Bacterial Cell Counts Research Articles

Inhibition of Staphylococcus aureus biofilm by quercetin combined with antibiotics

This study aimed to investigate the effects of combined quercetin and antibiotics on the bacteriostatic activity and biofilm formation of Staphylococcus aureus. Optimal concentrations of quercetin and antibiotics (tetracycline and doxycycline) for inhibiting biofilm formation were determined using the Fractional Inhibitory Concentration Index and Minimum Biofilm Inhibitory Concentration assays. The impact of the drug combinations on biofilm clearance at various formation stages was determined using crystal violet staining, scanning electron microscopy and confocal laser microscopy. The results indicated that quercetin enhanced the bactericidal effect of tetracycline antibiotics against S. aureus. The combination significantly reduced both the metabolic activity within S. aureus biofilms and the production of biofilm matrix components. Scanning electron microscopy and confocal laser microscopy confirmed that the combination treatment significantly reduced bacterial cell counts within the biofilm. Quercetin treatment significantly increased the sensitivity of biofilms to antibiotics, supporting its potential application as a novel antibiotic synergist.

Interaction between human dental microbiome and the host gingival model

Objective: Interactions between the dental biofilm and host are important to study infectious oral diseases such as dental caries and periodontitis. Previous studies have reported the reaction of a host gingival model co-cultured with bacteria or biofilms. However, these studies only report results of a gingival model, and a biofilm co-cultured with a gingival model has never been investigated although the microbiome of dental biofilms is associated with maintenance of health and onset of infectious oral diseases. Therefore, we aimed to investigate the reaction of a gingival model and experimental biofilm. An in situ dental biofilm model, which enables biofilm formation in the oral cavity, was used to prepare experimental biofilms. Methods: The in situ biofilm was co-cultured with a reconstructed gingival model (RHG). We performed 16S rRNA sequence analysis, metagenomic function prediction analysis, total bacterial cell counts using quantitative polymerase chain reaction, and confocal laser microscopic observation in addition to gene expression and histological investigation of RHG. Results: We found a change in Veillonella abundance in the in situ biofilms co-cultured with RHG. Metagenomic function prediction analysis revealed that the activity of certain pathways associated with amino acid production, lactic acid fermentation, and glycolysis system significantly increased in biofilms co-cultured with RHG. Conclusion: The microbiome and functions of dental biofilms can be affected by host gingival tissues. Our findings will contribute to the establishment of a host–biofilm interaction model to understand the pathophysiology of oral infectious diseases.

Insights into the role of hexa-bacterial consortium for bioremediation of soil contaminated with chlorantraniliprole

This study evaluates the efficacy of discrete bacterial consortia in bioremediating sandy loamy soil deliberately contaminated with 20 mg/kg of chlorantraniliprole (CAP). It monitors alterations in total bacterial populations and CO2 emissions, tracking residual CAP levels through UV scanning and HPLC analysis. Six active bacterial degraders (four Bacillus strains (B. subtilis subsp. subtilis AZFS3, B. pumilus AZFS5, B. mojavensis AZFS15, and B. paramycoides AZFS18), one Alcaligenes strain (A. aquatilis KZFS11), and one Pseudomonas strain (P. aeruginosa KZFS4)) were used in single or combined preparations and grown on trypticase soy broth for 24 h at 30 °C before preparing the inoculants and adjusting the bacterial cell count to 107 CFU/dwt g soil. The bacterial consortia were added to the CAP-contaminated soil and incubated for 20 days at 30 °C. The di-, tetra-, and hexa-bacterial consortia recorded the highest levels of viable bacteria, reaching their peak after 3 to 11 days of incubation. Then, they declined to the minimum levels at the end of the 20 days, which coincided with their complete removal of CAP from the soil. At the end of the incubation period (20 days), the CAP was mainly biodegraded, scoring biodegradation rates of 90.05%, 93.65%, and 98.65% for T3, T4, and T5, respectively. This concurred with the highest average CO2 production. Based on the results of the HPLC analysis, the hexa-bacterial consortium T5 demonstrated the highest rate of CAP biodegradation (99.33%) after a 20-day incubation period, resulting in the lowest residual level of CAP in the soil (0.67%). Bioinformatic analysis predicted that the CAP biodegradation pathway reached CO2 and H2O. Under optimized conditions, the hexa-bacteria consortium is the most effective CAP biodegraded and is recommended as an eco-friendly treatment for eliminating CAP pollution in the field.

