Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses. Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR. Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV. Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The responses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the ‘light-minus-dark’ difference spectrum of the chromatophores.The oxonols appear to respond to the light-induced ‘energization’ by shifting their absorption maxima. In the presence of K⁺, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift.The dye response has an apparent second-order rate constant of approx. 2 · 10⁶ M^?1 · s^?1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not posses intrinsic probes of potential.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.     

Mechanism for transport of nitro-blue tetrazolium into viable and non-viable leukocytes

The permeability of the membrane of phagocytic leukocytes (WBC) to the non-complexed nitro-blue tetrazolium (NBT) cation has not been well explored. In this study both complexed and non-complexed (or free) NBT have been incubated with viable cells. The entry of NBT into the WBC was determined by both colorimetric and infrared analyses. Very little free NBT enters the viable WBC compared with NBT complexed with serum factors. NBT entry was also studied in non-viable WBC by use of the infrared system. The entry of NBT into the non-viable cells was independent of serum complexing factors. Much larger quantities of both free and complexed NBT enter non-viable cells than enter viable WBC. It is concluded that the membrane of the viable WBC is quite impermeable to the free NBT cation. Ultrastructural studies suggest that the gaps in the plasma membrane of non-viable cells could allow the passage of relatively large amounts of both free and complexed NBT.     

Mechanism for transport of nitro-blue tetrazolium into viable and non-viable leukocytes

Summary The permeability of the membrane of phagocytic leukocytes (WBC) to the non-complexed nitro-blue tetrazolium (NBT) cation has not been well explored. In this study both complexed and non-complexed (or free) NBT have been incubated with viable cells. The entry of NBT into the WBC was determined by both colorimetric and infrared analyses. Very little free NBT enters the viable WBC compared with NBT complexed with serum factors. NBT entry was also studied in non-viable WBC by use of the infrared system. The entry of NBT into the non-viable cells was independent of serum complexing factors. Much larger quantities of both free and complexed NBT enter non-viable cells than enter viable WBC. It is concluded that the membrane of the viable WBC is quite impermeable to the free NBT cation. Ultrastructural studies suggest that the gaps in the plasma membrane of non-viable cells could allow the passage of relatively large amounts of both free and complexed NBT.     

Exploring the potential environmental functions of viable but non-culturable bacteria

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in “viable but non-culturable” (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.     

The counting of viable tubercle bacteria

Summary The publication ofChristensen, Robinson andWiddicombe (1953) was an occasion to draw renewed attention to the method of counting viable germs in rolling flasks since 1938 used in the Laboratory of Hygiene, State University at Utrecht and elsewhere (Julius, 1938). The way in which technique and medium are adapted to the viable counting of tubercle bacteria is described.     

Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein- and phycoerythrin-conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.     

Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study

Light scattering properties of hybridoma cells were examined with flow cytometry. Viable and dead cells form two distinct populations. The distribution of the two populations changes during a batch culture. the concentration of dead cells measured by flow cytometry correlates well to that measured by hemacytometer. The distribution based on small-angle light scattering is similar to the distribution based on volume as measured by Elzone particle counter. It thus appears that viable cells form the population with a larger mean cell volume. The results also indicate that the volume of viable cells decreases during the cultivation while that of dead cells remains relatively constant.     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets

Ethidium bromide monoazide (EMA) and propidium monoazide (PMA) were utilized to selectively allow real-time PCR (Rti-PCR) amplification of target DNA from viable but not heat killed cells from the mixed bacterial flora derived from cod fillets. A linear range of DNA amplification was exhibited from 3.2x10(2) to 1.0x10(5) genomic targets per Rti-PCR. Following the heat treatment of cell suspensions the surviving populations with the EMA and PMA Rti-PCR method were evaluated by comparison with plate counts and MPN assays following different heat exposures (45 to 95 degrees C) for 5 min. The percent of erroneous survival with PMA Rti-PCR was higher than with EMA treatment. Cellular leakage was examined by following the extracellular increase of 260 and 280 nm absorbing materials. Initial leakage of protein and nucleic acids occurred at 50 degrees C, the maximal amount of leakage occurred at 70 degrees C.     

