The viability and virulence of old cultures of tubercle bacilli; studies on 30-year-old broth cultures maintained at 37 degrees C.
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Bacillus↗
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Autofluorescence as a viability marker for detection of bacterial spores.
Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.
Bacillus anthracis↗
The tatC gene cluster is essential for viability in halophilic archaea.
In prokaryotes the twin-arginine translocase (Tat) is a unique transport system for the export of folded proteins. The Tat pathway is usually involved in the export of a small proportion of extracytoplasmic proteins. An exception is found in halophilic archaea, in which the majority of secretory proteins have been predicted to be Tat-dependent. All haloarchaea analysed to date contain two genes encoding homologues of the Tat-component TatC. In all of these cases both genes are located adjacently on the chromosome, indicating that they form a functional unit. We show that this gene cluster is essential for viability in haloarchaea, which is in complete contrast to all other prokaryotes that have been tested thus far.
DNA Primers↗
Survivability of Salmonella cells in popcorn after microwave oven and conventional cooking.
The survivability of Salmonella cells in popcorn preparation was determined for two distinct cooking methods. The first method used a standard microwave oven. The second method used conventional cooking in a pan. Prior to thermal processing in independent experiments, 12 suspensions in a range between 1x10(3) and 8x10(6) colony-forming units (CFU) per gram of Salmonella cells were inoculated in both raw microwave popcorn and conventional corn kernels. The influence of the initial concentration of Salmonella cells in the raw products and the lethal effects on Salmonella by thermal treatments for cooking were studied. Survival of Salmonella cells was determined in the thermally processed material by pre-enrichment and enrichment in selective medium, in accordance with the legislation for expanded cereals and cereals in flakes. Viable experimental contaminants were recovered from the conventionally cooked popcorn with initial inoculation concentrations of 9x10(4)cells/g or greater. Salmonella cell viability was significantly reduced after microwave oven treatment, with recoveries only from initial concentrations of 2x10(6)cells/g or superior.
Cooking↗
Acridine orange staining and viability of the coccoid form of Campylobacter upsaliensis.
Conversion of Campylobacter upsaliensis to the coccoid form during aerobic incubation at 37 degrees C was not prevented by treatment with chloramphenicol and was accompanied by severe decreases in isocitrate dehydrogenase activity and oxygen uptake. Although the coccoid forms fluoresced orange-red by acridine orange staining, agarose gel electrophoresis indicated an extensive degradation of the ribosomal RNA. This suggests that acridine orange staining may not be a good indicator of viability and that the coccoid form of C. upsaliensis at 37 degrees C is degenerative rather than part of the life cycle.
Acridine Orange↗
Bacterial adhesion to and viability on positively charged polymer surfaces.
Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than -7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than -8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80% of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60% of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than -8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.
Bacillus subtilis↗
Dentin enhances the effectiveness of bioactive glass S53P4 against a strain of Enterococcus faecalis.
OBJECTIVE: The aim of the current study was to test the impact of dentin powder on the antimicrobial efficacy of bioactive glass S53P4 (BAG). STUDY DESIGN: BAG was suspended (preincubated) in saline at 37 degrees C for different time periods with or without human dentin powder, hydroxylapatite, or decalcified dentin. Subsequently, Enterococcus faecalis ATCC 29212 cells were added to these suspensions and bacterial recovery measured with and without the use of gentle sonication. Furthermore, survival of bacteria in test and control suspensions was assessed over time. Supernatants of suspensions were analyzed for their element contents using atomic absorption spectrophotometry. The effects of pH, silica, and osmolarity on E faecalis viability were assessed using specifically prepared solutions. RESULTS: BAG preincubated with dentin powder caused a significant (P < .05) decrease in viability compared to pure BAG suspensions. This was not based on adherence of bacteria to solid particles or agglutination of the cells, because sonication did not increase bacterial yields. Hydroxylapatite and decalcified dentin did not increase BAG killing efficacy. The additive effect of BAG + dentin powder was dose dependent, occurred only with solids in suspension, and increased with suspension time. An augmented dissolution of glass components, especially silicon, was measured in BAG + dentin powder compared to pure BAG suspensions or counterparts containing hydroxylapatite or decalcified dentin. High osmolarity per se did not affect E faecalis viability, whereas high pH and silica levels did. CONCLUSION: The observed phenomenon was related to an increased BAG dissolution triggered by dentin powder, causing elevated local pH and silica levels.
Anti-Infective Agents, Local↗
In vitro effects of crude khat extracts on the growth, colonization, and glucosyltransferases of Streptococcus mutans.
