Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 mu g/ml reduced completely 24 h old biofilms while concentration of 25 mu g/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated

Genotypic and Phenotypic Characterization of Stenotrophomonas maltophilia Strains from a Pediatric Tertiary Care Hospital in Serbia

Background Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. Methods We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results. Results Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed. Conclusions High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates

Device for high throughput single-cell studies

The present invention concerns a microfluidic device or chip including at least one inlet for introducing at least one object into the device; an oil inlet for introducing an oil that supports droplet formation into the device or a droplet forming substance inlet for introducing a droplet forming substance into the device; a co-encapsulation area or structure where the at least one object is encapsulated by the droplet; a microfluidic tubing or channel for transporting the at least one object to an entrance of the co-encapsulation area or structure; an oil supporting droplet formation microchannel or droplet forming substance microchannel connected to the microfluidic tubing or channel to place a liquid of the microfluidic tubing or channel in direct contact with the oil that supports droplet formation or the droplet forming substance; and a droplet microchannel or tubing for transporting the droplet

Simplified Drop-seq workflow with minimized bead loss using a bead capture and processing microfluidic chip

Single-cell RNA-sequencing (scRNA-seq) has revolutionized biomedical research by enabling the in-depth analysis of cell-to-cell heterogeneity of tissues with unprecedented resolution. One of the catalyzing technologies is single cell droplet microfluidics, which has massively increased the overall cell throughput, routinely allowing the analysis of thousands of cells per experiment at a relatively low cost. Among several existing droplet-based approaches, the Drop-seq platform has emerged as one of the most widely used systems. Yet, this has surprisingly not incentivized major refinements of the method, thus restricting any lab implementation to the original Drop-seq setup, which is known to suffer from up to 80% bead loss during the process. In this study, we present a systematic re-engineering and optimization of Drop-seq: first, we re-designed the original dropleting device to be compatible with both air-pressure systems and syringe pumps, thus increasing the overall flexibility of the platform. Second, we devised an accompanying chip for post-encapsulation bead processing, which simplifies and massively increases Drop-seq's cell processing efficiency. Taken together, the presented optimization efforts result in a more flexible and efficient Drop-seq version

Stimulation of diesel degradation and biosurfactant production by aminoglycosides in a novel oil-degrading bacterium Pseudomonas luteola PRO23

Bioremediation is promising technology for dealing with oil hydrocarbons contamination. In this research growth kinetics and oil biodegradation efficiency of Pseudomonas luteola PRO23, isolated from crude oil-contaminated soil samples, were investigated under different concentrations (5, 10 and 20 g/L) of light and heavy crude oil. More efficient biodegradation and more rapid adaptation and cell growth were obtained in conditions with light oil. The 5 to 10 g/L upgrade of light oil concentration stimulated the microbial growth and the biodegradation efficiency. Further upgrade of light oil concentration and the upgrade of heavy oil concentration both inhibited the microbial growth, as well as biodegradation process. Aminoglycosides stimulated biosurfactant production in P. luteola in the range of sub-inhibitory concentrations (0.3125, 0.625 mu g/mL). Aminoglycosides also induced biofilm formation. The production of biosurfactants was the most intense during lag phase and continues until stationary phase. Aminoglycosides also induced changes in P. luteola growth kinetics. In the presence of aminoglycosides this strain degraded 82% of diesel for 96 h. These results indicated that P. luteola PRO23 potentially can be used in bioremediation of crude oil-contaminated environments and that aminoglycosides could stimulate this process

Phenol removal from four different natural soil types by Bacillus sp PS11

Biodegradation of phenol in four natural soils (loamy sand, sandy loam, sandy clay loam and loam) by indigenous microorganisms and in soils augmented by the Bacillus sp. PS11 was studied. During the laboratory soil microcosm experiments, the total removal of 2 g of phenol per kg of soil was achieved in all soil types in between 6 and 21 days. All biodegradation data was found to fit very well to saturation kinetics. The most efficient phenol removal was observed in the loamy woodland soil that contained the least amount of sand (42.5%) and the most silt and clay fraction (57.5%) in comparison to other three soil samples. However, amending sandy loam sample to contain more clay (from 13.5% to 30%) negatively affected the phenol removal rate, while increasing sand content (from 74.4% to 90%) resulted in the two times faster phenol removal in comparison to natural soil type. Bacillus sp. PS11 performed well in both pure culture and in the presence of soil microorganisms. Indigenous bacteria from sandy clay loam soil type possessed the ability of phenol bioremediation and almost whole amount of added phenol (2 g kg soil(-1)) was degraded within 9 days, whereas augmentation by Bacillus sp. PS11 improved the phenol removal by 20%. Carrying out small scale soil model experiments and amending soil granulometric properties by addition of clay or sand minerals is suggested as an effective and economically interesting way of enhancing bacterial soil bioremediation

Phenol removal from four different natural soil types by Bacillus sp PS11

Biodegradation of phenol in four natural soils (loamy sand, sandy loam, sandy clay loam and loam) by indigenous microorganisms and in soils augmented by the Bacillus sp. PS11 was studied. During the laboratory soil microcosm experiments, the total removal of 2 g of phenol per kg of soil was achieved in all soil types in between 6 and 21 days. All biodegradation data was found to fit very well to saturation kinetics. The most efficient phenol removal was observed in the loamy woodland soil that contained the least amount of sand (42.5%) and the most silt and clay fraction (57.5%) in comparison to other three soil samples. However, amending sandy loam sample to contain more clay (from 13.5% to 30%) negatively affected the phenol removal rate, while increasing sand content (from 74.4% to 90%) resulted in the two times faster phenol removal in comparison to natural soil type. Bacillus sp. PS11 performed well in both pure culture and in the presence of soil microorganisms. Indigenous bacteria from sandy clay loam soil type possessed the ability of phenol bioremediation and almost whole amount of added phenol (2 g kg soil(-1)) was degraded within 9 days, whereas augmentation by Bacillus sp. PS11 improved the phenol removal by 20%. Carrying out small scale soil model experiments and amending soil granulometric properties by addition of clay or sand minerals is suggested as an effective and economically interesting way of enhancing bacterial soil bioremediation

Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition

DisCo is a deterministic droplet microfluidics tool for single-cell analysis on low cell input samples, which is demonstrated to profile individual intestinal organoids and in vivo-derived small tissues. Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a(+) stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2(+)) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues

Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets

Pezoldt J, Wiechers C, Zou M, et al. Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets. Nature Communications. 2022;13(1): 7227.Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34(+) SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9(+) FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19(+) and Cxcl9(+) FRCs in LNs of Irf3(-/-) mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs. Lymph nodes in various locations of the body differ in their cell composition and gene expression signatures. Here authors show that the rapid postnatal expansion of lymph nodes is governed by CD34 (+) stromal cells and fibroblastic reticular stromal cell progenitors, distinguished by intrinsic, microbiome-independent core epigenetic blueprints