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PubMed2005

Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as part of a Laboratory Risk Assessment Program.

BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating/chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80 degrees C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.

Clinical Laboratory Techniques↗

PubMed2006

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon.

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30 degrees C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10(5)-10(7) CFU ml(-1)), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30 degrees C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg(-1) MP and 25 mg kg(-1) carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.

Biodegradation, Environmental↗

PubMed2006

Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine.

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.

Acetic Acid↗

PubMed2006

Survival, nodulation and N2 fixation ability of root nodule bacteria under different nutritional regimes.

Eleven strains of Rhizobium and five strains of Bradyrhizobium were examined for their viability as well nodulation and nitrogen fixation ability after storage under different conditions for two years. The storage conditions comprised lateritic soil, lateritic soil plus 1% mannitol, lateritic soil plus 0.1% yeast extract, lateritic soil plus 1% mannitol and 0.1% yeast extract, organic soil, organic soil plus 1% mannitol, organic soil plus 0.1% yeast extract, organic soil plus 1% mannitol and 0.1% yeast extract, and sterile distilled water. All the slow growing strains showed better viability than the fast growing strains in any of these conditions. The survived strains maintained their nodulation ability about 50-60% after one year and 40-50% after two years of preservation as compared to control, but the nodulation ability in sterile distilled water was very poor. Acetylene reduction activity in the nodules was found to be 70-90% and 50-70% after 12 and 24 months of preservation, respectively. The strains retained their phenotypic characters like antibiotic resistance and salt tolerance up to their highest survivability in respective nutritional condition.

Alkynes↗

PubMed2005

[Viability of a native isolate of Lactobacillus brevis in a fermented milky drink].

The objective of this research was to test the viability of a native isolate of Lactobacillus brevis in two fermented milky drinks: a drink without prebiotic ingredient and another drink with oat flour (0.5%, p/v) as prebiotic ingredient. The viability of L. brevis was tested at 7, 14, and 21 after inoculation. Drink pH and lactic acid concentration was measured as a function of time (2, 7, 14, and 21 days after inoculation). The isolate was viable in both drinks because its counting plates was higher than 10(6) CFU/mL until day 21st. Between the 7th and 14th day, the rate of growth was higher in the drink that included oat flour, however, at the day 21st the colony density was similar in both drinks.

Animals↗

PubMed2006

Influence of cranberry juice on glucan-mediated processes involved in Streptococcus mutans biofilm development.

Cranberry juice (CJ) has biological properties that may provide health benefits. In this study, we investigated the influence of CJ (pH 5.5) on several activities in vitro associated with the development of Streptococcus mutans UA159 biofilms. The ability of CJ to influence the adherence of S. mutans to either saliva- (sHA) or glucan-coated hydroxyapatite (gsHA), and to inhibit the glucan production by purified glucosyltransferases adsorbed to sHA was determined. For the adherence assays, we used both uncoated and saliva-coated bacterial cells. Furthermore, we examined whether CJ interferes with the viability, development, polysaccharide composition and acidogenicity of S. mutans biofilms. A solution containing equivalent amounts of glucose, fructose and organic acids at pH 5.5 was used as negative control. The adherence of S. mutans (uncoated and saliva-coated) to either sHA or gsHA treated with 25% CJ (v/v) was remarkably reduced (40-85% inhibition compared to control: p < 0.05), indicating that CJ effectively blocked the bacterial adherence to binding sites in salivary pellicle and in glucans. In contrast, when the bacterial cells alone were treated with CJ they adhered to the similar untreated surfaces. Cranberry juice (25%, v/v) also inhibited the activities of surface-adsorbed GTF B and C (70-80% inhibition compared to control, p < 0.05). The effect of CJ on the viability of microorganisms in biofilms was not significant. Biofilm formation and accumulation were significantly reduced by topical applications of 25% CJ (v/v) twice daily with 1-min exposures (p < 0.05). The biomass and insoluble glucan content of the biofilms in addition to its acidogenicity were significantly reduced by cranberry treatments (p < 0.05). Our data show that cranberry juice inhibited glucan-mediated biofilm development and acid production, and holds promise as a natural product to prevent biofilm-related oral diseases.

Analysis of Variance↗

PubMed2007

Biocidal effect of cathodic protection on bacterial viability in biofilm attached to carbon steel.

