Ketamine (KET) is an N-methyl-D-aspartate (NMDA) antagonist with rapid and long-lasting antidepressant effects, but how the drug shows its sustained effects is still a matter of controversy. The objectives were to evaluate the mechanisms for KET rapid (30 min) and long-lasting (15 and 30 days after) antidepressant effects in mice. A single dose of KET (2, 5, or 10 mg/kg, po) was administered to male Swiss mice and the forced swim test (FST) was performed 30 min, 15, or 30 days later. Imipramine (IMI, 30 mg/kg, ip), a tricyclic antidepressant drug, was used as reference. The mice were euthanized, separated into two time-point groups (D1, first day after KET injection; D30, 30 days later), and brain sections were processed for glycogen synthase kinase-3 (GSK-3), histone deacetylase (HDAC), brain-derived neurotrophic factor (BDNF), and glial fibrillary acidic protein (GFAP) immunohistochemical assays. KET (5 and 10 mg/kg) presented rapid and long-lasting antidepressant-like effects. As expected, the immunoreactivities for brain GSK-3 and HDAC decreased compared to control groups in all areas (striatum, DG, CA1, CA3, and mainly pre-frontal cortex, PFC) after KET injection. Increases in BDNF immunostaining were demonstrated in the PFC, DG, CA1, and CA3 areas at D1 and D30 time-points. GFAP immunoreactivity was also increased in the PFC and striatum at both time-points. In conclusion, KET changed brain BDNF and GFAP expressions 30 days after a single administration. Although neuroplasticity could be involved in the observed effects of KET, more studies are needed to explain the mechanisms for the drug’s sustained antidepressant-like effects.

RESUMO Este estudo teve como objetivo identificar isolados de Staphylococcus coagulase-negativa (SCN) isolados de mastite bovina, por meio de métodos fenotípicos e PCR-RFLP (reação em cadeia de polimerase - polimorfismo nos fragmentos de restrição), e compará-los com a técnica de tempo de voo de ionização/desorção por laser assistida por matriz de espectrofotometria de massa (MALDI-TOF MS). O método de PCR-RFLP, que utiliza uma parte conservada da sequência do gene groEL, é uma alternativa promissora, por ser reprodutível e confiável. Por outro lado, a técnica proteômica MALDI-TOF MS permite uma acurácia e um diagnóstico muito mais rápidos e tem sido cada vez mais empregada na identificação microbiológica. Os perfis fenogenotípicos de resistência aos beta-lactâmicos também foram investigados. Essa caracterização é importante, considerando-se que os antimicrobianos são os elementos-chave para o controle da mastite na produção leiteira. A concordância entre os testes fenotípicos, PCR-RFLP E MALDI-TOF MS na identificação foi de 77,5% (31/40). S. chromogenes foi a espécie mais frequentemente isolada. A resistência antimicrobiana foi relativamente baixa, apresentando valores de 25,5% para penicilina, 9,6% para oxacilina e 6,2% para cefoxitina. Resistência ao imipenem, à cefalotina e à amoxacilina + ácido clavulâncico não foi observada. O gene mecA e sua variante foram detectados em 7,6% e 4,1% dos isolados, respectivamente. O gene blaZ foi encontrado em 43,2% dos isolados resistentes à penicilina.

ABSTRACT This study aimed to identify Coagulase-Negative Staphylococci (CoNS) species isolated from bovine mastitis, through phenotypic and PCR-RFLP (Restriction Fragment Length Polymorphism-Polimerase Chain Reaction) methods and to compare both techniques to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique. Among them, the PCR-RFLP method, using a partially conserved sequence of the groEL gene, is a promising alternative, because of its reproducibility and reliability. On the other hand, the proteomic technique MALDI-TOF MS provides an accurate and much faster diagnosis and has been increasingly employed in microbiological identification. The pheno-genotypic profiles of beta-lactam resistance were also investigated, this characterization is important, considering that the use of antimicrobials is a key element for mastitis control in dairy farms. The concordance of the phenotypic, PCR-RFLP and MALDI-TOF MS assays to identify CoNS species was 77,5% (31/40). S. chromogenes was the species most frequently isolated. Antibiotic resistance rate was relatively low, registering values of 25.5% to penicillin, 9.6% to oxacillin and 6.2% to cefoxitin. Resistance to imipenem, cephalotin and amoxicillin+clavulanate was not observed. The mecA gene and its variant were detected in 7.6% and 4,1% of the isolates respectively. The blaZ gene was found in 43.2% of the strains resistant to penicillin.

RESUMO O presente estudo teve como objetivo detectar os principais genes de virulência e resistência antimicrobiana em Staphylococcus aureus oriundos de leite bovino mastítico e classificá-los de acordo com a tipagem do gene agr. Foram selecionados 55 isolados de seis unidades produtores no estado do Rio de Janeiro. Destas, o gene fbnA foi encontrado em 27,3% das cepas e 78,2% possuíam o gene fbnB. Em nenhuma cepa foi encontrado o gene cap5 e 3,6% possuíam o gene cap8. O gene hlA foi encontrado em 94,5% das cepas e 89,1% possuíam o gene hlB. O gene icaA foi encontrado em 67,3% das cepas e 87,3% possuíam o gene icaD. Com base nesses resultados, foi possível estabelecer 12 diferentes perfis de virulência. Prevalência do agr tipo II foi detectada em 81,8% dos isolados. Considerando-se a avaliação da resistência antimicrobiana, as cepas estudadas foram suscetíveis a todos os antibióticos exceto penicilina, sendo detectado um percentual de 83,6% de cepas resistentes. Nenhuma das cepas apresentou o gene mecA, contudo 40% das cepas apresentaram o gene blaZ. Vinte e três perfis diferentes foram estabelecidos por associação de dados de virulência e resistência. Essa grande diversidade de cepas mostra a ampla gama de estratégias bacterianas e o desafio da prevenção à mastite no gado bovino, considerando-se que, a despeito da suscetibilidade antimicrobiana, essas cepas apresentam genes que permitem sua persistência no rebanho.

ABSTRACT This study aims to detect the main virulence and antimicrobial resistance genes in Stapylococcus aureus from bovine mastitic milk as well as classifying them according to agr typing. A total of 55 strains from six dairy unities in the state of Rio de Janeiro were selected, of these 27.3% presented fbnA and 78,2% for fbnB genes, respectively. None of the strains tested were positive for cap5 gene, 3.6% were positive for cap8 gene. Additionally, 94.5% of strains had hlA gene and 89.1% had hlB gene while 67.3% of the strains had icaA gene and 87.3% had icaD gene. From these results it was possible to establish 12 different virulence profiles. Prevalence of agrII type was detected in 81.8% of the isolates. Concerning antimicrobial resistance evaluation, the studied strains were susceptible to all antibiotics tested except penicillin, 83.6% being resistant strains. None of the strains had mecA gene, however, 40% of the strains had blaZ gene. Associating virulence and resistance data made it possible to obtain 23 different profiles. This great diversity of strains shows wide array of bacterial strategies and the challenge of mastitis prevention in cattle. Despite antimicrobial susceptibility, these strains presented certain genes that allow its persistence in the herd.