Living Bacteria Cells Research Articles

Survival of Probiotic Bacterial Cells in the Upper Gastrointestinal Tract and the Effect of the Surviving Population on the Colonic Microbial Community Activity and Composition.

Many health-promoting effects have been attributed to the intake of probiotic cells. However, it is important that probiotic cells arrive at the site of their activity in a viable state in order to exert their beneficial effects. Careful selection of the appropriate probiotic formulation is therefore required as mainly the type of probiotic species/strain and the administration strategy may affect survival of the probiotic cells during the upper gastrointestinal (GIT) passage. Therefore, the current study implemented Simulator of the Human Microbial Ecosystem (SHIME®) technology to investigate the efficacy of different commercially available probiotic formulations on the survival and culturability of probiotic bacteria during upper GIT passage. Moreover, Colon-on-a-Plate (CoaP™) technology was applied to assess the effect of the surviving probiotic bacteria on the gut microbial community (activity and composition) of three human donors. Significantly greater survival and culturability rates were reported for the delayed-release capsule formulation (>50%) as compared to the powder, liquid, and standard capsule formulations (<1%) (p < 0.05), indicating that the delayed-release capsule was most efficacious in delivering live bacteria cells. Indeed, administration of the delayed-release capsule probiotic digest resulted in enhanced production of SCFAs and shifted gut microbial community composition towards beneficial bacterial species. These results thus indicate that careful selection of the appropriate probiotic formulation and administration strategy is crucial to deliver probiotic cells in a viable state at the site of their activity (distal ileum and colon).

Sandwich-type electrochemical aptasensor based on hemin-graphite oxide as a signal label and rGO/MWCNTs/chitosan/carbon quantum dot modified electrode for sensitive detection of Acinetobacter baumannii bacteria

Acinetobacter baumannii (A. baumannii) is a pathogenic bacterium that causes severe infections and its rapid and reliable diagnosis is essential for effective control and treatment. In this study, we present an electrochemical aptasensor based on a signal amplification strategy for the detection of A. baumannii, the high specificity and affinity of the aptamer for the target make it favorable for signal amplification. This allows for a highly sensitive and selective detection of the target. The aptasensor is based on a carbon screen-printed electrode (CSPE) that has been modified with a nanocomposite consisting of multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO), chitosan (CS), and a synthesized carbon quantum dot (CQD) from CS. Additionally, the self-assembled aptamers were immobilized on hemin-graphite oxide (H-GO) as a signal probe.The composition of the nanocomposite (rGO-MWCNT/CS/CQD) provides high conductivity and stability, facilitating the efficient capture of A. baumannii onto the surface of the aptasensor. Also, aptamer immobilized on Hemin-graphite oxide (H-GO/Aptamer) was utilized as an electrochemical signal reporter probe by H reduction. This approach improved the detection sensitivity and the aptamer surface density for detecting A. baumannii. Furthermore, under optimized experimental conditions, the aptasensor was demonstrated to be capable of detecting A. baumannii with a linear range of (10 – 1 × 107 Colony-forming unit (CFU)/mL) and a limit of detection (LOD) of 1 CFU/mL (σ = 3). One of the key features of this aptasensor is its ability to distinguish between live and dead bacteria cells, which is very important and critical for clinical applications. In addition, we have successfully detected A. baumannii bacteria in healthy human serum and skim milk powder samples provided using the prepared electrochemical aptasensor.The functional groups present in the synthetic CQD, rGO-MWCNT, and chitosan facilitate biomolecule immobilization and enhance stability and activity. The fast electron-transfer kinetics and high conductivity of these materials contribute to improved sensitivity and selectivity. Furthermore, The H-GO/Aptamer composite's large surface area increases the number of immobilized secondary aptamers and enables a more stable structure. This large surface area also facilitates more H loading, leading to signal amplification.

