RESUMO
Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.
Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/fisiologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/efeitos dos fármacos , Staphylococcus/fisiologia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos , Coagulase/análise , Farmacorresistência Bacteriana/genética , Feminino , Variação Genética , Mastite Bovina/tratamento farmacológico , Testes de Sensibilidade Microbiana/veterinária , Infecções Estafilocócicas/microbiologia , Staphylococcus/genéticaRESUMO
In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world.
Assuntos
Elementos de DNA Transponíveis/genética , Enterobacter/isolamento & purificação , Escherichia coli/isolamento & purificação , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Aztreonam/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases , Brasil , Conjugação Genética , Sequência Conservada , Enterobacter/efeitos dos fármacos , Enterobacter/genética , Enterobacter/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfomicina/farmacologia , Humanos , Índia , Testes de Sensibilidade Microbiana , Marrocos , Nepal , Plasmídeos , Reto/microbiologia , beta-Lactamases/genéticaRESUMO
Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.
Assuntos
Chaperonina 60/metabolismo , Coagulase/metabolismo , Mastite Bovina/microbiologia , Reação em Cadeia da Polimerase/veterinária , Infecções Estafilocócicas/veterinária , Staphylococcus/isolamento & purificação , Animais , Bovinos , Chaperonina 60/genética , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Polimorfismo de Fragmento de Restrição , Especificidade da Espécie , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificaçãoRESUMO
A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.