Influence of Bacterial Lysate Quality on Growth of Two Bacterioplankton Species

All physico-chemical parameters that affect bacterial growth rate will also affect bacterial molecular composition, which in turn influences the chemical composition of bacterial lysate and its turnover rate in the ecosystem. To produce qualitatively different lysates, Vibrio sp. cells were grown under different pH, salt, or temperature conditions in rich growth media and then washed and lysed by autoclaving. Both the absolute concentrations and the ratios between elements in the lysates varied with different growth conditions, implying differences in lysate quality. Either Pseudoalteromonas sp. or Vibrio sp. was grown on the lysates at non-limiting lysate concentrations. Different lysates supported growth rates of Pseudoalteromonas sp. in the range from 0.25 to 1.53 h⁻¹. On the other hand, growth rates of Vibrio sp. grown on its own lysates were around 0.4 h⁻¹ and were not dependent on lysate quality. Two orders of magnitude decrease in Zn concentration in Vibrio sp. cells grown on different lysates as compared to cells grown on rich growth medium suggested that Zn might be a factor limiting growth. In the simple microbial loop studied, the initial difference in lysate quality was preserved in Pseudoalteromonas sp., whereas Vibrio sp. decreased the initial differences in lysate quality, thereby neutralizing the primary effect of environmental conditions on carbon turnover.     

Environmental Stress Determines the Quality of Bacterial Lysate and Its Utilization Efficiency in a Simple Microbial Loop

Heterotrophic bacteria provide the critical link in the microbial loop by converting dissolved organic matter (DOM) into particulate form. In this study, DOM was prepared from recently isolated estuarine bacterial strain Vibrio sp. (DSM14379) grown at different salinities [0.2%, 0.5%, 3%, 5%, or 10% (w/v)], washed, concentrated, and lysed by autoclaving. The corresponding lysate-containing media were designated LM_0.2, LM_0.5, LM₃, LM₅, and LM₁₀. Vibrio sp. cells grown at different salinities had similar C/N/P ratios, but different C/S ratios, different trace element composition, and different 2D gel electrophoresis protein profiles. Pseudoalteromonas sp. (DSM06238) isolated from a similar environment was able to grow on all lysates, and its biomass production was dependent on lysate type. The highest growth rate and biomass production of Pseudoalteromonas sp. at saturation lysate concentrations were observed in LM₃. The biomass production at saturation lysate concentrations was about 3-fold higher as compared to LM_0.2 and LM₁₀. The initial respiration rate, intracellular adenosine triphosphate (ATP) levels, and ³H-Leu and ³H-TdR incorporation rates were lowest in LM₃. On the other hand, in LM_0.2 or LM₁₀ lysates the situation was reversed, the growth rates and biomass production were lowest, whereas ³H-Leu and ³H-TdR incorporation, respiration rates, as well as ATP levels, were highest. These results imply uncoupling of catabolism from growth in either high- or low-salinity lysates. The results also suggest that differences in organic carbon quality generated during Vibrio sp. growth at different NaCl concentrations were propagated through the simple microbial loop, which may have important ecological implications for higher trophic levels that depend on microbial grazing.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

Assessing DNA Barcoding as a Tool for Species Identification and Data Quality Control

In recent years, the number of sequences of diverse species submitted to GenBank has grown explosively and not infrequently the data contain errors. This problem is extensively recognized but not for invalid or incorrectly identified species, sample mixed-up, and contamination. DNA barcoding is a powerful tool for identifying and confirming species and one very important application involves forensics. In this study, we use DNA barcoding to detect erroneous sequences in GenBank by evaluating deep intraspecific and shallow interspecific divergences to discover possible taxonomic problems and other sources of error. We use the mitochondrial DNA gene encoding cytochrome b (Cytb) from turtles to test the utility of barcoding for pinpointing potential errors. This gene is widely used in phylogenetic studies of the speciose group. Intraspecific variation is usually less than 2.0% and in most cases it is less than 1.0%. In comparison, most species differ by more than 10.0% in our dataset. Overlapping intra- and interspecific percentages of variation mainly involve problematic identifications of species and outdated taxonomies. Further, we detect identical problems in Cytb from Insectivora and Chiroptera. Upon applying this strategy to 47,524 mammalian CoxI sequences, we resolve a suite of potentially problematic sequences. Our study reveals that erroneous sequences are not rare in GenBank and that the DNA barcoding can serve to confirm sequencing accuracy and discover problems such as misidentified species, inaccurate taxonomies, contamination, and potential errors in sequencing.     

