Mycobacterium Frederiksbergense Research Articles

Prevalence and species diversity of nontuberculous mycobacteria in drinking water supply system of Bahía Blanca City, Argentina.

There is evidence that tap water is the vehicle through which nontuberculous mycobacteria (NTM) infect or colonize the human body. The objective of this study was to determine the presence and diversity of NTM in the water distribution system of Bahía Blanca city, Argentina (sites S2/S3) and in the dike that supplies water to it (S1). Culture-dependent method, biochemical tests, and molecular method (16S rRNA sequencing gene) were combined to detect and identify NTM. NTM were isolated in 51.6% (64/124) of all the samples analyzed. Mycobacterium gordonae was the most frequently isolated organism (15/64) in all samples analyzed, followed by Mycobacterium peregrinum and Mycobacterium frederiksbergense. Significant differences were found in the residual chlorine values between sampling S2 and S3. In both sites, maximum counts were recorded but they did not correlate with low chlorine values. A concentration higher than 500 colony-forming unit/L of NTM was never found, which can be attributed to the negative effect caused by decontamination methods being a point to consider for the recovery of NTM. In 46.9% (30/64) of samples, both methods coincided in the identification, and the obtained sequences presented ≥99% identity. Identification at the species level was achieved in 50% (32/64) of the isolates. Nearly 17.2% (11/64) of the isolates showed a similarity <99%. It should be taken into account that sequencing of the 16S rRNA gene and biochemical tests are useful for the identification of several species, but it is necessary to incorporate other genes (hsp 65 and rpo B) to obtain accurate identification.

Mycobacteriosis in Farmed Sea Bream (Sparus aurata) Caused By Mycobacterium frederiksbergense in Turkey

Abstract Over the past decades, different mycobacteriosis agents have become important fish pathogens. The present study describes a different mycobacteriosis in farmed sea bream (Sparus aurata) caused by Mycobacterium frederiksbergense in Turkey. Affected 15 fish, weighing 15 to 20 g, showed lethargy, stunted growth, pale skin, dorsal fin necrosis, and a significant level of mortality (40 %) in fish stocks. Internally, no multifocal white-colored granulomas in the visceral organs were observed. Inoculation of the visceral organs onto Löwenstein-Jensen medium and Tryptic Soy Agar (1.5% NaCl) slants produced only fast-growing (2-3 weeks), orange to yellow-colored, photochromogenic acid-fast colonies. Ziehl-Nielsen positive bacterial isolates were identified using a commercially available line probe assay (Genotype Mycobacterium CM/AS assay) and with 16S rRNA gene sequencing analysis based on 16S rRNA gene sequencing, fifteen isolates were identified as Mycobacterium frederiksbergense. Histopathologically, epithelioid cell granulomas were not observed in any vicseral organs but acid-fast bacteria were detected in the liver, kidney, spleen, and heart tissue. This study shows that asystemic mycobacteriosis is observed in sea bream with high mortality.

Isolation of Mycobacterium frederiksbergense From Redundant Tap Water: A Case Report

Non-tuberculosis mycobacteria (NTM) are saprophytic bacteria in environmental resources such as water and soil. The presence of atypical mycobacteria in hospital resources may lead to infections and the spread of aerosol particles through ventilation systems, wind, and even drinking water. Therefore, control of contamination of environmental resources in hospitals is one of the most important approaches to reduce and manage NTM nosocomial infections. This study reported the isolation of Mycobacterium frederiksbergense from a tap water sample, which is considered important for clinical and biodegradation aspects. The isolated bacterium was identified using phenotypic features and 16S rRNA sequencing. This report verified the necessity to identify the presence of NTM in water and to find a solution for controlling such contaminations.

Metarhizium brunneum - An enzootic wireworm disease and evidence for its suppression by bacterial symbionts.

