Taurine-reduction Powers Rapid Growth of Bilophila wadsworthia: A Liquid Minimal-salts Medium for Clinical Research?

Clinical isolates of Bilophila wadsworthia grew rapidly (1–3 days) in the liquid taurine-minimal-salts medium developed for B. wadsworthia RZATAU, whereas growth on Bacteroides Bile Esculin Agar takes up to 1 week. Though rapid growth of B. wadsworthia was achieved, and no other pure cultures grew, the medium was not selective for the organism in human faeces or in intra-abdominal specimens. We hope, however, that our understanding of the physiological and biochemical characteristics of the organism supplies a tool for further research.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India

The diversity of cyanobacteria in the North-Eastern region of India has not been studied except for a few sporadic and inconclusive reports. Loktak Lake is a huge reservoir for various kinds of organisms, including cyanobacteria. The present study describes the isolation and molecular diversity of 72 filamentous, heterocystous cyanobacterial strains isolated from samples collected from Loktak Lake, its adjoining rice fields and rice fields in Indian Council of Agricultural Research (ICAR) complex, Shillong, Meghalaya, India. The isolated strains belonged to the genera Anabaena, Nostoc, Calothrix, Cylindrospermum and Mastigocladus. The molecular analysis of isolates revealed the occurrence of certain strains being present in the sample collected from the rice fields falling in the catchment area of Loktak Lake, Manipur and rice fields in ICAR complex, Shillong, Meghalaya both. A polyphasic approach based on morphological features and PCR based molecular polymorphism revealed enormous level of molecular diversity. Out of three primers targeted regions used for determining genetic polymorphism, STRR1A produced best fingerprint profile of cyanobacterial strains. The morphological diversity of isolates was assured by light microscope whereas PCR based multiple fingerprint profile was used for molecular characterization. Molecular typing using short tandemly repeated repetitive STRR1A sequences as primer provided strain specific fingerprint profiles of the isolates.     

Bilophila wadsworthia in Ear Infections: A Report of Three Cases

During a 3-year period routine anaerobic cultures were examined for the presence of Bilophila wadsworthia. Using taurine-supplemented Bacteroides–Bile–Esculin agar for isolation, we observed three cases of complicated polybacterial ear infections where B. wadsworthia was involved. The first case involved a 69-year-old female patient presenting an otitis externa following stapedectomy where B. wadsworthia was isolated. The second patient, 30-year-old woman, with a 2-decade history of otitis and otorrhoea presented with a cholesteatoma, complicated by brain abscess formation and B. wadsworthia was isolated from the purulent ear secretion as well as from the abscess material. The third case, a 39-year-old male patient suffering from cholesteatoma presented with otorrhoea and otalgia, B. wadsworthia was isolated from purulent ear secretion. In all cases, B. wadsworthia was part of mixed aerobic–anaerobic infections. Because this species was not found in 200 ear swabs from 100 healthy volunteers and was not detectable in throat swabs or saliva from of these patients, an exogenous origin of these outer and middle ear infections as well as an infection by fecal contamination seems more probable than ascending infections from the pharynx or the ear canal.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.     

Discrimination among the Clinical Isolates of Candida albicans by Amplification of the Repetitive Sequences,alts

A primer pair, PB and BSH, which amplified alts, a portion of Candida albicans-specific repetitive sequence, RPS, gave stable and reproducible fingerprint patterns of the strains by polymerase chain reaction (PCR). We applied this method to clinical isolates of C. albicans for strain discrimination. Using PCR fingerprint patterns, we could analyze the relatedness of C. albicans strains including those isolated from children with leukemia and their bedside parents. The results indicated that PCR analysis targeting an alt region gives rise to the same conclusion as the previous study obtained by SmaI RFLP analysis.     

Identification of Bilophila wadsworthia by specific PCR which targets the taurine:pyruvate aminotransferase gene

The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.     

