Representational Difference Analysis and Real-Time PCR for Strain-Specific Quantification of Lactobacillus sobrius sp. nov.

Lactobacillus sobrius sp. nov., which was recently isolated from the intestine of weaning piglets, has potential probiotic properties. To follow the fate of L. sobrius strain 001^T in dietary interventions, a novel and strain-specific quantitative detection procedure was developed. This procedure was based on the isolation of specific genomic fragments from the type strain by representational difference analysis and their detection by real-time PCR. The described strain-specific quantification approach may be used in studies aimed at tracking bacterial strains added to specific environments.     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

Design and verification of a pangenome microarray oligonucleotide probe set for Dehalococcoides spp

Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1

16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.     

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.     

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.

The molecular basis of strain variation in scrapie diseases is unknown. The only identified component of the agent is the posttranslationally modified host prion protein (PrPSc). The biochemical and physical properties of PrP from two strains of transmissible mink encephalopathy (TME), called hyper (HY) and drowsy (DY), were compared to investigate if PrP heterogeneity could account for strain diversity. The degradation rate of PrPTME digested with proteinase K was found to be strain specific and correlated with inactivation of the TME titer. Edman protein sequencing revealed that the major N-terminal end of HY PrPTME commenced at least 10 amino acid residues prior to that of DY PrPTME after digestion with proteinase K. Analysis of the brain distribution of PrPTME exhibited a strain-specific pattern and localization of PrPTME to the perikarya of specific neuron populations. Our findings are consistent with HY and DY PrPTME having distinct protein conformations and/or strain-specific ligand interactions that influence PrPTME properties. We propose that PrPTME conformation could play a role in targeting TME strains to different neuron populations in which strain-specific formation occurs. These data are consistent with the idea that PrPTME protein structure determines the molecular basis of strain variation.     

Strain-Specific Identification of Probiotic Lactobacillus rhamnosus with Randomly Amplified Polymorphic DNA-Derived PCR Primers

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.     

Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.     

Characterization of Iberian pig genotypes using AFLP markers

The use of the AFLP (amplified fragment length polymorphism) technique for the characterization of highly inbred Iberian pig breed genotypes and the detection of strain-specific polymorphisms is demonstrated. Twelve different primer combinations were used on individual DNA samples from animals belonging to two black hairless Iberian pig strains, Guadyerbas and Coronado. These amplification reactions allowed the detection of more than 1700 amplification products of which 26 were identified as strain-specific markers, present in all individuals of one strain and absent in the other. Comparison of male and female amplification products within one strain also allowed the identification of 8 male-specific amplified bands. AFLP showed a great power of marker detection due to a high multiplex ratio and high reproducibility. Comparison of similarity and co-ancestry coefficient matrices also showed the usefulness of AFLP markers to estimate genetic relationships between individuals pigs.     

The immunopathology of experimental allergic encephalomyelitis (EAE). III. Differential in situ expression of strain 13 Ia on endothelial and inflammatory cells of (strain 2 x strain 13)F1 guinea pigs with EAE

This study was undertaken to determine if there is differential expression of the relevant strain-specific Ia antigen on endothelial and parenchymal inflammatory cells in the central nervous systems (CNS) of guinea pigs (GP) with acute experimental allergic encephalomyelitis (EAE). Adult inbred GP were sensitized with GP spinal cord homogenate and complete Freund's adjuvant. Strain 13 GP and (2 x 13)F1 hybrids developed clinical disease within 2 to 3 wk after sensitization, whereas strain 2 GP did not, although all sensitized GP had CNS mononuclear inflammatory infiltrates. By using monoclonal antibodies to strain-specific and framework Ia epitopes with an immunoperoxidase technique, the distribution and amount of the strain 2 and strain 13 Ia were analyzed. Equivalent strain 2 and strain 13 Ia expression was found in normal tissues from F1 animals. Strain 2 and strain 13 GP sensitized for EAE had increased strain-specific Ia staining of CNS vessels and inflammatory cells over controls. However, F1 GP with EAE had markedly increased strain 13, but not strain 2, Ia on CNS parenchymal vessels and mononuclear inflammatory cells (p less than 0.001, for both). These results suggest for the first time that specific major histocompatibility complex gene products are selectively expressed on endothelial and inflammatory cells in situ in immune reactions in the target organ of individuals of heterogeneous immunogenetic composition.     

