The toxic mode of action of cyclic lipodepsipeptide fusaricidins, produced by Paenibacillus polymyxa, toward mammalian cells.

AIMS: Toxigenic strains of Paenibacillus polymyxa were isolated from buildings connected with the symptoms of ill health. Our aim was to identify the toxic compounds of Paenibacillus polymyxa and to describe their toxic actions. METHODS AND RESULTS: The toxins of Paenibacillus polymyxa were purified and analysed by HPLC and mass spectrometry. Toxic fusaricidins A and B, and LI-F05a with mass ions at m/z 883·7, 897·6 and 897·6, respectively, were found. The cytotoxicity of purified fusaricidins A and B was measured using boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts. The ion channel forming properties of fusaricidins were studied using the black lipid membrane (BLM) technique. Fusaricidins A and B depolarized the mitochondria of boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts at concentrations of 0·5-1 µg ml-1 and caused nuclear fragmentation and induced apoptosis at concentrations of 2·5-5 µg ml-1 . Furthermore, fusaricidins A and B induced K+ permeating single channels. CONCLUSIONS: It was concluded that fusaricidins were toxic to mitochondria and induced apoptosis in mammalian cells. It was proposed that the observed toxicity of fusaricidins is due their ion channel forming properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper revealed, for the first time, the mode of action of Paenibacillus polymyxa fusaricidins toxins towards mammalian cells. Fusaricidins, due to their potassium ionophoricity and mitochondria depolarizing impacts, may have contributed to the health damage observed at sites where the producer strains were isolated at high density.

Building-related symptoms are linked to the in vitro toxicity of indoor dust and airborne microbial propagules in schools: A cross-sectional study.

INTRODUCTION: Indoor microbial toxicity is suspected to cause some building-related symptoms, but supporting epidemiological data are lacking. OBJECTIVE: We examined whether the in vitro toxicity of indoor samples from school buildings was associated with work-related health symptoms (building-related symptoms, BRS). METHODS: Administrators of the Helsinki City Real Estate Department selected 15 schools for the study, and a questionnaire on symptoms connected to work was sent to the teachers in the selected schools for voluntary completion. The cellular toxicity of classroom samples was determined by testing substances extracted from wiped indoor dust and by testing microbial biomass that was cultured on fallout plates. Boar sperm cells were used as indicator cells, and motility loss was the indicator for toxic effects. The effects were expressed as the half maximal effective concentration (EC50) at which >50% of the exposed boar sperm cells were immobile compared to vehicle control. RESULTS: Completed symptom questionnaires were received from 232 teachers [median age, 43 years; 190 (82.3%) women] with a median time of 6 years working at their school. Samples from their classrooms were available and were assessed for cellular toxicity. The Poisson regression model showed that the impact of extracts of surface-wiped school classroom dust on teacher work-related BRS was 2.8-fold (95% CI: 1.6-4.9) higher in classrooms with a toxic threshold EC50 of 6µgml-1 versus classrooms with insignificant EC50 values (EC50 >50µgml-1); P<0.001. The number of symptoms that were alleviated during vacation was higher in school classrooms with high sperm toxicity compared to less toxic sites; the RR was 1.9 (95% CI: 1.1-3.3, P=0.03) for wiped dust extracts. CONCLUSIONS: Teachers working in classrooms where the samples showed high sperm toxicity had more BRS. The boar sperm cell motility inhibition assay appears promising as a tool for demonstrating the presence of indoor substances associated with BRS.

Boar spermatozoa as a biosensor for detecting toxic substances in indoor dust and aerosols.

The presence, quantity and origins of potentially toxic airborne substances were searched in moisture damaged indoor environments, where building related ill health symptoms were suspected and reference sites with no health complaints. Boar spermatozoa were used as the toxicity sensor. Indoor aerosols and dusts were collected from kindergartens, schools, offices and residences (n=25) by electrostatic filtering, vacuuming, wiping from elevated surfaces and from the interior of personal computers. Toxicity was measured from the ethanol or methanol extracts of the dusts and aerosols. EC(50) was expressed as the lowest concentration of the airborne substance that inhibited motility of >50% of the exposed sperm cells compared to vehicle control, within 30 min, 1 day or 3-4 days of exposure. Remarkably toxic aerosols (EC(50)

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin.

AIM: To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group. METHODS AND RESULTS: Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21-37 degrees C for B. subtilis and 11-21 degrees C for B. mojavensis. Both species produced amylosin in air as well as in 7-8% CO(2) with 8-9% O(2). CONCLUSIONS: Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus.

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate.