Wireworms (Coleoptera: Elateridae) are serious agricultural pests, with soil-dwelling larvae attacking subterranean tissues of crop plants and their fruit when in contact with the soil surface. Researchers collect wireworms for laboratory experiments to study their behaviour and test pest control agents but frequently lose them to Metarhizium Petch (Ascomycota: Hypocreales: Clavicipitaceae) infection. We found latent M. brunneum infection in 13-100% of live, asymptomatic Agriotes obscurus and A. lineatus wireworms acquired from agricultural fields and in wireworms maintained indoors, indicating its enzootic presence. M. brunneum DNA in the wireworms maintained indoors sometimes exceeded 250pg/ug total DNA (0.025% of whole-sample DNA mass). Expressed as copies of M. brunneum DNA/g, unadulterated soil levels of M. brunneum ranged from 4037 in agricultural field soil to 721,538 in soil harbouring a wireworm collection indoors, with the prevalence of latently-infected live wireworm specimens being directly related to soil levels. M. brunneum levels in live wireworms, when regressed against relative levels of 394 bacteria species in the microbiome, were proportionally related to only four: Pantoea agglomerans, Pandoraea pnomenusa, Nocardia pseudovaccinii, and Mycobacterium frederiksbergense. All four of these bacteria have previously been reported to express antimicrobial mechanisms. Consistent with occurrences of disease immunity reported for other pathogen-insect pairs, symbiotic bacteria may be suppressing M. brunneum-induced wireworm mortality. This would help explain why wireworms commonly succumb to infection after being brought into sterilized conditions, as well as the sometimes limited efficacy of M. brunneum when using it as a pest control agent in the field.

Heterogenic response of prokaryotes toward silver nanoparticles and ions is facilitated by phenotypes and attachment of silver aggregates to cell surfaces.

Tons of anthropogenic silver nanoparticles (AgNPs) are assumed to be released into the environment due to their use in many consumer products. AgNPs are known to be toxic toward microorganisms and thus may harm their specific functions in ecosystems. Here we explore the impact of AgNPs on functioning of single cells in microbial populations at doses typically found in anthropogenic environments. The response of single cells to AgNPs was analyzed by flow cytometry and using the fluorescent dyes propidium iodide and DiBAC4 (3) as markers for cell membrane disintegration and depolarization, respectively. The effects of 10-nm and 30-nm AgNPs on three bacterial species (Mycobacterium frederiksbergense, Pseudomonas putida, and Escherichia coli) showed that the populations split into affected cells and others not showing any malfunction, with varying abundances depending on strains and cell growth states. Further, the dissolution of AgNPs measured with 3 KDa ultrafiltration and inductively coupled plasma-mass-spectrometry to distinguish particle-related effects from toxicity of dissolved Ag revealed that Ag ions were the principal toxicant. AgNP aggregate formation was followed by dynamic light scattering and the aggregates' attachment to cell surfaces was visualized by transmission electron microscopy and scanning electron microscopy-energy dispersive X-ray spectroscopy. An increased AgNP-affected cell fraction relative to the Ag ion impact was identified. The study shows that individual cells in a population cope differently with AgNP induced stress by evolving heterogeneous phenotypes. The response is linked to cell death and cell energy depletion depending on cell type and cell growth states. The attachment of AgNP aggregates to cell surfaces seems to amplify the heterogeneous response. © 2017 International Society for Advancement of Cytometry.

R-strategist versus K-strategist for the application in bioremediation of hydrocarbon-contaminated soils

A laboratory study was undertaken to determine the degradative potentials of the pure bacterial strains Mycobacterium frederiksbergense IN53 (Actinobacteria, a K-strategist) and Acinetobacter sp. IN47 (Gammaproteobacteria, an r-strategist) as well as a combination of these strains under harsh environmental conditions (high hydrocarbon load: 50,883 and 46,682 mg kg−1 dry weight of soil for non-sterile and sterile soil samples, respectively; moisture content lower than 20%). A decrease in total petroleum hydrocarbons (TPH) and the activity of soil enzymes (dehydrogenase and lipase) indicated that the effects of M. frederiksbergense IN53 and the consortium were equivalent. However, these two microbial treatments showed significantly better degradative actions than Acinetobacter sp. IN47. Similar biodegradation yields were observed in sterile and non-sterile soil samples. These results suggest that bioaugmentation with M. frederiksbergense IN53 (K-strategist) provides a better approach for the treatment of hydrocarbon-contaminated sites in which the optimum remediation bioprocess conditions are difficult to maintain.

