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Environmental strains of Vibrio parahaemolyticus, a foodborne pathogen, were isolated from milkfish and grouper aquaculture facilities in southern Taiwan and characterized by pulsed-field gel electrophoresis ( PFGE) and polymerase chain reaction targeting on the virulence-associated and genomic island ( VPaI) marker genes. Analyses of 62 environmental isolates, including two putative pathogenic isolates, by NotI- PFGE revealed 11 pulse-type clusters with a similarity of 85%. Some of the T3 SS2 α-associated genes ( vopB2, vopC and vopT) were not present in all of these two putative pathogenic isolates. Marker genes of VPaI-1 ( MTase gene), VPaI-2 ( VP0636) and VPaI-3 ( VP1073 and VP1077) were detected in 14-100% of the environmental isolates examined, and the VP1073 and VP1077 of VPaI-3 marker genes were not detected. This study confirmed the high genetic variability of the environmental isolates including the putative pathogenic strains and identified some marker genes of VPaI in the environmental V. parahaemolyticus isolates. Significance and Impact of the Study Vibrio parahaemolyticus is a prevalent seafood-borne enteropathogen with the appearance of pandemic O3:K6 strains in 1996. This study characterized the environmental nontoxigenic and toxigenic isolates by pulsed-field gel electrophoresis and the presence of marker genes of genomic islands. Results showed that the T3 SS2 α-associated genes are not present in all environmental tdh + isolates, and the presence of movable elements may contribute to genetic variation in the environmental V. parahaemolyticus isolates. [ABSTRACT FROM AUTHOR]

Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR-based denaturing gradient-gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms. [ABSTRACT FROM AUTHOR]

Aims: Anaerobic sludge granules underpin high-rate waste-to-energy bioreactors. Granulation is a microbiological phenomenon involving the self-immobilization of several trophic groups. Low-temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low-temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low-temperature (R2 and R3, 15°C) laboratory-scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose-based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low-temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H2/CO2-mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high-rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented. [ABSTRACT FROM AUTHOR]

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx1, stx2, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx1 and stx2, and the flagellar antigen ( fliC) genes in nonmotile isolates. Five isolates were eae+ and stx−, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx1c stx2d-O118 (46·9%), stx1c (27·2%), stx2d-O118 (23·4%), and stx1c stx2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded. [ABSTRACT FROM AUTHOR]

Aims: Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars. Methods and Results: Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200. Conclusions: PFGE offers the advantages of simple, reliable and reproducible results. Significance and Impact of the Study: PFGE provides a convenient tool for the identification of clinical isolates. [ABSTRACT FROM AUTHOR]

Aims: The identification of a new compound active against Agrobacterium tumefaciens. Methods and Results: The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter’s antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml−1) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l−1 glucose, 15 g l−1 K2HPO4 and 5 g l−1 MgSO4 7H2O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato. Conclusion: The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens. Significance and Impact of the Study: The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease. [ABSTRACT FROM AUTHOR]

Aims: In this study, three facile repetitive-sequence PCR (rep-PCR) techniques have been compared with the pulsed-field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR-based methods, i.e. enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), BOX-PCR and repetitive extragenic palindromic-PCR (REP-PCR). When compared with PFGE, ERIC-PCR displayed a much lower discriminatory power, whereas BOX-PCR and REP-PCR had a comparable discriminatory power for close genetic-related isolates. Conclusion: BOX-PCR and REP-PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to assess the applicability of a combined approach of traditional and molecular epidemiology in order to detect salmonellosis outbreaks in the Piedmont region (Italy), characterized by high Salmonella prevalence. Methods and Results: Pulsed field gel electrophoresis (PFGE) was used in real-time and in combination with clinical surveillance to assess the relatedness of salmonellosis human cases; subsequently, PFGE profiles of clinical isolates were compared with those of isolates from food items collected during the same study period to identify putative food sources of Salmonella. The real-time subtyping approach allowed the identification of an outbreak (21 isolates), which was undetected by epidemiological surveillance. Conclusions: Traditional epidemiological investigation did not allow the formulation of hypotheses on food items possibly associated with the outbreak owing mainly to patients’ difficulties in remembering foods they ate, and the tendency of health-care professionals to direct patient’s suspicion towards specific food items. Significance and Impact of the Study: This finding highlighted the value of real-time molecular subtyping in salmonellosis outbreak identification. In order to improve national epidemiological investigations implementing public health agency network and planning, information campaigns for health-care professionals are required. [ABSTRACT FROM AUTHOR]