Comparative evaluation of surface roughness and bacterial adhesion on two bioactive cements: an in-vitro study

BackgroundDental restorative materials are recognized as artificial niches that facilitate the adherence and accumulation of oral microorganisms. To mitigate oral diseases and extend the lifespan of restorations, it is advantageous to use dental materials that exhibit low susceptibility to bacterial adhesion.ObjectiveTo evaluate and compare bacterial adhesion on two bioactive restorative materials, a glass hybrid restorative, and an alkasite with a nanohybrid resin composite as a positive control. The secondary objectives were to compare the surface roughness (SR) of the materials and determine the correlation between the bacterial adhesion and the SR.Materials and methodsThe samples consisted of 33 polished discs of each material: Group A: Tetric® N-Ceram (nanohybrid resin composite), Group B: Equia Forte™ HT Fil (glass hybrid restorative) and Group C: Cention N® (alkasite). Streptococcus mutans cultures were inoculated and after 24-hours of incubation, bacterial adhesion was measured by measuring optical density (OD) and number of colony forming units (CFUs). After 96-hours incubation, the bacterial cell count was determined using scanning electron microscopy (SEM). SR was assessed using surface profilometer.ResultsAlkasite had significantly lower OD and CFUs (p < 0.001 and p = 0.015 respectively). According to the SEM analysis, the glass hybrid restorative had lower mean bacterial cell count with no significant difference between the groups. The nanohybrid composite had the smoothest surface that was significantly lower than the alkasite and glass hybrid restorative (p = 0.002). None of the groups demonstrated a correlation between bacterial adhesion and SR.ConclusionAlkasite impedes bacterial adhesion better than the glass hybrid restorative and nanohybrid composite, while smoother surfaces are achieved with the nanohybrid composite.

Repurposing simvastatin for treatment of Klebsiella pneumoniae infections: in vitro and in vivo study

Simvastatin had minimum inhibitory concentrations of 32 to 128 µg/mL against Klebsiella pneumoniae isolates and hindered the biofilm-formation ability of 58.54% of the isolates. It considerably diminished the bacterial cell counts in the biofilms as revealed by scanning electron microscope. Also, qRT-PCR revealed a downregulation of the biofilm genes (bcsA, wza, and luxS) by simvastatin in 48.78% of the isolates. Moreover, simvastatin has significantly improved the survival of mice and decreased the burden of bacteria in the infected lungs. Also, the histological architecture was substantially improved in the simvastatin-treated group, as the alveolar sacs and bronchioles appeared normal with minimal collagen fiber deposition. The immunohistochemical studies exposed that the TNF-α, NF-kβ, and COX-2 immunostaining considerably declined in the simvastatin-treated group. Furthermore, ELISA exposed that both IL-1β and IL-6 were considerably diminished in the lungs of the simvastatin-treated group.

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

MMO-induced batch and pilot-scale electro-oxidation treatment of municipal wastewater.

The present research aimed to explore the durability of MMO electrodes through electro-oxidation (EO) in purifying secondary treated actual sewage wastewater using batch and pilot-scale setups. The main aim is to inactivate bacteria in sewage treatment plants before they are released into the environment, thus contaminating water and soil. Process parameters such as current density (j), NaCl dose (n), and treatment time (t) were optimized using response surface methodology in a lab-scale EO reactor under batch conditions. The results showed that optimization of current density at 5.90mA/cm2 and NaCl concentration at 1.31g/L led to 93.90% of bacterial inactivation (Q1) within 8min of treatment and 0.48 kWh/m3 energy consumption (Q2). Biological analysis was conducted to validate bacterial cell destruction and count coliform bacteria in the EO-treated sewage wastewater. XRD, cyclic voltammetry studies, and FE-SEM/EDS analysis were done to confirm the MMO anode's durability and stability after 100 recycles. The study prioritized bacterial inactivation along with organic matter degradation. Besides that, a small pilot-scale study on the actual sewage wastewater with a volume of 10-50 L was done in batch mode under previously optimized conditions to analyze the efficacy of the MMO anodes in terms of bacterial inactivation.

