Morphological alterations of a xerotolerant Acinetobacter baumannii under desiccation stress investigated by atomic force microscopy

Abstract

Multidrug-resistant(MDR) Acinetobacter baumannii has recently emerged as a challenging problem and major cause of healthcare-associated infections (HAIs) because of its antimicrobial resistance and propensity to cause multi- facility outbreaks. The ability to develop antimicrobial resistance mechanisms and tolerance to desiccation are at the basis of the epidemiological success of A. baumannii. Therefore, desiccation tolerance is thought to contribute significantly to the persistence for an extended period of time and the spread of these bacteria in the healthcare environment. Despite the relevance of long-time desiccation tolerance of A. baumannii, the molecular and morphological basis of this phenomenon remains elusive. Alternatively, desiccation resistance can vary between different strains of A. baumannii specimen. In this regard, it has recently been proven that the adapted strains to the hospital environment like A. baumannii ACICU cells can survive desiccation far better than the laboratory strains, however, most of the studies have been restricted to the laboratory strains such as A. baumannii ATCC19606. Considering the basic function of the bacterial cell membrane, as the outer layer of cell structure, that maintains the bacterial shape and protects the bacterial components against environmental stresses, it plays a significant role to tolerate desiccation. Thus, elucidating the structure and alterations of the cell envelope is important to unravel not only the complexities of cell morphology and maintenance of integrity but also how desiccation leads to cell damage and death. Therefore, this Ph.D. thesis was aimed to study the consequences of desiccation- induced stresses on A. baumannii ACICU bacterial cell envelope under the simulated-hospital conditions. The following main questions were asked: • What is the overall morphology of A. baumannii ACICU bacterial cells? • Is there any relation between the cell morphology changes and cell desiccation survival? • Which growth factors can significantly influence the shape of A. baumannii ACICU cells? • What is the effect of different degrees of desiccation stresses and long-time storage on bacterial cell morphology? To address these questions, the advanced Atomic Force Microscopy (AFM) technique was employed, which has produced a wealth of new opportunities in nanobiotechnology and has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of biological systems. The evolution of the bacterial cell under different growth phases, distinct cultural growth conditions, and in various growth media as the effective parameters on cell morphology changes were assessed. Then different degrees of environmental stresses on the bacterial cell membranes were examined to discover the cell membrane changes and resistant mechanism of A. baumannii ACICU. Meanwhile, the desiccation survival of the cells before and after drying, during prolonged desiccation, and different storage conditions were examined. Furthermore, a new AFM approach (SFMSF) was implemented to explore the specified-bacterial cell over time. The presented results in this thesis revealed growth phase-independent morphology for A. baumannii ACICU cells, displaying a coccus morphology. Conversely, the cell morphology was found to significantly depend on growth conditions concerning oxygen availability during cultivation. Taken together both results of morphology alterations and cell viability assessment demonstrated that the cells with minimal footprint and lower surface-to-volume ratio are more resistant to desiccation. The cell membrane pores and intracellular material leakage were observed as a sign of the cell death pathway. Additionally, deformation of the outer cell membrane, and reduction in cell volume was found to be dependent on the stresses caused by different drying methods. Moreover, it was observed that although the maintenance of the cells in water can help to retain their viability for long-time, osmotic stress causes cell membrane weaknesses.