Detection and molecular characterization of filamentous actinobacteria and thermoactinomycetes present in water-damaged building materials.

UNLABELLED: In this study the dominant filamentous actinobacteria occurring in water-damaged building materials were detected by culture and characterized by automated ribotyping and 16S rRNA gene sequencing. Fifty-two samples were taken from 20 water-damaged houses in four different countries. A total of 122 bacterial isolates were analyzed. Actinobacteria or thermoactinomycetes were present in 48% of the samples. The dominant genus was Streptomyces (58% of isolates), followed by Thermoactinomyces (23%), Laceyella (14%), Nocardiopsis (3%), Pseudonocardia (1%) and Saccharomonospora (1%). The most frequently detected species was the thermophilic Thermoactinomyces vulgaris (14 samples/4 countries). The most common streptomycetes were closely related to the heterogeneous species Streptomyces microflavus (7/2) or Streptomyces griseus (6/2). Automated ribotyping was a rapid tool for reliable characterization of these isolates. The spores of thermoactinomycetes and toxic substances of Nocardiopsis species and S. griseus may constitute a risk for human health. PRACTICAL IMPLICATIONS: Harmful microbes in indoor environments are a cause of public concern. To develop rapid and simple-to-use molecular biological methods to detect the presence of harmful actinobacterial species in water-damaged buildings more information about their occurrence in those materials is needed, which this study provides.

Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population.

AIM: To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria. METHODS AND RESULTS: Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR-DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR-DGGE 0.8 in the antibiotic vs 4.3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83%vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet(W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet(W) was observed in one of the susceptible strains. CONCLUSIONS: Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.

Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration.

Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

Comparison of three test media for antimicrobial susceptibility testing of bifidobacteria using the Etest method.

The performance of three test media for antimicrobial susceptibility testing of bifidobacteria using the Etest was compared. All Bifidobacterium strains (n=42) displayed good growth on trypticase-phytone-yeast extract agar (TPY). Most strains showed good growth on lactic acid bacteria susceptibility test medium supplemented with cysteine (LSM+cys); Bifidobacterium bifidum showed moderate growth. Growth of seven strains was inadequate on Brucella blood agar (BRU) and an additional eight strains showed moderate growth. The minimum inhibitory concentrations (MICs) for tetracycline were highest on BRU and lowest on LSM+cys (agreement 57%), whereas the MICs for streptomycin were lowest on BRU and highest on TPY (agreement 40%). Occasional mismatches (agreement 71-91%) between the test media were also detected for the beta-lactam antibiotics. This study describes test medium-dependent variation of MICs and the applicability of LSM+cys for antimicrobial susceptibility testing of bifidobacteria.

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1.

AIMS: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. METHODS AND RESULTS: Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. CONCLUSIONS: Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

Occurrence and characterization of actinobacteria and thermoactinomycetes isolated from pulp and board samples containing recycled fibres.

The aim of this study was to characterize the actinobacterial population present in pulps and boards containing recycled fibres. A total of 107 isolates was identified on the basis of their pigmentation, morphological properties, fatty acid profiles and growth temperature. Of the wet pulp and water sample isolates (n=87), 74.7% belonged to the genus Streptomyces, 17.2% to Nocardiopsis and 8.0% to thermoactinomycetes, whereas all the board sample isolates (n=20) were thermoactinomycetes. The identification of 53 isolates was continued by molecular methods. Partial 16S rDNA sequencing and automated ribotyping divided the Streptomyces isolates (n=31) into 14 different taxa. The most common streptomycetes were the mesophilic S. albidoflavus and moderately thermophilic S. thermocarboxydus. The Nocardiopsis isolates (n=11) belonged to six different taxa, whereas the thermoactinomycetes were mainly members of the species Laceyella sacchari (formerly Thermoactinomyces sacchari). The results indicated the probable presence of one or more new species within each of these genera. Obviously, the drying stage used in the board making processes had eliminated all members of the species Streptomyces and Nocardiopsis present in the wet recycled fibre pulp samples. Only the thermotolerant endospores of L. sacchari were still present in the final products. The potential of automated ribotyping for identifying actinobacteria was indicated, as soon as comprehensive identification libraries became available.

Polysaccharide-producing bacteria isolated from paper machine slime deposits.

Development of novel enzymatic methods for slime deposit control in paper mills requires knowledge of polysaccharide-producing organisms and the polysaccharide structures present in deposits. In this work, 27 polysaccharide-producing bacteria were isolated from slime samples collected from different parts of a paper machine. Most of the isolates produced polysaccharides in liquid culture and nine of them were selected for production of polysaccharides for characterisation. The selected isolates belonged to seven different genera: Bacillus, Brevundimonas, Cytophaga, Enterobacter, Klebsiella, Paenibacillus and Starkeya. Using ribotyping, partial 16S rDNA sequencing, physiological tests and fatty acid analysis, four of the nine isolates: Bacillus cereus, Brevundimonas vesicularis, K. pneumoniae and P. stellifer were identified to the species level. Production of polysaccharides by the selected isolates varied between 0.07 and 1.20 g L(-1), the highest amount being produced by B. vesicularis. The polysaccharides were heteropolysaccharides with varying proportions of galactose, glucose mannose, rhamnose fucose and uronic acids.

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.

Genetic heterogeneity and functional properties of intestinal bifidobacteria.

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.

Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis.

Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype 1/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE.

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres.

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.

Potential microbiological hazards in the production of refined paper products for food applications.