Tracing pesticide dynamics: High resolution offers new insights to karst groundwater quality

Generally, karst aquifers and springs are highly susceptible to contamination due to the high permeability and, therefore, groundwater flow velocities. The often thin soil cover, accompanied by dolines, can lead to fast infiltration of precipitation water loaded with mobilized contaminants such as pesticides and their transformation products. To date, continuous, temporally highly resolved in-situ monitoring to decipher concentration dynamics for a broad range of pesticides is missing. Therefore, a transportable HPLC-HRMS/MS system (MS2field) was positioned at two karst study sites in the Swiss Jura. Water samples were collected and analyzed for pesticides and their transformation products in-situ every 20 min for 6 weeks in 2021 and 8 weeks in 2022. During the spraying season in 2021, six rain events at site 1 and three at site 2 in 2022 were captured. Concurrently, the water quality parameters electrical conductivity, pH, nitrate, turbidity, and water level, were monitored continuously at high temporal resolution. Further, bacterial cell counts were monitored via online flow cytometry.In 2021, several pesticides and pesticide transformation products were detected in peak concentrations after rain events, of which metamitron showed the highest concentration of up to 1000 ng/L. In one rain event, the Swiss federal and EU drinking water limit of 100 ng/L was exceeded for up to 38 h. Compared with highly frequent MS2field samples collected every 20 min, 42-hours composite samples severely underestimated peak concentrations for all compounds, especially for labile ones. Therefore, it was demonstrated that exceedences of the regulatory limit would have been missed if just composite sampling would have been conducted. Peak concentrations of pesticides coincided with peaks in nitrate concentration and bacterial cell counts following rain events. The correlation analysis showed strong correlations between the three analyzed contaminants (pesticides, nitrate and bacteria), and the proxy parameters electrical conductivity, and pH. The investigation of a second spring revealed similar dynamics indicating that these can be expected in other karst aquifers as well.

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Microbial assessment of the ecological linkage between a red tide of Karenia brevis and bottom water anoxia off the coast of Fort Myers and Sanibel Island, Florida

Coastal ecosystem management necessitates a comprehensive understanding of the physical, chemical, and biological components and their interplay with socio-economic activities. Red tides of Karenia brevis reoccur almost annually on the West Florida Shelf and cause significant human and environmental health problems that often result in negative economic impacts. It is crucial that we understand the processes and dynamics of both phytoplankton and microbial communities as conditions necessary for the formation of hypoxia and anoxia in the coastal ecosystem. With the aim of identifying key microbial communities responsible for anoxia, we investigated the 2017–2018 Karenia bloom. Various water quality parameters, phytoplankton, and microbial community compositions were studied at eight sites on the West Florida Shelf. K. brevis densities were highest at the site of the plume of the Caloosahatchee River. At the site with the highest K. brevis abundance (6.3 ×106 cells/L), dissolved oxygen (DO) was supersaturated (303 %) at the surface, and the chlorophyll a concentration was 118 µg/L. The highest bacterial cell count (9.0 ×106 cells/mL) and the lowest bacterial diversity were also observed at this site. The bacterial community determined by high-throughput amplicon sequencing at this bloom site was unique and dominated by Bacteroidetes, which occupied 65 % of the relative abundance of the microbial community. The bottom water was generally hypoxic (mean ± SD: 0.98 ± 1.13 mg/L), with select stations having DO as low as 0.06 mg/L (anoxic). The anoxic bottom water harbored unique microbial taxa that were associated with the low oxygen conditions. Significant negative correlations were found between DO and the relative abundance of Deltaproteobacteria, Firmicutes, Spirochaetes, and unclassified Proteobacteria. Our investigation lays the groundwork for subsequent studies delving into the responses of phytoplankton and microbial communities to disturbances caused by Karenia blooms, as well as future strategies for mitigation and ecosystem restoration.

Meta-Genomic Analysis of Different Bacteria and Their Genomes Found in Raw Buffalo Milk Obtained in Various Farms Using Different Milking Methods.