AIMS: To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms. METHODS AND RESULTS: Hyphal extracts of an indoor fungus, identified as the cycloheximide-tolerant species Acremonium exuviarum, inhibited motility of boar spermatozoa (EC(50) 5 +/- 2 microg of crude solids ml(-1)) and caused cytolysis of murine neuroblastoma cells (MNA) and feline fetal lung cells (FL). The responsible substances were purified and identified as two structurally similar, heat-stable, novel, toxic peptaibols, 1726 Da and 1740 Da, respectively, with amino acid sequences of Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Aib-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH and Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Val-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH. Purified acrebol inhibited motility of boar sperm, depleted ATP half-content in 1 day (EC(50) of 0.1 microg ml(-1), 60 nmol l(-1)) depolarised the mitochondria after 2 days, but did not affect the cellular content in NADH. This indicates mitochondrial toxicity. Plate-grown biomass of A. exuviarum BMB4 contained 0.1-1% (w/w) of acrebol, depending on the culture medium. CONCLUSIONS: Acrebol paralysed the energy generation of mammalian cells suggesting that mitochondria were its target of action. SIGNIFICANCE AND IMPACT OF THE STUDY: Acremonium exuviarum, as an indoor fungus, is potentially hazardous to health because of the toxic peptaibols that it produces.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato.

AIM: To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. METHODS AND RESULTS: In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. CONCLUSIONS: Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.

Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk.

To elucidate the occurrence of heat-stable toxin-producing strains among mastitic Bacillus isolates, 100 milk samples of mastitic cows from different parts of Finland were screened. Bacillus was identified as the major organism in 23 samples. Toxinogenic Bacillus isolates identified by sperm cell motility inhibition assay were isolated from six samples. Four isolates belonged to the species Bacillus pumilus and two to Bacillus licheniformis. The toxic substances were heat-stable and soluble to methanol thus being of non-protein nature. The methanol extracted substances disrupted the sperm cell plasma membrane permeability barrier at exposure concentrations of 1-15 microg ml(-1) (B. pumilus) or 20-30 microg ml(-1) (B. licheniformis). The toxic properties of the two mastitic B. licheniformis strains were similar to those of B. licheniformis strains known to produce the lipopeptide lichenysin A and the synthetase genes lchAA, lchAB and lchAC for lichenysin were found in the mastitic strains by PCR. Toxin synthetase genes for the syntheses of lichenysin or surfactin were searched but not found in the toxic B. pumilus strains. The ribopatterns of the mastitic B. pumilus and B. licheniformis isolates were similar to those of the toxinogenic strains described earlier from food poisoning incidents and contaminated indoor air. B. licheniformis and B. pumilus survive pasteurization and other heat treatments as spores. Toxin-producing strains of these species in the dairy production chain may thus be of food safety concern.

Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization.

Pig mycobacteriosis is the most common animal mycobacterial disease in Finland with a long-term average prevalence of 0.34% and temporary peaks as high as 0.85%. In the current study Mycobacterium-specific real-time qPCR and 16S rRNA sandwich hybridization were utilized for culture-independent detection and measurement of potentially infectious mycobacteria in selected piggeries. Participating herds (n=5) were selected according to prevalence of tuberculous lesions (>4%) in slaughtered carcasses. When DNA extracted from piggery bedding materials was analyzed by Mycobacterium-targeted qPCR using the SYBR green I dye for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA were detected in unused bedding materials and 10(8) to 10(10)g(-1) dry weight in used bedding materials. When Mycobacterium-specific hybridization probes were used for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA g(-1) dry weight were detected in unused bedding materials in four out of the five piggeries studied and up to 10(8) cell equivalents in used bedding material. The results were confirmed by the Mycobacterium-specific 16S rRNA sandwich hybridization assay. The present results show, that mycobacteria occur in organic materials commonly used on pig farms, and may proliferate in bedding materials during use. We also show that DNA- and RNA-based methods may be utilized for detection of environmental reservoirs of mycobacteria causing porcine and human infection.

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili.

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

Use of antimicrobials and risk of type 1 diabetes in a population-based mother-child cohort.