Biofilm formed on carbon steel by various species of bacterial cells causes serious problems such as corrosion of steel, choking of flow in the pipe, deterioration of the heat-transfer efficiency, and so on. Cathodic protection is known to be a reliable method for protecting carbon steel from corrosion. However, the initial attachment of bacteria to the surface and the effects of cathodic protection on bacterial viability in the biofilm have not been clarified. In this study, cathodic protection was applied to an artificial biofilm containing Pseudomonas aeruginosa (PAO1), a biofilm constituent, on carbon steel. The aims of this study were to evaluate the inhibition effect of cathodic protection on biofilm formation and to reveal the inhibition mechanisms. The viability of PAO1 in artificial biofilm of 5 mm thickness on cathodically protected steel decreased to 1% of the initial cell concentration. Analysis of pH distribution in the artificial biofilm by pH microelectrode revealed that pH in proximity to carbon steel increased to approximately 11 after cathodic protection for 5 h. Moreover, 99% of region in the artificial biofilm was under the pH conditions of over nine. A simulation of pH profile was shown to correspond to experimental values. These results indicate cells in the artificial biofilm were killed or damaged by cathodic protection due to pH increase.

Biofilms↗

PubMed2006

Survival of Vibrio fluvialis in seawater under starvation conditions.

The viability of Vibrio fluvialis in seawater microcosms, with and without sediment was investigated. The strain survived as culturable bacteria for at least 1 year and the expression of its virulence factors was maintained. In microcosms containing sediment Vibrio fluvialis was more stable. Viable but nonculturable (VBNC) cells of Vibrio fluvialis were able to resuscitate to the culturable state up to 6 years of incubation in marine sediment. These cells recuperate their initial biochemical characteristics after 3 months of incubation in marine broth. Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA) was used to confirm that it is the same strain of Vibrio fluvialis which resists in all microcosms during a long period of time.

Colony Count, Microbial↗

PubMed2006

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake, viability, ergosterol and production of non-protein thiols.

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10mum Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.

Ascomycota↗

PubMed2006

Low-intensity photosensitization may enhance RecA production.

Three bacterial strains-Escherichia coli, Acinetobacter calcoaceticus, and the A. calcoaceticus RecA- mutant-underwent photosensitization by a low-concentration (0.73 micromol/L) tetramethyl pyridyl porphine (a cationic hydrophylic photosensitizer) and a 4-J/cm2 dose of 407 to 420 nm blue light. The viability of the first two strains decreased by approximately 60%. and that of the RecA- strain decreased by 90%. Increasing the amount of photosensitizer to 14.6 micromol/L at the same dose of blue light resulted in a 95% to 98% decrease in viability of the three strains. Very little damage to the bacterial DNA was observed after this treatment. Increasing the concentration photosensitizer under the same illumination conditions also resulted in very little damage to the DNA. Western blotting demonstrated that the low photosensitization procedures enhance RecA production for mending the damaged chromosomal DNA. RecA production as a result of low-dose photosensitization was confirmed and demonstrated by immunofluorescent staining and gold immunolabeling. Although DNA is not the primary target for photosensitization, this process of RecA production may provide a certain degree of DNA mending and may also affect the survival of bacterial cells on low-intensity photosensitization.

Acinetobacter calcoaceticus↗

PubMed2006

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress.

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

Amino Acid Sequence↗

PubMed2006

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria.

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

Anti-Bacterial Agents↗

PubMed2006

Survival, elongation, and elevated tolerance of Salmonella enterica serovar Enteritidis at reduced water activity.

Growing microorganisms on dry surfaces, which results in exposure to low water activity (a(w)), may change their normal morphology and physiological activity. In this study, the morphological changes and cell viability of Salmonella enterica serovar Enteritidis challenged to low a(w) were analyzed. The results indicated that exposure to reduced a(w) induced filamentation of the cells. The amount of filamentous cells at a(w) 0.94 was up to 90% of the total number of cells. Surviving filamentous cells maintained their membrane integrity after exposure to low a(w) for 21 days. Furthermore, cells prechallenged to low a(w), obtained with an ionic humectant, demonstrated higher resistance to sodium hypochlorite than control cells. These resistant cells are able to survive disinfection more efficiently and can therefore cause contamination of foods coming in contact with surfaces. This points to the need for increased attention to cleaning of surfaces in household environments and disinfection procedures in processing plants.