This study sought to investigate the significance of raw materials (starch-based glues, raw material papers) at different microbiologically critical stages in the manufacturing process of refined paper products. The study examined the occurrence of microorganisms in the process and in end-product samples. Microbiological surveys verified that the production and use of pasteurized starch-based glue was the most important factor threatening the process hygiene and product safety. Subsequently, the production and use of starch-based glue was changed, and a follow-up programme targeting the microbiological quality of glue was developed as part of a hygiene and safety management system. A total of 33 spore-forming bacterial and 15 enterobacterial isolates were ribotyped, and 22 and 10 different ribogroups (ribotypes), respectively, were generated. These isolates from starch-based glue, raw material paper and end products were atypical and, thus, in many cases physiological, chemotaxonomic (FAME) and molecular (partial 16S rDNA) results did not correspond. The most common spore-forming bacteria (55% of the isolates) were Paenibacillus sp. and within this genus several new species were also proposed. The most common enterobacteria (87%) were Enterobacter cloacae and Citrobacter freundii belonging to bacteria in hazard group 2, or species closely related to them. It was demonstrated that the same spore-forming bacteria (ribotypes) were present in both the glue samples and the end products (45% of isolates). All RiboPrint patterns were saved at the VTT identification library for future use.

Improved synthesis and characterization of 1,3,4,6-tetra-O-acetyl-2-(N-acetylacetamido)-2-deoxy-beta-D-glucopyranose.

A method from the 1960s to synthesize the N,N-diacetyl derivative of peracetylated beta-D-glucosamine was improved by assistance of molecular sieves. The melting point of the title compound was revised and the structure determined by means of X-ray diffraction.

Dry sausage fermented by Lactobacillus rhamnosus strains.

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.

Flavour profiles of dry sausages fermented by selected novel meat starter cultures.

Probiotic or bioprotective Lactobacillus rhamnosus strains GG, LC-705 and E-97800 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 were studied for their ability to act as main fermenting organisms in the manufacturing process of dry sausages. In the preliminary tests, their abilities to produce lactic acid and biogenic amines, histamine or tyramine, were studied in MRS broth and analysed by high-performance liquid chromatography. The strains produced higher or equal amounts of lactic acid compared to control and were amine negative. During the actual fermentation process of dry sausages the numbers of inoculated bacteria increased from the level 6.5-7.0 log cfu/g to 8.0-9.0 log cfu/g. The most fast growing strains were P. pentosaceus E-90390 and the control while the growth of L. plantarum E-98098 and L. rhamnosus LC-705 were the slowest. The pH value of the sausages decreased from 5.6 to 4.9-5.0. The presence of these experimental strains as major organisms in the sausages after fermentation and ripening was confirmed on the bases of their genetic fingerprints. The flavour profiles of the experimental sausages produced by these probiotic or protective strains were similar with that produced by the commercial meat starter culture and commercial North European dry sausage recipe.

Identification of pediococci by ribotyping.

Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library.

The effect of lactose derivatives on intestinal lactic acid bacteria.

Nine strains of lactic acid bacteria were studied for growth and fermentation end products on lactulose, lactitol, and lactobionic acid. In addition, human fecal and biopsy isolates were screened for new potential by probiotic strains utilizing lactose derivatives, and one new isolate of Lactobacillus rhamnosus was enriched. The utilization of lactose derivatives and the effect on the fermentation end products were dependent on strain. Typical mixed-acid fermentations were observed with Lb. rhamnosus and Lactococcus lactis. Microbiota enriched from fecal and biopsy samples using modified MRS medium consisted mainly of enterococci and streptococci. The adhesion of tested strains to Caco-2 cells was not dependent on carbon source. The new Lb. rhamnosus strain VTT E-97800 has potential for further probiotic studies.

Purity of recycled fibre-based materials.

In order to study the purity of recycled fibre-based materials, products containing recycled fibre as well as recycled pulp were examined with regard to their chemical impurities, toxicity and microbiological quality. The study was carried out to clarify both qualitatively and quantitatively the variations in microbiological quality. The levels of several classes of chemical substances were analysed and semi-volatile and volatile substances identified. The toxicity and mutagenicity of virgin fibre and recycled fibre materials were screened using the Photobacter toxicity test and the Ames Salmonella mutagenicity test. Preliminary chemical characterization of the mutagens was carried out. Identification of the compounds found in the mutagenic fractions was performed by gas chromatography/mass spectrometry (GC/MS). The concentration of various substances analysed was found to be low, although the variety of substances present appeared to be very broad. Preliminary chemical characterization revealed that some samples contained compounds known to have mutagenic or other toxic activity. Also, the recycled fibre pulps contained large amounts of various microbes, the microbial load consisting mainly of aerobic spore-forming bacteria. The paper-making process was found to clearly have reduced the total microbial counts.

Microscopic study of migration of microbes in food-packaging paper and board.

The microbiological barrier properties of food-packaging paperboards, coated with polyethylene, mineral pigment or a biodegradable polymer and of high-density paper were examined with confocal laser scanning microscopy. The results show that the spatial distribution of microscopically observable bacterial cells was uneven inside the paperboard. The concentration in the interface between the polyethylene coating and the cellulose fibers was 100-200 times higher than inside the cellulose matrix. The bacteria in the interface and the mineral coating layer grew in response to access to food and moisture, whereas no growth was observed inside the fiber web, not even after extended exposure for up to 90 days. The paper and paperboards studied contained soluble nutrients (C:N:P 54:9:1 to 309:3:1) and no measurable antimicrobial activity. The factor limiting growth and migration of bacteria inside the fiber web was most likely limited access to free water, even under conditions of extensive wetting. The studied paperboards functioned as efficient barriers against translocation of microbes. The microbes residing between the paperboard and its polymer coating facing food, was the only potential site from which microbes could leak into food. This emphasizes the need for high hygienic quality of surface-sizing chemicals. Mineral-coating pigments were a source of microbes and their application behind the PE coating facing food is contraindicated.