Biomedical subjects

Anthony C Hilton

Publications and source records attributed to Anthony C Hilton.

8 recordsLinked to original sources

PubMed2005

Description and critical appraisal of principal components analysis (PCA) methodology applied to pulsed-field gel electrophoresis profiles of methicillin-resistant Staphylococcus aureus isolates.

Principal components analysis (PCA) has been described for over 50 years; however, it is rarely applied to the analysis of epidemiological data. In this study PCA was critically appraised in its ability to reveal relationships between pulsed-field gel electrophoresis (PFGE) profiles of methicillin-resistant Staphylococcus aureus (MRSA) in comparison to the more commonly employed cluster analysis and representation by dendrograms. The PFGE type following SmaI chromosomal digest was determined for 44 multidrug-resistant hospital-acquired methicillin-resistant S. aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK). Strain relatedness was determined using Dice band-matching with UPGMA clustering and PCA. The results indicated that PCA revealed relationships between MRSA strains, which were more strongly correlated with known epidemiology, most likely because, unlike cluster analysis, PCA does not have the constraint of generating a hierarchic classification. In addition, PCA provides the opportunity for further analysis to identify key polymorphic bands within complex genotypic profiles, which is not always possible with dendrograms. Here we provide a detailed description of a PCA method for the analysis of PFGE profiles to complement further the epidemiological study of infectious disease.

Cluster Analysis↗

PubMed2005

Molecular analysis of methicillin-resistant Staphylococcus aureus reveals an absence of plasmid DNA in multidrug-resistant isolates.

The number, diversity and restriction enzyme fragmentation patterns of plasmids harboured by 44 multidrug-resistant hospital-acquired methicillin-resistant Staphylococcus aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK) were compared. In addition, pulsed-field gel electrophoresis (PFGE) type following SmaI chromosomal digest and SCCmec element type assignment were ascertained for each isolate. All MR-HA-MRSA and MR-CA-MRSA isolates possessed the type II SCCmec, harboured no plasmid DNA and belonged to one of five PFGE types. Forty-three out of 50 HA-MRSA isolates and all 34 CA-MRSA isolates possessed the type IV SCCmec and all but 10 of the type IV HA-MRSA isolates and nine CA-MRSA isolates carried one or two plasmids. The 19 non-multidrug-resistant isolates (NMR) that did not harbour plasmids were only resistant to methicillin whereas all the NMR isolates harbouring at least one plasmid were resistant to at least one additional antibiotic. We conclude that although plasmid carriage plays an important role in antibiotic resistance, especially in NMR-HA-MRSA and CA-MRSA, the multidrug resistance phenotype from HA-MRSA is not associated with increased plasmid carriage and indeed is characterised by an absence of plasmid DNA.

Anti-Bacterial Agents↗

PubMed2005

Mechanisms of resistance in Salmonella enterica adapted to erythromycin, benzalkonium chloride and triclosan.

The potential for adaptive resistance of S. enterica serovar Enteritidis, Typhimurium and Virchow to increasing sub-lethal concentrations of erythromycin, benzalkonium chloride and triclosan was investigated to identify mechanisms underlying resistance. Permeability changes of the outer membrane, including LPS, cell surface charge, hydrophobicity and the presence of an active efflux in the adapted strain compared with the parent were studied. Examination of the outer membrane and LPS did not reveal any significant changes, although most of the pre-adapted strains were notably less hydrophobic than resistant strains. More than one type of active efflux was identified in all strains investigated, on the basis of restored sensitivity in the presence of the inhibitors reserpine and carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cell surface hydrophobicity and the presence of active efflux could contribute to the resistance of S. enterica to the antibacterial agents studied here.

Adaptation, Physiological↗

PubMed2003

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD.

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists.

Buffers↗

PubMed2003

Representational difference analysis: critical appraisal and method development for the identification of unique DNA sequences from prokaryotes.

Representational difference analysis (RDA) has great potential for preferential amplification of unique but uncharacterised DNA sequences present in one source such as a whole genome, but absent from a related genome or other complex population of sequences. While a few examples of its successful exploitation have been published, the method has not been well dissected and robust, detailed published protocols are lacking. Here we examine the method in detail, suggest improvements and provide a protocol that has yielded key unique sequences from a pathogenic bacterial genome.

DNA, Bacterial↗

PubMed2003

RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript.

Base Sequence↗

PubMed2002

Isolation of Salmonella from urban wild brown rats (Rattus norvegicus) in the West Midlands, UK.

A 6-month study was undertaken to investigate the prevalence of Salmonella enterica in wild urban brown rats (Rattus norvegicus) in the West Midlands. Samples were obtained of faecal droppings (n = 100) and from rectal swabs (n = 50) of rat carcases collected from active infestation sites. A subset of the rats (n = 25) had additional swab samples taken of the fur, paws and tail. Five (10%) of the rectal swabs were positive for Salmonella by direct plating onto XLD media. No further samples were positive following pre-enrichment and selective culture. A total of eight (8%) faecal samples were positive for Salmonella; two by direct plating and a further six following enrichment. All positive faecal samples were fresh or moist upon collection. None of the samples obtained from the outer surfaces of the rat were positive. Additionally, rat faeces were spiked with Salmonella and sampled periodically to determine survival in drying faeces exposed to a typical indoor environment. Salmonella could be recovered by direct culture up to 86 days. These results demonstrate a regional variability in the carriage of Salmonella in urban rats compared to other studies and that Salmonella longevity in faecal pellets is sufficient to present a potential contamination risk in the absence of an active infestation.

Animals↗

PubMed2001

Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates.

Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E. coli. Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains. Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone. Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.

Bacterial Typing Techniques↗