AIMS/HYPOTHESIS: The aim of this study was to investigate whether the use of antimicrobials is associated with the risk of childhood type 1 diabetes. MATERIALS AND METHODS: The study population included all children born in Finland between 1996 and 2000 who were diagnosed with type 1 diabetes by the end of 2002. For each case (n=437), four matched controls were selected. Data on diabetes and the maternal use of antimicrobials was derived from nationwide registries. RESULTS: Maternal use of phenoxymethyl penicillins (odds ratio [OR]=1.70, 95% CI 1.08-2.68, p=0.022) or quinolone antimicrobials (OR=2.43, 95% CI 1.16-5.10, p=0.019) before pregnancy was associated with an increased risk of type 1 diabetes in the child, whereas the use of other specific antimicrobials was not related to the risk. The risk was also higher among mother-child pairs where macrolides were used both by the mother before pregnancy and by the child, compared with pairs where neither used macrolides (OR=1.76, 95% CI 1.05-2.94, p=0.032). Maternal use of antimicrobials during pregnancy was not associated with an increased risk. The high use of antimicrobials by the child (more than seven vs seven or less purchases) was related to greater risk (OR=1.66, 95% CI 1.24-2.24, p=0.001). CONCLUSIONS/INTERPRETATION: Overall, the use of antimicrobials before pregnancy, during pregnancy or during childhood was not related to the risk of childhood type 1 diabetes. However, the use of some specific antimicrobials by the mother before pregnancy and by the child may be associated with an increased risk. Further studies are needed to confirm these associations and to elucidate the underlying mechanisms of action.

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project.

An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.

Description of heterotrophic bacteria occurring in paper mills and paper products.

AIMS: To isolate aerobic mesophilic bacilli and thermophilic bacteria from different paper mill samples and to evaluate their potential harmfulness. METHODS AND RESULTS: A total of 109 mesophilic and 68 thermophilic isolates were purified and characterized by automated ribotyping and partial 16S rDNA sequencing. The mesophilic isolates belonged to the genera Bacillus (13 taxa), Brevibacillus (three taxa) and Paenibacillus (five taxa). The thermophilic bacteria represented seven taxa of Bacillus, Geobacillus or Paenibacillus, four of proteobacteria and one of actinobacteria. The most frequently occurring bacteria were Bacillus cereus, B. licheniformis, Pseudoxanthomonas taiwanensis and bacteria closely related to Paenibacillus stellifer, P. turicensis or Leptothrix sp. One mill was contaminated throughout with bacteria of a novel mesophilic genus most closely related to Brevibacillus centrosporus and another with bacteria of a novel thermophilic genus most closely related to Hydrogenophilus thermoluteolus. One B. cereus isolate producing haemolytic diarrhoeal enterotoxin was detected and all the tested B. licheniformis isolates produced a metabolite toxic to boar sperm cells. CONCLUSIONS: The bacilli and thermophilic bacteria isolated represent species which should not present occupational hazards in paper mill environments. The most harmful bacterium detected was B. licheniformis and potentially also B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the microbial diversity in a paper mill provides a rational basis for development of an effective controlling programme. A database constructed from the fingerprints generated using automated ribotyping helps to identify and trace the contamination routes of bacteria occurring in paper mills.

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food.

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography-ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 microg ml(-1) or g(-1) wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (> 99.5% N2), less than 0.05 microg of cereulide ml(-1) or g(-1) wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l(-1)) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16-24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

A new method for in vitro detection of microbially produced mitochondrial toxins.

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Colored moderately thermophilic bacteria in paper-machine biofilms.

Biofilms cause several problems in papermaking. This report describes a microbiological survey of colored biofilms in six paper and board machines, including two case studies of outbreaks of colored slimes in which the causative bacteria were found. A total of 95 pink-, red-, orange- or yellow-pigmented strains were isolated. Nearly all (99%) of the strains grew at 52 degrees C, 72% grew at 56 degrees C, but only 30% grew at 28 degrees C, indicating that most of the strains were moderately thermophilic. Biofilm formation potential and biocide susceptibility of the strains were analyzed with a microtiter plate assay. In the presence of 5 ppm of methylene bisthiocyanate or 2,2-dibromo-3-nitrilopropionamide in paper-machine water, 55 strains formed biofims. Moreover, 39 strains increased biofilm production by 5-753% in the presence of biocide, suggesting that biocide concentrations inhibitory to planktonic but not to surface-attached cells may actually promote biofouling. The cells may have inactivated a portion of the biocides, as the cell density in this assay was high, corresponding to the highest cell densities occurring in the circulating waters. Four groups of colored bacteria that were isolated from several mills were identified. Pink-pigmented Deinococcus geothermalis and red-pigmented Meiothermus silvanus occurred as common primary biofilm-formers in paper machines. This report is the first description of the involvement of Meiothermus species in red-slime formation in the paper industry. The third group of bacteria (putative new species related to Roseomonas) contained strains that were not biofilm formers, but which were commonly found in slimes of neutral or alkaline machines. The fourth group contained red-pigmented biofilm-forming strains representing a novel genus of alpha- Proteobacteria related to Rhodobacter. Many colored paper-machine bacteria are species previously known from microbial mats of hot springs. Some characteristics of the bacterial groups are described here in order to facilitate their recognition in future cases of colored-slime outbreaks in the paper industry.