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In the present study, polyphasic analysis [cultivation, combined with the fingerprinting of individual isolates, and denaturing gradient gel electrophoresis (DGGE)] was applied to study whether similar features concerning the diversity and temporal stability of selected bacterial groups could be detected intra-individually in two different niches – the oral cavity and the colon – from ten adult volunteers consuming probiotics. The predominant bacterial microbiota, Clostridium coccoides–Eubacterium rectale group and bifidobacterial populations, were generally stable in salivary and faecal samples, with the greater diversity seen in faeces. Furthermore, different species predominated at the two different sites. Lactobacillus group DGGE profiles were unstable, yet the intra-individual profiles from faecal and salivary samples collected at the same time resembled each other. The ingested probiotic product did not affect the stability of the bacterial groups studied. The culture-based analysis showed that most subjects harboured identical indigenous Lactobacillus genotypes in saliva and faeces (Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus paracasei and Lactobacillus plantarum group). Thus, identical indigenous lactobacilli were able to inhabit both ends of the orogastrointestinal tract, whereas the composition of the other bacterial groups studied varied between the two sites.

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.

The efficiencies of potassium persulphate, isopropanol, hydrogen peroxide and peracetic acid, quaternary ammonium compound, hypochlorite, sodium dichloroisocyanurate, ethanol and phenol derivatives, tertiary alkylamines and dimethyl alamine betaine‐based disinfectants and a hypochlorite‐based disinfecting cleaning agent were evaluated against eight Listeria monocytogenes strains representing three different ribotypes. All the disinfectants were effective in a suspension test with an exposure time of 30 s at the lowest concentrations recommended by the manufacturer. The efficiencies on surfaces were reduced. However, on clean surfaces all the agents were considered effective when the exposure time was 5 min and the concentration was the average recommended by the manufacturer. Five of nine disinfectants and the disinfecting cleaning agent were considered effective in soiled conditions in the surface test. The most efficient agent was isopropanol‐based and the least effective was the disinfectant containing tertiary alkylamine and dimethyl alamine betaine. Differences in bactericidal efficiencies of disinfectants against different L. monocytogenes strains on meat soiled surfaces were found.

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.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

A total of 179 non-spore-forming bacteria aerobically growing on Nutrient Agar, Plate Count Agar or in specific enrichment conditions for salmonella, campylobacteria, listeria, yersinia or staphylococci, were isolated from 16 untreated paper mill pulps. After phenotypical screening the isolates were characterised by automated ribotyping and partial sequencing of the 16S rRNA gene. They could be divided into seven taxonomical classes representing 63 taxa (species): actinobacteria (11 species), bacilli (7), flavobacteria (3) alphaproteobacteria (10), betaproteobacteria (5), gammaproteobacteria (25) and sphingobacteria (2). Most of the gammaproteobacteria were enterobacteria, mainly species of the genera Enterobacter (7 species, 7 samples/3 mills) and Klebsiella (5 species, 6 samples/3 mills). Other commonly occurring bacteria were most closely related to Microbacterium barkeri (7 samples/3 mills), Cloacibacterium normanense (6 samples/2 mills), Pseudoxanthomonas taiwanensis (5 samples/2 mills) and Sphingobacterium composti (5 samples/1 mill). Sporadic isolates of Listeria innocua, L. monocytogenes, Enterococcus casseliflavus and Staphylococcus warneri were detected, from which only L. monocytogenes is considered to be a food pathogen. No isolates of the genera Campylobacter, Salmonella or Yersinia were detected. The detected bacteria may be harmful in process control, but the load of food pathogens with recycled fibres to paper machines is insignificant. Faecal contamination of the pulp samples was not indicated.

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.

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.

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.

Current cultivation-based methods used by most breweries for microbiological quality control reveal possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and nonspoilage microbes or allow the detection of viable but noncultivable cells or tracing of contamination sources. In recent years, several promising molecular biological applications have been described for the detection, characterization, and identification of brewery contaminants. Group-, genus-, and species-specific PCR tests have been designed and evaluated for brewery contaminants, i.e., for lactic and acetic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCycler(™) PCR, standard PCR) have been set up for most of these organisms. In order to detect the very low amounts of microbes in brewing samples, an enrichment method has been devised. Practical pre-PCR treatment methods, including collection of cells, DNA extraction, and removal of PCR inhibitors, have been developed for filterable (such as bright beer and process water) and nonfilterable (pitching yeast, wort, fermenting wort) samples. Modular PCR kits for standard PCR and real-time PCR have also become available from several companies. All developed PCR applications are, despite the enrichment step, more rapid and specific than the cultivation methods. Currently, PCR without prior cultivation is less sensitive and more expensive to use than cultivation. Denaturing gradient gel electrophoresis (DGGE) for separation of bacterial or eukaryotic ribosomal DNA amplicons is a valuable tool for characterizing microbial communities in specific environmental niches. This technique has also been applied to study and compare microbial communities during beer, wine, and whisky production. The automated ribotyping system (RiboPrinter® System DuPont Qualicon, U.S.A.) is a rapid molecular biological method for the identification and characterization of bacteria to species or even below species level. A comprehensive identification database has been created for brewery contaminants, such as Lactobacillus spp., Pediococcus spp., Pectinatus spp., M. cerevisiae, and Obesumbacterium proteus, using three different restriction enzymes. In addition to identification and renaming of bacteria, the database has been applied to tracing of contamination sources and detection of troublesome house flora in industrial processes.