Milking methods have significant impacts on the microbiological composition, which could affect the quality of raw buffalo milk. Hence, the current study was conducted on the impact of milking methods on microorganisms in buffalo tank raw milk from 15 farms in Guangxi, China. The farms were divided into two groups based on the milking method: mechanical milking (MM, n = 6) and hand milking (HM, n = 9). Somatic cell counts, bacterial cell counts and nutrients of the raw buffalo milk samples were analyzed. The comparison of raw buffalo milk samples was analyzed using metagenomic sequencing to detect any differences between the two groups. There was no significant difference in the basic nutritional compositions and somatic cell count of raw buffalo milk between the two milking methods. However, the HM samples had significantly higher bacterial counts and diversity compared to the MM samples. The results showed that Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the major microbes present in canned raw buffalo milk. However, the differences between the two milking methods were the relative abundance of core microorganisms and their potential mastitis-causing genera, including the content of antibiotic-resistance genes and virulence genes. Our study revealed that Staphylococcus spp. and Streptococcus spp. were significantly more abundant in the MM group, while Klebsiella spp. was more abundant in the HM group. Regardless of the milking method used, Pseudomonas spp. was identified as the primary genus contributing to antibiotic resistance and virulence genes in canned raw buffalo milk. These findings affirm that there are differences in the microbial and genomic levels in canned raw milk. To prove the functional roles of the discovered genes and how these genes affect milk quality, further research and experimental validation are necessary.

Safeguarding reverse osmosis membrane integrity in drinking water production: A pilot study using 2 native bacterial communities and viruses in surface water as integrity indicators

Water treatment train including a reverse osmosis barrier is capable of producing high-quality water coupled with microbial safety. However, analytically proving this with log removal value (LRV) > 3 has remained a challenge. Conservative techniques that quantify salt or sulfate ion passage as virus surrogates are currently used to credit the membrane system for its microbial removal capacity. These methods are insensitive, and it is now understood that the transport mechanism correlation between these surrogates with the actual passage of viruses is weak. There is also a huge gap between the current log credit assigned (using spiking of surrogates in feed) and the actual log removal that can be proven in the drinking water plant. In this paper, we introduce two techniques that exploit the high concentration of microbial communities in fresh surface water by quantifying bacteria through flow cytometry (FC) and native viruses through qPCR assays. These two methods are compared to LRV of conductivity, TOC, and fluorescent markers. That high concentration of bacterial community allowed for LRV > 4 by flow cytometry in an intact module. After inflicting damage with a 1.5 mm defect, the FC method detected the damage, and a bacterial cell count >103 cell count/ml was measured in the permeate, while conductivity lacked sensitivity to monitor any integrity loss. Furthermore, after chlorine exposure to the membrane, both bacteria and virus detected no leakage, proving the membrane's intactness and also demonstrating the effect of chlorination on the passage of small solutes. The implementation of the two microbial quantifying methods in full-scale water treatment plants facilitates higher sensitivity in membrane integrity loss than currently used methods.

3D Melt-Extrusion Printing of Medium Chain Length Polyhydroxyalkanoates and Their Application as Antibiotic-Free Antibacterial Scaffolds for Bone Regeneration.

