Novel groups of Gammaproteobacteria catalyse sulfur oxidation and carbon fixation in a coastal, intertidal sediment

The oxidation of hydrogen sulfide is essential to sulfur cycling in marine habitats. However, the role of microbial sulfur oxidation in marine sediments and the microorganisms involved are largely unknown, except for the filamentous, mat‐forming bacteria. In this study we explored the diversity, abundance and activity of sulfur‐oxidizing prokaryotes (SOP) in sulfidic intertidal sediments using 16S rRNA and functional gene sequence analyses, fluorescence in situ hybridization (FISH) and microautoradiography. The 16S rRNA gene analysis revealed that distinct clades of uncultured Gammaproteobacteria are important SOP in the tidal sediments. This was supported by the dominance of gammaproteobacterial sequences in clone libraries of genes encoding the reverse dissimilatory sulfite reductase (rDSR) and the adenosine phosphosulfate reductase (APR). Numerous sequences of all three genes grouped with uncultured autotrophic SOP. Accordingly, Gammaproteobacteria accounted for 40–70% of all ¹⁴CO₂‐incorporating cells in surface sediments as shown by microautoradiography. Furthermore, phylogenetic analysis of all three genes consistently suggested a discrete population of SOP that was most closely related to the sulfur‐oxidizing endosymbionts of the tubeworm Oligobrachia spp. FISH showed that members of this population (WS‐Gam209 group) were abundant, reaching up to 1.3 × 10⁸ cells ml^?1 (4.6% of all cells). Approximately 25% of this population incorporated CO₂, consistent with a chemolithoautotrophic metabolism most likely based on sulfur oxidation. Thus, we hypothesize that novel, gammaproteobacterial SOP attached to sediment particles may play a more important role for sulfide removal and primary production in marine sediments than previously assumed.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

Molecular characterization of sulfate-reducing bacteria in a New England salt marsh

Sulfate reduction, mediated by sulfate-reducing bacteria (SRB), is the dominant remineralization pathway in sediments of New England salt marshes. High sulfate reduction rates are associated with the rhizosphere of Spartina alterniflora when plants elongate aboveground. The growth process concurrently produces significant amounts of new rhizome material belowground and the plants leak dissolved organic compounds. This study investigated the diversity of SRB in a salt marsh over an annual growth cycle of S. alterniflora by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Because the dsrAB gene is a key gene in the anaerobic sulfate-respiration pathway, it allows the identification of microorganisms responsible for sulfate reduction. Conserved dsrAB primers in polymerase chain reaction (PCR) generated full-length dsrAB amplicons for cloning and DNA sequence analysis. Nearly 80% of 380 clone sequences were similar to genes from Desulfosarcina and Desulfobacterium species within Desulfobacteraceae. This reinforces the hypothesis that complete oxidizers with high substrate versatility dominate the marsh. However, the phylotypes formed several clades that were distinct from cultured representatives, indicating a greater diversity of SRB than previously appreciated. Several dsrAB sequences were related to homologues from gram-positive, thermophilic and non-thermophilic Desulfotomaculum species. One dsrAB lineage formed a sister group to cultured members of the delta-proteobacterial group Syntrophobacteraceae. A deeply branching dsrAB lineage was not affiliated with genes from any cultured SRB. The sequence data from this study will allow for the design of probes or primers that can quantitatively assess the diverse range of sulfate reducers present in the environment.     