Susceptibility Testing of &amp;lt;i&amp;gt;Mycobacterium frederiksbergense&amp;lt;/i&amp;gt; Strains Isolated from Alfalfa Plants against Antibacterial Compounds

Mycobacteria constitute vast population of bacteria that are found in animals, plants and outer environments. Tuberculosis mycobacteria are a group that causes very dangerous diseases for human and animals. Nontuberculous mycobacteria are mainly environmental and are pathogenic in certain circumstances. There are reports of finding mycobacteria in plant tissues, which can be effective to survey in case of different infectious disease issues. In a previous study, we found mycobacteria strains in alfalfa vascular tissues and identified them to species level using 16S rRNA gene sequence analysis, causing some symptoms in plant tissues and aerial parts. As these rapidly growing mycobacteria (RGM) have the potential to cause opportunistic infections in human tissues, one of the most important issues for dealing with these agents is to find the best and most effective drug to treat them. Here, we conducted some assays to investigate the MIC of 7 antibiotics and two antibacterial agents against eight Mycobacterium frederiksbergense alfalfa isolated strains, in order to find the susceptible and resistant ones. We performed susceptibility testing based on broth microdilution method using Mueller hinton broth as the medium used. Results were different among the strains and different resistant and susceptible reactions were observed among different strains against each drug tested, but ethambutol resulted in similar high MIC for all of them. This variability could be attributed to many factors, such as plant tissue composition and developmental stage from which the strains have been isolated, existence and nature of drug targets and other factors such as test conditions and environmental causes. These results can bring us some insights into design and develop new drugs for treating these kinds of organisms, based on the variations in susceptibility reactions and the plant composition effect on them.

Bacterial community of biofilms developed under different water supply conditions in a distribution system

In order to understand the bacterial community characteristics of biofilms developed under different finished water supply histories in drinking water distribution systems (DWDS), biofilm samples on different type of iron corrosion scales in a real DWDS were collected and systematically investigated using 454 pyrosequencing of 16S rRNA gene. The richness and diversity estimators showed that biofilms formed in DWDS transporting finished groundwater (GW) had the lowest level of bacterial diversity. From phylum to genus level, the dominant bacterial groups found in the biofilms under finished surface water (SW) and GW conditions were distinct. Proteobacteria was the dominant group in all biofilm samples (in the range of 40%–97%), but was relatively higher in biofilms with GW. The relative abundance of Firmicutes in biofilms with SW (28%–35%) was significantly higher (p<0.01) than that in biofilms with GW (0.5%–2.88%). Statistical analysis (Spearman's rank) revealed that alkalinity and chemical oxygen demand (CODMn) positively correlated with the relative abundance of Proteobacteria and Firmicutes, respectively. The abundance of sequences affiliated to iron-reducing bacteria (mainly Bacillus) and iron-oxidizing bacteria (mainly Acidovorax) were relatively higher in biofilms with SW, which might contribute to the formation of much thicker or tubercle-formed corrosion scales under SW supply condition. Several potential opportunistic pathogens, such as Burkholderia fungorum, Mycobacterium neoaurum, Mycobacterium frederiksbergense were detected in the biofilms.