DNA Polymorphism Revealed by Arbitrary Primers Polymerase Chain Reaction Among Blastocystis Strains Isolated from Humans, a Chicken, and a Reptile

DNA polymorphisms of different strains of Blastocystis isolated from humans, a chicken, and a reptile were examined by an arbitrary primer PCR method. Two strains of Blastocystis hominis isolated from humans in the USA and Japan yielded nearly identical PCR products. However, one strain of B. hominis (isolated from a human in Singapore) yielded quite different PCR products. Blastocystis sp. isolated from a chicken yielded PCR products similar to those of the former two strains, while Blastocystis lapemi, isolated from a reptile, shared no bands with any of the other isolates. These results indicate the possibility that our isolate from the chicken is a zoonotic strain, and that there is intraspecific variation of Blastocystis hominis.     

利用扩增片段长度多态性分析鉴别炭疽和蜡样芽孢杆菌

目的:利用扩增片段长度多态性(AFLP)分析建立鉴别炭疽芽孢杆菌和蜡样芽孢杆菌的分子生物学方法。方法:3株炭疽芽孢杆菌和3株蜡样芽孢杆菌基因组经限制性内切酶EcoRⅠ和MseⅠ酶切后与对应接头连接,通过预扩增和选择性扩增获得特异性DNA片段,将片段进行毛细管电泳,并利用GeneScan和BioNumerics软件对电泳数据进行分析。结果:选择性扩增最佳引物组合为EcoRⅠ-G/MseⅠ-A,其扩增片段在100~500 bp范围内的有效数量为40~50条;比较炭疽芽孢杆菌和蜡样芽孢杆菌的AFLP特征峰值图和DNA指纹图谱,确定了5个有明显差异的片段区。结论:利用AFLP分析可对芽孢杆菌属中相近的炭疽芽孢杆菌和蜡样芽孢杆菌进行鉴别,该方法可作为炭疽芽孢杆菌传统鉴定方法的补充。     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.     

Recovery of Bilophila wadsworthiafrom Clinical Specimens in Italy

This report is the first survey in Italy to evaluate the incidence of recovery of Bilophila wadsworthia in clinical situations. The survey was carried out at the departments of Microbiology in two Northern Italian hospitals over a one-year period. Tests for B. wadsworthia were carried out on a range of specimens from different body sites, when etiology by anaerobes was suspected. Out of a total of 350 samples examined, 67% were positive in bacteriological tests. Mixed anaerobic infections were detected in 53 specimens, corresponding to 23% of all cases. Strains of B. wadsworthia were isolated from 12 samples, equivalent to 5% and 22% of total and mixed/anaerobic infections, respectively. Bilophila wadsworthia was always isolated in mixed infections, mainly from the large intestine (67% of cases). The infectious process of B. wadsworthia was often complicated by abscess formation, regardless of body site. Interestingly, a strain was isolated from one case of bacteremia. The microorganisms most frequently isolated with B. wadsworthia were Escherichia coli for facultative species (38%), and Bacteroides fragilis, from anaerobic isolates (25%). Production of beta-lactamases by B. wadsworthia isolates was found in ten strains (83%), which appeared to be penicillin G resistant at concentration equal to or greater than the break-point (4 microg/mL). Epidemiological and clinical data from this and previous studies point to the involvement of B. wadsworthia in mixed infections. To assess the specific contribution of the species to the disease, studies of pathogenetic factors are to be considered in parallel. Nonetheless, production of beta-lactamases by most B. wadsworthia isolates could easily interfere with the therapeutical approach to infections involving the new species. The addition of a selective medium to culture specimens from the abdominal cavity should be considered in order to detect the presence of B. wadsworthia.     

Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting

PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.     

Diaphorobacter nitroreducens gen nov,sp nov,a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge

Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized. All of the isolates had almost identical phenotypic characteristics. They were motile gram-negative rods with single polar flagella and grew well with simple organic compounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions. However, none of the sugars tested supported their growth. The cellular fatty acid profiles showed the presence of C16:1omega7cis and C16:0 as the major components and of 3-OH-C10:0 as the sole component of hydroxy fatty acids. Ubiquinone-8 was detected as the major respiratory quinone. A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of beta-Proteobacteria, but formed no monophyletic cluster with any previously known species of this family. The closest relative to our strains was an unidentified bacterium strain LW1 (=DSM 13225) (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium. DNA-DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50%. The G+C content of genomic DNA of the new strains was 64 to 65 mol%. Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we propose the name Diaphorobacter nitroreducens. The type strain is strain NA10B (=JCM 11421=CIP 107294). We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter.     