THE ROLE OF LACTOBACILLUS RHAMNOSUS GG AS A MODIFIER OF THE HUMAN BREAST CANCER CELLS PROLIFERATION AND APOPTOTIC PROGRAM

Summary. Aim: to investigate the influence of human microbiota element Lactobacillus rhamnosus on the viability, proliferation and apoptosis of breast cancer (BC) cells with various degrees of malignancy in vitro. Objects and methods: human breast cancer cells of the MCF-7, T47D and MDA-MB-231 lines, as well as live and pasteurized bacteria cells of Lactobacillus rhamnosus or their secreted metabolites, were used as research objects. The expression of proteins associated with the proliferation and apoptosis regulation in breast cancer cells was assessed by immunocytochemical analysis. Results: the direct interaction of Lactobacillus rhamnosus live cells and BC cells leads to the most significant effect on the viability and proliferation of malignant cells, compared to the action of their secreted metabolites (contactless cocultivation in the Insert system) or pasteurized microorganisms. Incubation of luminal-type breast cancer cells with Lactobacillus rhamnosus was accompanied by suppression of the proliferation marker Ki-67expression. Co-cultivation of T47D cells and Lactobacillus rhamnosus led to increased expression of both the pro-apoptotic proteins Bax and p21WAF1, as well as the anti-apoptotic protein Bcl-2. In MCF-7 cells lactobacilli increased the expression of p21WAF1, but did not affect the expression of Bcl-2. In MDA-MB-231 cells, which are characterized by the highest degree of malignancy, Lactobacillus rhamnosus caused only an increase in the anti-apoptotic marker Bcl-2 expression. Conclusions: in human ВС cells with various degrees of malignancy after their cultivation with Lactobacillus rhamnosus, various vector mechanisms of modification of the vital activity system of malignant cells by changing their proliferative activity and initiating their apoptotic program were revealed. Key words: microbiota, Lactobacillus, breast

Abundance of Live and Dead Bacteriopsammon Inhabiting Sandy Ecosystems of Recreational Marine Beaches of the Southern Baltic Sea

The study was carried out on four non-tidal sandy marine beaches located on the Polish part of the southern Baltic Sea coast. We applied a LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Invitrogen™) method to determine the abundance of live and dead bacteriopsammon. Live psammon bacteria cells constituted 31–53% of the total number of bacteria inhabiting sand of the studied beaches. Abundance of live and dead psammon bacteria generally differed along the horizontal profile in all beaches. The maximum density of bacteria was noted in the dune and the middle part of the beach (dry zones) and the minimum in wet zones, i.e., under seawater surface and at the swash zone. Generally along the vertical profile, the highest numbers of two studied bacterial groups were noted in the surface sand layer, while with increasing sediment depth their numbers significantly decreased. The abundance of live and dead bacteria showed a distinct seasonal variation.

Antimicrobial and antibiofilm potential of Curcuma longa Linn. Rhizome extract against biofilm producing Staphylococcus aureus and Pseudomonas aeruginosa isolates.

More than 65% of all human bacterial infection are associated with biofilm. Bacteria in such biofilms are 10 to 1000-fold more resistant to antibiotics than free living bacteria cells. Organisms such as S. aureus and P. aeruginosa are responsible for a significant number of biofilm related infections. In this study, we investigated the antimicrobial and anti-biofilm activity of C. longa L. rhizome extract against biofilm producing S. aureus and P. aeruginosa isolates. The results of MIC and MBC demonstrated promising antibacterial activity of the rhizome extract. TLC and column chromatography detected various curcuminoids while phytochemical analysis also reveals presence of number of bioactive compounds such as alkaloids, flavonoids, phenolics, terpenoids, etc. Micro titer plate assay indicated significant inhibition of biofilm formation in clinical isolates treated with turmeric extract. Thus, on basis of our results turmeric extracts can be considered as natural antibiofilm and antibacterial agent.

Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.

As an effective programmable DNA targeting tool, CRISPR–Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.

Physicochemical and Biocidal Properties of Nickel–Tin and Nickel–Tin—Titania Coatings

The morphology and composition of Ni–Sn and Ni–Sn–TiO2 coatings have been studied by scanning electron microscopy and energy-dispersive X-ray analysis. The electrochemical behavior of the obtained coatings in a 3% NaCl solution has been examined. The influence of the inclusion of titania in the Ni–Sn coating on the mechanical and antibacterial properties has been revealed. Dependences of the influence of incubation time and exposure to UV radiation on the concentration of living bacteria cells on the surface of the Ni–Sn–TiO2 coating have been established. In bacterial tests with Ni–Sn–TiO2 coatings deposited from the electrolyte containing 2 g/L TiO2, the concentration of viable Staphylococcus aureus decreases from 130 to 90 CFU/mL and from 70 to 30 CFU/mL without and with UV irradiation, respectively.

Antibacterial Activity of N,O-Acylated Chitosan Derivative.