Pyrene Biodegradation by Mycobacterium Frederiksbergense Using an Encapsulated Oil System

Polycyclic aromatic hydrocarbons (PAHs) belong to a large group of organic pollutants and are persistent in nature. Negligible bioavailability due to low aqueous phase solubility of these compounds largely limits their biodegradation. The present study investigated oil encapsulation method for delivery and enhancement of bioavailability in biodegradation of PAHs using Mycobacterium frederiksbergense. Using pyrene as the model PAH compound, different organic solvents were tested for their solubilizing ability and biocompatibility with pyrene. Solubility of pyrene in silicone oil, paraffin oil, hexadecane, and isopropyl myristate (IPM) were found to be 2.8, 25.5, 40, and 72 g/l, respectively. Besides, paraffin oil and silicone oil showed relatively high degrees of biocompatibility. However, based on both pyrene solubilizing ability and biocompatibility, paraffin oil was further selected and pyrene-containing paraffin oil was successfully encapsulated within chitosan-coated alginate-polyvinyl alcohol (PVA) microspheres with more than 99% encapsulation efficiency. Nearly complete degradation of 20 g/l pyrene by M. frederiksbergense was achieved within seven days employing the novel paraffin oil encapsulated PAH delivery system.

Surfactant aided biodegradation of pyrene using immobilized cells of Mycobacterium frederiksbergense

Low aqueous phase solubility is the major limiting factor in successful biodegradation of pyrene and other polycyclic aromatic hydrocarbons (PAH), which can, however, be overcome by using a suitable surfactant. Biodegradation of pyrene by immobilized cells of Mycobacterium frederiksbergense in presence of non-ionic surfactant Tween 80 was evaluated. For cell immobilization, beads were prepared using calcium alginate as the immobilizing material based on immobilized cell viability and mechanical stability of the beads. Complete degradation of pyrene was achieved employing the immobilized cells in batch shake flask experiments for all four different initial concentrations of the PAH at 100 mg l−1, 200 mg l−1, 400 mg l−1 and 1000 mg l−1. The experimental results of biodegradation of pyrene at very high initial concentration of 1000 mg l−1 using the cell immobilized beads was further investigated in a 3 l fermentor operated at controlled conditions of 150 rpm, 28 °C, pH 7 and 1.5 l min−1 aeration. The results confirmed complete degradation of the PAH with a very higher degradation rate of 250 mg l−1 d−1, which is so far the highest value reported for pyrene biodegradation.

Chitosan-coated alginate-polyvinyl alcohol beads for encapsulation of silicone oil containing pyrene: a novel method for biodegradation of polycyclic aromatic hydrocarbons

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are potential hazards in the environment owing to their toxic, carcinogenic and recalcitrant nature. Biodegradation of these compounds, although effective compared with other treatment techniques, is problematic owing to its low aqueous solubility and negligible bioavailability. The present study reports a novel method for biodegradation of PAHs using an encapsulated form of the pollutant in chitosan-coated alginate–polyvinyl alcohol (PVA) beads. RESULTS: A suitable combination of 3% (w/v) PVA, 100 g L−1 non-ionic surfactant Brij 30 and 0.3 silicone oil fraction in the formulation was found to be optimal in the preparation of stable emulsion. The emulsion obtained was admixed with alginate (3% w/v) to prepare suitably sized microspheres by an emulsion gelation technique, which were later coated with chitosan to yield a maximum pyrene encapsulation efficiency of 90.7%. Pyrene in silicone oil at concentration as high as 2 g L−1, when delivered through the chitosan coated alginate–PVA beads, was completely degraded by Mycobacterium frederiksbergense within 10 days without any significant lag phase. CONCLUSION: Using chitosan-coated alginate–PVA beads sustained release of pyrene and subsequent biodegradation by M. frederiksbergense were achieved. Using the present system, complete degradation of pyrene was attained even at its very high initial concentration and within a short time period. Further advantage offered by this system seems to be negligible toxic effect of pyrene and solvents on the degrading microorganisms since these were in an encapsulated form and were not in direct contact with the organism. Copyright © 2010 Society of Chemical Industry

An Immobilized Cell System for Biodegradation of Pyrene by Mycobacterium Frederiksbergense

Pyrene biodegradation by immobilized cells of Mycobacterium frederiksbergense was investigated in the present study. For immobilizing the cells, ion tropic gelation method using sodium alginate and CaCl2 as the immobilizing materials was applied. Concentration of sodium alginate was optimized based on immobilized cell growth and mechanical stability of the resulting beads. Based on the results obtained beads prepared using 5% sodium alginate and 3.5% CaCl2 was chosen for pyrene biodegradation by M. frederiksbergense in which encapsulated silicone oil containing pyrene was used for delivery of pyrene. For a very high pyrene concentration of 2000 mg/l in the oil phase, nearly 90% degradation of pyrene could be achieved by the immobilized cell system without any significant lag phase in degradation. Further, the study indicated the possibility of using non-biocompatible and bio-available non aqueous phase liquid (NAPL) in two phase partitioning bioreactor system for pyrene biodegradation by the immobilized microorganism present in the aqueous phase.