Enumeration and identification of dominant types of sulfate-reducing bacteria in pulp from a paper-recycling plant: a multiphasic approach

Multiple independent approaches were applied for monitoring the abundance and identity of sulfate-reducing bacteria (SRB) in pulp of a paper-recycling plant suffering from excessive sulfide emission. The methods applied included most-probable-number (MPN) enumeration of cultivable SRB, rate measurements, FISH and PCR-based retrieval of the functional marker genes dsrA and B (encoding the two major subunits of dissimilatory bisulfite reductase) and 16S rRNA genes. The SRB community was composed of phylogenetically highly different lineages all of low abundance relative to the total microbial community in the pulp, which hampered the applicability of FISH. It was also demonstrated that dsrA- or B -targeted PCR primers commonly used for denaturing gradient gel electrophoresis and real-time PCR analyses were biased. However, using a novel approach combining MPN-PCR and terminal restriction fragment length polymorphism analysis of dsrAB amplicons generated from serially diluted DNA extracts allowed the enumeration and identification of the quantitatively most important members of the SRB community. For fast quantification of SRB in the pulp, the dsrAB -MPN-PCR assay and sulfate reduction rate measurements were found to be most suitable.     

Effect of using a stand-off pad on Campylobacter jejuni strain diversity in a herd of dairy cows

Aim:  To determine the effect of stand-off pad (SOP) use on the prevalence and strain diversity of Campylobacter jejuni in a small herd of dairy cows.
Methods and Results:  Faecal samples were collected from 21 cows on four sampling occasions (events), one in each season, over 1 year. The cows usually grazed on pasture but during winter they spent 18 h a day on a SOP. Campylobacter prevalence ranged from 48–52% on pasture but was 62% on the SOP. The diversity of 386 C. jejuni isolates was determined using Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (ERIC/PCR). There were 11 ERIC types identified for the herd over the course of the study. Of those 11, four to seven (per event) were present when the cows were grazing pasture but only two during SOP use.
Conclusions:  The use of the SOP was associated with an increase in prevalence and a reduction in diversity of C. jejuni.
Significance and Impact of the Study:  The reduction in ERIC types on the SOP indicated an increase in transfer of only some strains of C. jejuni among the cows. One of these strains persisted throughout the study. The zoonotic potential of this strain warrants further investigation.     

Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.     

Diversity of the dsrAB (dissimilatory sulfite reductase) gene sequences retrieved from two contrasting mudflats of the Seine estuary, France

The diversity of sulfate-reducing microorganisms was investigated in two contrasting mudflats of the Seine estuary, by PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta subunits of dissimilatory sulfite reductase (dsrAB). One site is located in the mixing-zone and shows marine characteristics, with high salinity and sulfate concentration, whereas the other site shows freshwater characteristics, with low salinity and sulfate concentration. Diversity and abundance of dsrAB genes differed between the two sites. In the mixing-zone sediments, most of the dsrAB sequences were affiliated to those of marine Gram-negative bacteria belonging to the order of Desulfobacterales, whereas in the freshwater sediments, a majority of dsrAB sequences was related to those of the Gram-positive bacteria belonging to the genus Desulfotomaculum. It is speculated that this is related to the salinity and the sulfate concentration in the two mudflats.     

Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers

Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.     

Physiological adaptation to low temperatures of strains of Rhizobium leguminosarum bv. viciae associated with Lathyrus spp.(1)

Phylogenetic analyses of ribosomal RNA gene sequences (rDNAs) retrieved from an Australian desert soil sample (Sturt National Park) revealed the presence of a number of clones which branched deeply from the high GC Gram-positive division line of descent. The most abundant group of these clones were related to Rubrobacter. An oligonucleotide probe was designed to have broad specificity to Rubrobacter and relatives. This probe was used to interrogate eight rDNA libraries representing four distinct land forms within the Australian arid zone. Relative abundance of Rubrobacter-relatives in these samples ranged from 2.6 to 10.2%. Clones from these libraries were selected for sequence analysis on the basis of a heteroduplex mobility assay to maximise the diversity represented in the sample. Phylogenetic analyses of these rDNA clones and Rubrobacter-related clones reported in the literature show strong support for three distinct groups. Database-searching revealed 'Rubrobacteria' were relatively abundant in a number of published soil rDNA libraries but absent from others. A PCR assay for group-1 'Rubrobacteria' was used to test for their presence in 21 environmental samples. Only marine and arid-zone soil samples gave positive PCR results. Taken together these results indicate 'Rubrobacteria' are a widespread group of variable abundance and diversity.     

