Detecció, identificació de Mycobacterium tuberculosis i caracterització molecular de la resistència als principals antituberculosos en mostra clínica directa mitjançant piroseqüenciació: aplicabilitat en l’estudi de la clonalitat de les soques.

L’augment de soques de Mycobacerium tuberculosis multiresistents i l’aparició de soques extremadament resistents, ha posat de manifest la necessitat de disposar de nous mètodes de detecció precoç de M.tuberculosis i les seves resistències als principals fàrmacs antituberculosos, així com l’estudi eficaç dels brots, per a poder dur a terme un control eficient de la malaltia. L’objectiu de l’estudi va ser determinar la capacitat de la piroseqüenciació per a detectar i identificar micobacteris a partir d’aïllats clínics i mostra directa, determinar el seu patró de resistència a Rifampicina, Isoniacida, Etambutol, Fluoroquinolones, Canamicina i Capreomicina i explorar la seva potencialitat per a ser emprada en el tipatge molecular de les soques. Mitjançant la optimització de la tecnologia de la piroseqüenciació pels gens analitzats, i el disseny de primers específics, hem verificat la utilitat de la piroseqüenciació per al maneig de la tuberculosi. S’ha pogut estudiar la presència de mutacions relacionades amb resistències a fàrmacs de primera línia en el 96.5% de les posicions analitzades en soca clínica i en el 70.4% en mostra directa. Van concordar amb el resultats obtinguts per altres mètodes fenotípics i/o fenotípics el 97.1% i el 98.2% del resultats obtinguts en soca clínica i mostra directa respectivament. La piroseqüenciació ens ha permet analitzar la presència de mutacions relacionades amb resistències a antituberculosos de segona línia, servint com a mètode de confirmació en l’anàlisi d’altres mètodes genotípics. La tècnica ens ha permès també identificar els principals micobacteris no tuberculosos implicats en la infecció humana, sòls o en coinfecció. Resultats preliminars mostren que l’anàlisi amb piroseqüenciació pot ser d’utilitat per l’estudi clonal de les soques de M.tuberculosis. Les nostres observacions mostren la piroseqüenciació com una eina valuosa en l’àmbit clínic, ja que permet una reducció de la demora del diagnòstic, estudi filogenètic i detecció de resistències M.tuberculosis, i per tant una millora de l’aplicació del tractament adequat, ajudant al control de la malaltia.

High-Resolution Mass Spectrometry applied to the identification of new metabolites and transformation products of quinolones in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from Enrofloxacin (ENR), Ciprofloxacin (CIP), Difloxacin (DIF) and Sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and Multiple Mass Defect Filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.

High-Resolution Mass Spectrometry applied to the identification of transformation products of quinolones from stability studies and new metabolites of enrofloxacin in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from Enrofloxacin (ENR), Ciprofloxacin (CIP), Difloxacin (DIF) and Sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and Multiple Mass Defect Filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.

High-resolution mass spectrometry applied to theidentification of transformation products of quinolones from stability studies and new metabolites of enrofloxacin in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from Enrofloxacin (ENR), Ciprofloxacin (CIP), Difloxacin (DIF) and Sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and Multiple Mass Defect Filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.

Evidence of an efflux pump in Serratia marcescens

Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10-7 to 10-9. Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug

Evidence of an efflux pump in Serratia marcescens

Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10-7 to 10-9. Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug