Avaliação da formação de células persistentes em Acinetobacter baumannii

A persistência é um fenótipo de tolerância a fármacos antimicrobianos com bases moleculares pouco entendidas. Tais células tolerantes (chamadas de persisters) correspondem a pequenas parcelas da população bacteriana e são capazes de sobreviver a concentrações elevadas do fármaco, mesmo sem possuir mecanismos de resistência específicos. O impacto clínico das células persisters se dá pela capacidade destas células de retomar seu crescimento e restabelecer a infecção após os níveis do fármaco diminuírem no sítio da infecção, podendo ser responsáveis pela reincidência e pelo aspecto crônico de certas doenças infecciosas. Este fenótipo de sobrevivência já foi observado em inúmeras espécies, porém relatos envolvendo o A. baumannii são escassos. Portanto, este trabalho teve por objetivo avaliar o fenótipo de persistência a drogas antimicrobianas em isolados nosocomiais de A. baumannii, bem como verificar a possível participação do gene sodB envolvido no controle do estresse oxidativo e do gene pmrC, determinante de resistência às polimixinas, em células persisters formadas a partir da exposição à polimixina B.Para tal, isolados clínicos de A. baumannii foram expostos a concentrações elevadas de tobramicina e polimixina B por 6 h e o número de células sobreviventes foi averiguado em intervalos de 1,5 h. Além disso, as expressões gênicas a nível de transcrição de dois isolados foram avaliadas após a exposição à polimixina B por 5 h. Uma grande heterogeneidade na capacidade de formação de células persisters foi observada dentre os isolados, não havendo correlação entre a fração de persisters após o tratamento com tobramicina e polimixina B. Estes dados podem indicar variações genéticas ou epigenéticas determinantes para o desenvolvimento desta característica, as quais não são relacionadas entre drogas de diferentes classes. Além disso, os resultados preliminares dos ensaios de expressão gênica podem sugerir que o mecanismo de resistência às polimixinas não participa diretamente na persistência à polimixina B, e que não há a participação de sodB no desenvolvimento e manutenção deste fenótipo nas condições testadas. O estudo desta característica e de seus determinantes moleculares são de suma importância para o desenvolvimento de novas opções terapêuticas.Persistence is an antimicrobial tolerance phenotype with an unclear molecular background. Such tolerant cells (called persisters) correspond to small portions of the bacterial population and are capable of surviving excessive concentrations of the drug, even though they do not possess any specific resistance mechanism. The clinical impact of persisters lies on their capacity to resume multiplication and reestablish the infection after the concentration of the drug diminishes in the infection site, possibly being responsible for the re-incidence and for the chronic aspect of certain infectious diseases. This survival phenotype has been observed in several species; however reports involving A. baumannii are scarce. Therefore, this work aimed to evaluate the persistence phenotype to antimicrobial drugs in nosocomial isolates of A. baumannii, as well as verifying the possible contribution of the sodB gene, which is involved in the control of oxidative stress, and the pmrC gene, a determinant of polymyxins resistance, in persister cells formed upon polymyxin B exposure. In order to do so, clinical isolates of A. baumannii were exposed to high concentration of tobramycin and polymyxin B for 6 h and the number of surviving cells were estimated every 1. 5 h.In addition, the gene expressions at transcription level of two isolates were evaluated after the exposure to polymyxin B for 5 h. A high heterogeneity in the ability to form persister cells was observed among the isolates, presenting no correlation between the fractions of persisters after tobramycin and polymyxin B treatments. These data may indicate genetic or epigenetic variations that are determinant to the development of this characteristic, and that are not related among drugs of different classes. Moreover, the preliminary results of the gene expression assays may suggest that the mechanism involved in the polymyxin B resistance phenotype does not directly participate in persistence to polymyxin B, nor indicate the participation of sodB in this phenotype. In conclusion, the molecular mechanisms for persistence are still to be determined, and this characterization is highly important to assist in the development of new therapeutic options

Heterogeneous persister cells formation in Acinetobacter baumannii.

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy

Correlation between polymyxin B and tobramycin persister cells in the sample population.

<p>Correlation between polymyxin B and tobramycin persister cells in the sample population.</p

Frequency distribution of the persister fractions.

<p>Persister producer strains were grouped in 6 classes (defined by the square root of sample size) according to their persister fraction in the population after polymyxin B (a) or tobramycin (b) exposure. Most isolates presented persister fractions that corresponded to less than 1.2% and 0.5% of the total population for polymyxin B and tobramycin, respectively.</p