Millions of Yemenites, East Africans, and immigrants to Western countries chew khat daily for its amphetamine-like effects. There is little information in the literature concerning the possible effects of the habit on oral microbiota. Our objective was to study in vitro crude khat extract effects on Streptococcus mutans growth and sucrose-dependent colonization, and on its glucosyltransferase (GTF) activity and production. Three khat cultivars were used. Lyophilized crude aqueous khat extracts were applied to the different assays at concentrations of 0-1% (w/v). Sucrose-dependent colonization was assessed as the ability of Streptococcus mutans UA159 to form adherent biofilms in glass culture tubes. Colony forming units (CFUs) in the planktonic phase served as a measure of bacterial growth, while CFUs in the biofilm phase were used to quantify viability in the biofilms. GTFs activity was tested by incubating a crude GTFs preparation with sucrose and determining the amount of water-soluble and water-insoluble glucans formed. GTFs production was assayed by comparing intensities of GTF bands in Western blots of extracts from control and khat-containing cultures. The khat extracts effectively inhibited biofilm formation. The minimum biofilm inhibitory concentration (MBIC) varied among the cultivars (0.25-1%). The extracts also inhibited synthesis of both glucan types, particularly insoluble glucans (average 85% inhibition at 1%), with significant differences among the cultivars. However, khat increased bacterial growth and at sub-MBIC also viability within biofilms; there were no inter-cultivar differences. It is shown that khat leaves contain water-soluble constituents that inhibit some cariogenic properties of S. mutans in vitro.
Bacterial Adhesion↗
Intrinsic tolerance of Bifidobacterium species to heat and oxygen and survival following spray drying and storage.
AIMS: This study examined the tolerance of various species of the genus Bifidobacterium to heat and oxygen and evaluated the survival of selected strains following spray drying and during storage. METHODS AND RESULTS: Nine Bifidobacterium species were considered to be relatively tolerant to both heat and oxygen and mostly segregated into two clusters within the 16S rDNA phylogenetic tree. Four species were tolerant to oxygen and 12 species were considered sensitive to oxygen and heat. Using a skimmed milk-based carrier good survival following spray drying and storage at 4 degrees C correlated with tolerance to heat and oxygen. Viability was inversely related to storage temperature and at 15 degrees C and 25 degrees C, a significant decline was observed for all species. The inclusion of gum acacia had no significant affect on survival or viability. However, using a fluidized-bed spray dryer viability was greatly improved. CONCLUSIONS: A group of closely related species tolerant to heat and oxygen had high survival following spray drying and maintained viability during prolonged storage at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray drying is a suitable method for the production of skimmed milk powder enriched with high numbers of viable bifidobacteria.
Bifidobacterium↗
Effect of storage temperature on survival of Chlamydia trachomatis after lyophilization.
Lyophilized preparations of Chlamydia trachomatis were made to investigate how well they would survive storage at four relevant incubation temperatures for 1 week and 1 month. Good viability was maintained by storage at either 4 degrees C or 20 degrees C for 1 week. If the ambient temperature is not too high, short-term transportation of C. trachomatis is achievable through lyophilization.
Chlamydia trachomatis↗
Effect of additives on the viability of bifidobacteria loaded in alginate poly-l-lysine microparticles during the freeze-drying process.
Bifidobacteria-loaded alginate poly-l-lysine microparticles (bap microparticles) were prepared using an air atomization method and then freeze-dried. The viability of the bap microparticles was investigated as a function of the amount of the bifidobacteria cultures, and the addition of a yeast extract, cryoprotectants, antioxidants and neutralizer. The size of the bap microparticles with and without the bifidobacteria was 84.8 +/- 28.5 microm (mean +/- standard deviation) and 113.1 +/- 38.5 microm, respectively. The surface morphology was slightly ellipsoid and wrinkled regardless of the incorporating bifidobacteria. The viability gradually decreased with increasing freeze-drying time. Free-flowing powdered bap microparticles were obtained at least 12 h after freeze-drying the wetted slurry of bap microparticles. However, the particles tended to aggregate when either lactose or ascorbic acid was added. The addition of a yeast extract, cryoprotectants (glycerol and lactose), antioxidants (NaHSO3 and ascorbic acid) and neutralizer (Mg3(PO4)2) resulted in a significantly higher viability of the bifidobacteria in the bap microparticles after freeze-drying (0.34-1.84 log) compared with the culture alone.
Alginates↗
Viability and molecular authentication of Coccidioides immitis strains from culture collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.
Twenty Coccidioides immitis strains were evaluated. Only 5 of the 20 strains kept under mineral oil maintained their viability while all 5 subcultures preserved in water remained viable and none of the 13 subcultures kept in soil were viable. A 519 bp PCR product from the csa gene confirmed the identity of the strains.
Brazil↗
Interaction of CmeABC and CmeDEF in conferring antimicrobial resistance and maintaining cell viability in Campylobacter jejuni.
