Le traitement des infections dues aux bactéries multirésistantes aux antibiotiques est un défi médical majeur du 21ème siècle. Ce défi a incité la recherche de cibles ayant des fonctions essentielles pour le développement de nouveaux antibiotiques. Les enzymes de la voie FASII, responsables de la synthèse des acides gras (AG), sont considérées comme essentielles et de nombreux antibiotiques, appelés anti-FASII, ont été développés pour lutter contre des pathogènes du phylum des Firmicutes. Cependant, notre laboratoire a montré que plusieurs pathogènes contournent l’inhibition des anti-FASII par l’utilisation des AGs exogènes abondants chez l’hôte (sang, organes, aliments). Ce contournement compromet l’utilisation des anti-FASII en traitement. Le statut du pathogène majeur, Staphylococcus aureus, est néanmoins resté en débat. Il synthétise un AG non disponible chez l’hôte, et donc, d’après la littérature, ne pourrait pas être compensé. La question de cette thèse est de comprendre les mécanismes utilisés par S. aureus lui permettant de contourner les anti-FASII. Deux mécanismes sont mis en évidence: I- Des mutations à haute fréquence du gène fabD surviennent et permettent à la bactérie d’utiliser des AGs exogènes. Ce type de mutation favorise la disponibilité de la protéine ACP permettant l’utilisation des AGs exogènes. II- Une stratégie sans mutation décelable survient en présence de fluides hôtes tels que le sérum. Elle comprend une première étape de "dormance" d’environ 8 à 10 heures pendant laquelle des AGs sont incorporés. Durant cette adaptation les bactéries semblent bloquées dans la division cellulaire, et subissent des changements morphologiques. Cette étape est suivie par une reprise de croissance « normale » où S. aureus utilise librement des AGs exogènes et reste insensible aux anti-FASII. Dans nos conditions, une étude microscopique « time-lapse », a permis de visualiser qu’environ 3% de la population bactérienne adaptée aux anti-FASII émerge. Nos résultats pointent vers un mécanisme d’adaptation dans lequel le sérum diminuerait le stress bactérien et augmenterait ainsi la disponibilité de l’ACP et des AGs exogènes, facilitant leur utilisation pour la synthèse des phospholipides. Les AGs exogènes peuvent donc remplacer totalement les endogènes. Ce résultat va à l’encontre de l’hypothèse couramment acceptée qu’un AG endogène de S. aureus est conservé et essentiel. Nous avons poursuivi cette étude par des analyses protéomiques et par le criblage d’une banque de 2000 mutants de S. aureus en cherchant des loci impliqués dans l’adaptation aux anti-FASII. Des fonctions de la réponse au stress, la division cellulaire et le métabolisme des lipides semblent être impliqués dans cette adaptation. Pour conclure, cette étude a permis de clarifier les étapes impliquées dans la réponse adaptative de S. aureus aux anti-FASII. Même si nos résultats prouvent que S. aureus contourne les anti-FASII, une approche combinatoire pourrait être envisagée où l’anti-FASII serait couplé avec un deuxième inhibiteur qui bloquerait sa capacité à sortir de la dormance. / Treatment of infections caused by multidrug-resistant bacteria is a major medical challenge of the 21st century, which has stimulated the search for essential bacterial functions as potential antimicrobial drug targets. The fatty acid synthesis (FASII) pathway enzymes are considered essential, and numerous antibiotics called (anti-FASII), have been developed to eliminate pathogens of the Firmicutes phylum. However, our laboratory has shown that several pathogens bypass FASII inhibitors by incorporating exogenous fatty acids (FAs), which are abundant in the host (in blood, organs and foods). FASII bypass thus compromises the use of FASII-based antibiotics. The status of the major pathogen, Staphylococcus aureus, has remained in debate. S. aureus synthesizes an FA not produced by the host and according to the literature, is required and would not be available in the host. However, work in my lab showed that indeed bypasses FASII. The goal of my research project is to understand the mechanisms used by S. aureus to bypass FASII antibiotics. Two mechanisms are highlighted: I- High frequency mutations of the fabD gene allow S. aureus to use exogenous FAs; our study indicates that higher availability of ACP in these mutants facilitates FA utilization. II- A strategy without detectable mutation occurs in the presence of host fluids such as serum. It comprises a first "dormancy" step of about 8 to 10 hours, followed by outgrowth; FAs are incorporated throughout these steps. The latency phase appears to be due to a division block, during which cells undergo morphological changes. During "normal" growth recovery, S. aureus freely uses exogenous FAs and remains insensitive to anti-FASII. Using « time-lapse » microscopic study, we showed in our test conditions that about 3% of the bacterial population adapted to FASII antibiotics. Ours results point to an adaptation mechanism in which serum decreases bacterial stress, leading to increased availability of ACP and exogenous FAs. These substrates can then be used for phospholipid synthesis. These results resolve the debate by showing that S. aureus can replace endogenous FAs with exogenous FAs when FASII is blocked. Contrary to current dogma, FASII is not essential in S. aureus. To identify the loci involved in adaptation to anti-FASII, we performed proteomic analyses and also screened a S. aureus mutant library. Functions of stress response, cell division, and lipid metabolism appear to be involved in this adaptation. To conclude, this study clarified the steps leading to S. aureus adaptation to FASII antibiotics. Although our results show that S. aureus bypasses anti-FASII, a combinatorial approach could be considered in which FASII antibiotics could be coupled to a second inhibitor that would prevent exit from the dormancy.

