Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:34:28Z No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:34:34Z (GMT) No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) / Made available in DSpace on 2017-06-30T18:34:43Z (GMT). No. of bitstreams: 1 DIS_BRUNA_KERN_DONAMORE_PARCIAL.pdf: 256997 bytes, checksum: a87e4fb183ebf7c784b824c77b71092c (MD5) Previous issue date: 2016-03-31 / Acinetobacter calcoaceticus-baumannii complex opportunistic human pathogens are responsible for several diseases affecting specially intensive care units and immunocompromised patients. These bacteria may also present the persistence phenotype, where a small population of susceptible bacteria survives after a high antimicrobial concentration treatment ? which the fraction of surviving cells may be affected by environmental conditions present in the medium. Thus, the aim of this study was to evaluate the influence of oxygen availability and galactose, sodium citrate and sheep blood presence in the formation of these cells upon meropenem and tobramycin exposure. For this, 10 clinical isolates, sent by the Clinical Pathology Laboratory of S?o Lucas Hospital, had their susceptibility characterized regarding the minimum inhibitory concentration (MIC) to tobramycin and meropenem. The influence of different environmental conditions was analyzed by exposing late exponential-phase cultures to meropenem and tobramycin for 48 h in the presence of sodium citrate, galactose and sheep blood; or in aerated or static condition. For all evaluations, aliquots were removed at determined time points, followed by serial decimal dilutions and drop plating technique on nutrient agar, to estimate the fraction of surviving cells. Aerated condition promoted a reduction of persister cells fraction ? independent of antimicrobial used ?, and, in addition, concentrations of tobramycin higher than 10X MIC provided even lower levels of these cells after 48 h of treatment. Regarding the presence of different sources of nutrients, it was observed that sodium citrate presence in 48 h of meropenem treatment promoted lower fraction of surviving cells when compared to this same antibiotic associated with galactose; whereas tobramycin exposure in the presence of galactose provided a reduced formation of persister cells in the first 6 h of treatment. Sheep blood, however, did not affect the fraction of surviving cells after 48 h of treatment, independently of the antimicrobial used. A remarkable heterogeneity in the behavior of all 10 isolates used in the study was present, regardless the conditions that have been exposed. Thus, it is possible that the conditions imposed in the experiments have influenced in the formation of persister cells by enhanced production of reactive oxygen species, and through the availability of more targets for antibiotic action by the bacterial growth stimulation. / Os pat?genos oportunistas do complexo Acinetobacter calcoaceticus-baumannii s?o respons?veis por diversas enfermidades, acometendo principalmente pacientes de unidades de tratamento intensivo e imunocomprometidos. Estas bact?rias podem ainda apresentar o fen?tipo de persist?ncia, onde uma pequena popula??o de bact?rias suscet?veis sobrevive ap?s o tratamento com elevadas concentra??es de antimicrobianos - podendo essa fra??o de c?lulas sobreviventes ser afetada pelas condi??es ambientais presentes no meio. Desta forma, o objetivo deste trabalho foi avaliar a influ?ncia da disponibilidade de oxig?nio e da presen?a de galactose, citrato de s?dio e sangue de carneiro na forma??o destas c?lulas frente ? exposi??o ao meropenem e ? tobramicina. Para isto, 10 isolados cl?nicos, cedidos pelo Laborat?rio de Patologia Cl?nica do Hospital S?o Lucas da PUCRS, tiveram sua suscetibilidade caracterizada quanto ? concentra??o inibit?ria m?nima (CIM) de tobramicina e de meropenem. A influ?ncia das diferentes condi??es ambientais foi analisada ao expor culturas de fase exponencial tardia ao meropenem e ? tobramicina por 48 h na presen?a de citrato de s?dio, galactose ou sangue de carneiro; ou em uma condi??o aerada ou est?tica. Para todas as observa??es, al?quotas foram removidas em tempos determinados, seguidas de dilui??es decimais seriadas e semeadura pela t?cnica da gota em ?gar nutriente, para estimar a fra??o de c?lulas sobreviventes. A condi??o aerada promoveu uma diminui??o na fra??o de c?lulas persisters - independente do antimicrobiano utilizado -, e tamb?m verificarmos que concentra??es superiores a 10X a CIM de tobramicina proporcionaram n?veis ainda menores destas c?lulas ap?s 48 h de tratamento. Quanto ? presen?a de diferentes fontes de nutrientes, foi observado que a presen?a de citrato de s?dio no tratamento de 48 h com meropenem proporcionou uma menor fra??o de c?lulas sobreviventes comparado a quando este mesmo antimicrobiano estava associado ? galactose; enquanto a exposi??o ? tobramicina na presen?a de galactose promoveu uma menor forma??o de c?lulas persisters nas primeiras 6 h de tratamento. O sangue de carneiro, entretanto, n?o afetou a fra??o de c?lulas sobreviventes ap?s 48 h de tratamento, independente do antimicrobiano utilizado. P?de-se ainda constatar uma not?vel heterogeneidade no comportamento de todos os 10 isolados utilizados no estudo, independente das condi??es as quais foram expostos. Desta forma, ? poss?vel que as condi??es avaliadas nos experimentos tenham influenciado a forma??o c?lulas persisters atrav?s da maior produ??o de esp?cies reativas de oxig?nio, e por disponibilizar mais alvos para a a??o dos antimicrobianos ao estimular o crescimento bacteriano.