The antibacterial activity of N,O-acylated chitosan derivative with linoleic acid (CH_LA) was tested by disc and well diffusion, agar impregnation and microdilution methods against Staphylococcus aureus, Escherichia coli and Helicobacter pylori strains. Hydrophobically modified chitosan (HMC) was expected to exhibit enhanced antibacterial activity and specific mucin interactions. Although diffusion tests have not indicated the antibacterial potential of chitosan (CH) or CH_LA, the results of the microdilution method demonstrated that tested polymers significantly reduced the amount of living bacteria cells in different concentrations depending on the microorganism. Additionally, CH_LA was characterized by enhanced antibacterial activity compared to CH, which may suggest a different mechanism of interaction with S. aureus and H. pylori. Furthermore, the UV-VIS analysis revealed that the amphiphilic character of derivative led to strong CH_LA–mucin interactions. The study proved the high potential of CH_LA in antibacterial applications, especially for the gastrointestinal tract.

Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.

Histidine kinases (HK) switch between conformational states that promote kinase and phosphatase activities to regulate diverse cellular processes. Past studies have shown that these functional states can display heterogeneity between cells in microbial communities and can vary at the subcellular level. Methods to track and correlate the kinase conformational state with the phenotypic response of living bacteria cells will offer new opportunities to interrogate bacterial signaling mechanisms. As a proof of principle, we incorporated both mClover3 (donor) and mRuby3 (acceptor) fluorescent proteins into the Caulobacter crescentus cell-cycle HK CckA as an in vivo fluorescence resonance energy transfer (FRET) sensor to detect these structural changes. Our engineered FRET sensor was responsive to CckA-specific input signals and detected subcellular changes in CckA signal integration that occurs as cells develop. We demonstrated the potential of using the CckA FRET sensor as an in vivo screening tool for HK inhibitors. In summary, we have developed a new HK FRET sensor design strategy that can be adopted to monitor in vivo changes for interrogation of a broad range of signaling mechanisms in living bacteria.

Toward Predicting Gene Expression and Metabolic Functions from Label-Free Raman Imaging of Living Cells

Current label-free imaging methods do not allow to estimate or predict the gene expression and specific metabolic functions directly from living cells. We hypothesized that the spectral signatures of molecules obtained by Raman spectroscopy are linked to the gene expression through the complex metabolic/proteomic network of the cell. We developed a unique approach to predict, in an accurate manner, the gene expression of thousands of living cells observed by microscopic spectral imaging, without any labeling nor destructive treatment. Using a trained AI model, our multi-omic approach enabled the prediction of the full transcriptome of living bacteria cells from new spectral data of unknown strain with high accuracy. In addition, the model parameters highlighted metabolic functions with cell-line specificity corresponding to the phenotype of the cells, demonstrating the model does match biological information. Escherichia coli strains exhibiting antibiotic resistance are used as a case study. This work is a milestone toward a revolution of the use of Raman spectral imaging in biomedical and industrial fields.

Rapid Light-Triggered Spatial Reorganization of Proteins in Living Bacteria Cells

In gram-negative bacteria, the cytoplasm is surrounded by two lipid bilayers that encapsulate the peptidoglycan cell wall. Due to a lack of membrane-enclosed organelles within the cytoplasm, the proper formation and spatiotemporal regulation of subcellular protein structures and scaffolds are critical to the cell's ability survive and function. Here we demonstrate, for the first time, that the CRY2/CIB1 system from Arabidopsis thaliana can be used to induce the fast reorganization of proteins inside living bacterialcells. We further show that this system can be used to rapidly inhibit cytokinesis in Escherichia coli cells.

Fluorescence assay to predict activity of the glycopeptide antibiotics.

Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.

A Novel Nanocomposite with Superior Antibacterial Activity: A Silver‐Based Metal Organic Framework Embellished with Graphene Oxide