Bakgrund:  Multiresistenta bakterier (MRB) förekommer i hela världen och klassas  som ett globalt folkhälsoproblem. Kunskapen om multiresistenta bakterier är låg både hos  patienter och sjukvårdspersonal. För att minska risken för spridning behövs mer kunskap och om vikten av följsamhet till basala hygienrutiner. Överdriven användning av antibiotika är en  bidragande orsak till den ökade antibiotikaresistensen i världen.  Sjukvårdspersonal upplever svårigheter med att vårda patienter med MRB och bristande kunskap hos kan ses som bidragande faktor till detta. Att undvika smittspridning är en stor del i hur patienterna ska hanteras inom vården.  Syfte: Att sammanställa patienters upplevelser av att leva med multiresistenta bakterier såsom Meticillinresistenta Staphylococcus aureus (MRSA), Vancomycinresistenta enterokocker (VRE) och Extended Spectrum Beta-Lactamase (ESBL).  Metod: Studien har genomförts som en litteraturöversikt baserat på 15 vetenskapliga artiklar. Artiklarna är av kvalitativ, kvantitativ och mixad ansats. Databaser som använts är CINAHL, PubMed och Scopus.  Resultat: Resultatet presenteras i fyra olika kategorier: Upplevelser av MRB i vardagen, Upplevelser av att få bristande information, Upplevelser av bemötande från sjukvårdspersonal och Upplevelser av stigmatisering. Konklusion: Stigmatisering, oro och ångest genomsyrade resultatet av denna litteraturöversi kt. Sjukvårdspersonalens bemötande spelar en viktig roll i hur patienten hanterar att bli diagnostiserad med multiresistenta bakterier och sen att leva med multiresistenta bakterier.  Okunnig sjukvårdspersonal gav upphov till ökat lidande för patienterna. / Background: Multi-resistant bacteria (MRB) occur worldwide and are classified as a global public health problem. Knowledge of multi-resistant bacteria is low in both patients and healthcare professionals. To reduce the risk of spreading, more knowledge and the importance of adherence to basic hygiene routines are needed. Excessive use of antibiotics is a contributing cause of the increased antibiotic resistance in the world. Healthcare professionals experience difficulties in caring for patients with MRB and lack of knowledge can be seen as a contributing factor. Avoiding the spread of infection is a major part of how patients should be managed in healthcare situations.  Aim: To compile patients' experiences of living with multi-resistant bacteria such as Multiresistant staphylococcus aureus, Vancomycin-resistant enteroccal and Extended-spectrum betalactamase  Method: The study has been conducted as a literature review based on 15 scientific articles.  The articles are of qualitative, quantitative, and mixed method. Databases used in the search  are CINAHL, PubMed and Scopus.  Results: The results are presented in four different categories: Experiences of multi-resistant bacteria in everyday life, Experiences of lack of information, Experiences of treatment from healthcare professionals and Experiences of stigmatization. Conclusion: Stigmatization, anxiety and concern permeated the results of this literature review. The care of healthcare personnel plays an important role in how the patient handles being diagnosed with and then living with multidrug resistant bacteria. Unskilled healthcare  professionals caused increased suffering for patients.

