Enclosed are the abstract and outlook sections of my master’s thesis, developed over the recent months, focusing on the dissemination of antibiotic resistances through wastewater, facilitated by natural transformation processes in Acinetobacter baumannii. This study endeavors to contribute to the existing body of knowledge regarding microbial resistance mechanisms and their environmental implications. Should you have any inquiries or require further clarification on the research findings, please feel free to reach out.

Abstract
Introduction: Bacteria of the Acinetobacter genus have gained increasing attention in recent years. This is partly due to their potential to cause severe nosocomial infections. Additionally, their growing significance is attributed to the increasing discovery of isolates that are resistant to common antibiotics, making them difficult to treat. The success of A. baumannii, the most well-known representative of this genus, is largely due to its ability to acquire various genetic elements through horizontal gene transfer, of which natural transformation is a key process, involving the active uptake of free DNA by competent bacterial cells.

Material and methods: The investigated Acinetobacter isolates originate from environmental and livestock-related settings and were initially tested for their natural transformation competence. Two competent strains were then transformed with DNA from various sources, including DNA from clinical wastewater, and the resulting transformants were analyzed for changes in resistance patterns and DNA profiles.

Results: It was demonstrated that divalent cations, as well as the concentration of donor DNA, significantly influence the transformation frequency of A. baumannii isolates. Moreover, the results suggest that clinical A. baumannii isolates exhibit higher transformation rates compared to those from livestock and environmental sources. Importantly, chromosomal alteration appears to be a critical resistance mechanism during transformation, and plasmids may be reconstituted less frequently than previously assumed. This finding warrants further investigation, especially considering that wastewater is a significant driver of horizontal gene transfer, potentially contributing critically to the development of resistance in Acinetobacter spp.

Outlook
This study reinforces the importance and significance of natural transformation in the dissemination of antibiotic resistance through wastewater. It has been demonstrated that factors such as the presence of divalent cations and the concentration of donor DNA affect the frequency of transformation. Furthermore, it was illustrated that clinical Acinetobacter isolates, likely due to specific selective pressures in their environment, exhibit higher transformation rates compared to isolates from other sources. Additionally, upon completion of this work, there is a suspicion that chromosomal integration as a process of resistance development may occur more frequently than previously assumed. This alteration of the bacterial chromosome, for example, through the incorporation of transcription promoters like ISAba1 and ISAba9, promotes a long-term shift towards more resistant Acinetobacter spp.

These mechanisms require further investigation, as wastewater is considered a significant driver of horizontal gene transfer. The treatment of antibiotic-resistant pathogens is increasingly restricted. Targeted wastewater purification and more judicious use of antibiotics, both in human medicine and in animal husbandry, can impact the spread of antibiotic-resistant bacteria. Nonetheless, it is essential to decode the exact mechanisms and processes behind the dissemination of antibiotic resistances. Employing modern methods, such as the use of flow cytometric techniques in combination with fluorescent proteins, can refine data collection. Additional experiments must be conducted to understand the processes of natural transformation in Acinetobacter spp. The interplay between wastewater, DNA, and bacterial populations must be considered, necessitating a naturally designed experimental setup. Further investigations could potentially reduce the spread of antibiotic-resistant Acinetobacter baumannii.