Aims: To investigate the presence and partial characterization of ecto-ATPase in Balamuthia mandrillaris. Methods and Results: In vitro assays were used to demonstrate that live B. mandrillaris hydrolyses extracellular ATP. Using nondenaturing polyacrylamide gel electrophoresis, B. mandrillaris exhibited a single ecto-ATPase band of molecular mass of more than 545 kDa. This ecto-ATPase was insensitive to ouabain, levamisole, sodium azide and sodium orthovanadate but stimulated by MgCl2. The ecto-ATPase was heat stable, but labile to detergent, sodium dodecyl sulphate. Suramin, an antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, inhibited B. mandrillaris binding to and cytotoxicity of HBMEC (human brain microvascular endothelial cello), in vitro. Conclusions: For the first time, we describe that live B. mandrillaris hydrolyses extracellular ATP and exhibits a >545kDa ecto-ATPase. Significance and Impact of the Study: This surface enzyme may play a role in the salvage of purines from the extracellular medium and may be important for the pathogenesis of B. mandrillaris. [ABSTRACT FROM AUTHOR]

Aims: To compare three methods for DNA extraction from Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium. Methods and Results: The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis, and of a 1700 bp fragment of FR300 region in M. avium avium. Conclusions: Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex. Significance and Impact of the Study: The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis. [ABSTRACT FROM AUTHOR]

Aims: The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. Methods and Results: Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l−1 of Ca2+ markedly enhanced both CMCase and avicelase activities of the enzyme. Conclusions: We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities. Significance and Impact of the Study: Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation. [ABSTRACT FROM AUTHOR]

Aims: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. Methods and Results: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. Conclusions: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. Significance and Impact of the Study: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance. [ABSTRACT FROM AUTHOR]

Aims: To assess of the genotypic diversity of Candida albicans isolated from removable prosthesis wearers, with and without denture-related stomatitis (DRS). The occurrence of different genotypes in pathological and control cases was investigated. Methods and Results: One hundred and sixty-four isolates of C. albicans obtained from different oral cavity locations were compared by randomly amplified polymorphic DNA (RAPD). The coherence of this analysis was confirmed by genotyping a selected group of isolates with pulsed field gel electrophoresis (PFGE). Among the 164 isolates, 150 were grouped into seven groups on the basis of their RAPD patterns. Three of these groups (comprising 54 isolates) had significant ( α < 0·10) predominance of clinical or control cases. For the other isolates, no significant differences were observed between control and DRS cases. Occasionally, more than one genotype was found in the same person. These findings were sustained by PFGE analysis. No relevant associations between the genotypic patterns and pathology level were found. Conclusions: This study evidenced that C. albicans with similar genotypes may be found in individuals with DRS and in control cases. Significance and Impact of the Study: This conclusion hints the involvement of other aetiological factors that alone or in association with C. albicans may trigger the emergence of DRS. [ABSTRACT FROM AUTHOR]

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion-exchange chromatography in DEAE-Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml−1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml−1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications. [ABSTRACT FROM AUTHOR]

Aims:  The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. Methods and Results:  Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 106 CFU ml−1 in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8·5 to 9·5 units during the last 50 days. Conclusion:  STEC O26 strains were able to survive in cow slurry for an extended period. Significance and Impact of the Study:  Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26. [ABSTRACT FROM AUTHOR]

Aims: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. Methods and Results: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65°C and pH 4·5, respectively. Activity remained stable after incubation at 60°C and pH 4·5 for 3 h. Conclusions: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. Significance and Impact of the Study: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications. [ABSTRACT FROM AUTHOR]

Aim: DNA fingerprinting using (GTG)5 oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. Methods and Results: RAPD analysis using the (GTG)5 oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. Conclusions: Laboratory RADP typing using the (GTG)5 primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. Significance and Impact of the Study: RAPD typing using (GTG)5 is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method. [ABSTRACT FROM AUTHOR]