Plasmids are small, circular, extrachromosomal double-stranded genetic elements present in bacteria. Plasmids can replicate independently of the bacterial chromosome and play an important role as a transmitter of antibiotic resistance genes between bacteria. Antibiotic resistance genes have been shown to be selected for even in the presence of subinhibitory levels of antibiotics, but the effect of antibiotics on conjugation is not as well understood. To study this, we designed a novel conjugation tracking system utilizing a Cre-expressing plasmid and a chromosomal floxed blue fluorescent protein (BFP) gene. We found that our model worked opposite as intended as cells expressed BFP before conjugation and lost BFP expression upon recombination. An issue with the system was isolated to the direction of the single loxP site remaining after recombination. Both loxP sites were inverted but this did not restore the intended expression of BFP after recombination. Subsequently the system was modified to increase the space between the promoter region and the single loxP site remaining after recombination. This extension produced the desired result as BFP expression now increased upon recombination. Still, further work needs to be done to construct a Cre-expressing plasmid, tune expression of BFP, and show expression of yellow fluorescent protein (YFP) in our model before the system can be applied to clinical isolates.

Antibiotics have been one of the greatest scientific discoveries for humanity. Their great success has been hindered by the increasing of antibiotic resistance events. Conventional wisdom considers antibiotic resistance as a strictly clinical issue. In reality, more and more studies have proven that non-clinical environments, especially aquatic environments, are critical factors for the spread of antibiotic resistance. Antibiotic resistance genes (ARGs) are usually located on mobile genetic elements, which can disseminate among taxonomically unconnected species. Therefore, environmental bacteria can serve as hotspots for the development and dissemination of antibiotic resistance. The aim of this thesis is to determine how human originated pollution affects the antibiotic resistance abundance in the surrounding water environments. The case studies were three example environments, wastewater treatment plants (WWTPs), receiving river bodies and combined sewer overflows (CSOs). Molecular microbiology methods have been applied to analyse the resistance levels of bacterial communities from WWTP, CSOs and wastewater affected environments at different European geographical locations. Additionally, a novel approach consisting in an amplicon sequencing is described, in order to be able to investigate and asses the composition of a significant amount of integron gene cassettes. Findings consistently indicate that effluents originated from WWTPs and CSOs have a significant impact on the levels of ARGs of the receiving water bodies. Moreover, this thesis suggests that gene blaOXA-58, could be utilized as a proxy to investigate the spread of ARGs. Its occurrence has been reported to show, consistently throughout the chapters, lower concentrations upstream, but at higher concentrations in the WWTP effluent, CSOs and downstream of the effluent. In conclusion, although antibiotic resistance genes and integrons are part of the environmental resistome, water environments that are affected by anthropogenic wastewater display high levels of the above-mentioned genetic elements. These findings clearly suggest the need to limit pollution levels, as well as the need to establish a more responsible policy in antibiotic prescriptions. This must take place in order to be able to perform efficient risk assessments and to establish acceptable levels of antibiotic and ARGs pollution.:List of Figures VII List of Tables IX List of Abbreviations X 1. Introduction 11 1.1 Antibiotic resistance: an increasing problem 11 1.2 Intrinsic antibiotic resistance and horizontal gene transfer 13 1.2.1 Transformation 13 1.2.2 Transduction 13 1.2.3 Conjugation 14 1.3 Gene transfer elements 14 1.4 Antibiotic resistance and anthropogenic impact on surrounding environments 16 1.5 Study goals 17 2. Genetic variations of the resistome of wastewater treatment plants driven by the seasonality of antibiotic prescription 18 2.1 Introduction 18 2.2 Materials and methods 19 2.3 Results and discussion 20 3. Assessment of inter-laboratory variations of quantitative analyses of antibiotic resistance genes 27 3.1 Introduction 27 3.2 Materials and methods 27 3.3 Results and discussion 30 3.4 Conclusions 36 4. Antibiotic resistance genes in treated wastewater and in the receiving water bodies: A pan-European survey of urban settings 37 4.1 Introduction 37 4.2 Materials and methods 38 4.3 Results 44 4.4 Discussion 49 4.5 Conclusions 53 5. Analysis of integron gene cassettes composition in treated wastewater 55 5.1 Introduction 55 5.2 Materials and methods 55 VI 5.3 Results 56 5.4 Discussion 57 6. Role of combined sewer overflows in the dissemination of antibiotic resistance in surrounding environments 59 6.1 Introduction 59 6.2 Materials and methods 60 6.3 Results 61 6.4 Discussion 66 7. Closing Conclusions 68 References 70 List of publications 88 Note on the commencement of the doctoral procedure 89 Acknowledgments 90

