The aim of this study will be to evaluate the association between antimicrobial use (UAM) and resistance of Streptococcus uberis causing clinical mastitis (CM) in Brazilian herds. The specific objectives will be: (a) determine the distribution of the bacterial species previously identified as Strep. uberis by conventional phenotypic identification and Matrix Associated Laser Desorption-Ionization - Time of Flight (MALDI-TOF MS); and, (b) evaluate the susceptibility of Strep. uberis to antimicrobials with a high frequency of use for the treatment of CM in Brazilian herds. The MALDI score cut off values will be applied to each measurement as follows: (score of >2) indicate species-level identification; (scoree1.7 and <2) genus-level identification; and (score<1.7) indicate that no reliable identification can be made. Isolates with result discrepancy between microbiologic techniques (MALDI vs. conventional culture) will be submitted for 16S rRNA gene sequencing. Subsequently, the antimicrobial susceptibility of isolates identified as Streptococcus spp. by MALDI-TOF MS will be tested in vitro for the following antimicrobials: amoxicillin, ampicillin, cephalexin, ceftiofur, ciprofloxacin, gentamicin, oxytetracycline, tetracycline, sulfa + trimetropin, penicillin, enrofloxacin and florfenicol. Analysis of minimal inhibitory concentration (MIC) will be performed using the microdilution test. Results will be determined by visual inspection of the plates' wells, in which the lowest antimicrobial concentrations required to inhibit bacterial growth will be recorded. The association between the UAM and resistance profile of isolates will be performed by multivariate logistic regression models, in which the frequency of resistant isolates will be considered as the dependent variable and antimicrobial consumption as an independent variable in the models created for each tested compound. Antimicrobial consumption data will be obtained from a previous study on the use of antimicrobials (intramammary and systemic) recorded for the treatment of CM in Brazilian dairy herds (Tomazi, 2017). The conduction of the present study will advance the etiological and pharmaco-epidemiological understanding of Strep. uberis causing CM in dairy herds in Brazil. In addition, the association analysis between antimicrobial consumption and resistance profile of Strep. uberis will allow greater understanding on the development of bacterial resistance in dairy herds, which may encourage producers for the rational use of antimicrobials for mastitis control.
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