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.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Andressa Liberal Santos - 2018.pdf: 2603632 bytes, checksum: 0a1312a46fb6a806189f5479682576a2 (MD5) Previous issue date: 2018-06-28 / Enterobacteria are microorganisms involved with bacteria and the health of women with care. Treatment of bacterial infections is most often done with the use of antibiotics and one of the major classes of antimicrobials is one of the β-lactams. Among the main mechanisms of resistance to the observational β-lactam antibiotics: alteration of the antimicrobial target; alteration of β-lactam permeability; Flow pumps and the entry of enzymatic signals that destroy the β-lactate totally or with the development of an alternative metabolic pathway. These enzymes are known as beta-lactamases and are encoded by specific genes. Thus, the objective of this study was to correlate the resistance profile of enterobacteria, using phenotype methodologies and identifying 14 genes that encode as beta-lactamase enzymes: blaOXA genes; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGim and blaSHV. The phenotypic methodologies used were the Antimicrobial Sensitivity Test for Disk-Diffusion (antibiogram), and complementary tests for the detection of resistance mechanisms of beta-lactamases (ESBL, MBL, AmpC and Carbapenemase). The molecular methodology used was Real Time PCR using the Sybr Green system. Among the results found in the tests it was observed that 74.28% were resistant to ampicillin, 34.28% were resistant to aztreonam, 62.85% were resistant to amoxicillin associated with clavalunate, 51.42% were resistant to ceftazidime, 41 , 42% were resistant to cefoxitin, 54.28% were resistant to cefazolin, 44.28% were resistant to cefepime, 41.42% were resistant to cefuroxime, 8.57% were resistant to cefuroxime, 35.71% were resistant an imipenem and 41.42% were resistant to piperacillin associated with tazobactam. Among the total samples, the mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of genera were blaGIM and blaSIM (66.66% of the samples). The gene that was amplified in a smaller number of samples was blaVIM (16.66%). It is concluded that although there is a low correlation between the methodologies analyzed, the levels of antimicrobial resistance in enterobacteria are high and worrying, and a way to minimize the accelerated emergence of resistance includes the development or improvement of techniques that generate diagnoses with high efficiency and speed. / As enterobactérias são microrganismos envolvidos com infecções bacterianas adquiridas na comunidade e nos ambientes dos cuidados com a saúde. O tratamento das infecções bacterianas na maioria das vezes é realizado com a utilização de antibióticos e uma das maiores classes de antimicrobianos é a dos β-lactâmicos. Entre os principais mecanismos de resistência aos antimicrobianos β-lactâmicos observa-se: alteração do alvo antimicrobiano; alteração da permeabilidade ao β-lactâmico; bombas de e-fluxo e a presença de mecanismos enzimáticos que destroem o β-lactâmico totalmente ou parcialmente com desenvolvimento de uma via metabólica alternativa. Essas enzimas são conhecidas como beta-lactamases e são codificadas por genes específicos. Dessa forma, o objetivo deste estudo foi o de correlacionar o perfil de resistência das enterobactérias, utilizando metodologias fenotípicas e identificar 14 genes que codificam as enzimas beta-lactamases: genes blaOXA; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGIM e blaSHV. As metodologias fenotípicas utilizadas foram o Teste de Sensibilidade aos Antimicrobianos por disco-difusão (antibiograma), e testes complementares para detecção dos mecanismos de resistência das beta-lactamases (ESBL, MBL, AmpC e Carbapenemase). A metodologia molecular utilizada foi a PCR em Tempo Real, utilizando o sistema Sybr Green. Entre os resultados fenotípicos encontrados nas bactérias observou-se que 74,28% foram resistentes a ampicilina, 34,28% foram resistentes a aztreonam, 62,85% foram resistentes a amoxicilina associado ao clavalunato, 51,42% foram resistentes a ceftazidima, 41,42% foram resistentes cefoxitina, 54,28% foram resistentes a cefazolina, 44,28% foram resistentes a cefepime, 41,42% foram rsistentes a ceftriaxona, 8,57% foram resistentes a cefuroxima, 35,71% foram resistentes a imipenem e 41,42% foram resistentes a piperacilina associada tazobactam. Entre o total de amostras, o mecanismo de resistência que apresentou maior expressão foi o ESBL (17,14%). Os genes estudados que foram detectados em um maior número de gêneros foram o blaGIM e o blaSIM (66,66% das amostras). O gene amplificado em menor número de amostras foi o blaVIM (16,66%). Conclui-se que apesar de haver baixa correlação entre as metodologias analisadas, os níveis de resistência a antimicrobianos em enterobactérias são altos e preocupantes e uma maneira de minimizar a acelerada emergência de resistência é desnvolver e aprimorar técnicas de diagnósticos com alta eficiência e rapidez.