Parabacteroides distasonis (Pdis) is the type species for the new Parabacteroides genus, and a gut commensal of the Bacteroidetes phylum. Emerging reports (primarily based on reference ...strain/ATCC-8503) concerningly propose that long-known opportunistic pathogen Pdis is a probiotic. We posit there is an urgent need to characterize the pathogenicity of Pdis strain-strain variability. Unfortunately, no methods/insights exist to classify Bacteroidetes for this purpose. Herein, we developed a virulence gene-based classification system for Pdis and Bacteroidetes to facilitate pathogenic-vs-probiotic characterization. We used DNA in silico methods to develop a system based on the virulence (lipopolysaccharide/bacterial wall) 'rfbA O-antigen-synthesis gene'. We then performed phylogenetic analysis of rfbA from fourteen Pdis complete genomes (21 genes), other Parabacteroides, Bacteroidetes, and Enterobacteriaceae; and proposed a PCR-based Restriction-Fragment Length Polymorphism method. Cluster analysis revealed that Pdis can be classified into four lineages (based on gene gaps/insertions) which we designated rfbA-Types I, II, III, and IV. In context, we found 14 additional rfbA-types (I-XVIII) interspersed with numerous Bacteroidetes and pathogenic Enterobacteriaceae forming three major "rfbA-superclusters." For laboratory rfbA-Typing implementation, we developed a PCR-primer strategy to amplify Pdis rfbA genes (100%-specificity) to conduct MboII-RFLP and sub-classify Pdis. In-silico primers for other Bacteroidetes are proposed/discussed. Comparative analysis of lipopolysaccharide/lipid-A gene lpxK confirmed rfbA as highly discriminant. In conclusion, rfbA-Typing classifies Bacteroidetes/Pdis into unique clusters/superclusters given rfbA copy/sequence variability. Analysis revealed that most pathogenic Pdis strains are single-copy rfbA-Type I . The relevance of the rfbA strain variability in disease might depend on their hypothetical modulatory interactions with other O-antigens/lipopolysaccharides and TLR4 lipopolysaccharide-receptors in human/animal cells.
► nosZ gene was in the range of 104–106 and nirS gene with 104–105 copy number per ml of sludge. ► Betaproteobacteria were found to numerically dominate the sludge habitat. ► Some isolates showed ...efficient denitrification, while others accumulated nitrite during denitrification. ► The results also indicate presence of different truncated denitrifiers with high numbers in sludge habitats.
The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 104–105, nosZ with 104–106 and 16S rRNA gene in the range 109–1010 copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.
The epidemiology and public health significance of Cryptosporidium species and genotypes were investigated in Beni-Suef Governorate, Egypt. A total of 610 animal fecal samples (480 from cattle and ...130 from buffaloes) beside 290 stool samples from humans were collected in the period between January and December 2014. Based on the microscopic examination, the overall estimated prevalence of Cryptosporidium spp. in cattle, buffaloes, and humans was 10.2, 12.3, and 19 %, respectively. The highest detection rates were in calves less than 2 months of age (17.1 %) and diarrheic animals (13.0 %). Likewise in humans, the highest prevalence of Cryptosporidium was in infants (31.3 %) and diarrheic individuals (21.1 %). The gender distribution in humans denoted that Cryptosporidium was reported more frequently in males (21.7 %) than females (14.5 %). Based on the molecular characterization of Cryptosporidium, Cryptosporidium oocyst wall protein (COWP) and gp60 genes were successfully amplified in 36 out of 50 samples subjected to genotyping. Restriction fragment length polymorphism (RFLP) analysis of the COWP fragments revealed that Cryptosporidium parvum was the only species detected in cattle (12 isolates) and buffaloes (4 isolates), while in humans, the detected species were Cryptosporidium hominis (15 isolates) and C. parvum (5 isolates). Sequence analysis of the gp60 gene identified the subtype IIdA20G1 within C. parvum isolated from both animals and humans. The common occurrence of zoonotic subtypes of C. parvum in cattle and buffaloes highlights the potential role of these animals as significant reservoirs of infection to humans. Also, the presence of C. hominis and C. parvum in humans indicates that both anthroponotic and zoonotic pathways are expected.
The economically most important honey bee species,
Apis mellifera, was formerly considered to be parasitized by one microsporidian,
Nosema apis. Recently, Higes, M., Martín, R., Meana, A., 2006.
...Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93–95 and Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A
Nosema ceranae isolate from the honeybee
Apis mellifera. Apidologie 38, 30–37 used 16S (SSU) rRNA gene sequences to demonstrate the presence of
Nosema ceranae in
A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between
N. apis and
N. ceranae based on PCR–RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (
N =
9 isolates gave
N. apis RFLPs and sequences,
N =
20 isolates gave
N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze
N =
115 isolates from across the world. Our data, combined with
N =
36 additional published sequences demonstrate that (i)
N. ceranae most likely jumped host to
A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii)
N. ceranae is now a parasite of
A. mellifera across most of the world. The rapid, long-distance dispersal of
N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.
Background:
Caenorhabditis elegans is a major model system in biology, yet very little is known about its biology outside the laboratory. In particular, its unusual mode of reproduction with ...self-fertile hermaphrodites and facultative males raises the question of its frequency of outcrossing in natural populations.
