The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.

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Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

/pdf/determination-of-bacterial-load-by-real-time-pcr-using-a-56oienigs3.pdf

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain(23F)-1 clonal complex of Streptococcus pneumoniae, "population snapshots" of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.

eBURST: Inferring Patterns of Evolutionary Descent among Clusters of Related Bacterial Genotypes from Multilocus Sequence Typing Data

Helicobacter pylori infection. Implications for ulcer therapy in older patients.

Background. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. Methods. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Results. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P = .01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. Conclusions. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising ≥10 reference spectra per bacterial species and a ≥1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

/pdf/ongoing-revolution-in-bacteriology-routine-identification-of-2n18ddyl1y.pdf

Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

IgG antibody subclass responses determined by immunoblot in infants' sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine.

Method for detecting Campylobacter by PCR detection of DNA sequence, highly conserved between species lari, coli, jejuni and upsaliensis. Speciation between these four is possible as the PCR product is differentially cleaved by restriction endonucleases.

Detection and speciation of campylobacter

A previously unrecognized bacterial flagellar structure present in Campylobacter jejuni and Campylobacter fetus is described. The structure is located just below the insertion of the flagellar apparatus through the bacterial wall. It is disc shaped, approximately 100 nm in diameter and has eleven spokes radiating from a central hole. The function of this new structure and its possible importance in the taxonomy of campylobacters and other spiral micro-organisms are discussed.

A new bacterial flagellar structure found in campylobacters.

The development of an inhibition enzyme-linked immunosorbent assay to detect total antibody to the P1.16 subtype antigen of Neisseria meningitidis in human sera

Andrew J. Fox

Papers

IgG antibody subclass responses determined by immunoblot in infants' sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine.

Detection and speciation of campylobacter

A new bacterial flagellar structure found in campylobacters.

The development of an inhibition enzyme-linked immunosorbent assay to detect total antibody to the P1.16 subtype antigen of Neisseria meningitidis in human sera

The surface polysaccharide seen on Neisseria gonorrhoeae after treatment with CMP-NANA contains sialyl groups.