OBJECTIVES: To determine the role of CmeDEF in conferring antimicrobial resistance in Campylobacter jejuni and examine the interaction of CmeABC and CmeDEF in mediating antimicrobial resistance and maintaining cell viability. METHODS: Single and double mutants of cmeF and cmeB were generated in multiple strains using insertional mutagenesis. The mutants were compared with their wild-type strains for antimicrobial susceptibility and growth characteristics. Transcription fusion was used to quantify the expression of cmeDEF and cmeABC. Ethidium bromide (EB) accumulation assay was used to measure the efflux function. RESULTS: Insertional mutagenesis of the cmeF gene in C. jejuni NCTC 11168 resulted in a 2-fold decrease in the resistance to ampicillin, polymyxin B and EB, whereas the same mutation in C. jejuni 81-176 and 21190 led to a 2-4-fold increase in the resistance to multiple antimicrobials and toxic compounds. The increased resistance in the cmeF mutants of 81-176 and 21190 was associated with the elevated efflux in the mutants. Compared with the cmeB mutant, the cmeF/cmeB double mutants of 81-176 and 21190 showed further decrease in the resistance to various antimicrobials and toxic compounds. Transcription fusion assay indicated that the expression level of cmeF was substantially lower than that of cmeB. Notably, the cmeB/cmeF double mutation, not the single mutations, impaired cell viability in Campylobacter. CONCLUSIONS: CmeDEF interacts with CmeABC in conferring antimicrobial resistance and maintaining cell viability in C. jejuni. CmeABC is the predominant efflux pump in C. jejuni, whereas CmeDEF plays a secondary role in conferring intrinsic resistance to antimicrobials.
Anti-Bacterial Agents↗
A comparison among different methods for evaluating the biomass activity in activated sludge systems: preliminary results.
In order to improve activated sludge plant operation (achieving higher efficiency and cost savings) beside influent and effluent characteristics and working parameters (e.g. dissolved oxygen, total and volatile suspended solids, pH, recirculation flow rate, etc.), the biomass activity should be monitored, the bacteria being responsible for the pollutant degradation. Since conventional cultivation based methods are inadequate to quantify environmental microorganisms (due to scarce number of cultivable microorganisms and time-consuming procedures) several "non-conventional" techniques were applied in this study, in order to compare the obtainable information and their routine feasibility. Different measurements (VSS concentration, Oxygen Uptake Rate, microbial counts by cultural and biomolecular methods--MPN-PCR, ATP content, dehydrogenase activity, microbial cell viability and enzymatic activity) were carried out on mixed liquor samples, taken from a municipal activated sludge plant (440,000 p. e.). The preliminary results of the research are presented in this paper.
Adenosine Triphosphate↗
A model system for increasing the intensity of whole-cell biocatalysis: investigation of the rate of oxidation of D-sorbitol to L-sorbose by thin bi-layer latex coatings of non-growing Gluconobacter oxydans.
We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.
Bioreactors↗
The nonredundant roles of two 4'-phosphopantetheinyl transferases in vital processes of Mycobacteria.
Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4'-phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4'-phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and nonribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Guérin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs.
Bacterial Proteins↗
Impact of surface energy and roughness on cell distribution and viability.
The aim of this study was to assess the respective impacts of the surface energy and surface roughness of bare and coated steels on biofouling and sanitisation. Bioadhesion of Staphylococcus aureus CIP 53.154 was studied on two stainless steel surfaces with smooth or specific micro-topography. Two coatings were also studied: silicon oxide (hydrophilic) and polysiloxane (hydrophobic). On smooth surfaces, adhesion was reduced on an apolar coating and cell viability increased with the surface polarity. A specific micro-topography decreased the level of bacterial adhesion on bare surfaces by a factor ten. On this surface, only single adherent cells were observed, contrasting with cells in clusters on smoother surfaces. As a consequence, cell repartition influenced bacterial viability. Most isolated adherent cells were dead whereas cells in clusters were still alive. In addition, the quaternary ammonium chloride used in sanitisation, acted at once both as a tensio-active molecule and a biocide. It only displaced adherent cells but did not remove them.
Bacterial Adhesion↗
Biosynthesis of Ribose-5-Phosphate-Metabolic Regulator of Escherichia coli Viability.
Biosynthesis of ribose-5-phosphate (R5P) underlies all biosynthetic processes associated with biomass growth. Actively dividing cells continuously require building blocks for genome replication, synthesis of ribosomes and other derivatives containing R5P as a carbohydrate backbone. The main source of R5P in the cell is the pentose phosphate pathway (PPP), which is an anabolic sensor designed to coordinate the level of pentose phosphates and reduced NADPH required for anabolic processes. This review is devoted to a comparative analysis of R5P biosynthesis pathways among different domains of microorganisms, the features of PPP regulation in bacterial cells depending on physiological conditions, as well as genetic modifications of PPP and their effect on cell viability. We emphasize that ribose metabolism is a factor in the consolidation of cellular homeostasis under conditions of intensive biomass growth and the discrepancy between the processes of ribose synthesis and consumption is marked by spontaneous cell death.
Escherichia coli↗