Results: We describe the first analysis of
C. elegans individuals sampled directly from natural populations.
C. elegans is found predominantly in the dauer stage and with a very low frequency of males versus hermaphrodites. Whereas
C. elegans was previously shown to display a low worldwide genetic diversity, we find by comparison a surprisingly high local genetic diversity of
C. elegans populations; this local diversity is contributed in great part by immigration of new alleles rather than by mutation. Our results on heterozygote frequency, male frequency, and linkage disequilibrium furthermore show that selfing is the predominant mode of reproduction in
C. elegans natural populations but that infrequent outcrossing events occur, at a rate of approximately 1%.
Conclusions: Our results give a first insight in the biology of
C. elegans in the natural populations. They demonstrate that local populations of
C. elegans are genetically diverse and that a low frequency of outcrossing allows for the recombination of these locally diverse genotypes.
Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the ...difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25 ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65 cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.
•Toxoplasma gondii SAG3 was detected from 6.25 ng of DNA and 200 parasites/mL blood.•Fidelity of the AmpliTaq Gold™ polymerase was 100%.•Nested PCR amplification did not show evidence of the generation of artifacts.•The error rate of the bacterial cloning system was calculated as 1 × 10−4.•Cloning in a bacterial system did not produce artificial recombinant sequences.
The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF) gene polymorphisms with diabetic foot ulcer (DFU) susceptibility in a Chinese Han ...population.Around 88 type 2 diabetes mellitus (T2DM) patients without DFU and 97 T2DM patients with DFU were enrolled in this study. A total of 103 age and gender matched healthy individuals were recruited as healthy control. VEGF gene polymorphisms rs699947 and rs13207351 were analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) with 95% confidence interval (CI) was calculated to assess the association between VEGF gene polymorphisms and DFU risk.The frequency of AA and AC genotypes of rs699947 were lower in DFU patients than that in healthy controls (P = .020, P = .031), suggesting that AC and AA genotypes were negatively associated with DFU risk originating from healthy individuals (OR = 0.496, 95%CI = 0.274-0.899; OR = 0.130, 95%CI = 0.015-1.112). Significantly decreased trend of rs699947 A allele was observed in DFU patients when compared to the controls (P = .004), suggesting A allele was distinctly correlated with decreased DFU risk (OR = 0.490, 95%CI = 0.298-0.804). But no significant differences were detected in rs13207351 genotype and allele distributions between patients and control groups (P > .05).Individuals carrying VEGF rs699947 A allele show low susceptibility to DFU in the Chinese Han population.
Cellulose is the most abundant biopolymer in terrestrial ecosystems and is degraded by microbial communities in soils. However, relatively little is known about the diversity and function of soil ...prokaryotes that might participate in the overall degradation of this biopolymer. The active cellulolytic and saccharolytic Bacteria in an agricultural soil were evaluated by 16S rRNA ¹³C-based stable isotope probing. Cellulose, cellobiose and glucose were mineralized under oxic conditions in soil slurries to carbon dioxide. Under anoxic conditions, these substrates were converted primarily to acetate, butyrate, carbon dioxide, hydrogen and traces of propionate and iso-butyrate; the production of these fermentation end-products was concomitant with the apparent reduction of iron(III). ¹³C-cellulose was mainly degraded under oxic conditions by novel family-level taxa of the Bacteroidetes and Chloroflexi, and a known family-level taxon of Planctomycetes, whereas degradation under anoxic conditions was facilitated by the Kineosporiaceae (Actinobacteria) and cluster III Clostridiaceae and novel clusters within Bacteroidetes. Active aerobic sub-communities in oxic ¹³C-cellobiose and ¹³C-glucose treatments were dominated by Intrasporangiaceae and Micrococcaceae (Actinobacteria) whereas active cluster I Clostridiaceae (Firmicutes) were prevalent in anoxic treatments. A very large number (i.e. 28) of the detected taxa did not closely affiliate with known families, and active Archaea were not detected in any of the treatments. These collective findings suggest that: (i) a large uncultured diversity of soil Bacteria was involved in the utilization of cellulose and products of its hydrolysis, (ii) the active saccharolytic community differed phylogenetically from the active cellulolytic community, (iii) oxygen availability impacted differentially on the activity of taxa and (iv) different redox guilds (e.g. fermenters and iron reducers) compete or interact during cellulose degradation in aerated soils.
Drug efficacy trials monitor the continued efficacy of front-line drugs against falciparum malaria. Overestimating efficacy results in a country retaining a failing drug as first-line treatment with ...associated increases in morbidity and mortality, while underestimating drug effectiveness leads to removal of an effective treatment with substantial practical and economic implications. Trials are challenging: they require long durations of follow-up to detect drug failures, and patients are frequently reinfected during that period. Molecular correction based on parasite genotypes distinguishes reinfections from drug failures to ensure the accuracy of failure rate estimates. Several molecular correction "algorithms" have been proposed, but which is most accurate and/or robust remains unknown. We used pharmacological modeling to simulate parasite dynamics and genetic signals that occur in patients enrolled in malaria drug clinical trials. We compared estimates of treatment failure obtained from a selection of proposed molecular correction algorithms against the known "true" failure rate in the model. Our findings are as follows. (i) Molecular correction is essential to avoid substantial overestimates of drug failure rates. (ii) The current WHO-recommended algorithm consistently underestimates the true failure rate. (iii) Newly proposed algorithms produce more accurate failure rate estimates; the most accurate algorithm depends on the choice of drug, trial follow-up length, and transmission intensity. (iv) Long durations of patient follow-up may be counterproductive; large numbers of new infections accumulate and may be misclassified, overestimating drug failure rate. (v) Our model was highly consistent with existing
data. The current WHO-recommended method for molecular correction and analysis of clinical trials should be reevaluated and updated.
Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial ...communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-Evar) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-Evar). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.
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