Colorimetry as Quality Control Tool for Individual Inkjet-Printed Pediatric Formulations

Printing technologies were recently introduced to the pharmaceutical field for manufacturing of drug delivery systems. Printing allows on demand manufacturing of flexible pharmaceutical doses in a personalized manner, which is critical for a successful and safe treatment of patient populations with specific needs, such as children and the elderly, and patients facing multimorbidity. Printing of pharmaceuticals as technique generates new demands on the quality control procedures. For example, rapid quality control is needed as the printing can be done on demand and at the point of care. This study evaluated the potential use of a handheld colorimetry device for quality control of printed doses of vitamin Bs on edible rice and sugar substrates. The structural features of the substrates with and without ink were also compared. A multicomponent ink formulation with vitamin B₁, B₂, B₃, and B₆ was developed. Doses (4 cm²) were prepared by applying 1–10 layers of yellow ink onto the white substrates using thermal inkjet technology. The colorimetric method was seen to be viable in detecting doses up to the 5th and 6th printed layers until color saturation of the yellow color parameter (b*) was observed on the substrates. Liquid chromatography mass spectrometry was used as a reference method for the colorimetry measurements plotted against the number of printed layers. It was concluded that colorimetry could be used as a quality control tool for detection of different doses. However, optimization of the color addition needs to be done to avoid color saturation within the planned dose interval.     

Biological Control as a Tool for Ecosystem Management

Biological control is proposed as a tool useful for ecosystem management and compatible with the goals of often competing interests regarding the restoration and maintenance of ecosystems. We summarize the effects of introduced species on ecosystems in three broad groups: insects, vertebrates, and weeds. We then discuss the role of biological control for each of these groups in the context of ecosystem management and realistic outcomes. Of the three groups, we show that biological control of weeds appears to have the best chance for success in ecosystem management. We provide two case studies to support our ideas and finally discuss future needs and trends including fiscal considerations, cost/benefits associated with biological weed control, and potential funding sources.     

LogViewer: A Software Tool to Visualize Quality Control Parameters to Optimize Proteomics Experiments using Orbitrap and LTQ-FT Mass Spectrometers

Visualization tools that allow both optimization of instrument''s parameters for data acquisition and specific quality control (QC) for a given sample prior to time-consuming database searches have been scarce until recently and are currently still not freely available. To address this need, we have developed the visualization tool LogViewer, which uses diagnostic data from the RAW files of the Thermo Orbitrap and linear trap quadrupole-Fourier transform (LTQ-FT) mass spectrometers to monitor relevant metrics. To summarize and visualize the performance on our test samples, log files from RawXtract are imported and displayed. LogViewer is a visualization tool that allows a specific and fast QC for a given sample without time-consuming database searches. QC metrics displayed include: mass spectrometry (MS) ion-injection time histograms, MS ion-injection time versus retention time, MS² ion-injection time histograms, MS² ion-injection time versus retention time, dependent scan histograms, charge-state histograms, mass-to-charge ratio (M/Z) distributions, M/Z histograms, mass histograms, mass distribution, summary, repeat analyses, Raw MS, and Raw MS². Systematically optimizing all metrics allowed us to increase our protein identification rates from 600 proteins to routinely determine up to 1400 proteins in any 160-min analysis of a complex mixture (e.g., yeast lysate) at a false discovery rate of <1%. Visualization tools, such as LogViewer, make QC of complex liquid chromotography (LC)-MS and LC-MS/MS data and optimization of the instrument''s parameters accessible to users.     

Denaturing Gradient Gel Electrophoresis as a Tool To Determine Batch Similarity of Probiotic Cultures of Porcine Cecal Bacteria

A continuous-flow porcine cecal bacterial culture has been used experimentally as treatment against enterotoxigenic Escherichia coli in weanling pigs. Periodically, the cultures must be started from frozen stock. Our results indicate that denaturing gradient gel electrophoresis can be applied as an indirect indication of culture similarity for each new batch generated from frozen stock.     

Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp²¹⁴, Trp⁶¹), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp⁶¹ and iodide Trp²¹⁴. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp²¹⁴ and moxifloxacin near Trp⁶¹. These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.     

