Andrew J. Fox

Bio: Andrew J. Fox is an academic researcher from Manchester Royal Infirmary. The author has contributed to research in topics: Campylobacter jejuni & Multilocus sequence typing. The author has an hindex of 44, co-authored 96 publications receiving 7655 citations. Previous affiliations of Andrew J. Fox include Health Protection Agency & University College London.

Multilocus Sequence Typing System for Campylobacter jejuni

Abstract: The gram-negative bacterium Campylobacter jejuni has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194 C. jejuni isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (http://mlst.zoo.ox.ac.uk), forming a virtual isolate collection which could be continually expanded. These data indicated that C. jejuni is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of C. jejuni epidemiology to be established and will permit more detailed studies of the population genetics of this organism.

Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR

Abstract: A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR

Abstract: A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains

Abstract: Objectives Recently, a CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. Methods A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. Results One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. Conclusions We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.