Feeding of Lactobacillus sobrius reduces Escherichia coli F4 levels in the gut and promotes growth of infected piglets

The microbial community in the guts of mammals is often seen as an important potential target in therapeutic and preventive interventions. The aim of the present study was to determine whether enterotoxigenic Escherichia coli (ETEC) F4 infection in young animals might be counteracted by a probiotic treatment with Lactobacillus sobrius DSM 16698. The experiment was conducted in three randomized consecutive replications, each consisting of 16 piglets, and including a control group and an L. sobrius fed group, both experimentally challenged with ETEC. During the entire trial, the animals' health status, body weight, and microbial parameters were monitored periodically. Probiotic supplementation containing L. sobrius significantly reduced the levels of ETEC in the ileum when fed directly to piglets after weaning. In contrast, the number of days when the piglets had an increased faecal water content was significantly higher in the probiotic group. Nevertheless, an improved daily weight gain was also observed in the animals that received probiotic L. sobrius relative to the control fed group. The data indicate that L. sobrius may be effective in the reduction of the E. coli F4 colonization and may improve the weight gain of infected piglets.     

DNA microarray for discrimination between pathogenic 0157:H7 EDL933 and non-pathogenic Escherichia coli strains

The primary technique currently used to detect biological agents is based on immunoassays. Although sensitive and specific, currently employed immunoassays generally rely on the detection of a single epitope, and therefore often cannot discriminate subtle strain-specific differences. Since DNA microarrays can hybridize hundreds to thousands of genomic targets simultaneously and do not rely on phenotypic expression of these genetic features for identification purposes, they have enormous potential to provide inexpensive, flexible and specific strain-specific detection and identification of pathogens. In this study, pathogenic Escherichia coli O157:H7-specific genes, non-pathogenic K12-specific genes, common E. coli genes, and negative control genes were polymerase chain reaction-amplified and spotted onto the surface of treated glass slides. After labeled bacterial cDNA samples were hybridized with probes on the microarray, specific fluorescence patterns were obtained, enabling identification of pathogenic E. coli O157:H7 and non-pathogenic E. coli K12. To test the utility of this microarray device to detect genetically engineered bacteria, E. coli BL21 (a B strain derivative with antibiotic resistance gene, ampR) and E. coli JM107 (a K12 strain derivative lacking the gene ompT) were also employed. The array successfully confirmed the strain genotypes and demonstrated that antibiotic resistance can also be detected. The ability to assess multiple data points makes this array method more efficient and accurate than a typical immunoassay, which detects a single protein product.     

Tryptic peptide disparity between serologically indistinguishable guinea pig Ia.3,5 antigens

Serological studies have suggested that the DHCBA strain guinea pig expresses an I region which is identical to that of strain 13. However, chemical characterization of Ia.3,5 molecules isolated from these two strains has indicated that these serologically indistinguishable Ia molecules are actually chemically distinct. Ia.3,5 molecules biosynthetically labeled with either [3H]leucine, [3H]arginine, or [3H]lysine were purified by ricin affinity chromatography and isolated by indirect immunoprecipitation with specific alloantisera. Initial examination of the two Ia.3,5 molecules by SDS-PAGE, isoelectric focusing, and two-dimensional gel analyses revealed no strain-specific differences. Furthermore, comparative peptide mapping of the DHCBA and strain 13 radiolabeled Ia.3,5 alpha-chains demonstrated complete peptide homology. In contrast, tryptic peptide maps of amino acid radiolabeled beta-chains revealed two peptides unique to the strain 13 beta-chain and one peptide unique to the DHCBA beta-chain. Analysis of [3H] mannose-labeled beta-chain tryptic peptides verified that the peptide differences observed using 3H-amino acids were not due to variation in N-linked glycosylation. However, strain-specific variability was also noted in the profiles of [3H]mannose-labeled beta-chains. These data indicate that the strain 13 and DHCBA alpha-chains are probably structurally identical, while the beta-chains show strain specific alterations in their chemical structure.     

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.     

Identification of Rhizobium spp. in peat-based inoculants by DNA hybridization and PCR and its application in inoculant quality control.

Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants. Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company. The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R. leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains. DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR. The hybridization probes were total DNAs from pure cultures of R. galegae HAMBI 1207 and R. leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R. galegae HAMBI 1174. The total DNA probes distinguished inoculants containing R. galegae or R. leguminosarum, and the strain-specific probe distinguished inoculants containing R. galegae HAMBI 1174. The hybridization results for R. galegae were verified in a PCR experiment by amplifying an R. galegae species-specific fragment and an R. galegae HAMBI 1174 strain-specific fragment in the same reaction. When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants.     

Real-time PCR approach for detection of environmental sources of Campylobacter strains colonizing broiler flocks

Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 10(2) to 3.7 x 10(2) CFU/ml in bacterial cultures and 6.6 x 10(2) CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.