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Pesquisa do Estado do Rio Grande do Sul (FAPERGS) / Acinetobacter calcoaceticus-baumannii (ACB) complex comprises opportunistic and emerging pathogens that are responsible for several diseases, mainly affecting immunocompromised patients and those hospitalized in intensive care units. Therapeutic options for ACB infections are restricted, since these microorganisms are often resistant to most antimicrobials. In addition, these bacteria may also form persister cells, which constitute a small population of susceptible cells able to survive lethal concentrations of bactericidal antimicrobials and other stressors. This phenotype is associated with failure in antimicrobial therapy, especially in chronic and recurrent infections. Therefore, the aim of this study was to evaluate the presence of persister cells from ACB complex isolates exposed to meropenem and/or polymyxin B, in biofilm and planktonic cells, as well as to analyze these cells regarding their morphology and identify expression patterns of proteins possibly involved in the formation and maintenance of persistence. For this, 20 clinical isolates were characterized for the ability to form biofilm on polystyrene surface, and for meropenem and polymyxin B susceptibility, by the assessment of Minimum Inhibitory Concentration (MIC). All isolates were exposed to meropenem at different concentrations above the MIC, while five isolates were exposed to polymyxin B for the assessment of the persisters presence. For all experiments, in order to estimate the fraction of remaining cells, aliquots were removed at determined time points, followed by serial decimal dilutions and drop plating technique on nutrient agar. All isolates presented persisters at different proportions, in both culture conditions when exposed to meropenem or polymyxin B after 48 h. The higher fractions were verified in biofilm for both antimicrobials in comparison with planktonic cultures. Meropenem concentrations did not influence persisters levels. However, after polymyxin B exposure, persister cells fractions were dependent on the concentration employed. After 24 h polymyxin B exposure, a growth resumption of surviving cells was observed. These cells were again evaluated for susceptibility to this antimicrobial, remaining susceptible with MIC of 1 ?g/ mL. Moreover, integrity of polymyxin B in the supernatant of the cultures was verified by chromatographic assay, demonstrating that polymyxin B is not significantly degraded after 48 h exposure. On the other hand, when meropenem and polymyxin B were associated at different concentrations, no resumption of cell growth was observed, as well as this combination was able to eradicate persister cells from A. baumannii (Acb-1) cultured in late exponential phase. Furthermore, Nano-Liquid Chromatography Coupled to Tandem Mass Spectrometry was employed for the identification and relative quantification of proteins possibly associated with persistence in A. baumannii, after exposure to meropenem. Different patterns of expression were identified between persister cells present in planktonic and biofilm cultures, suggesting that persistence may be regulated by different mechanisms. Proteins involved in the cell division and DNA replication were overexpressed in planktonic persisters, in agreement with the electron scanning microscopy images that presented dividing cells in this culture condition. Overexpression of glucose dehydrogenase (GDH), NADH dehydrogenase 1 (NDH-1), succinate dehydrogenase and ATP synthase indicates the electron transfer from the GDH-catalyzed reaction to the electron transport chain as a possible energy source for persisters, supporting the presence of cell division observed in planktonic culture. Conversely, proteins involved in amino acid metabolism, as well as major elongation factors were underexpressed in Acb-1 persister cells, suggesting that protein synthesis is reduced, even though many proteins were overproduced. Increased expression of several membrane-related proteins has also been observed, indicating possible changes in its composition and function, although proteins related to lipid metabolism were underexpressed. Overall, proteomic analysis of the persister cells showed that these cells could be physiologically distinct when cultured under different conditions, as well as overtime in the same condition. Therefore, considering the different behaviors of Acb-1 when exposed alone to meropenem or polymyxin B, as well as when exposed to these drugs in combination, it was concluded that each antimicrobial might act as a different stressor, possibly leading and/or selecting distinct tolerance mechanisms among persisters, which enabled their eradication when the drugs were combined. / Os pat?genos pertencentes ao complexo Acinetobacter calcoaceticus-baumannii (ACB) s?o considerados oportunistas e emergentes, respons?veis por ocasionar diversas enfermidades, acometendo principalmente pacientes imunocomprometidos e internados em unidades de tratamento intensivo. As op??es terap?uticas para o tratamento de infec??es