Isothermal titration calorimetry — A new method for the quantification of microbial degradation of trace pollutants

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2 -3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4 -(2 ,4 -dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis–Menten kinetics. The half-saturation constant k D and the degradation rate r D Max were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.

A two liquid phase partitioning bioreactor system for the biodegradation of pyrene: Comparative evaluation and cost–benefit analysis

AbstractBACKGROUND: Biodegradation of pyrene, a novel polycyclic aromatic hydrocarbon (PAH) of environmental concern, was investigated employing a two‐liquid phase partitioning bioreactor (TPPB) using Mycobacterium frederiksbergense. After initial screening of solvents, silicone oil was chosen as the biocompatible and economic solvent for use in this TPPB system with an initial pyrene concentration of 400 mg L−1. The efficiency of the TPPB system developed was also compared with the results of pyrene degradation by the microorganism in slurry phase and surfactant‐aided systems. A cost–benefit analysis comparison of the three systems evaluated in the work further revealed very good potential of the TPPB system in pyrene biodegradation.RESULTS: Using silicone oil as the non‐aqueous phase liquid in the TPPB system, pyrene was found to be completely degraded by M. frederiksbergense within 6 days with a degradation rate of 140 mg L−1 d−1, which was at least seven times more than the values obtained in the slurry phase or surfactant‐aided systems. Further cost–benefit analysis comparison of the three systems revealed TPPB to be the most effective with a minimum cost of $ 0.62 per mg of pyrene degraded.CONCLUSION: Performance of TPPB system, in terms of both efficiency and economics of pyrene degradation by M. frederiksbergense was found to be superior to slurry phase or surfactant aided systems. Copyright © 2010 Society of Chemical Industry

Clinical Management of Rapidly Growing Mycobacterial Cutaneous Infections in Patients after Mesotherapy

Increasing numbers of patients are expressing an interest in mesotherapy as a method of reducing body fat. Cutaneous infections due to rapidly growing mycobacteria are a common complication of such procedures. We followed up patients who had developed cutaneous infections after undergoing mesotherapy during the period October 2006-January 2007. Sixteen patients were infected after mesotherapy injections performed by the same physician. All patients presented with painful, erythematous, draining subcutaneous nodules at the injection sites. All patients were treated with surgical drainage. Microbiological examination was performed on specimens that were obtained before and during the surgical procedure. Direct examination of skin smears demonstrated acid-fast bacilli in 25% of the specimens that were obtained before the procedure and 37% of the specimens obtained during the procedure; culture results were positive in 75% of the patients. Mycobacterium chelonae was identified in 11 patients, and Mycobacterium frederiksbergense was identified in 2 patients. Fourteen patients were treated with antibiotics, 6 received triple therapy as first-line treatment (tigecycline, tobramycin, and clarithromycin), and 8 received dual therapy (clarithromycin and ciprofloxacin). The mean duration of treatment was 14 weeks (range, 1-24 weeks). All of the patients except 1 were fully recovered 2 years after the onset of infection, with the mean time to healing estimated at 6.2 months (range, 1-15 months). This series of rapidly growing mycobacterial cutaneous infections highlights the difficulties in treating such infections and suggests that in vitro susceptibility to antibiotics does not accurately predict their clinical efficacy.