<Emphasis Type="Italic">Trichophyton rubrum</Emphasis> and <Emphasis Type="Italic">Trichophyton interdigitale</Emphasis>: Genetic Diversity Among Species and Strains by Random Amplified Polymorphic DNA Method

Onychomycosis is a common condition that represents up to 50% of all nail problems and 30% of all cases of dermatophytoses. Trichophyton rubrum and Trichophyton interdigitale are the most common agents involved in this condition. In cases of recurrent post-treatment onychomycosis, strain fingerprinting could reveal whether the original isolate is responsible, a new strain has been acquired or if multiple strains are involved. The aim of this study was to evaluate the efficacy of the RAPD method for species and strain differentiation of T. rubrum and T. interdigitale obtained from patients with subungeal distal-lateral onychomycosis. A set of 86 strains of onychomycosis causative dermatophytes were submitted to species differentiation and strain typing by RAPD method with two previously described primers. Both primers proved capable of strain differentiation when tested for each species. Nineteen molecular profiles were configured for T. rubrum isolates with primers 1 and 6. For T. mentagrophytes, ten molecular profiles were configured with primer 1 and twenty-one with primer 6. We found that T. interdigitale and T. rubrum species were grouped in different clusters when both primers were analyzed together. This study shows that these primers are valuable tools for strain differentiation with T. rubrum and T. intedigitale.     

Verification of strain identity in Brazilian soybean inoculants by using the Polymerase Chain Reaction

The standards for meeting inoculant quality in Brazil include a requirement of strain verification as well as the necessity for a minimal number of viable bacterial cells. This requirement stimulated us to develop a rapid and simple PCR-based method to distinguish the four Brazilian strains of Bradyrhizobium, SEMIA 587, SEMIA 5019 (29W), SEMIA 5079 (CPAC-15) and SEMIA 5080 (CPAC-7), recommended for the inoculation of soybean. PCR reactions using the random amplified polymorphic DNA (RAPD) PCR primer CRL-7 together with a modified buffer resulted in four different fingerprint patterns, which permitted the Brazilian soybean strains to be distinguished from each other. The fingerprint patterns obtained were reproducible irrespective of the source of templates. Comparisons with fingerprint patterns obtained with soybean bradyrhizobia from the USDA Culture Collection led to the conclusion that two of the Brazilian inoculant strains (SEMIA 587 and SEMIA 5019) were characteristic for B. elkanii, while the other two (SEMIA 5079 and SEMIA 5080) were more typical of B. japonicum. Also, we obtained evidence that the RAPD analysis with primer CRL-7 may be useful for distinguishing variants of the same strain.     

Application of Rep-PCR fingerprint in rapid identification of beer-spoilage bacteria

The application potential of rep-PCR in typing beer-spoilage isolates was studied. The effects of different factors, including DNA templates and primers, on the quality and reproducibility of fingerprints were investigated. The CATB protocol was shown to be the feasible method for DNA extraction. Primers BOXA1R and (GTG)₅ were used in rep-PCR, and the PCR products were sequenced to identify strains isolated from two breweries. Rep-PCR fingerprint profiles were obtained using GelCompar II software. Cluster analysis showed that the isolates belonging to Lactobacillus brevis, L. buchneri, L. casei/paracasei, and L. plantarum are divided into 2 or 3 subgroups. In addition, the two rep-PCR fingerprint profiles complemented each other in typing these isolates. By combining the similarity coefficient cut-off (SCC) of species, 9 unknown isolates were rapidly identified using both fingerprint databases. The results indicate that rep-PCR is a simple, reliable, and promising method for the rapid identification of beer-spoilage bacteria.     

Rapid detection and identification of the free-living nitrogen fixing genus <Emphasis Type="Italic">Azospirillum</Emphasis> by 16S rRNA-gene-targeted genus-specific primers

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263 bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787^T) and Azospirillum irakense (DSM 11586^T) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10² CFU g⁻¹ in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species.     

Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.