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O setor de produtos lácteos transforma-se constantemente, uma vez que exigências feitas tanto pelo consumidor como por órgãos fiscalizadores, requerem produtos inócuos e com elevado padrão de qualidade, provocando mudanças significativas em toda a cadeia produtiva. Para tanto, medicamentos veterinários, em especial os antimicrobianos, têm sido amplamente utilizados na pecuária, quer para tratamento e prevenção de doenças, quer para incremento da produção. No entanto, quando utilizados em desrespeito às boas práticas, colocam em risco tanto a saúde animal como a humana, predispondo a população a eventuais reações alérgicas e expressão de resistência microbiana. Neste contexto, o presente trabalho teve como objetivo pesquisar a presença de resíduos de antimicrobianos de uso veterinário em amostras de diferentes tipos de leite comercializados na cidade de São Paulo, verificando se a freqüência de ocorrência observada para essa contaminação correspondia à estimada de 1%. Foram analisadas, através do Delvotest® SP, 1.500 amostras de leite, durante os meses de abril de 2003 a março de 2004 e obteve-se dez amostras positivas, o que significa uma freqüência de ocorrência de antimicrobiano no leite de 0,66%, com intervalo de confiança entre 0,32 a 1,22%, valor este, menor que o estimado / The dairy industry have been transformed day by day, because the consumer and the competent inspection groups that have been demanded good products without contamination and them have to be a increasing quality, so, this facts provokes significants chances in all dairy segment. About that, the antimicrobians had to been used in large scala in animals to terapy and to increase the weight quickly, so is necessary to be careful to use those products because theirs residues may be cause irritatation or allerrgy and bacteria resistense. The presense of the antimicrobian in the milk is a very important theme since the pecuary untill the public health, because that, this assignment (work) had like objetive research the presense of residues of veterinary antimicrobian in milk sample salled in the Sao Paulo city and check if the observaded frequence is similar to the stimated frequence like 1%. The results was ten positives samples of the 1.500, that means occurence of frequency 0,66% with break between 0,32 to 1,22%, this result was smaller than hope for

O setor de produtos lácteos transforma-se constantemente, uma vez que exigências feitas tanto pelo consumidor como por órgãos fiscalizadores, requerem produtos inócuos e com elevado padrão de qualidade, provocando mudanças significativas em toda a cadeia produtiva. Para tanto, medicamentos veterinários, em especial os antimicrobianos, têm sido amplamente utilizados na pecuária, quer para tratamento e prevenção de doenças, quer para incremento da produção. No entanto, quando utilizados em desrespeito às boas práticas, colocam em risco tanto a saúde animal como a humana, predispondo a população a eventuais reações alérgicas e expressão de resistência microbiana. Neste contexto, o presente trabalho teve como objetivo pesquisar a presença de resíduos de antimicrobianos de uso veterinário em amostras de diferentes tipos de leite comercializados na cidade de São Paulo, verificando se a freqüência de ocorrência observada para essa contaminação correspondia à estimada de 1%. Foram analisadas, através do Delvotest® SP, 1.500 amostras de leite, durante os meses de abril de 2003 a março de 2004 e obteve-se dez amostras positivas, o que significa uma freqüência de ocorrência de antimicrobiano no leite de 0,66%, com intervalo de confiança entre 0,32 a 1,22%, valor este, menor que o estimado / The dairy industry have been transformed day by day, because the consumer and the competent inspection groups that have been demanded good products without contamination and them have to be a increasing quality, so, this facts provokes significants chances in all dairy segment. About that, the antimicrobians had to been used in large scala in animals to terapy and to increase the weight quickly, so is necessary to be careful to use those products because theirs residues may be cause irritatation or allerrgy and bacteria resistense. The presense of the antimicrobian in the milk is a very important theme since the pecuary untill the public health, because that, this assignment (work) had like objetive research the presense of residues of veterinary antimicrobian in milk sample salled in the Sao Paulo city and check if the observaded frequence is similar to the stimated frequence like 1%. The results was ten positives samples of the 1.500, that means occurence of frequency 0,66% with break between 0,32 to 1,22%, this result was smaller than hope for

Fluoroquinolones are a class of antibiotics used clinically to treat a wide array of bacterial infections. These therapeutics act by targeting a bacterial enzyme required for cell viability, bacterial type-II topoisomerases. Fluoroquinolones act by forming a ternary complex with bacterial type II topoisomerases and cleaved DNA; religation of DNA is subsequently blocked, therefore leading to bacterial cell death. In ternary complex the keto-acid moiety of the fluoroquinolone is complexed with a divalent magnesium ion, forming a drug-magnesium-water bridge to a serine and an aspartate (or glutamate) residue on helix-4 of the topoisomerase enzyme. A major issue with fluoroquinolones is the rise in bacterial resistance. Resistance arises through substitutions of the serine or aspartate/glutamate residue, therefore preventing formation of the magnesium-water bridge and dramatically diminishing the overall antibiotic activity of the fluoroquinolone. Quinazoline-2,4-diones are structurally similar to fluoroquinolones; diones also form a ternary complex similar to fluoroquinolones, however, these complexes are less active due to lack of a potent magnesium-water bridge interaction in helix-4. While quinazoline-2,4-diones are therefore less potent antibiotics, their non-reliance on the magnesium water bridge generally affords equipotent activity with wild-type and fluoroquinolone-resistant strains of bacteria. The first objective of this work was to probe the helix-4 interaction of the bacterial type-II topoisomerase by quinazoline-2,4-dione modification, specifically at the N3 and C4 positions of the quinazoline-2,4-dione scaffold to afford potentially new binding contacts. These modified quinazoline-2,4-diones will provide deeper understanding of the helix-4 interaction and potentially afford potent novel quinazoline-2,4-dione scaffolds, against both wild-type and resistant bacteria, for iterative drug design. Metabolism is one of the primary sources of detoxification, inactivation, and clearance of drugs from the body and is a critical consideration for all early stage therapeutic development. Clinically used fluoroquinolones, i.e. Moxifloxacin and Ciprofloxacin, historically are metabolically stable, and are not known to be metabolized by Phase I and/or Phase II drug metabolizing enzymes. However, major modifications to the Moxifloxacin and Ciprofloxacin scaffolds, due to the development of next generation antibiotics, may display different metabolic stability profiles. Moreover, metabolism of quinazoline-2,4-diones, developed for fluoroquinolone-resistant bacteria, is not extensively studied and may be subject to different metabolic liabilities that may render the quinazoline-2,4-dione an ineffective potential antibiotic. The second objective of this work was to determine the in vitro Phase I and Phase II metabolic stabilities of fluoroquinolone and quinazoline-2,4-dione scaffolds to determine any structural features that render the potential therapeutic a metabolic liability. The results from these two objectives have led to the discovery of a novel bacterial type-II topoisomerase catalytic inhibitor and the acquisition of initial metabolic stability data of fluoroquinolone and quinazoline-2,4-dione scaffolds. These findings further promote research into quinazoline-2,4-diones as bacterial topoisomerase targets, and provide metabolic considerations for both fluoroquinolone and quinazoline-2,4-dione therapeutic development, which is severely underrepresented in the field of quinolone antibiotics.

Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal ++ / plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic ++ / biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.