ocasionadas por estes pat?genos s?o restritas, uma vez que estes apresentam frequentemente resist?ncia ? maioria dos antimicrobianos. Al?m disso, essas bact?rias podem ainda formar c?lulas persisters, que constituem uma pequena popula??o de c?lulas suscet?veis capazes de tolerar concentra??es letais de antimicrobianos bactericidas e outros agentes estressores. Este fen?tipo est? associado a falhas na terapia antimicrobiana, especialmente nas infec??es cr?nicas e recorrentes. Desta forma, o objetivo deste trabalho foi avaliar a presen?a de c?lulas persisters formadas por isolados do complexo ACB frente ? exposi??o ao meropenem e/ou ? polimixina B, na condi??o de biofilme e em cultivo planct?nico, assim como analisar estas c?lulas morfologicamente e identificar padr?es de express?o de prote?nas que pudessem estar envolvidos na forma??o e manuten??o da persist?ncia. Para tanto, 20 isolados cl?nicos foram caracterizados quanto ? capacidade em formar biofilme em superf?cie de poliestireno, e ? suscetibilidade ao meropenem e ? polimixina B, que foi avaliada por meio da determina??o da Concentra??o Inibit?ria M?nima (CIM) a estes f?rmacos. Todos os isolados foram submetidos ? exposi??o ao meropenem em diferentes concentra??es acima da CIM, enquanto que cinco foram expostos ? polimixina B para a avalia??o da presen?a de c?lulas persisters. Para todos os experimentos, a fim de estimar a fra??o de c?lulas remanescentes, al?quotas foram removidas em tempos determinados, efetuando-se dilui??es decimais seriadas e semeadura pela t?cnica da gota em ?gar nutriente. C?lulas persisters, em diferentes fra??es, foram encontradas nos cultivos de todos os isolados, tanto em biofilmes como na condi??o planct?nica, ap?s a exposi??o por 48 h ao meropenem e ? polimixina B, sendo as fra??es mais elevadas encontradas na condi??o de biofilme para ambos os antimicrobianos. As diferentes concentra??es de meropenem avaliadas n?o influenciaram os n?veis de c?lulas persisters; entretanto, frente ? exposi??o ? polimixina B, a fra??o de c?lulas mostrou-se dependente da concentra??o empregada. Ap?s exposi??o ? polimixina B por 24 h, foi observada retomada de crescimento das c?lulas sobreviventes, que foram avaliadas novamente quanto ? suscetibilidade a este antimicrobiano, mantendo-se suscet?veis com CIM de 1 ?g/mL, bem como foi verificada a integridade do antimicrobiano no sobrenadante destes cultivos por ensaios cromatogr?ficos, demonstrando que o mesmo n?o sofre degrada??o ap?s 48 h de exposi??o. Entretanto, quando se associou meropenem ? polimixina B em diferentes concentra??es, al?m de n?o ter sido observada a retomada de crescimento das c?lulas remanescentes, ocorreu a erradica??o das c?lulas persisters de um isolado de A. baumannii (Acb-1) cultivado em fase exponencial tardia. Al?m disso, a t?cnica de Nano-Cromatografia L?quida acoplada ? Espectrometria de Massas em Tandem foi empregada para a identifica??o e quantifica??o relativa de prote?nas possivelmente associadas ? persist?ncia em A. baumannii, ap?s a exposi??o ao meropenem. Diferentes padr?es de express?o foram identificados entre as c?lulas persisters presentes em cultivo planct?nico e em biofilme, sugerindo que a regula??o da persist?ncia possa ser realizada por mecanismos diferentes. Observou-se express?o aumentada de prote?nas envolvidas nos processos de divis?o celular e replica??o de DNA, especialmente no cultivo planct?nico, em concord?ncia com a presen?a de divis?o celular observada nas imagens obtidas a partir da microscopia eletr?nica de varredura nesta condi??o de cultivo. O aumento de express?o da glicose desidrogenase (GDH), NADH desidrogenase (NDH-1), succinato desidrogenase e ATP sintase indicam a transfer?ncia de el?trons a partir da rea??o catalisada por GDH para a cadeia de transporte de el?trons como uma poss?vel fonte de energ?tica para as persisters, corroborando a observa??o da presen?a de divis?o celular observada no cultivo planct?nico. Em contraste, prote?nas envolvidas no metabolismo de amino?cidos, bem como os principais fatores de elonga??o apresentaram express?o diminu?da em c?lulas persisters de Acb-1, sugerindo que a s?ntese proteica esteja reduzida, mesmo que muitas prote?nas tenham sido identificadas com express?o aumentada. Muitas prote?nas relacionadas ? membrana apresentaram a sua express?o aumentada, indicando poss?veis altera??es em sua composi??o e fun??o, embora prote?nas relacionadas ao metabolismo de lip?deos tenham apresentado express?o diminu?da. A an?lise prote?mica das c?lulas persisters, sobretudo, mostrou que estas c?lulas podem ser fisiologicamente distintas quando cultivadas em condi??es diferentes, bem como ao longo do tempo em uma mesma condi??o. Desta forma, considerando os distintos comportamentos do Acb-1 quando exposto isoladamente ao meropenem ou ? polimixina B, bem como quando exposto a estes f?rmacos ao mesmo tempo, pode-se concluir que cada antimicrobiano pode ter atuado como um diferente estressor, possivelmente, levando a e/ou selecionando mecanismos de toler?ncia diferentes entre as persisters, o que possibilitou a sua erradica??o quando os f?rmacos foram combinados.