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. © 2005 Elsevier B.V. All rights reserved.

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease XbaI, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsα and Gi2α genes

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsα, Gi2α and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsα, and Gi2α contribute little if any to cranipharyngioma development.

Biodiversity of the surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen cheeses

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Modified lipids from LDL in the blood during mid-life increase blood brain barrier permeability

Low density lipoprotein levels (LDL) are consistently elevated in cardiovascular disease. It has been suggested that those with high circulating LDL levels in mid-life may be susceptible to develop neurodegenerative diseases in later life. In the circulation, high levels of LDL are associated with increased oxidative modification (oxLDL) and nitration. We have investigated the hypothesis that disruption of blood brain barrier function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention in mid-life can mitigate the neurodegenerative effects of hyperlipidaemia. Blood from statin-naïve, normo- and hyperlipidaemic subjects (n=10/group) was collected at baseline. Hyperlipidaemic subjects received statin-intervention whereas normolipidaemic subjects did not prior to a second blood sampling, taken after 3 months. The intervention will be completed in June 2013. Plasma was separated by centrifugation (200g, 30min) and LDL was isolated by potassium bromide density gradient ultracentrifugation. Total homocysteine, LDL cholesterol, 8-isoprostane F2α levels were measured in plasma using commercial kits. LDL were analysed by agarose gel electrophoresis. LDL-lipids were extracted by partitioning in 1:1 chloroform:methanol (v/v) and conjugated to fatty acid free-BSA in serum-free EGM-2 medium (4hrs, 370C) for co-culture with human microvascular endothelial cells (HMVEC). HMVEC were maintained on polycarbonate inserts for two weeks to create a microvascular barrier. Change in barrier permeability was measured by trans-endothelial electrical resistance (TER), FITC-dextran permeability and immunohistochemistry. HMVEC glutathione (GSH) levels were measured after 2 hours by GSH-glo assay. LDL isolated from statin-naïve hyperlipidaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and plasma 8-isoprostane F2α (43.5±8.42 pg/ml) compared to control subjects (0.46±0.05 and 24.2±5.37 pg/ml; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hyperlipidaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased GSH (18.5 nmol/mg v 12.3 nmol/mg; untreated cells 26.2±3.6 nmol/mg).