Almost two hundred years ago, Dr John Snow identified the faecal contaminated water as a source of bacterial infections during a severe cholera outbreak. Several years later, we have developed many weapons on our arsenal to reduce the bacterial infections, from simple ones such as public hygiene measures (e.g. frequent showers & hand washing, clean water), to specialised ones such as the use of antibiotics. The antibiotics inhibit the bacterial growth, thus their use has effectively helped to treat many bacterial infections, revolutionizing medicine. Successful recovery from surgical operations would be seldom and would last exponentially without their use. Yet, antimicrobial resistance (AMR) has increased globally threatening to render antibiotics useless. However, the “golden era” of novel antibiotics development, when many novel antibiotics were discovered in a few years, belongs to the past. The bacteria developed resistance mechanisms to every single one of the antibiotics and rendered them useless. This could be reflected to an increase in the death rates, but more importantly to the increased health-care costs, which might compromise the treatment for other diseases. The Covid-19 pandemic provided such a clear paradigm on the straining of health care systems during massive parallel hospitalisation of patients. While, the misuse of antibiotics for human and veterinary was the main contributor of the increased AMR levels, other anthropogenic activities greatly contributed to AMR spread as well. Specifically, the wastewater treatment plants are considered as hotspots for AMR and agricultural practices, such as manure amendment, have been show to clearly promote AMR. Thus, the scientific community across clinical settings, environmental and agricultural sectors intensively researches on AMR, in an attempt to fully understand the AMR phenomenon. Nevertheless, the AMR is not the only problem that currently occurs in our society. The climate change, the urbanisation and the ever increasing human population has caused an increasing freshwater scarcity. The demand for treated wastewater (TWW) irrigation has increased due to this freshwater scarcity, and is expected to increase more. Since the TWW contains a high load of antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), the irrigation with TWW has raised concerns regarding AMR spread in the environment. Many studies have attempted to investigate the impact of TWW on AMR spread in crops and soil; however, the impact on deeper lying environments remains not yet elucidated. This should raise concerns, since groundwater remains the most valuable drinking water source globally. Here in this thesis, I attempted to gain further understanding on whether TWW irrigation promotes the AMR spread in the soil and the so-far neglected deeper-lying subsurface environments. My outmost desire is that the present work will contribute to a framework of minimising the potential risks during TWW irrigation, rendering TWW irrigation as a safe and sustainable alternative for freshwater resources depletion.

L’émergence et la dissémination des bactéries résistantes aux antibiotiques sont préoccupantes. L’infection causée par les bactéries multi-résistantes (BMR) aggrave le pronostic des malades infectés et augmente les dépenses liées à leur prise en charge. Parmi les BMR, les bactéries à Gram négatif (BGN), plus particulièrement les entérobactéries productrices de béta-lactamase à spectre étendu (E-BLSE) sont les plus fréquemment isolées. La résistance aux antibiotiques pourrait avoir un impact sur la morbidité et la mortalité dans les pays à revenu faible ou intermédiaire (PRFI) en raison du potentiel d’émergence et de diffusion de bactéries résistantes aux antibiotiques, et du fardeau des infections bactériennes dans ces pays. Cependant, les données sur la résistance bactérienne sont rares et très majoritairement hospitalières dans les PRFI. De plus, dans ces pays, les infections bactériennes néonatales sévères (septicémies, pneumonies et méningites) représentent encore les principales causes de décès chez les nouveau-nés. Les entérobactéries sont majoritairement responsables de ces infections néonatales. Ainsi, investiguer la transmission d'E-BLSE chez le nouveau-né permettrait de proposer des stratégies de prévention. Ce travail de recherche s’est appuyé sur le programme BIRDY (Bacterial Infections and antibiotic Resistant Diseases among Young children in low-income countries). Le premier objectif était d’estimer la prévalence de la colonisation par des E-BLSE chez les femmes enceintes à Madagascar ainsi que les facteurs favorisant cette colonisation. Les résultats ont montré une prévalence globale de colonisation de 18.5% [IC à 95% 14.5-22.6]. Des facteurs reflétant un niveau socioéconomique plus élevé comme l’accès privatif à l’eau de boisson et avoir une maison individuelle sont associés à la colonisation. Le second objectif de ce travail était d'étudier l'incidence de la première colonisation par des E-BLSE chez les nouveau-nés en milieu communautaire et d'identifier les facteurs de risque d'acquisition. Les résultats révèlent une incidence globale d'acquisitions d'E-BLSE de 10.4 pour 1000 nouveau-nés-jours [IC à 95% : 8.0; 13.4]. Par ailleurs, nous avons mis en évidence que le faible poids à la naissance HR ajusté 2.7 [IC à 95% 1.2 ; 5.9], l'accouchement par césarienne HR ajusté 3.4 [IC à 95% 1.7 ; 7.1], la prise maternelle d'antibiotique à l'accouchement HR ajusté 2.2 [IC à 95% 1.1 ; 4.5] étaient des facteurs de risque d'acquisition d'E-BLSE. Le troisième objectif était de documenter les infections néonatales. Nous avons trouvé une incidence d'infections néonatales de 30.6 cas pour 1000 naissances vivantes [IC à 95%: 23.4 ; 40.1].Nos résultats montrent que les mesures de santé publique devraient axer sur l’amélioration de la prise en charge de la grossesse et sur le diagnostic précoce des infections néonatales. / The emergence and spread of antibiotic-resistant bacteria is a concern. Infection caused by multidrug-resistant bacteria (MDR) worsens the prognosis of infected patients and increases the costs associated with their management. Among the MDRs, Gram-negative bacteria (GNB), especially extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) are the most frequently isolated. Antibiotic resistance may have an impact on morbidity and mortality in low- and middle-income countries (LMICs) because of the potential for emergence and spread of antibiotic-resistant bacteria, and the burden of bacterial infections in these countries. However, data on bacterial resistance are scarce or came from the hospital, for the great majority, in LMICs. In these settings, severe neonatal bacterial infections (sepsis, pneumoniae and meningitis) still represent the leading causes of death in newborns. Enterobacteriaceaeare responsible for a great part of these neonatal infections. Thus, investigating the transmission of ESBL-PE in newborns would make it possible to propose prevention strategies. This work was based on the BIRDY program (Bacterial Infections and Antibiotic Resistant Diseases among Young Children in Low-Income Countries). The first objective was to estimate the prevalence of colonization by ESBL-PE in pregnant women in Madagascar as well as the risk factors of this colonization. The results showed an overall colonization prevalence of 18.5% [95% CI 14.5-22.6]. Factors reflecting a higher socioeconomic level such as private access to drinking water and having a house are associated with colonization. The second objective of this work was to study the incidence of ESBL-PE colonization in community-based infants and to identify acquisition risk factors. The results reveal an overall incidence of ESBL-PE acquisition of 10.4 per 1000 newborn-days [95% CI: 8.0; 13.4]. In addition, we found that low birth weight adjusted HR 2.7 [95% CI 1.2; 5.9], cesarean section delivery adjusted HR 3.4 [95% CI 1.7; 7.1], maternal intake of antibiotic at delivery adjusted HR 2.2 [95% CI 1.1; 4.5] were risk factors for the acquisition of ESBL-PE. The third objective was to document neonatal infections. We found an incidence of neonatal infections of 30.6 cases per 1000 live births [95% CI: 23.4; 40.1]. Our results suggest that public health measures should focus on the improvement of pregnancy follow-up and early diagnosis of neonatal infections.

Antibiotics are substances that inhibit the growth of microorganisms and are widely used in modern medicine. High consumption of antibiotics correlates with their elevated occurence in sewage systems, from where they are further released into the environment. The threat of their occurrence in the environment is in triggering the formation and spread of antibiotic resistance. This thesis focuses on the removal of selected antibiotics and partly on the antibiotic resistance from wastewater by advanced oxidation processes (AOPs). AOPs are based on the creation of highly reactive hydroxyl radicals, which are able to oxidize even highly persistent substances. The thesis focuses on the purification of effluents from municipal wastewater treatment plants (WWTP), which are identified as the main source of pharmaceutical pollution in the environment, by AOPs mainly based on ozonation. These processes were tested under laboratory conditions on model water as well as under real conditions as the tertiary stage of the treatment at Brno-Modřice WWTP under various operating states. As the results of the performed experiments show, it could be concluded that the studied AOPs are capable of effective degradation of studied antibiotics from the treated medium. Under real conditions, the antibiotics sulfamethoxazole, trimethoprim, azithromycin and clarithromycin were monitored. Output concentrations in each operational state were achieved to be below the limit of detection for all four antibiotics. The application of AOPs based on ozonation as the tertiary step of the treatment also had a positive effect on the reduction of microbial contamination and antibiotic resistance. Within the pilot plant experiments, a reduction of up to 4 orders of magnitude of E.coli, coliform microorganisms and a reduction of their resistant strains in the range of 1.4 - 4.0 logs were observed, compared to the effluent from the WWTP.

