Single-Cell Phenotypic Analysis and Digital Molecular Detection Linkable by a Hydrogel Bead-Based Platform.

Cell heterogeneity, such as antibiotic heteroresistance and cancer cell heterogeneity, has been increasingly observed. To probe the underlying molecular mechanisms in the dynamically changing heterogeneous cells, a high throughput platform is urgently needed to establish single cell genotype-phenotype correlations. Herein, we report a platform combining single-cell viability phenotypic analysis with digital molecular detection for bacterial cells. The platform utilizes polyethylene glycol hydrogel that cross-links through a thiol-Michael addition, which is biocompatible, fast, and spontaneous. To generate uniform nanoliter-sized hydrogel beads (Gelbeads), we developed a convenient and disposable device made of needles and microcentrifuge tubes. Gelbead-based single cell viability and molecular detection assays were established. Enhanced thermal stability and uncompromised efficiency were achieved for digital polymerase chain reaction (PCR) and digital loop-mediated isothermal amplification (LAMP) within the Gelbeads. Reagent exchange for in situ PCR following viability phenotypic analyses was demonstrated. The combined analyses may address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. The platform promises unique perspectives in mechanism elucidation of environment-evolution interaction that may be extended to other cell types for medical research.

Characterization of purified antimicrobial peptide produced by Pediococcus pentosaceus LJR1, and its application in preservation of white leg shrimp.

The bacteriocinogenic lactic acid bacterium Pediococcus pentosaceus LJR1 isolated from rumen liquor of goat had strong anti-bacterial activity toward Listeria monocytogenes in vitro. This antibacterial activity was lost on treatment with protease indicating that the bacteriocin is proteinaceous in nature. The bacteriocin LJR1 produced by P. pentosaceus was purified following a three step procedure consisting of ammonium sulphate precipitation, gel filtration chromatography and reverse phase-high performance liquid chromatography. The molecular weight of purified bacteriocin was determined to be 4.6 kDa using Tricine SDS-PAGE. Further, we found that the proteinaceous bacteriocin was stable at 100 °C as well as 121 °C for 30 min and 15 min respectively and also at different pH ranging from 4 to 10 when stored for 15 min at 37 °C. Its minimum inhibitory concentration for S. typhi MTCC134 and L. monocytogenes MTCC 1143 was 7.81 µg/ml and 15.63 µg/ml respectively. Scanning electron microscopy analysis of the surface of S. typhi treated with the bacteriocin showed the presence of craters; while in the case of treated L. monocytogenes blebs were observed. The addition of the bacteriocin to shrimp (white leg shrimp) has led to reduction of about 1 log units of L. monocytogenes on day 1 and maintained for 7 days on storage at 4 °C. It is clear that the purified bacteriocin has good potential as a bio preservative for application in food industry.

Asymmetric Membrane for Digital Detection of Single Bacteria in Milliliters of Complex Water Samples.

In this work, we introduce an asymmetric membrane as a simple and robust nanofluidic platform for digital detection of single pathogenic bacteria directly in 10 mL of unprocessed environmental water samples. The asymmetric membrane, consisting of uniform micropores on one side and a high density of vertically aligned nanochannels on the other side, was prepared within 1 min by a facile method. The single membrane covers all the processing steps from sample concentration, purification, and partition to final digital loop-mediated isothermal amplification (LAMP). By simple filtration, bacteria were enriched and partitioned inside the micropores, while inhibitors typically found in the environmental samples ( i.e., proteins, heavy metals, and organics) were washed away through the nanochannels. Meanwhile, large particles, indigenous plankton, and positively charged pollutants in the samples were excluded by using a sacrificial membrane stacked on top. After initial filtration, modified LAMP reagents, including NaF and lysozyme, were loaded onto the membrane. Each pore in the asymmetric membrane functioned as an individual nanoreactor for selective, rapid, and efficient isothermal amplification of single bacteria, generating a bright fluorescence for direct counting. Even though high levels of inhibitors were present, absolute quantification of Escherichia coli and Salmonella directly in an unprocessed environmental sample (seawater and pond water) was achieved within 1 h, with sensitivity down to single cell and a dynamic range of 0.3-10000 cells/mL. The simple and low-cost analysis platform described herein has an enormous potential for the detection of pathogens, exosomes, stem cells, and viruses as well as single-cell heterogeneity analysis in environmental, food, and clinical research.

Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing.

Microarray technology to isolate living cells using external fields is a facile way to do phenotypic analysis at the cellular level. We have used alternating current dielectrophoresis (AC-DEP) to drive the assembly of live pathogenic Salmonella typhi (S.typhi) and Escherichia coli (E.coli) bacteria into miniaturized single cell microarrays. The effects of voltage and frequency were optimized to identify the conditions for maximum cell capture which gave an entrapment efficiency of 90% in 60 min. The chip was used for calibration-free estimation of cellular loads in binary mixtures and further applied for rapid and enhanced testing of cell viability in the presence of drug via impedance spectroscopy. Our results using a model antimicrobial sushi peptide showed that the cell viability could be tested down to 5 µg/mL drug concentration under an hour, thus establishing the utility of our system for ultrafast and sensitive detection.

[Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

Antagonistic activity of Lactobacillus isolates against Salmonella typhi in vitro.

BACKGROUND: Enteric fever is a global health problem, and rapidly developing resistance to various drugs makes the situation more alarming. The potential use of Lactobacillus to control typhoid fever represents a promising approach, as it may exert protective actions through various mechanisms. METHODS: In this study, the probiotic potential and antagonistic activities of 32 Lactobacillus isolates against Salmonella typhi were evaluated. The antimicrobial activity of cell free supernatants of Lactobacillus isolates, interference of Lactobacillus isolates with the Salmonella adherence and invasion, cytoprotective effect of Lactobacillus isolates, and possibility of concurrent use of tested Lactobacillus isolates and antibiotics were evaluated by testing their susceptibilities to antimicrobial agents, and their oxygen tolerance was also examined. RESULTS: The results revealed that twelve Lactobacillus isolates could protect against Salmonella typhi infection through interference with both its growth and its virulence properties, such as adherence, invasion, and cytotoxicity. These Lactobacillus isolates exhibited MIC values for ciprofloxacin higher than those of Salmonella typhi and oxygen tolerance and were identified as Lactobacillus plantarum. CONCLUSION: The tested Lactobacillus plantarum isolates can be introduced as potential novel candidates that have to be subjected for in vivo and application studies for treatment and control of typhoid fever.

In vitro effects of azithromycin on Salmonella typhi: early inhibition by concentrations less than the MIC and reduction of MIC by alkaline pH and small inocula.

To explain good clinical results of azithromycin in patients with typhoid fever, 10 strains of Salmonella typhi were grown in cation-adjusted Mueller-Hinton broth. MICs of azithromycin were 4-16 mg/L. At a sub-MIC of 2 mg/L, early inhibition of growth was shown at 2, 4 and 8 h of incubation, but at 24 and 48 h growth to turbidity occurred. At 4 mg/L, inhibition occurred up to 8 h, after which growth towards turbidity followed. Elongated curved bacilli formed in broth containing 4 mg/L after 24-48 h. Adjusting the pH of the broth with phosphate-citrate buffer to 7.5 and 8.0 caused reductions in MICs to 0.25-0.5 mg/L. Large inocula of 10(6) cfu/mL resulted in median MICs four- to six-fold greater than with inocula of 10(1)-10(3) cfu/mL. An inoculum of 10 bacteria per mL in broth at pH 7.5 resulted in an MIC of 0.13 mg/L. Clinical benefits in patients may occur because of early inhibition by sub-MIC concentrations of azithromycin, and due to lower MICs at alkaline pH and lower MICs with small inocula that may correspond to the low-grade bacteraemia in typhoid fever.