In this work, we investigated, for the first time, the possibility of developing scaffolds for bone tissue engineering through three-dimensional (3D) melt-extrusion printing of medium chain length polyhydroxyalkanoate (mcl-PHA) (i.e., poly(3-hydroxyoctanoate-co-hydroxydecanoate-co-hydroxydodecanoate), P(3HO-co-3HD-co-3HDD)). The process parameters were successfully optimized to produce well-defined and reproducible 3D P(3HO-co-3HD-co-3HDD) scaffolds, showing high cell viability (100%) toward both undifferentiated and differentiated MC3T3-E1 cells. To introduce antibacterial features in the developed scaffolds, two strategies were investigated. For the first strategy, P(3HO-co-3HD-co-3HDD) was combined with PHAs containing thioester groups in their side chains (i.e., PHACOS), inherently antibacterial PHAs. The 3D blend scaffolds were able to induce a 70% reduction of Staphylococcus aureus 6538P cells by direct contact testing, confirming their antibacterial properties. Additionally, the scaffolds were able to support the growth of MC3T3-E1 cells, showing the potential for bone regeneration. For the second strategy, composite materials were produced by the combination of P(3HO-co-3HD-co-HDD) with a novel antibacterial hydroxyapatite doped with selenium and strontium ions (Se-Sr-HA). The composite material with 10 wt % Se-Sr-HA as a filler showed high antibacterial activity against both Gram-positive (S. aureus 6538P) and Gram-negative bacteria (Escherichia coli 8739), through a dual mechanism: by direct contact (inducing 80% reduction of both bacterial strains) and through the release of active ions (leading to a 54% bacterial cell count reduction for S. aureus 6538P and 30% for E. coli 8739 after 24 h). Moreover, the composite scaffolds showed high viability of MC3T3-E1 cells through both indirect and direct testing, showing promising results for their application in bone tissue engineering.

The Interaction between Canine Semen Bacteria and Semen Quality Parameters.

Assessing canine semen quality helps to detect infertility in males, but identifying factors that influence canine semen quality is a complicated task. The objective of this study was the assessment of the potential influence of bacteria found in canine semen samples on the characteristics of dogs' semen. In this study, semen samples were collected manually from 30 dogs and subjected to a comprehensive examination. The results of sperm motility, concentration, viability, and morphology were statistically analysed in relation to the number of bacteria in the semen (CFUs/mL) and the seminal microbiota. Samples with an increased bacterial count per millilitre were associated with lower-quality sperm motility (p < 0.05). The most frequently isolated bacterial genera from the analysed semen samples were Staphylococcus spp. (26.0%), Corynebacterium spp. (17.8%), and Streptococcus spp. (16.4%). The presence of β-haemolytic Escherichia coli bacteria was linked to suboptimal semen samples, characterised by significantly reduced semen viability and a lower proportion of morphologically normal spermatozoa (p < 0.05). Corynebacterium spp. was associated with reduced bacterial load and superior semen quality (p < 0.01). These findings highlight the importance of bacterial cell counts and microbiota diversity in relation to various factors influencing canine semen quality, providing a more comprehensive understanding of canine reproductive well-being.

Migrating ripples create streambed heterogeneity altering microbial diversity and metabolic activity

AbstractSandy sediments of lowland streams are typically transported at low flow in the form of migrating ripples. In these bedforms, microbial communities spanning all trophic guilds (heterotrophic bacteria, fungi, photoautotrophic and phagotrophic protists) are exposed to highly frequent moving–resting cycles of sediment grains. Up to date, it is unknown to what extent ripple migration impacts community metabolism and composition as well as the vertical zonation of sediment‐associated multitrophic microbial communities compared to stationary sediments. We hypothesize that, as a result of mechanical abrasion and limited light supply, migrating ripple sediments have lower microbial abundance, diversity, metabolism and resource acquisition and no vertical zonation compared to stationary sediments. We collected samples from five lowland streams in north‐eastern Germany between May and June 2020. The coarser and better sorted sediments of migrating ripples had a higher oxygen concentration and less organic matter than stationary sediments. Photosynthetic pigments, potential extracellular enzyme activities, bacterial cell counts, and fungal gene copies were lower in migrating ripples than in stationary sediments. In contrast, cell‐specific bacterial production was higher in migrating ripples. Metabarcoding revealed that bedform migration was important in shaping the community structure of bacteria, fungi, and phagotrophic protists. Dry mass‐related net community production, respiration, and bacterial production were higher in superficial compared to underlying layers irrespective of sediment transport. By modulating the abundance, diversity, and structure of different trophic guilds of microbial communities and their resource acquisition, migrating bedforms create streambed heterogeneity, shaping regional biodiversity and the flow of matter through the benthic food web.

