Direct antimicrobial susceptibility testing (dAST) is not a standardized method and there is no recommendations regarding the volume of blood culture media to be used. The aim of the study was to optimize the method of dAST by determining a volume of blood culture media in µL to be used and to test the reproducibility of the volume optimized method. The dASTs (n=160) were performed on blood culture bottles (n=40) inoculated with reference bacteria (n=4), using four test volumes (50 µL, 75 µL, 100 µL and 125 µL). The optimized method was implemented on frozen and fresh bacterial isolates (n=120) derived from blood cultures. Susceptibility tests according to EUCASTs method for disk diffusion was also performed. Deviations in SIR-categorical agreement was calculated. The optimal volume of blood culture media for dAST was 75 µL. All dASTs were approved for three out of four reference bacteria whereas for the fourth reference bacteria eight out of ten dASTs was approved. Testing the reproducibility, the optimized method showed a sensitivity and specificity of 100 %. No deviation in categorical agreement of SIR-categorization was observed. The result shows a possibility of implementing a standardized method for dAST regarding the volume of blood culture media. / Vid direktresistensbestämning används blododlingsmedium för tillverkning av bakteriesuspensioner. Metoden är inte standardiserad och det finns ingen rekommenderad volym blododlingsmedium som bör användas. Syftet med studien var att optimera metoden för direktresistensbestämning genom att bestämma en volym blododlingsmedium i µL som används för tillverkning av bakteriesuspensioner. Den optimerade metodens prestanda utvärderades därefter på kliniska patientisolat. Metoden optimerades genom att direktresistensbestämningar (n=160) utfördes baserat på fyra volymer (50 µL, 75 µL, 100 µL och 125 µL) blododlingsmedium (n=40), som var inokulerade med fyra olika referensstammar. Den optimerade metodens reproducerbarhet testades på frysta och färska patientisolat (n=120) genom jämförelse av SIR-kategorisering mellan direktresistensbestämning samt resistensbestämning enligt EUCASTs metod för diskdiffusion. Den optimala volymen blododlingsmedium med kända referensstammar fastställdes vara 75 µL då samtliga direktresistensbestämningar godkändes för tre av fyra referensstammar, för den fjärde referensstammen godkändes åtta av tio. Då metoden implementerades på kliniska patientisolat från positiva blododlingar var känsligheten och specificiteten 100 % avseende kategorisk överensstämmelse enligt SIR-systemet. Inga avvikelser avseende SIR-kategorisering observerades. Resultaten visar att det är möjligt att optimera metoden avseende volymen blododlingsmedium som används för direktresistensbestämning på blododlingsflaskor. Då den optimerade metodens känslighet och specificitet var 100 %, är det möjligt att implementera metoden i rutinarbetet.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: A alta frequência de Estafilococos Coagulase-Negativa (CoNS) na pele de indivíduos saudáveis e em doenças associadas ao sangue, associados à seleção de cepas resistentes devido a uso indiscriminado de antimicrobianos, tornou mais estreitos os limites entre o ambiente hospitalar e o comunitário quanto à distribuição de cepas. Objetivou-se, com este estudo, caracterizar isolados de CoNS de origem hospitalar e comunitária da cidade de Botucatu-SP quanto ao perfil clonal, analisar os aspectos de resistência à oxacilina pela aferição de metodologia de detecção, e investigar os determinantes de heterorresistência à vancomicina nessas cepas. As espécies estudadas incluíram S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans e S. xylosus. O teste de disco-difusão (TDD) com discos de oxacilina e cefoxitina, fitas de Etest impregnadas com oxacilina e pesquisa do gene mecA por PCR em tempo real foram realizadas. A triagem em ágar com 6 e 8 µg/ml de vancomicina, microdiluição em caldo para aferição da Concentração Inibirtória Mínima (MIC), microscopia eletrônica de transmissão para verificar espessamento da parede celular e alterações fenotípicas por testes bioquímicos foram realizadas. O perfil clonal foi determinado por PFGE (Pulsed-Field Gel Eletrophoresis) e para clones de S. epidermidis, o MLST (Multilocus Sequence Typing). S. epidermidis apresentou alta diversidade clonal, mas presença de clusters no ambien... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The high frequency of Coagulase-Negative Staphylococci (CoNS) on the skin of healthy individuals and in bloodstream infections, together with the selection of resistant strains, has narrowed the boundaries between the hospital and the community environment for the distribution of strains. This study aimed to characterize CoNS isolated from clinical and colonization specimens of patients and individuals from Botucatu-SP, to compare their clonal profile, to analyze the determination of oxacillin resistance by the evaluation of the methodology of detection, and to investigate the determinants of reduced susceptibility to vancomycin in those strains. CoNS species included S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans and S. xylosus. The disc diffusion test (DDT) using oxacillin and cefoxitin discs was employed, Etest strips impregnated with oxacillin and mecA gene detection by real-time PCR were used. An agar screening with 6 and 8 µg/ml of vancomycin, the broth microdiluition method for the Minimal Inhibitory Concentration (MIC), the transmission eletronic microscopy for evaluation of cellwall thickening and phenotypic modifications by biochemical tests were performed. Clonal profile was determined by PFGE (Pulsed-Field Gel Eletrophoresis) and, for S. epidermidis clones, MLST (Multilocus Sequence Typing). S. epidermidis presented high clonal diversity, despite some clusters circulating within hospi... (Complete abstract click electronic access below) / Doutor