Global metabolite profiling of human colorectal cancer xenografts in mice using HPLC-MS/MS.

Reversed-phase gradient LC-MS was used to perform untargeted metabonomic analysis on extracts of human colorectal cancer (CRC) cell lines (COLO 205, HT-29, HCT 116 and SW620) subcutaneously implanted into age-matched athymic nude male mice to study small molecule metabolic profiles and examine possible correlations with human cancer biopsies. Following high mass accuracy data analysis using MS and MS/MS, metabolites were identified by searching against major metabolite databases including METLIN, MASSBANK, The Human Metabolome Database, PubChem, Biospider, LipidMaps and KEGG. HT-29 and COLO 205 tumor xenografts showed a distribution of metabolites that differed from SW620 and HCT 116 xenografts (predominantly on the basis of relative differences in the amounts of amino acids and lipids detected). This finding is consistent with NMR-based analysis of human colorectal tissue, where the metabolite profiles of HT-29 tumors exhibit the greatest similarity to human rectal cancer tissue with respect to changes in the relative amounts of lipids and choline-containing compounds. As the metabolic signatures of cancer cells result from oncogene-directed metabolic reprogramming, the HT-29 xenografts in mice may prove to be a useful model to further study the tumor microenvironment and cancer biology.

Methodological considerations in the development of HPLC-MS methods for the analysis of rodent plasma for metabonomic studies.

A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.

UPLC-MS-based analysis of human plasma for metabonomics using solvent precipitation or solid phase extraction.

A study of the factors involved in obtaining valid global metabolite profiles from the LC-MS of human plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were either precipitated with 3 vol of organic solvent (methanol or acetonitrile) or subjected to solid phase extraction (SPE) on a C18-bonded phase. For chromatography, a reversed-phase gradient system, based on acidified water/methanol, was used. Ultra performance liquid chromatography (UPLC) was performed on a C18-bonded stationary phase using sub 2 microm particles packed into a 2.1 x 100 mm column. The eluent from the column was subjected to analysis by positive electrospray ionization using a time-of-flight mass spectrometer. To obtain reproducible results for solvent-precipitated plasma, the "conditioning" of the system with injections of matrix prior to the main analytical run was essential. The repeatability of the methodology was improved significantly when the sample preparation was performed using solid phase extraction.

Frequency of biocide resistance genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates.

OBJECTIVES: To detect genes conferring resistance to biguanides, quaternary ammonium compounds, beta-lactams and fluoroquinolones in clinical methicillin-resistant Staphylococcus aureus (MRSA) and to demonstrate whether reduced susceptibility is spread clonally and if the presence of any of the detected genes links to a specific epidemic MRSA. Finally, to identify if exposure to chlorhexidine may cause reduced susceptibility to antibiotics and chlorhexidine. METHODS: In total, 120 clinical MRSA isolates were isolated. qacA/B, qacG, qacH, norA, smr and blaZ genes were amplified by PCR. MICs of eight antibiotics were determined and PFGE was used for typing. Surface disinfection and residue tests were performed for chlorhexidine and a selection of isolates. RESULTS: qacA/B (8.3%), qacH (3.3%), norA (36.7%), smr (44.2%) and blaZ (97.5%) were prevalent within the population but qacG was not detected. EMRSA-15 (19.2%), EMRSA-16 (15%), P3 (15%) and H (12.5%) were the most common PFGE types. Clinical isolates demonstrated various degrees of susceptibility to chlorhexidine in the surface disinfection [mean microbiocidal effect (ME) = 0-1.91] and biocide residue (mean ME = 0.29-3.74) tests. Increases in post-exposure MICs were observed in both EMRSA-16 and the susceptible S. aureus control. CONCLUSIONS: In our study, isolates resembling PFGE type EMRSA-16 harboured more biocide resistance genes than other types. The observed reduction in susceptibility of clinical isolates to chlorhexidine may mean that a selective pressure is being exerted by residues in the clinical environment, and highlights the importance of efficacy testing on clinical strains and good infection control practices. The development of reduced microbial susceptibility to biocides represents a serious cause for concern in the clinical environment.