During 1998/1999 a total of 36 surveys were carried out in six meat, five seafood and two poultry processing plants. A total of 2522 samples, including processing lines and environment, personnel, raw materials and products (raw or ready-to-eat, RTE), were analysed for the presence of Listeria monocytogenes. The environmental and personnel samples were taken after cleaning and after the process had been running for 2 h. Samples of food products were taken after every important step during processing and samples of poultry carcasses were taken immediately after production and after storage for 21 days. The overall incidence of L. monocytogenes in meat processing plants varied from 0% to 15.1%, in poultry plants from 20.6% to 24.1% and in seafood plants from 5.9% to 22.1%. In raw products the average incidence was 15.6% for meat, 22.2% for poultry and 39.0% for seafood products. The heating steps during the production of RTE products eliminated Listeria. On average, 2.3% of RTE meat and 4.8% of RTE seafood products were recontaminated with L. monocytogenes. In the seafood sector almost all Listeria positive samples also included L. monocytogenes (91.1% of the positive samples), whereas in the meat and poultry sectors other Listeria species (mainly L. innocua) dominated. In most plants, the implemented cleaning procedures were insufficient to eliminate Listeria. The results clearly indicated the problematic sites, which in most plants were conveyer belts and other transporting equipment, floors and drains, but also raw material in the meat industry, cutting boards in the poultry industry and cooking equipment in the seafood industry. In order to solve the problems observed in this study, there is a need for close co-operation between the suppliers of equipment and cleaning agents, the staff of cleaning companies and hygiene specialists from the food industry.

Contamination of brewing process by spoilage organisms may lead to production problems and quality defects in final product. Present quality control methods based on cultivation reveal the possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and non-spoilage microbes nor allow the detection of fastidious or injured organisms. In recent years, several promising molecular biological applications have been described for the detection, characterization and identification of brewery contaminants. Group, genus and species specific PCR tests have been developed for contaminants present in brewing process, i.e. for lactic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, acetic acid bacteria and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCyclerTM PCR, standard PCR) have been set-up for most of the beer spoilage organisms. Pre-PCR treatment methods have been developed for filterable samples, such as bright beer and process water, and for unfilterable samples, such as pitching yeast, fermenting wort and unfilterable beer. Due to the very low amounts of microbes in brewing samples, they need to be enriched in a suitable medium prior to the PCR. Practical DNA extraction methods and removal of PCR inhibitors have been developed for different sample types. For example, a commercial system containing magnetic particles for DNA collection and purification can be used for this purpose. Modular PCR kits for standard PCR and real-time PCR are available from several companies. The developed PCR applications are, despite of the enrichment step, more rapid and specific than conventional methods. Without prior enrichment, sensitivity of PCR methods is lower and they are at the moment expensive to use compared to cultivation methods. An alternative to PCR, a commercial fluorescent in situ hydridization (FISH) method has been developed for beer-spoilage bacteria. It is based on the use of fluorescently labelled gene probes and epifluorescence microscope. The automated ribotyping system (RiboPrinter®System DuPont Qualicon,USA) is rapid molecular biological method for identification and characterization of bacteria to species or even below species level. The comprehensive identification database using three different restriction enzymes has been created to the brewery contaminants including Lactobacillus spp., Pediococcus spp, Pectinatus spp., Megasphaera cerevisiae and Obesumbacterium proteus. In addition to identification purposes and renaming of bacteria, the database has been applied in tracing contamination sources and in detection of troublesome "house flora" in industrial processes.

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.

A bacterial gene encoding a-acetolactate decarboxylase, isolated from Klebsiella ternigena or Enterobacteraerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase(PGKI) or the alcohol dehydrogenase (ADHI) promoter and were integrated by gene replacement byusing cotransformation into the PGKI or ADHI locus, respectively, of a brewer's yeast. The expression levelof the aL-acetolactate decarboxylase gene of the PGKI integrant strains was higher than that of the ADHIintegrants. Under pilot-scale brewing conditions, the a-acetolactate decarboxylase activity of the PGKIintegrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and nolagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, andthe quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.

Four brewer's yeast strains carrying the α- ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes ) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The α- ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae . In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.

The microbiological and sensory quality of blood pancakes and vacuum-packed cooked ring sausages were studied one day after production, on the sell-by date and one week after the sell-by date. Samples were taken straight from 5 manufacturing plants, stored at 4°C and analyzed by 3 different laboratories using similar methods. Blood pancakes contained high numbers of aerobic organisms and yeasts. The principal sensory defects were visible mould spots, rancid odour and musty taste. A correlation between taste and microbiological counts was observed. Bacillus cereus was observed in some samples and may constitute a potential health hazard when temperature abuse occurs. Cooked ring sausages showed good microbiological and sensory quality after production. Aerobic plate counts and lactobacilli numbers increased during storage. The main defects in sensory analysis were fermented, sour aroma and taste. No correlation between taste and microbiological counts could be determined. The store shelf-lives (sell-by periods) of blood pancakes and cooked ring sausages set by the manufacturers, 7–11 and 20–28 days, respectively, can be considered overly optimistic. Differences between laboratories were observed in the sensory analyses.