Colony Count, Microbial↗

PubMed2005

A combination of direct viable count and fluorescent in situ hybridization for estimating Helicobacter pylori cell viability.

The viable but non-culturable (VBNC) stage of Helicobacter pylori may represent a problem of public health concern, since these cells cannot be detected by traditional culture methods. In this study, the direct viable count method (DVC) was modified and adapted to H. pylori analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. The DVC procedure was combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of H. pylori (DVC-FISH). Incubation with 0.5 microg/ml of novobiocin for 24 h provided the optimal conditions for obtaining 3-5 times the original size of Helicobacter viable cells. Field work performed with various types of water (freshwater and seawater) using the DVC-FISH approach enabled us to confirm the presence of VBNC H. pylori cells in 16 of the 45 analyzed samples. The combination of the modified DVC procedure with FISH can provide a rapid and specific method to detect and identify viable cells of H. pylori in environmental samples.

Colony Count, Microbial↗

PubMed2006

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

Animals↗

PubMed2006

Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato.

Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3-2.6, oxidation-reduction potential 1007-1025 mV, and free active chlorine concentration 27-35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4-8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide.

Disinfectants↗

PubMed2026

Homologous recombination mutants cause differing lethality between h- and h+ Schizosaccharomyces pombe strains due to mat1 heterochromatin.

Homologous recombination (HR) is generally considered dispensable in yeast and vertebrates, yet mounting evidence indicates that its essentiality depends on cellular context. Here, we dissect the basis of this context dependency in Schizosaccharomyces pombe. In the homothallic h90 strain, regarded as wild type, mating-type switching (MTS) occurs every other cell division and requires HR to repair programmed double-strand breaks (DSBs) at the mat1 locus. We show that the widely used heterothallic h-S strain is likewise dependent on HR for viability. HR-deficient h-S mutants (rad51Δ, rad52Δ, or rad54Δ), still frequently employed in the literature, survive only when carrying secondary suppressor mutations that abolish mat1 DSB formation, such as smt-0, swi1Δ, or fml1Δ. In contrast, HR is dispensable in the h+N strain, where duplication of the mat2/3 region into mat1 introduces the cenH and REIII elements. These elements nucleate H3K9 methylation and heterochromatin spreading across the imprint site, blocking imprintosome recruitment and thereby preventing both imprinting and DSB formation. Disruption of this heterochromatin, via deletion of cenH or key chromatin modifiers, restores DSB formation in h+N cells and reinstates HR essentiality in the absence of the Clr4 methyltransferase. Collectively, our findings demonstrate that HR is indispensable for S. pombe survival due to its critical role in repairing mat1 DSBs, except under genetic or epigenetic conditions that suppress their formation.

Homologous Recombination↗

PubMed2006

Survival and stress induced expression of groEL and rpoD of Campylobacter jejuni from different growth phases.

Although Campylobacter jejuni is the leading cause of bacterial diarrhoeal disease in humans worldwide, its potential to adapt to the stressful conditions and survive in extra-intestinal environment is still poorly understood. We tested the effect of heat shock (55 degrees C, 3 min) and oxidative stress (3 mM H2O2 for 10 min or prolonged incubation at atmosphere oxygen concentration) on non-starved and starved cells of Campylobacter jejuni from different growth phases. Viability as assessed with the Bacterial Viability Kit LIVE/DEAD BacLighttrade mark dying before fluorescent microscopy and culturability of the cells (CFU ml(-1)) from both growth phases showed that starvation increased heat but not oxidative resistance. High temperature and oxidative stress invoked quick transformation from culturable spiral shaped to nonculturable spiral and coccoid cells. Despite physiological changes of the cells we were not able to document clear differences in the expression of heat shock and starvation genes (dnaK, htpG, groEL), oxidative (ahpC, sodB), virulence (flaA) and housekeeping genes (16S rRNA, rpoD) after heat treatment (55 degrees C, 3 min) or oxidative stresses applied. When starving, no induction of expression of any of these genes was noticed, chloramphenicol had no influence on their gene expression. Quantitative real-time PCR analyses showed that at least 10-20 min of heat shock was necessary to evidently increase the amount of groEL and rpoD transcripts.

Adaptation, Physiological↗