Optimized Novel Antibacterial Production from Geobacillus kaustophilus Tm6T2 (a) as Treatment for Salmonella typhimurium

Geobacillus sp. is recognized for its potential to produce bacteriocins, antibacterial substances that hold promise in addressing gastrointestinal illnesses. This study aimed to optimize the medium and pH conditions for producing antibacterial substances by Geobacillus kaustophilus Tm6T2 (a). The research employed a descriptive and experimental methodology. Growth studies were conducted in Mueller Hinton Broth with CaCl2 and MgSO4 and Nutrient Broth with KCl and MgCl2 across 6, 7, and 8 pH values. Subsequently, antibacterial substance production was achieved at the late logarithmic phase and was assessed against the gastrointestinal pathogen Salmonella typhimurium. Interestingly, findings indicated that antibacterial substance production might not solely correlate with bacterial cell count. Despite a lower bacterial cell count, the highest inhibition zone against S.typhimurium was observed at 13.11 mm in NB salt at pH 8. Analytical results show that the variation of pH and both mediums significantly affects the presence of the inhibition zone (p &lt; 0.10). This finding suggests the complexity of factors influencing antibacterial activity. Overall, the optimum condition for antibacterial production in G.kaustophilus Tm6T2(a) was identified at pH 8 using NB salt. These findings have potential implications for developing antibacterial solutions targeting gastrointestinal pathogens.

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole

The presence and accumulation of both, plastics and antibiotics in soils may lead to the colonization, selection, and propagation of soil bacteria with certain metabolic traits, e.g., antibiotic resistance, in the plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and degrade 13C-labeled sulfamethoxazole (SMX). Using single-cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polyethylene (PE) and polystyrene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse microorganisms were colonizing both plastic types, with a slight preference for PE substrate. CARD-FISH bacterial cell counts on PE and PS surfaces formed under SMX amendment ranged from 5.36 × 103 to 2.06 × 104, and 2.06 × 103 to 3.43 × 103 hybridized cells mm−2, respectively. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom%, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 ‰). Independent Mann-Whitney U test showed a significant difference between the control plastisphere samples incubated without SMX and those in 13C-SMX incubations (P < 0.001). Our results provide direct evidence demonstrating, at single-cell level, the capacity of bacterial colonizers of plastics to assimilate 13C-SMX from contaminated soils. These findings expand our knowledge on the role of soil-seeded plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

Tissue Factor Maintains Lung Epithelial Barrier Integrity in Acute Lung Injury Through Cell Adhesion

Background. Acute Respiratory Distress Syndrome (ARDS) is a common cause of acute respiratory failure. While loss of lung epithelial barrier integrity is a pathologic mechanism of ARDS, little is known about the factors that regulate epithelial barrier integrity in the lung. Our previously published mouse studies showed that deletion of alveolar type II epithelial cell Tissue Factor (TF) caused impaired lung barrier integrity in models of Acute Lung Injury (ALI). TF is an integral membrane protein that both initiates the extrinsic coagulation cascade and serves several non-coagulant functions. Notably, TF promotes cell adhesion through interactions with cell surface integrin heterodimers, comprised of individual α and β integrin subunits. As such, we hypothesize that epithelial TF is necessary for maintaining lung barrier integrity and that its overexpression is protective in ALI through increased cell adhesion. Methods. To determine whether supraphysiologic overexpression of TF in the lung epithelium can enhance barrier integrity we created a novel transgenic mouse in which TF was inducibly overexpressed in the lung epithelium (TFEpi+) using a CMV-TetO promoter and crossed with SPC-rtTA59 mice. High alveolar epithelial TF expression compared to wild-type littermates (WT) was confirmed through immunohistochemistry and western blot analysis after one week of doxycycline in drinking water. To induce ALI, mice were intranasally infected with 2000 colony forming units of Klebsiella pneumoniae (KP) or PBS control. At 24-hours post infection, mice were euthanized, lung tissue was collected, and a bronchoalveolar lavage (BAL) was performed. BAL protein, clot time, and leukocyte influx were measured. Lung wet-to-dry weight ratios and bacterial burden were measured. To better understand TF regulation of β1 integrin function, TF-knockout A549 cells were created using CRISPR and cultured on specific β1 integrin ligands (Collagen I, IV, Laminin-511, and Fibronectin). On-cell western blots and crystal violet were used to assess cell surface β1 integrin expression and cell adhesion. Results. TFEpi+ mice showed higher lung TF protein expression (median 38031 [IQR 25033, 57948] vs 1801 [IQR 804.3, 2083] expression normalized to total protein; p=0.0016) compared to WT. TFEpi+ mice infected with KP showed lower BAL protein (mean 248.51 [SD 98.03] vs 366.3 [SD 159.8] μg/ml; p=0.0035) and lung wet-to-dry weight ratios (mean 4.27 [SD 0.37] vs 5.29 [SD 0.75]; p=0.0152) compared to WT. Bacterial burden and BAL inflammatory cell counts were higher, while BAL clot time was lower in infected WT mice compared to control. However, these outcomes did not differ between infected TFEpi+ and WT mice. Loss of TF in alveolar epithelial cells reduced cell surface β1 integrin expression and cell adhesion to Laminin-511. Conclusion: Alveolar epithelial TF overexpression is protective for maintaining lung barrier integrity independent of coagulation and inflammation. Further, cell adhesion studies suggest that this is potentially mediated through increased a6β1 epithelial cell adhesion. AHA Postdoctoral Fellowship, National Institute of Health Grants (HL150783, I01BX002288). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rigorous Donor Selection for Fecal Microbiota Transplantation in Active Ulcerative Colitis: Key Lessons From a Randomized Controlled Trial Halted for Futility

