An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes

PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.     

Misuse of PCR assay for diagnosis of mollusc protistan infections

Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.     

Global biogeography of highly diverse protistan communities in soil

Protists are ubiquitous members of soil microbial communities, but the structure of these communities, and the factors that influence their diversity, are poorly understood. We used barcoded pyrosequencing to survey comprehensively the diversity of soil protists from 40 sites across a broad geographic range that represent a variety of biome types, from tropical forests to deserts. In addition to taxa known to be dominant in soil, including Cercozoa and Ciliophora, we found high relative abundances of groups such as Apicomplexa and Dinophyceae that have not previously been recognized as being important components of soil microbial communities. Soil protistan communities were highly diverse, approaching the extreme diversity of their bacterial counterparts across the same sites. Like bacterial taxa, protistan taxa were not globally distributed, and the composition of these communities diverged considerably across large geographic distances. However, soil protistan and bacterial communities exhibit very different global-scale biogeographical patterns, with protistan communities strongly structured by climatic conditions that regulate annual soil moisture availability.     

PCR fingerprinting for epidemiological studies of Staphylococcus aureus

Staphylococcus aureus isolates (n = 126), collected during two different periods from patients hospitalised in pediatric wards, were analysed using polymerase chain reaction (PCR) mediated genotyping. These isolates were compared with 29 isolates from individuals attending the out-patient clinic of the same hospital and 13 isolates from pediatric hospital personnel. Within a group of 99 isolates gathered from 48 individuals during surveillance period I, 22 distinct genotypes were identified by application of two PCR assays. Among the 58 isolates collected in surveillance period II from pediatric and out-clinic patients, 25 genotypes were detected by a single PCR assay only. Based on these results it was demonstrated that patients can be colonised with multiple strains that may persist in a certain anatomical location for prolonged periods of time. It is shown that persistence of a S. aureus strain in a pediatric ward can be deduced from the PCR genotyping studies. As such PCR can be used for longitudinal monitoring of bacterial infections in hospital departments, analysis of patient-to-patient and personnel-to-patient transmission and for detection of genetic variation in general in S. aureus. Also, isolate-specific DNA probes can be generated for S. aureus by PCR genotyping. The probes can be used for the recognition of re-emerging S. aureus epidemics.     

Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 x 10(-2) cell per 10-microl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities

Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.     

A ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil

A direct molecular method for assessing the diversity of specific populations of rhizobia in soil, based on nested PCR amplification of 16S-23S ribosomal RNA gene (rDNA) intergenic spacer (IGS) sequences, was developed. Initial generic amplification of bacterial rDNA IGS sequences from soil DNA was followed by specific amplification of (1) sequences affiliated with Rhizobium leguminosarum "sensu lato" and (2) R. tropici. Using analysis of the amplified sequences in clone libraries obtained on the basis of soil DNA, this two-sided method was shown to be very specific for rhizobial subpopulations in soil. It was then further validated as a direct fingerprinting tool of the target rhizobia based on denaturing gradient gel electrophoresis (DGGE). The PCR-DGGE approach was applied to soils from fields in Brazil cultivated with common bean (Phaseolus vulgaris) under conventional or no-tillage practices. The community fingerprints obtained allowed the direct analysis of the respective rhizobial community structures in soil samples from the two contrasting agricultural practices. Data obtained with both primer sets revealed clustering of the community structures of the target rhizobial types along treatment. Moreover, the DGGE profiles obtained with the R. tropici primer set indicated that the abundance and diversity of these organisms were favoured under NT practices. These results suggest that the R. leguminosarum-as well as R. tropici-targeted IGS-based nested PCR and DGGE are useful tools for monitoring the effect of agricultural practices on these and related rhizobial subpopulations in soils.     

T-track PCR fingerprinting for the rapid detection of genetic polymorphism

The diversity of DNA sequences can be analyzed by comparing randomly amplified polymorphic DNA, or restriction fragment length polymorphism fragments of DNA. Such analyses are dependent on the selection of appropriate restriction enzyme(s) and/or primers. We have investigated a simpler approach to providing sensitive and specific genotyping. Cyclic extension of target sequences with dideoxythymidine generates PCR products with variable lengths. We analyzed these variable PCR products by scoring the number of variable bands and comparing the scores (numerical profiles) to establish similarities. We found that the polymorphic lengths of the PCR products were comparable among serologically defined strains. It suggests that this single PCR reaction followed by a one-step electrophoresis yields easily analyzable data that can be compared with data from other gels.     

Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labeling the PCR primers

Culture independent molecular methods have emerged as indispensable tools for studying microbial community structure and dynamics in natural habitats, since they allow a closer look at microbial diversity that is not reflected by culturing techniques. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one of the informative and widely used techniques for such studies. However, the method has a few limitations to predict microbial community structure with significant accuracy. One of the major limitations is variation in real Terminal Restriction Fragment (TRF) length and observed TRF length. In the present study we report the generation of TRF length variations using different fluorescent dyes to label the PCR primers. T-RFLP profiles generated from primers labeled with different dyes varied significantly and led to inconsistent microbial species identification. Occurrence of such variations can have serious consequences on interpretation of the T-RFLP profiles from environmental samples representing complex microbial community. Therefore, in a T-RFLP study, the primers and labeling dye system should be carefully evaluated and optimized for an individual community under investigation. Further, it would be recommended to establish a target gene library in parallel with T-RFLP analysis to facilitate the accurate prediction of microbial community structure.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

Variability of protistan and bacterial communities in two Arctic fjords (Spitsbergen)

Krossfjorden and Kongsfjorden are Arctic fjords on the western side of Spitsbergen. These fjords share a common mouth to the open sea, and both are influenced by the input of sediment-rich glacial meltwater leading to decreased surface salinity, increased turbidity and decreased light penetration during summer. Earlier classical taxonomic studies had described the pelagic protistan composition of the Kongsfjorden during summer, revealing the dominance of flagellates of often unresolved taxonomic origin. Only little information existed on microbial eukaryote composition of the Krossfjorden as well as the bacterial composition of both fjords. The aim of the present study was to analyze and compare surface summertime protistan and bacterial communities in both fjords, using molecular approaches (16S and 18S rRNA DGGE, sequencing). Samples were collected three times a week from the central Kongsfjorden over a 1-month period. Additionally, 10 marine and 2 freshwater sites were sampled within a 1-week period in both Kongsfjorden and Krossfjorden. The central Kongsfjorden revealed a relatively stable protistan community over time with dinoflagellates, chlorophytes and small heterotrophs dominating. In contrast, the bacterial community varied over time and appeared to be correlated with the inflow of glacial meltwater. The Kongsfjorden and Krossfjorden were found to harbor distinctive bacterial and eukaryotic communities. We speculate that differences in glacial meltwater composition and fjord bathymetry affect the surface water properties and therefore the observed spatial variability in the community fingerprints.     

松江湿地沉积物细菌T-RFLP分析中PCR体系的建立与优化

细菌是微生物的重要组成部分,其对保持湿地生态系统平衡起着重要作用,在沉积物细菌T-RFLP(terminal restriction fragment length polymorphism)分析中建立与优化PCR体系对进一步研究沉积物细菌群落组成具有重要意义。此次研究以松江湿地沉积物细菌基因组DNA为模板,采用正交和单因素试验对PCR体系中各因素(模板DNA、Mg2+、dNTPs、引物、Taq酶)进行优化,系统分析各因素对PCR扩增结果的影响,建立最佳PCR反应体系。在20μL反应体系中,DNA模板30 ng、Mg2+2.0 mmol/L、dNTPs 0.2 mmol/L、引物0.2μmol/L、Taq酶0.5 U。该优化体系确保了沉积物细菌T-RFLP分析过程中PCR产物的质量与效果。     

A direct PCR approach to accelerate analyses of human-associated microbial communities

Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.     

An isoclinal approach to the comparative statics of biological communities

An understanding of the comparative statics of biological communities is important both as a means of explaining the long-term effects of changes in environmental conditions, and as a framework for viewing community time trajectories. A general formulation of community dynamics is presented here which, given full information about a particular community's dynamic behavior, describes the impact of a change in environmental conditions on the community steady state. However, since such full information is often lacking in studies of biological communities, various approaches to partial information analysis of comparative statics are presented and compared, including a generalized protocol for isocline analysis. The suggested isocline protocol is shown to be a useful tool for both full and partial information analyses, as well as for both general and partial equilibrium studies. This work was supported over several years through funding from the International Biological Program, the Oregon State University Sea Grant College Program, the U.S. Environmental Protection Agency, the U.S. Forest Service, the Electric Power Research Institute, and the Northwest and Alaska Fisheries Center.     

An approach to the prediction of temperate freshwater fish communities

The organization and structure of temperate fish communities is discussed. A measure of organization based on the Shannon measure of entropy is applied. It is concluded that the way organization changes with species number reflects the underlying structure of the community. The prediction of fish assemblages by the analysis of individual species requirements is also investigated using fish data sets from regions considered to be undergoing acidification. The species set within a habitat is generated by a set of rules termed the operational niche. It was concluded that an analysis based on physico-chemical conditions within the habitats gave reasonable correspondence between observed and predicted.     

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with logCRPconcentration across a 0.5-50nM range and associated with a limit of detection of 176pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed.     

T-RFLP analysis of bacterial communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand

This study investigated bacterial community structures in the midguts of Apis mellifera and Apis cerana in Thailand to understand how bacterial communities develop in Apis species. The bacterial species present in replicate colonies from different locations and life stages were analysed. PCR amplification of bacterial 16S rRNA gene fragments and terminal restriction fragment length polymorphism analyses revealed a total of 16 distinct terminal restriction fragments (T-RFs), 12 of which were shared between A. mellifera and A. cerana populations. The T-RFs were affiliated to Beta- and Gammaproteobacteria, Firmicutes and Actinomycetes. The Gammaproteobacteria were found to be common in all stages of honey bee, but in addition, the Firmicutes group was found to be present in the worker bees. Bacterial community structure showed no difference amongst the replicate colonies, but was affected to some degree by geographical location, life stage and species of honey bees.