Co-infection dynamics of a major food-borne zoonotic pathogen in chicken.

A major bottleneck in understanding zoonotic pathogens has been the analysis of pathogen co-infection dynamics. We have addressed this challenge using a novel direct sequencing approach for pathogen quantification in mixed infections. The major zoonotic food-borne pathogen Campylobacter jejuni, with an important reservoir in the gastrointestinal (GI) tract of chickens, was used as a model. We investigated the co-colonisation dynamics of seven C. jejuni strains in a chicken GI infection trial. The seven strains were isolated from an epidemiological study showing multiple strain infections at the farm level. We analysed time-series data, following the Campylobacter colonisation, as well as the dominant background flora of chickens. Data were collected from the infection at day 16 until the last sampling point at day 36. Chickens with two different background floras were studied, mature (treated with Broilact, which is a product consisting of bacteria from the intestinal flora of healthy hens) and spontaneous. The two treatments resulted in completely different background floras, yet similar Campylobacter colonisation patterns were detected in both groups. This suggests that it is the chicken host and not the background flora that is important in determining the Campylobacter colonisation pattern. Our results showed that mainly two of the seven C. jejuni strains dominated the Campylobacter flora in the chickens, with a shift of the dominating strain during the infection period. We propose a model in which multiple C. jejuni strains can colonise a single host, with the dominant strains being replaced as a consequence of strain-specific immune responses. This model represents a new understanding of C. jejuni epidemiology, with future implications for the development of novel intervention strategies.

Multiplex real-time single nucleotide polymorphism detection and quantification by quencher extension.

Multiplex quencher extension (multiplex-QEXT) is a novel closed tube single-step method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The principle of multiplex-QEXT is that 5' reporter-labeled probes are 3' single-base-extended with TAMRA dideoxy nucleotides if the respective SNP alleles are present. TAMRA can serve as either an energy acceptor (quencher-based detection) or donor [fluorescence resonance energy transfer (FRET)-based detection] for a wide range of different reporter fluorochromes. The extension can therefore be recorded by the respective reporter fluorescence change. We evaluated multiplex-QEXT, analyzing four different SNP loci in the Listeria monocytogenes inlA gene. Probes labeled with the reporters 6-FAM, TET, VIC, and Alexa Fluor 594 were used. Responses for the fluorochromes 6-FAM, TET, and VIC were detected by quenching (decreased fluorescence), while the response for Alexa Fluor 594 was detected by FRET (increased fluorescence). We evaluated the SNP-allele pattern in 252 different L. monocytogenes strains. Multiplex-QEXT gave a good resolution, detecting seven major and five minor groups of L. monocytogenes. Comparison with serotyping showed that multiplex-QEXT gave better resolution. We also evaluated the quantitative aspects of multiplex-QEXT. Quantitative information was obtained for all the fluorochrome/probe combinations in the sample pools. The detection limits for 6-FAM, TET and Alexa Fluor 594 were the presence of the 10% target SNP alleles (P < 0.05), while the detection limit for VIC was the presence of the 5% target SNP alleles (P < 0.05). Currently, overlap in the fluorescence emission spectra is the limiting factor for the multiplexing potential of QEXT. With the emergence of new fluorochromes with narrow emission spectra, we foresee great potential for increasing the multiplex level in the future.

Quencher extension for single nucleotide polymorphism quantification in bacterial typing and microbial community analyses.

Quencher extension is a novel single-step closed tube real-time method to quantify single nucleotide polymorphisms (SNPs) in combination with primer extension. A probe with a 5'-reporter is single-base extended with a dideoxy nucleotide containing a quencher if the target SNP allele is present. The reaction is measured from the quenching (reduced fluorescence) of the reporter. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermocycling reaction. The quencher extension protocol presented was developed for SNP allele quantification in Listeria monocytogenes and for microbial community analyses.

Alignment-independent comparisons of human gastrointestinal tract microbial communities in a multidimensional 16S rRNA gene evolutionary space.

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities.

Alignment-independent bilinear multivariate modelling (AIBIMM) for global analyses of 16S rRNA gene phylogeny.

Alignment-independent phylogenetic methods have interesting properties for global phylogenetic reconstructions, particularly with respect to speed and accuracy. Here, we present a novel multimer-based alignment-independent bilinear mathematical modelling (AIBIMM) approach for global 16S rRNA gene phylogenetic analyses. In AIBIMM, jackknife cross-validated principal component analyses (PCA) are used to explain the variance in nucleotide n-mer frequency data. We compared AIBIMM with alignment-based distance, maximum-parsimony and maximum-likelihood phylogenetic methods, analysing taxa belonging to the Proteobacteria (n=82), Actinobacteria (n=30) and Archaea (n=7). These analyses indicated an attraction between the Actinobacteria and Archaea for the traditional methods, with the two taxa Acidimicrobium and Rubrobacter at the root of the tree. AIBIMM, on the other hand, showed that the Actinobacteria was tightly clustered, with Acidimicrobium and Rubrobacter within a distinct subgroup of the Actinobacteria. The application of AIBIMM was further evaluated, analysing full-length 16S rRNA gene sequences for 2818 taxa representing the prokaryotic domains. We obtained a highly structured description of the prokaryote diversity. Sample-to-model (Si) distances were also determined for taxa included in our work. We determined Si distances for models of the six major subgroups of taxa detected in the global analyses, in addition to nested subgroups within the Alphaproteobacteria. The Si-distance evaluation showed a very good separation of the taxa within the models from those outside. We conclude that AIBIMM represents a novel phylogenetic framework suitable for accommodating the current exponential growth of 16S rRNA gene sequences in the public domain.