Comparative recoveries of airborne fungus spores by viable and non-viable modes of volumetric collection

Summary The suitability of viable and non-viable volumetric collectors as prevalence indicators for potentially allergenic airborne fungi was studied during 124 paired exposures of the Burkard (Hirst) spore trap and a modified, wind-oriented Andersen sampler. Overall, viable recoveries of several Cladosporium form species varied directly with microscopic spore counts (p0.0001). However, as spore levels rose, culture plate data progressively underestimated prevailing concentrations (recoveries falling below 5% at levels above 500 spores/M³). Viable collections yielded low estimates of prevalence (20–40%) even at modest Cladosporium levels (< 100 spores/M³) and substantially understated the abundance and regularity in air of several additional taxa. Spores typical of Penicillium and Aspergillus form species were not sought in spore trap deposits. Careful examination of these failed to reveal typical arthrospores or Fusarium macrospores despite substantial recoveries of corresponding growth in culture. Correlations in the occurrence patterns of arthrospore-forming and non-sporulating colonies with those of Coprinus and other basidiospores (excluding Ganoderma) were noted.This work was supported by Research Grant AI-10181 from the National Institute of Allergy & Infectious Diseases, National Institutes of Health, United States Public Health Service.     

ApoC-I and ApoC-III as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke

Early diagnosis and immediate therapeutic interventions are crucial factors to reduce the damage extent and the risk of death. Currently, the diagnosis of stroke relies on neurological assessment of the patient and neuro-imaging techniques including computed tomography and/or magnetic resonance imaging scan. An early diagnostic marker of stroke, ideally capable to discriminate ischemic from hemorrhagic stroke would considerably improve patient acute management. Using surface-enhanced laser desorption/ionization (SELDI) technology, we aimed at finding new early diagnostic plasmatic markers of stroke. Strong anionic exchange (SAX) SELDI profiles of plasma samples from 21 stroke patients were compared to 21 samples from healthy controls. Seven peaks appeared to be differentially expressed with significant p values (p < 0.05). Proteins were stripped from the SAX chips, separated on a one-dimensional electrophoresis (1-DE) gel and stained using mass spectrometry (MS)-compatible silver staining. Following in-gel tryptic digestion, the peptides were analyzed by MS. Four candidate proteins were identified as apolipoprotein CI (ApoC-I), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), and antithrombin-III fragment (AT-III fragment). Assessment of ApoC-I and ApoC-III levels in plasma samples using a sandwich enzyme-linked immunosorbent assay (ELISA) allowed to distinguish between hemorrhagic (n = 15) and ischemic (n = 16) stroke (p < 0.001). To the best of our knowledge, ApoC-I and ApoC-III are the first reported plasmatic biomarkers capable to accurately distinguish between ischemic and hemorrhagic stroke in a small number of patients. It requires further investigation in a large cohort of patients.     

Study of airborne fungus spores by viable and non-viable methods in Havana,Cuba

The Havana aeromycobiota diversity was studied from November 2010 to October 2011 using two complementary volumetric methods. A total of 35 fungal genera were characterised, 26 of them were recognised only by non-viable methods, six with viable methodology and the other three with both sampling methods. Furthermore, 47 species were identified by cultivation and the spores collected with the non-viable methodology. These could not be included in a specific genus, and thus, were categorised into five fungal types. In general, the main, spread worldwide, mitosporic fungi also predominated the Havana atmosphere. The predominant species were Cladosporium cladosporioides, Aspergillus flavus and Penicillium citrinum. Moreover, several Zygomycetes (Syncephalastrum racemosum, Rhizopus stolonifer and Rhizopus oryzae), Ascomycetes (Chaetomium globosum) and Basidiomycetes such as Coprinus or Ganoderma were isolated. In the present paper, the review of the airborne fungi conducted in previous studies in Cuba was completed by the detection of two new genera and the first isolation of ten new records in the Cuban atmosphere. Most of the fungi detected showed a diurnal pattern with high spore peak at 11.00–12.00.     