Rigorous donor preselection on microbiota level, strict anaerobic processing, and repeated fecal microbiota transplantation (FMT) administration were hypothesized to improve FMT induction of remission in ulcerative colitis (UC). The RESTORE-UC trial was a multi-centric, double-blind, sham-controlled, randomized trial. Patients with moderate to severe UC (defined by total Mayo 4-10) were randomly allocated to receive 4 anaerobic-prepared allogenic or autologous donor FMTs. Allogenic donor material was selected after a rigorous screening based on microbial cell count, enterotype, and the abundance of specific genera. The primary endpoint was steroid-free clinical remission (total Mayo ≤2, no sub-score >1) at week 8. A pre-planned futility analysis was performed after 66% (n= 72) of intended inclusions (n= 108). Quantitative microbiome profiling (n= 44) was performed at weeks 0 and8. In total, 72 patients were included, of which 66 received at least 1 FMT (allogenic FMT, n= 30 and autologous FMT, n= 36). At week 8, respectively, 3 and 5 patients reached the primary endpoint of steroid-free clinical remission (P= .72), indicating no treatment difference of at least 5% in favor of allogenic FMT. Hence, the study was stopped due to futility. Microbiome analysis showed numerically more enterotype transitions upon allogenic FMT compared with autologous FMT, and more transitions were observed when patients were treated with a different enterotype than their own at baseline (P= .01). Primary response was associated with lower total Mayo scores, lower bacterial cell counts, and higher Bacteroides 2 prevalence at baseline. The RESTORE-UC trial did not meet its primary endpoint of increased steroid-free clinical remission at week 8. Further research should additionally consider patient selection, sterilized sham-control, increased frequency, density, and viability of FMT prior to administration. gov, Number: NCT03110289.

Influence of Season on Biodegradation Rates in Rivers.

Biodegradation plays a key role in the fate of chemicals in the environment. The variability of biodegradation in time can cause uncertainty in evaluating the environmental persistence and risk of chemicals. However, the seasonality of biodegradation in rivers has not yet been the subject of environmentally relevant testing and systematic investigation for large numbers of chemicals. In this work, we studied the biodegradation of 96 compounds during four seasons at four locations (up- and downstream of WWTPs located on two Swedish rivers). Significant seasonality (ANOVA, p < 0.05) of the first-order rate constant for primary biodegradation was observed for most compounds. Variations in pH and total bacterial cell count were not the major factors explaining the seasonality of biodegradation. Deviation from the classical Arrhenius-type behavior was observed for most of the studied compounds, which calls into question the application of this relationship to correct biodegradation rate constants for differences in environmental temperature. Similarities in magnitude and seasonality of biodegradation rate constants were observed for some groups of chemicals possessing the same functional groups. Moreover, reduced seasonality of biodegradation was observed downstream of WWTPs, while biodegradation rates of most compounds were not significantly different between up- and downstream.