<p>  </p> <p>Infections of dermal wounds is a growing burden for the healthcare industry, with a 2017 market exceeding $17.5 USD. As the number of patients with severe infections continues to increase year after year, there is an alarming downward trend in efficacy for traditional antibiotic treatments. In large part, this is due to the increasing development of antibiotic resistance within common bacteria strains. As microbes evolve to protect themselves from previously effective drugs, there is a growing need for new antimicrobial therapies. While alternatives exist in the market, they are largely impaired by non-selective toxicity which can cause further damage to the cells in the wound bed, as is the case with silver and other strong antiseptics, or the need for high energy, specialized equipment, as with cold atmospheric surface treatments. Gaseous ozone is a promising alternative therapy for treating these wound infections. Because ozone is a strong natural oxidant, it exhibits significant antimicrobial properties, and has also been shown to help stimulate natural wound healing in many cases. Herein is presented the design of a portable system for the topical delivery of gaseous ozone as an antimicrobial treatment for infected dermal wounds. This includes the design and characterization of the portable system and a custom ozone application dressing, the characterization of the safety and efficacy of the system using <em>in vitro</em> and <em>in vivo</em> models, and a disposable system for wound infection monitoring. The system utilizes a portable corona discharge generator to produce gaseous ozone from the ambient environment. The ozone gas is delivered through a dressing engineered to have a hydrophobic interface at the wound bed and disperse the ozone gas across the patch surface for more uniform application. The antimicrobial strength and biocompatibility of the system was optimized at varying ozone output levels. Additionally, an adjunct therapy of topical antibiotics was shown to significantly increase the strength of the treatment without leading to greater cytotoxicity. This synergistic effect between ozone and antibiotics was shown to circumvent natural bacterial resistances to antibiotics, which will have a major impact on the wound care industry. This adjunct treatment was then validated on a porcine animal model for safety and pilot results for efficacy testing. Finally, the pH sensor which can be incorporated with use of the ozone therapy enables objective monitoring of wound condition and is able to signal when appropriate infection therapy should begin. As it stands, this portable ozone wound treatment system shows great promise as an alternative therapy to improve the quality of live for millions of patients.</p>

Antibiotic resistance is critical to global health. This thesis examines the relationship between antibiotic resistance and genomic variations in E. coli from wastewater. E. coli is of interest as it causes urinary tract and other infections. Wastewater is a good source because it is a melting pot for E. coli from diverse origins. The research delves into two key aspects: including or excluding antibiotic resistance data and the level of granularity in representing genomic variations. The former is important because there is more genomic data than antibiotic resistance data. Consequently, relying solely on genomic data, this thesis studies positive selection in E. coli to identify mutations and genes favored by evolution. This study demonstrates the preferential selection of known antibiotic resistance genes and mutations, particularly mutations located on functionally important locations of outer membrane porins, and may hence have a direct effect on structure and function. Encouraged by these results, the study was expanded to include antibiotic resistance data and to examine genomic variations at three resolution levels: single mutations, unitigs (genome words) that may contain multiple mutations, and whole coding genome using machine learning classifier models that capture dependencies among multiple mutations and other genomic variations. Representation of single mutations detects well-known resistance mutations as well as potentially novel mechanisms related to biofilm formation and translation. By exploring larger genomic units such as genome words, the analysis confirms the findings from single mutations and additionally uncovers joint mutations in both known and novel genes. Finally, machine learning models, including AI language models, were trained to predict antibiotic resistance based on the whole coding genome. This achieved an accuracy of over 90% in predicting antibiotic resistance when sufficient data were available. Overall, this thesis unveils new antibiotic resistance mechanisms, conducts one of the largest studies of positive selection in E. coli, and stands out as one of the pioneering studies that utilizes AI language models for antibiotic resistance prediction.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.