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Previous issue date: 2017-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Funda??o de Amparo ? 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.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/7566 |
| Date | 27 March 2017 |
| Creators | Gallo, Stephanie Wagner |
| Contributors | Oliveira, Silvia Dias de, Ferreira, Carlos Alexandre Sanchez |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, Brasil, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 500, 500, 500, 600, 600, 36528317262667714, -1634559385931244697, 2075167498588264571, -4379409248623720768 |
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