Molecular Analysis of the Diversity of Sulfate-Reducing and Sulfur-Oxidizing Prokaryotes in the Environment, Using aprA as Functional Marker Gene

The dissimilatory adenosine-5′-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria.     

Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.     

Genetic diversity of Babesia bovis in virulent and attenuated strains

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.     

Molecular ecology of a facultative swine waste lagoon

Aims:  To investigate the microbial ecology of three facultative swine waste lagoons.
Methods and Results:  Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum , Verrucomicrobia , Acholeplasma , Turicibacter , Clostridium and Bacteroides . PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons.
Conclusions:  All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme . The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling.
Significance and Impact of the Study:  This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.     

Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples

The flagellate parasite Giardia duodenalis is a major cause of diarrhoea in humans and in animals worldwide. Molecular techniques are particularly useful for studying the taxonomy, the population structure, the zoonotic potential of animal isolates, and the correlation between the genetic variability of the parasite and the range of clinical symptoms observed in humans. In this work, a new PCR assay that targets the beta-giardin gene was tested on 21 Giardia duodenalis reference strains representing Assemblages A, B and E, which are associated with infections of humans and other mammals. The assay was then applied to 30 faecal samples collected from Italian persons. The sequence analysis of 31 PCR products from both reference strains and clinical samples showed that each Assemblage is clearly distinct from the others on the basis of specific substitutions; the sequence diversity was approximately 5%, and all substitutions occurred at the third codon positions of the gene. The analysis of the intra-Assemblage variability allowed for the identification of three genotypes within Assemblage A, and of four genotypes within Assemblage B. Interestingly, two genotypes were identified only in the clinical samples and not in reference strains. Finally, a simple PCR-restriction fragment length polymorphism method was developed for the rapid discrimination of Assemblages and applied for the direct genetic analysis of cysts present in human faecal samples.     

Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.     

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with "Minimum SNPs" software. "Minimum SNPs" selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D=1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.     

Erwinia amylovora novel plasmid pEI70: complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.     

添加有扩增内标的副溶血弧菌PCR检测方法

【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌(Vibrio parahaemolyticus)基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物(vp1332L/vp1332R),同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102cfu/mL。人工污染实验表明,起始染菌量为1.24cfu/25g样品时经8h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

Culture-independent analysis of desulfovibrios in the human distal colon of healthy, colorectal cancer and polypectomized individuals

The production of hydrogen sulphide, an end product of metabolism by the sulphate-reducing bacteria (SRB) has been cited as a potential aetiological agent in gastrointestinal disease. Quantitative PCR (Q-PCR) assays to enumerate desulfovibrios from two gastrointestinal disease groups: colorectal cancer (CRC) n =27 and polypectomized individuals (PP) n =27, and two healthy control groups, elderly (H1) n =8 and young adults (H2) n =30 was performed. Analysis of Desulfovibrio sp. diversity using the dissimilarity sulphite reductase ( dsrAB ) gene as a molecular marker was also undertaken. Q-PCR detected Desulfovibrio sp. in all samples and no significant difference was observed for PP, H1, H2 with gene copy numbers of Desulfovibrio sp. averaging at 10⁶ g⁻¹ of faeces. Significantly reduced numbers of Desulfovibrio sp. were observed for CRC (10⁵ g⁻¹) compared with both PP and H2 groups ( P <0.05). Diversity analysis indicated that a low Desulfovibrio sp. diversity and the predominance of Desulfovibrio piger was a feature of both healthy and disease groups. In addition, a dsrAB gene sequence distantly related to a Gram-positive SRB was also recovered, highlighting the importance of cultivation-independent techniques for furthering our understanding of the diversity of the human gastrointestinal ecosystem.