evaluated based on acetate production rates and microbial growth. Maximum acetate production rates of 0.12, 0.09 and 0.04 mM h-1 were achieved in the controls without sulfide at pH 7, pH 6 and pH 5. The half-maximal inhibitory concentration (IC50 qAc ) was 0.86, 1.16 and 1.36 mM [TDS] for pH 7, pH 6 and pH 5. At [TDS] above 3.33 mM, acetate production and microbial growth were completely inhibited at all pHs. 16S rRNA gene amplicon sequencing revealed major community composition transitions that could be attributed to both pH and [TDS]. Based on the observed toxicity levels, treatment approaches for incoming industrial CO2 streams can be determined.

Oral biofilms exposure to chlorhexidine results in altered microbial composition and metabolic profile.

Oral diseases (e.g., dental caries, periodontitis) are developed when the healthy oral microbiome is imbalanced allowing the increase of pathobiont strains. Common practice to prevent or treat such diseases is the use of antiseptics, like chlorhexidine. However, the impact of these antiseptics on the composition and metabolic activity of the oral microbiome is poorly addressed. Using two types of oral biofilms-a 14-species community (more controllable) and human tongue microbiota (more representative)-the impact of short-term chlorhexidine exposure was explored in-depth. In both models, oral biofilms treated with chlorhexidine exhibited a pattern of inactivation (>3 log units) and fast regrowth to the initial bacterial concentrations. Moreover, the chlorhexidine treatment induced profound shifts in microbiota composition and metabolic activity. In some cases, disease associated traits were increased (such as higher abundance of pathobiont strains or shift in high lactate production). Our results highlight the need for alternative treatments that selectively target the disease-associated bacteria in the biofilm without targeting the commensal microorganisms.

Synergistic Exposure of Return-Sludge to Anaerobic Starvation, Sulfide, and Free Ammonia to Suppress Nitrite Oxidizing Bacteria.

A key step toward energy-positive sewage treatment is the development of mainstream partial nitritation/anammox, a nitrogen removal technology where aerobic ammonium-oxidizing bacteria (AerAOB) are desired, while nitrite-oxidizing bacteria (NOB) are not. To suppress NOB, a novel return-sludge treatment was investigated. Single and combined effects of sulfide (0-600 mg S L-1), anaerobic starvation (0-8 days), and a free ammonia (FA) shock (30 mg FA-N L-1 for 1 h) were tested for immediate effects and long-term recovery. AerAOB and NOB were inhibited immediately and proportionally by sulfide, with AerAOB better coping with the inhibition, while the short FA shock and anaerobic starvation had minor effects. Combinatory effects inhibited AerAOB and NOB more strongly. A combined treatment of sulfide (150 mg S L-1), 2 days of anaerobic starvation, and FA shock (30 mg FA-N L-1) inhibited AerAOB 14% more strongly compared to sulfide addition alone, while the AerAOB/NOB activity ratio remained constant. Despite no positive change being observed in the immediate-stress response, AerAOB recovered much faster than NOB, with a nitrite accumulation ratio (effluent nitrite on nitrite + nitrate) peak of 50% after 12 days. Studying long-term recovery is therefore crucial for design of an optimal NOB-suppression treatment, while applying combined stressors regularly may lead toward practical implementation.