AbstractSilver‐based nanomaterials have attracted considerable attention due to their antimicrobial activities. In this work, a silver (Ag)‐based metal organic framework (Ag‐MOF) is embellished with graphene‐oxide (GO), leading to the fabrication of a novel Ag‐based nanocomposite (GO‐Ag‐MOF) whose biocidal activity is higher than those of Ag‐MOF and GO nanomaterials. The nanocomposite is characterized using X‐ray photoelectron spectroscopy, transmission electron microscopy, scanning electron microscope, Fourier transform infrared spectra, ultraviolet−visible absorption spectra, X‐ray powder diffraction, dynamic light scattering, and nitrogen gas adsorption/desorption. The characterization shows that the Ag‐MOF nanoparticles are uniformly dispersed on the GO nanosheets surfaces without any agglomeration. Toxicities of GO‐Ag‐MOF, Ag‐MOF, and GO are assessed against the Gram‐negative bacteria, Escherichia coli and the Gram‐positive bacteria, Bacillus subtilis using the growth curve, fluorescence imaging, and flow cytometry methods. GO‐Ag‐MOF shows an outstanding antibacterial activity (higher than those of the Ag‐MOF and GO alone). The interaction of GO‐Ag‐MOF and Ag‐MOF with the bacteria leads to the extirpation of 95 and 85% of live bacteria cells, respectively. This study indicates that GO‐Ag‐MOF is a promising antibacterial nanocomposite, especially for biomedical applications.

Bacterial adsorption with graphene family materials compared to nano-alumina

The present study investigates the bio-activity and bio-adsorption properties of graphene family materials (GFMs) in comparison to nano-Al2O3 (12 nm, gamma phase). This is the first study that determines the type of solid adsorbent (GFMs or nano-Al2O3) preferred by living bacteria cells. The invest igated GFMs were: graphene, graphene oxide (GO) and reduced graphene oxide (RGO). The SEM images indicated that bacterial preferences depended on the particular strain, the type of GFM and the composition of the suspension. Generally, bacteria preferred to colonize the GFMs surfaces in the presence of nano-Al2O3 which resulted in no changes of zeta potential (ζ) measured for bacteria cells due to the fact that carbon is known as a good nourishment for microorganisms. There were however few exceptions from that rule i.e. the Sarcina bacteria preferred to adsorb on the surface of nano-Al2O3 when suspended with RGO flakes, and the E. coli preferred to colonize the surface of nano-Al2O3 when suspended with graphene sheets. For this suspension, ζ of bacteria cells decreased suggesting their starving state on the surface of nano-Al2O3. We also found disruptions in morphology of almost all E. coli cells, when attached to all investigated adsorbents.

Nanoporous gold as a suitable substrate for preparation of a new sensitive electrochemical aptasensor for detection of Salmonella typhimurium

Nowadays, achievement to easy, fast, reliable and cost-effective techniques and tools is one of the most important challenges in detection of microorganisms. Salmonella typhimurium is one of the most important pathogenic bacteria that affect the human health and environmental infections. In this work we report a new, stable, biocompatible and cost-effective platform for construction of an aptasensor based on nanoporous gold (NPG) for detection of S. typhimurium. Nanoporous gold is electrochemically synthesized using Au-Cu alloy at the surface of Au/GCE. Thiol functionalized aptamer is used for effective linking to the surface of NPG/Au/GCE via self-assemble monolayers (SAMs) formation. Morphological and voltammetric characterizations of the fabricated aptasensor were performed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The purposed aptasensor is capable to detect S. typhimurium in a wide linear dynamic range 6.5×102 to 6.5×108CFUmL−1 with a LOQ of 6.5×101CFUmL−1 and LOD of 1CFUmL−1. The prepared aptasensor showed some significant advantages such as sensitivity, selectivity, repeatability and ability to distinguish between live and dead bacteria cells. The prepared biosensor is successfully applied for salmonella detection in real samples such as egg.

Investigating the Dynamics of Vibrio Cholerae Virulence Initiation by Stics and Single Molecule Tracking

Single molecule imaging techniques have provided insight into a vast realm of new biological processes. However, many of these techniques rely on capturing a sufficiently high number of emitted photons to localize a molecule and can thus fail when the photon count is low. In a system where a large fraction of the fluorescent labels are very dim and/or moving quickly, localization methods can fail or be biased toward the slowest and brightest molecules in the system. Fortunately, correlation-based methods can provide insight into dynamics in these systems. Spatio-Temporal Image Correlation Spectroscopy (STICS) uses the full spatiotemporal correlation of a fluorescent image time series to provide information such as the diffusion constants and the densities of fluorescent molecules. STICS does not rely on the emitters positions and thus operates with higher precision in systems where sub-diffraction limited localization fails. We have previously developed STICS for imaging single molecules in living bacteria cells, and here we apply STICS to the specific case of the V. cholerae virulence signaling pathway. In particular, the membrane-bound TcpP, which controls downstream expression of the cholera enderotoxin, is a dynamic protein whose behavior is believed to be affected by various environmental signals including temperature and specific small molecule hormones. Here, we examine the fluorescent signals from single molecules of TcpP fused at the native locus to the photoactivatable fluorescent protein PAmCherry. We analyze these data by STICS and single molecule tracking to compare these two techniques in realistic imaging conditions. Because each technique operates with higher precision in a different imaging regime, we conclude that these approaches used together can provide highly complementary information about the dynamics of TcpP.

BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter.

BackgroundBacterial surface display technique enables the exogenous proteins or polypeptides displayed on the bacterial surface, while maintaining their relatively independent spatial structures and biological activities. The technique makes recombinant bacteria possess the expectant functions, subsequently, directly used for many applications. Many proteins could be used to achieve bacterial surface display, among them, autotransporter, a member of the type V secretion system of gram-negative bacteria, has been extensively studied because of its modular structure and apparent simplicity. However, autotransporter has not been widely used at present due to lack of a convenient genetic vector system. With our recently characterized autotransporter BrkA (Bordetella serum-resistance killing protein A) from Bordetella pertussis, we are aiming to develop a new autotransporter-based surface display system for potential wide application.ResultsHere, we construct a bacterial surface display system named as BrkAutoDisplay, based on the structure of autotransporter BrkA. BrkAutoDisplay is a convenient system to host exogenous genes. In our test, this system is good to efficiently display various proteins on the outer membrane surface of Escherichia coli, including green fluorescent protein (GFP), various enzymes and single chain antibody. Moreover, the displayed GFP possesses green fluorescence, the enzymes CotA, EstPc and PalA exhibit catalytic activity 0.12, 6.88 and 0.32 mU (per 5.2 × 108 living bacteria cells) respectively, and the single chain antibody fragment (scFv) can bind with its antigen strongly. Finally, we showed that C41(DE3) is a good strain of E. coli for the successful functionality of BrkAutoDisplay.ConclusionsWe designed a new bacterial display system called as BrkAutoDisplay and displayed various exogenous proteins on E. coli surface. Our results indicate that BrkAutoDisplay system is worthy of further study for industrial applications.

Real sequence effects on the search dynamics of transcription factors on DNA.

Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF-search times self-consistently depend heavily on whether or not an auxiliary operator (an accessible sequence similar to the main operator) is present in the genome section. Importantly, within our model the extent to which the interconversion rates between search and recognition states depend on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning several orders of magnitude. Auxiliary operators are shown to act as funnels facilitating target detection by TFs.

Electrodes Microfluidics System for Microbio Object Analysis

We have designed and fabricated an electrode microfluidics system for microbio object analysis. Two parallel-plate electrodes were fabricated using the soft lithography technique integrated with a polydimethylsiloxane (PDMS) microfluidics channel. Gold (Au) material was decomposed to fabricate the electrodes. The voltage response through the charging and discharging of the electrodes was observed using an oscilloscope. For a constant dc voltage of 5 V we obtained a time constant for the electrodes of 3.6 ms. On the other hand, it requires 850 ms to discharge completely without an external load. The capacitance of the electrodes in air (room environment) is 0.39 pF. Due to the higher dielectric constant of distilled water (80.1), in the water medium the electrode capacitance is 0.77 pF. We also measured the capacitance of the electrodes by changing the medium to microbio objects such as yeast cells (5 pF) and live bacteria cells (30 pF). The results showed that bacteria have a higher electrical capacitance than yeast.

Top‐Hat and Asymmetric Gaussian‐Based Fitting Functions for Quantifying Directional Single‐Molecule Motion

Single-molecule fluorescence permits super-resolution imaging, but traditional algorithms for localizing these isolated fluorescent emitters assume stationary point light sources. Proposed here are two fitting functions that achieve similar nanometer-scale localization precision as the traditional symmetric Gaussian function, while allowing, and explicitly accounting for, directed motion. The precision of these methods is investigated through Fisher information analysis, simulation and experiments, and the new fitting functions are then used to measure, for the first time, the instantaneous velocity and direction of motion of live bacteria cells. These new methods increase the information content of single-molecule images of fast-moving molecules without sacrificing localization precision, thus permitting slower imaging speeds, and our new fitting functions promise to improve tracking algorithms by calculating velocity and direction during each image acquisition.