Bacteria In Buffer Research Articles

Heat resistance of Lactobacillus brevis, Pediococcus acidilactici and Enterococcus faecium in buffer (pH 4), alcoholic and alcohol-free beer

Why was the work done: To measure the heat resistance of three vegetative bacteria in buffer (pH 4), alcoholic and alcohol-free beer. To verify that Pediococcus acidilactici ATCC 8042 and Lactobacillus brevis BSO 566 are as heat resistant as previously reported and to establish if Enterococcus faecium NRRL B-2354 could have application in validation studies for the pasteurisation of beer. How was the work done: : The heat resistance of L. brevis, P. acidilactici and E. faecium in buffer, alcoholic and alcohol-free beer was determined using two approaches - capillary test tubes and flask method. What are the main findings: E. faecium was the most heat resistant microorganism in all three liquids. D values were significantly greater, and z-values were similar or significantly greater than the corresponding values of L. brevis and P. acidilactici. Why is the work important: E. faecium is used in the food industry as a pathogenic surrogate for the validation of thermal and non-thermal processes. The work reported here suggests that E. faecium can also be used for the validation of pasteurisation of beer. Its high z-value suggests that at higher pasteurisation temperatures (>65°C) it may be more resistant than yeast ascospores and could therefore be used as an indicator for flash pasteurisation.

Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis.

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

Viability assessment of bacteria using long-range surface plasmon waveguide biosensors

We demonstrate that long-range surface plasmon waveguide biosensors are useful to monitor the quiver of immobilized live bacteria in buffer and in human urine. First, the biosensor captures bacteria selectively, based on gram, using antibodies against gram adsorbed on the surface of the waveguide through Protein G coupling. Then, analysis of the noise present on the optical output signal reveals quiver of bacteria immobilized on the waveguide. Live bacteria produce a noisy signature compared to baseline levels. The standard deviation over time of the optical power output from the biosensor increased by factors of 3–60 over that of the baseline level for Staphylococcus epidermidis and Escherichia coli immobilized selectively on waveguides.

Optimization of Pathogen Capture in Flowing Fluids with Magnetic Nanoparticles.

Magnetic nanoparticles have been employed to capture pathogens for many biological applications; however, optimal particle sizes have been determined empirically in specific capturing protocols. Here, a theoretical model that simulates capture of bacteria is described and used to calculate bacterial collision frequencies and magnetophoretic properties for a range of particle sizes. The model predicts that particles with a diameter of 460 nm should produce optimal separation of bacteria in buffer flowing at 1 L h(-1) . Validating the predictive power of the model, Staphylococcus aureus is separated from buffer and blood flowing through magnetic capture devices using six different sizes of magnetic particles. Experimental magnetic separation in buffer conditions confirms that particles with a diameter closest to the predicted optimal particle size provide the most effective capture. Modeling the capturing process in plasma and blood by introducing empirical constants (ce ), which integrate the interfering effects of biological components on the binding kinetics of magnetic beads to bacteria, smaller beads with 50 nm diameters are predicted that exhibit maximum magnetic separation of bacteria from blood and experimentally validated this trend. The predictive power of the model suggests its utility for the future design of magnetic separation for diagnostic and therapeutic applications.

Antimicrobial activity of chitosan attached to ethylene copolymer films

AbstractEthylene copolymer film was coated with chitosan by attachment of the polymer to the corona‐treated surface of the film, and the composite film was analysed for antimicrobial activity. The film was active against bacteria in 0.625 mM phosphate buffer; it reduced colony counts of Escherichia coli 25922 and of Listeria monocytogenes Scott A by 5 and 2–3 log10, respectively after 24 h exposure. The activity of the chitosan‐coated film against bacteria in buffer was increased when silver ions were incorporated into the films as demonstrated by complete killing of Escherichia coli O157:H7 DD3795 and the chicken exudate isolate Stenotrophomonas maltophilia within 2 h in buffer. The film was active against L. monocytogenes Scott A in 0.5% buffered peptone water up to a pH of about 7.0. Tests on beef and chicken meat exudates revealed antimicrobial activity of the film against Escherichia coli O157:H7 and L. monocytogenes Scott A of about 2 and 1–2 log10 reductions in colony‐forming units, respectively. The antimicrobial activity of the film against L. monocytogenes Scott A was also tested on turkey breast, and a log10 reduction of about 1.7 log10 units after 10 days and 1.2 log10 after 15 days at 4°C was achieved. Exposure to chitosan‐coated film and 350 MPa of pressure, 55°C or 1% sodium diacetate resulted in a synergistic effect. Copyright © 2008 John Wiley & Sons, Ltd

Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.

Escherichia coli derived factors modulate human granulosa cell steroidogenesis

In order to identify bacterial compounds which directly affect granulosa cell steroidogenesis, the effect of culture-supernatants and extracts from Escherichia coli were tested in an in vitro granulosa cell culture model. Samples were drawn from the culture-supernatant of E. coli ATCC25922 cultured in Luria-broth medium. Bacterial extract was prepared by incubation of freshly harvested bacteria in buffer. The bacterial culture-supernatants and extracts were added to human granulosa cells in culture. The granulosa cells collected from the follicular aspirates from women undergoing in-vitro fertilization were cultured for 17-53 h and progesterone or oestradiol was assayed in the spent culture medium. The E. coli culture-supernatant stimulated the basal granulosa cell progesterone production demonstrating its maximum activity reached after 200-240 min of bacterial growth. The heat denaturated bacterial extract as well as its low-molecular-weight fraction (<10 kDa) stimulated both the basal and the hCG-stimulated progesterone production; the oestradiol production was slightly inhibited. Stimulation of progesterone production was time dependent increasing from 125 +/- 18% of control within the first 3 h to 205 +/- 35% within 17-53 h. The high-molecular-weight fraction (>30 kDa) of the bacterial extract inhibited progesterone production. The inhibitory activity was significantly diminished by heat denaturation. The present study demonstrates the existence of various compounds which are secreted by E. coli and could also be extracted out of E. coli bacteria. These bacterial compounds modulate ovarian steroidogenesis. Further studies are needed to clarify how far these compounds contribute to menstrual disturbance observed in chronic pelvic inflammation.

X-Ray-Induced Single-Strand Breaks in DNA of E. coli B / r: Effect of Irradiation and Postirradiation Conditions

Alkaline sucrose-density gradient results indicate that aerobic irradiation of log phase E. coli B/r CSH is 4.5 times more effective than anoxic irradiation in producing single-strand breaks in DNA. If 0.02 M EDTA, presumed to inhibit the rejoining enzyme, is present during irradiation, the yield of breaks is increased 6-fold in air and 10-fold in nitrogen, thus aerobic conditions are still more effective than anoxic in producing breaks. Holding bacteria at 0°C in buffer after aerobic or anoxic radiation resulted in 80-90% of the observed breaks being rejoined. Since rejoining takes place during irradiation, the yield of breaks is dose-rate dependent. The oxygen effect on single-strand breaks in log phase E. coli B/r may be related to radiation survival: however, survival is not improved by lowering the dose rate nor by postirradiation holding at 0°C conditions which result in the rejoining of breaks. Holding irradiated bacteria in buffer at 37°C results in only a small amount of rejoining, while consider...