Tolerância e persistência à enrofloxacina em Staphylococcus spp. de mastite caprina: caracterização, determinação de fatores de virulência e genômica comparativa

Abstract

Goat milk is an important source of food for human nutrition and contamination by bacteria from intramammary infections, especially Staphylococcus spp., poses a public health risk. Although antibiotic therapy is the therapy of choice, microorganisms can survive if they are resistant, tolerant or persistent. The identification of isolates with these profiles can help control complications caused by mastitis. The aim of this study was to characterize isolates of Staphylococcus spp. goat mastitis, as to the tolerance/persistence profile and associated virulence factors and identify possible genetic factors related to persistence. The taxonomic identification of twelve isolates of Staphylococcus spp. was carried out through the MALDI-TOF. TDTest was applied for tolerance/persistence screening, and through broth microdilution, minimal inhibitory (MIC) and bactericidal (CBM) concentrations were obtained for enrofloxaxin. Death curves were performed using 2xCIM of the antimicrobial, analyzing the surviving bacterial population according to the minimum duration of death, as well as if it is dose or time dependent. Quantification of the biofilm was carried out through the microplate adherence test. The genomes of the eight Isolates of Staphylococcus warneri before (4) and after (4) enrofloxacin treatment were sequenced. The phylogenomic relationship and genetic plasticity were determined. Additionally, there was the search for genes of resistance and virulence, as well as the presence polymorphisms. Bacterial isolates have been identified as Staphylococcus aureus (n=4) and Staphylococcus warneri (n=8). The bacteria showed MIC and CBM varying between 0.12 and 1.95 μg / mL, concentration below the breakpoint (4μg / mL). On the death curve, S. aureus isolates were tolerant and S. warneri were classified as persistent. It was found that in selected isolates, persistence was time dependent on type I. All isolates were biofilm producers. In the sequencing of S. warneri isolates, the complete sequences of three genomes and five in incomplete format were determined. The isolates were considered clonal and in the pangenomic analysis, 2017 central genes were found, and between 176 to 336 accessory genes. However, no single genes were found. According to COG analyzes, genes are involved in processes of general functions, transcription, replication and recombination repair. Three phages and six genomic islands were predicted and fully shared by all S. warneri genomes. Similarly, seven resistance genes and six virulence genes were also shared. Similarly, seven resistance genes and six virulence genes were shared. The polymorphism analysis detected 13 variants. It is concluded that tolerance and persistence in Staphylococcus spp. was a determining factor in the survival of enrofloxacin. Tolerance was confirmed for S. aureus, while persistence in S. warneri. This indicates that it may be a species-specific phenomenon in this case. Biofilm production by species may have significantly favored antibiotic tolerance. Comparative genomics analyzes showed similarity between persistent S. warneri isolates, indicating the potential for dissemination among animals and consequently a risk to public health. This study allowed totrace the genetic profile of persistent S. warneri isolates and the presence of genes that may be related to important processes for bacterial persistence