Batch biodegradation of PAHs in mixture by Mycobacterium frederiksbergense: analysis of main and interaction effects

Biodegradation is an effective technique to remediate soil and water contaminated with polycyclic aromatic hydrocarbons (PAHs). In the present experimental study, Mycobacterium frederiksbergense, a fast growing mycobacterium, was used to degrade anthracene, naphthalene and pyrene in mixture each at initial concentrations varying between 1 and 50 mg l−1. Experiments were conducted according to the 23 factorial design at the low (1 mg l−1) and high (50 mg l−1) levels of the PAHs in combination, to identify the main and interaction effects of the compounds on their biodegradation. The results showed that the PAH removals varied 54–81% when each PAHs were at low concentrations in the mixture and 67–89% at their higher concentration combinations. Statistical analysis of the results in the form of ANOVA and Student t test indicated significant role played by the main effects of pyrene on its degradation. Similarly on anthracene degradation, interaction effect with pyrene was found to be highly significant with P value less than 0.1.

PYRENE ENCAPSULATED ALGINATE BEAD TYPE FOR SUSTAINED RELEASE IN BIODEGRADATION: PREPARATION AND CHARACTERISTICS

A novel method for preparation of pyrene encapsulated alginate beads for controlled release delivery of pyrene is reported in this study. Five different bead types that varied on the organic phase for pyrene delivery, presence or absence of polyvinyl alcohol (PVA) and boric acid treatment were prepared. Based on the pyrene release profile, silicone oil encapsulated in PVA-alginate bead was selected where encapsulation efficiency was more than 99% without any solvent leakage. Silicone oil encapsulation was further confirmed in microscopy observations. Pyrene release behavior from the beads was explained by a diffusion controlled first order release (R2 > 0.96). Reusability of this bead was demonstrated, where pyrene was loaded from an external medium in a non-destructive way. Application of the system was finally established in a biodegradation study using Mycobacterium frederiksbergense, where simultaneous release and removal of pyrene along with appearance of pyrene metabolites was observed.

SYNCHRONOUS FLUORESCENCE AS A SELECTIVE METHOD FOR MONITORING PYRENE IN BIODEGRADATION STUDIES

A simple and effective method based on synchronous fluorescence spectroscopy for quantifying pyrene and its metabolites in a biodegradation study using Mycobacterium frederiksbergense is reported in this work. An optimum excitation-emission wavelength offset of 39 nm and a corresponding excitation wavelength of 335 nm was found to be suitable for the determination of pyrene. Interference due to naphthalene and anthracene in the analysis were found to be 11 and 4.7%, respectively. Batch biodegradation experiments in a 3L fermentor revealed a high pyrene degradation rate of 10.22 mg L− 1 d− 1 by the mycobacterium in presence of the surfactant tween 20. Tween 20 was, however, found preferentially utilized over pyrene, which resulted in a lag of 120 h in pyrene degradation by the culture. In the absence of the surfactant, pyrene degradation rate was found to be only 6.1 mg L− 1 d− 1, but without any lag in its degradation.

Biodegradation of pyrene by Mycobacterium frederiksbergense in a two-phase partitioning bioreactor system

Biodegradation of pyrene by Mycobacterium frederiksbergense was studied in a two-phase partitioning bioreactor (TPPB) using silicone oil as non-aqueous phase liquid (NAPL). The TPPB achieved complete biodegradation of pyrene; and during the active degradation phase, utilization rates of 270, 230, 139, 82 mg l −1 d −1 for initial pyrene loading concentrations (in NAPL) of 1000, 600, 400 and 200 mg l −1, respectively, were obtained. The degradation rates achieved using M. frederiksbergense in TPPB were much higher than the literature reported values for an ex situ PAH biodegradation system operated using single and pure microbial species. The degradation data was fitted to simple Monod, logistic, logarithmic, three-half-order kinetic models. Among these models, only exponential growth form of the three-half-order kinetic model provided the best fit to the entire degradation profiles with coefficient of determination ( R 2) value >0.99. From the experimental findings, uptake of pyrene by the microorganism in TPPB was proposed to be a non-interfacial based mechanism.

Limits of propidium iodide as a cell viability indicator for environmental bacteria

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.