Modulation of the endocannabinoid system in viable and non-viable first trimester pregnancies by pregnancy-related hormones

Background

In early pregnancy, increased plasma levels of the endocannabinoid anandamide (AEA) are associated with miscarriage through mechanisms that might affect the developing placenta or maternal decidua.

Methods

In this study, we compare AEA levels in failed and viable pregnancies with the levels of the trophoblastic hormones (beta-human chorionic gonadotrophin (beta-hCG), progesterone (P4) and (pregnancy-associated placental protein-A (PAPP-A)) essential for early pregnancy success and relate that to the expression of the cannabinoid receptors and enzymes that modulate AEA levels.

Results

The median plasma AEA level in non-viable pregnancies (1.48 nM; n = 20) was higher than in viable pregnancies (1.21 nM; n = 25; P = 0.013), as were progesterone and beta-hCG levels (41.0 vs 51.5 ng/mL; P = 0.052 for P4 and 28,650 vs 6,560 mIU/L; P = 0.144 for beta-hCG, respectively, but were not statistically significant). Serum PAPP-A levels in the viable group were approximately 6.8 times lower than those in the non-viable group (1.82 vs 12.25 mg/L; P = 0.071), but again these differences were statistically insignificant. In the spontaneous miscarriage group, significant correlations between P4 and beta-hCG, P4 and PAPP-A and AEA and PAPP-A levels were observed. Simultaneously, immunohistochemical distributions of the two main cannabinoid receptors and the AEA-modifying enzymes, fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), changed within both the decidua and trophoblast.

Conclusions

The association of higher AEA levels with early pregnancy failure and with beta-hCG and PAPP-A, but not with progesterone concentrations suggest that plasma AEA levels and pregnancy failure are linked via a mechanism that may involve trophoblastic beta-hCG, and PAPP-A, but not, progesterone production. Although the trophoblast, decidua and embryo contain receptors for AEA, the main AEA target in early pregnancy failure remains unknown.     

The use of fluorogenic esters to detect viable bacteria by flow cytometry

The ability of flow cytometry (FCM) to detect viable bacteria after staining with a range of fluorogenic esters was investigated with several bacterial species. The dyes studied were the fluorescein diacetate (FDA) derivatives carboxyfluorescein diacetate, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester and calcein acetoxymethyl ester, as well as ChemChrome B, a commercially-available stain for the detection of viable bacteria in suspension. No one dye was found to be universal but ChemChrome B dye stained the widest number of Gram-positive and Gram-negative species, whereas the FDA derivatives preferentially stained Gram-positive bacteria. The use of ChemChrome B to detect viable bacteria in environmental samples was investigated further by studying the survival of Klebsiella pneumoniae in lakewater. During survival studies, a higher number of viable bacteria were detected both by direct viable counts and FCM after staining with rhodamine 123 and ChemChrome B than by colony-forming units, suggesting the presence of viable but nonculturable cells. These results demonstrate the potential use of FCM to enumerate viable bacteria in natural waters.     

Monitoring of membrane potential by means of fluorescent dyes and time-resolved fluorescence spectroscopy

This work was supported by the NATO Linkage Grant H TECH.LG 930 686.     

Response of bioluminescent bacteria to sixteen azo dyes

Recombinant bioluminescent bacteria were used to monitor and classify the toxicity of azo dyes. Two constitutive bioluminescent bacteria,Photobacterium phosphoreum andEscherichia coli, E. coli GC2 (lac::luxCDABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminescentE. coli, DPD2794 (recA::luxCDABE), a DNA damage sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classification of some azo dyes, in the field, may be possible using these recombinant bioluminescent bacteria.