Proteomics as a Tool to Identify New Targets Against Aspergillus and Scedosporium in the Context of Cystic Fibrosis

Mycopathologia - Cystic fibrosis (CF) is a genetic disorder that increases the risk of suffering microbial, including fungal, infections. In this paper, proteomics-based information was collated...     

Metabarcoding of PalEnDNA as An Efficient Tool to Recover Ancient Bacterial Diversity

Past bacterial diversity of a paleosol was reconstructed using metabarcoding of paleo environmental DNA (PalEnDNA). The paleosol was subsampled from a sediment core which was excavated from a palaeo beach-ridge located 2.6?km away from present sea shore and identified that it was deposited under marine influence ～6000?years ago, using geological proxies. The bacterial community contained 37 bacterial phyla and dominated by Proteobacteria, followed by Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia, and Chloroflexi. The bacterial community was a mix-up of marine and terrestrial population, and thereby diversity was higher than marine populations. The result shows metabarcoding of PalEnDNA can effectively reconstruct past bacterial community structure.     

LIBS: A Quality Control Tool for Food Supplements

In the present paper the ability of calibration free laser induced breakdown spectroscopy (CF-LIBS) as a quality control tool to monitor the composition of different minerals present in food supplement samples belonging to Indian brands (brand-A and brand-B) has been demonstrated. LIBS spectra of these two food supplements (brand-A and brand-B) available in the form of tablet have been recorded. As reported by manufacturers of these two food supplements, LIBS spectra of brand-A contains the spectral signatures of minerals like Ca, Mg, C, P, Zn, Fe, Cu, and Cr whereas LIBS spectra of brand-B shows the presence of spectral lines like Ca, Mg and C. The spectral signatures of Na and K are also found in both brands whereas spectral signature of Ti is observed only in brand-B but these elements are not mentioned on the nutritional label of the brands. The quantitative analysis of mineral contents in food supplements has been done using CF-LIBS for brand A and brand B to verify the content of the minerals reported by the manufacturer of the food supplements. Our results show that Ca and Mg are the main matrix elements of these brands. The concentration of minor and trace elements estimated using CF-LIBS technique is found in agreement with the reported nutritional values of both the brands. The concentration of major elements Ca and Mg are also estimated from Atomic Absorption Spectroscopy which is in close agreement with CF-LIBS result.     

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.     

A Short Term Quality Control Tool for Biodegradable Microspheres

Accelerated in vitro release testing methodology has been developed as an indicator of product performance to be used as a discriminatory quality control (QC) technique for the release of clinical and commercial batches of biodegradable microspheres. While product performance of biodegradable microspheres can be verified by in vivo and/or in vitro experiments, such evaluation can be particularly challenging because of slow polymer degradation, resulting in extended study times, labor, and expense. Three batches of Leuprolide poly(lactic-co-glycolic acid) (PLGA) microspheres having varying morphology (process variants having different particle size and specific surface area) were manufactured by the solvent extraction/evaporation technique. Tests involving in vitro release, polymer degradation and hydration of the microspheres were performed on the three batches at 55°C. In vitro peptide release at 55°C was analyzed using a previously derived modification of the Weibull function termed the modified Weibull equation (MWE). Experimental observations and data analysis confirm excellent reproducibility studies within and between batches of the microsphere formulations demonstrating the predictability of the accelerated experiments at 55°C. The accelerated test method was also successfully able to distinguish the in vitro product performance between the three batches having varying morphology (process variants), indicating that it is a suitable QC tool to discriminate product or process variants in clinical or commercial batches of microspheres. Additionally, data analysis utilized the MWE to further quantify the differences obtained from the accelerated in vitro product performance test between process variants, thereby enhancing the discriminatory power of the accelerated methodology at 55°C.     

Rapid Determination of Bacteriological Water Quality by Using Limulus Lysate

The Limulus lysate assay was used to measure the endotoxin content in stream water and was found to reflect the degree of bacterial contamination as measured by coliform, enteric, gram-negative, and heterotrophic bacteria. The firm-clot method was found to be a less sensitive and reproducible technique for the detection of endotoxin than was the spectrophotometric modification of the Limulus lysate assay. Bound endotoxin, as determined by the spectrophotometric modification of the Limulus lysate assay, was found to be a better measure of the endotoxin associated with bacterial cells than was total endotoxin.