Aims: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. Methods and Results: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52–64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. Conclusions: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations. Significance and Impact of the Study: PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease. [ABSTRACT FROM AUTHOR]

n.r. mcewan, l. abecia, m. regensbogenova, c.l. adam, p.a. findlay and c.j. newbold. 2005. This work was carried out to determine if there was a difference in the microbial population of the rumen associated with daylength at which sheep are housed. Denaturing gradient gel electrophoresis (DGGE) was used to study the ciliate and bacterial diversity in the rumen of Soay rams kept in long day (16 h light) or short day (8 h light) photoperiods. Bacterial diversity varied according to the daylength conditions where the host animal was housed, as did total volatile fatty acids (VFA) concentrations. No differences associated with daylength were detected in ciliate diversity, branched VFA concentrations or the ruminal ammonia concentrations. As diets had identical composition, yet voluntary intakes levels were higher during long days, it is proposed that the differences in bacterial populations arise because of the differences in amount of food consumed. The outcome of this study demonstrated that factors beyond dietary composition must be taken into account when trying to study microbial populations, even in what can be considered a fairly constant environment. [ABSTRACT FROM AUTHOR]

r. bibiloni, m.a. simon, c. albright, b. sartor and g.w. tannock. 2005. To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations (‘ Bacteroides sp.’, Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent ( Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis. [ABSTRACT FROM AUTHOR]

m. kawanishi, a. kojima, k. ishihara, h. esaki, m. kijima, t. takahashi, s. suzuki and y. tamura. 2005.To investigate the existing antimicrobial susceptibility and genetic characteristics ofLactococcus garvieaeisolates from culturedSeriolain Japan.Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44·1%) were simultaneously resistant to erythromycin (EM) (MIC ≥ 2 μg ml−1), lincomycin (LCM) (MIC ≥ 128 μg ml−1) and oxytetracycline (OTC) (MIC ≥4 μg ml−1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessedermBandtet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI andSmaI), did not exceed 3.The present resistance pattern observed withermBandtet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics ofL. garvieaeisolates from a wide area in Japan in 2002 and ATCC strains were closely related.This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and thatL. garvieaestrains with same origin have spread amongSeriolaspp. in Japan since 1974. [ABSTRACT FROM AUTHOR]

t.s. edrington, m.e. hume, m.l. looper, c.l. schultz, a.c. fitzgerald, t.r. callaway, k.j. genovese, k.m. bischoff, j.l. mcreynolds, r.c. anderson and d.j. nisbet. 2004. To examine the variability in faecal shedding of Salmonella and Escherichia coli O157:H7 in healthy lactating dairy cattle and to evaluate the genetic relatedness of Salmonella isolates. Faecal samples were obtained from lactating Holstein dairy cattle on four commercial farms in the southwestern US. All farms were within an 8-km radius and were sampled in August 2001, January 2002 and August 2002 (60 cows per farm per sampling; n = 720 total samples). Samples were cultured for E. coli O157:H7 and Salmonella and a portion of the recovered Salmonella isolates were examined for genetic relatedness using pulsed-field gel electrophoresis (PFGE). Faecal shedding of E. coli O157:H7 and Salmonella varied considerably between farms and at the different sampling times. Large fluctuations in the percentage of positive animals were observed from summer to summer for both of these pathogens. Similarly, Salmonella serotype and serotype prevalence varied from farm to farm and within farm from one sampling time to another. Multiple Salmonella genotypes were detected for a number of serotypes and identical genotypes were found on different farms with one genotype of Salmonella Senftenberg identified on three of the four farms. This study demonstrated the wide variability in pathogen shedding within and among dairy farms all located in a small geographical region and highlights the complexity of pathogen control at the farm level. [ABSTRACT FROM AUTHOR]

c. andrighetto, g. marcazzan and a. lombardi. 2004. This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time. [ABSTRACT FROM AUTHOR]

s. giacomazzi, f. leroi, c. l'henaff and j.-j. joffraud. 2004. To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. Methods and Results: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. Conclusions: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. Significance and Impact of the Study: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone. [ABSTRACT FROM AUTHOR]