[The criterion of cell division synchronization for assessing the stressor exposure of a Salmonella typhi population]. / Kriterii sinkhronizatsii deleniia kletok dlia otsenki stressornogo vozdeistviia na populiatsiiu Salmonella typhi.

Criterion of the synchronization (CS) of cells division for S. typhi population is proposed. The criterion is based on the assumption of the normal distribution of cells with different generation time in the population after stressor (shock) action. CS is equal to the ratio of the dispersion of the generation time of cells in the population to the average generation time of the whole population and determined from the parameters of the mathematical model. The quantitative values of the parameters of the mathematical model were obtained by the minimization of error between the calculation and experimental data. CS was used for the evaluation and choice of the optimum stressor action in the synchronization of the division of S. typhi.

[The vital activities of enterobacteria in an aqueous medium in the absence of sources of organic nutrition]. / O zhiznedeiatel'nosti énterobakterii v vodnoi srede pri otsutstvii istochnikov organicheskogo pitaniia.

Heterotrophic bacteria were found to be capable of proliferation in physiological saline and distilled water. In 1988-1989 experimental studies were made with a view to establish the role of the gaseous phase of atmospheric air and the products of the autolysis of dead bacteria as the sources of organic nutrition. The studies revealed that the complete removal of atmospheric air from vials with bacterial suspension completely stopped the stimulation of reproduction. In vials with a higher concentration of dead bacterial bodies the proliferation rate was 2- to 400-fold (on the average, 118-fold) higher. The products of the autolysis of bacterial bodies proved to be of no importance as an independent source of organic nutrition for heterotrophic bacteria. The mechanism of the assimilation of autolysis products is "switched on" by biologically active geomagnetic disturbances. The mechanisms of the increase of bacterial biomass remain unclear.

Induction of stable L-forms of Salmonella typhi and Listeria monocytogenes.

Stable L-forms of Salmonella typhi and Listeria monocytogenes were produced using penicillin (4500 units/ml) as inducer, and sucrose, normal horse serum and Mg++ as stabilizers. Stable L-forms were produced after 100 and 56 passages, then adapted to grow and multiply in a medium free of inducer and stabilizers so that they did not revert to parental forms even after 12 continuous passages.

[Characteristics of the morphology and ultrastructure of the typhoid bacteria under limited and excess glucose conditions]. / Osobennosti morfologii i ul'trastruktury tifoznykh bakterii v usloviiakh limita i izbytka gliukozy.

The authors present the results of the study of morphology and physico-biochemical indices of periodic and continuous population of typhoid bacilli under conditions of glucose limit and excess in the medium. Changes in the parameters of cell distribution by length proved to reflect the physiologico-biochemical processes in the population. The results of the study of the ultrastructure of typhoid bacilli of the "aerobic" and "anaerobic" population in continuous cultivation are presented. A possibility of application of morphological tests for the assessment of stability, homogeneity, and viability of the population is discussed.

Morphology of Salmonella typhi in different inactivation techniques.

Heat-phenol treatment of Salmonella typhi results in a denaturation of the cytoplasm and a smoothing of the cell wall with numerous ruptures. Following acetone inactivation the cell wall changes similarly, the cytoplasm is shrunken and its structures remain intact. Destruction is more intensive following heat-phenol treatment than after acetone precipitation. In both cases three contrasting layers of the cell wall can be seen.

[Characteristics of the hydrodynamic and biological properties of flagellar preparations isolated from submerged cultures of S. typhi]. / Kharakteristika gidrodinamicheskikh i biologicheskikh svoistv preparatov zhgutikov, vydelennykh iz glubinnykh kul'tur S. typhi

A study was made of the hydrodynamic and biological properties of the flagella from the S. typhi 4446 cultures, isolated by the methods of differential centrifugation, in crude condition and following depolimerization and denaturing by the action of chemical and physical agents. Molecular parameters of the slow and rapid components of the flagella and subunits of the flagellin were compared. The greatest antigenic activity was possessed by the high molecular fraction of the flagella isolated in the 60% sucrose density gradient.