Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.

The Gram positive bacterium Staphylococcus aureus is a major cause of human disease in industrialized countries. This multifaceted pathogen is adapted to thrive in a variety of host niches, including the intracellular compartment. S. aureus rapidly develops antibiotic resistance, and infections due to resistant clones pose a global threat, calling for novel therapeutic approaches. The ability to exploit host nutrients and efficiently metabolize these resources for growth is paramount for bacterial pathogenesis. Understanding the nutritional and metabolic determinants that underpin bacterial virulence may lead to the identification of novel antimicrobial targets. This thesis investigates carbon nutrition and metabolism of community-acquired methicillin resistant S. aureus (CA-MRSA) USA300, a widely spread, hyper virulent multi-resistant strain. The dependence of S. aureus on carbohydrates for growth was considered first. In vitro studies in supplemented chemically defined media showed that sugar phosphates, such as hexose phosphates and glycerol phosphates, promote staphylococcal growth more efficiently than glucose. Deletion mutations were introduced to the two putative sugar phosphate transporter genes present in the S. aureus genome, uhpT (hexose phosphate permease) and glpT (glycerol phosphate permease). Phenotypic analysis of USA300 mutants and heterologous expression of the transporters in a previously described Listeria monocytogenes Δhpt mutant, totally unable to use sugar phosphates, confirmed that S. aureus UhpT and GlpT have different substrate specificities. Whilst both can transport glycerol monophosphate (excluding glycerol-2-phosphate) and the organophosphate antibiotic fosfomycin, hexose monophosphates are only imported via UhpT. Since sugar phosphates are only present in significant amounts inside living tissues, particularly the intracellular compartment, the role of S. aureus UhpT and GlpT in pathogenesis was investigated by constructing a double deletion mutant. The ΔuhpTΔglpT USA300 mutant was used to infect several relevant mammalian cell lines. In the conditions tested, it was found that UhpT and GlpT played no role in the intracellular replication of S. aureus. By contrast, Listeria exploits sugar phosphates from the host cell cytosol via the homologous hexose phosphate transporter, Hpt, to maximise replication and enhance virulence. The distinct requirement of sugar phosphates for intracellular proliferation may reflect intrinsic differences in carbon nutrient dependence between the two organisms. It was confirmed that S. aureus can efficiently use other readily available carbon sources for growth, such as amino acids. In contrast, Listeria is strictly dependent upon sugar-derived carbon for growth, due to an incomplete tricarboxylic acid cycle. Whilst the double ∆uhpT∆glpT mutation had no effect in S. aureus, expression of staphylococcal uhpT or glpT restored wild-type intracellular growth in the L. monocytogenes ∆hpt mutant. Taken together, the results illustrate that sugar phosphate permeases have a contextual role in bacterial virulence, where the background in which the genes are expressed determine their contribution as a virulence factor. The intracellular dynamics of S. aureus was also explored using immunofluorescence microscopy. It was observed that, during epithelial cell infection, USA300 remains enclosed in a membrane-bound vacuole. This localisation may form a barrier to cytosolic sugar phosphates and potentially explain the absence of effect of the sugar phosphate permease deletions in intracellular proliferation. Preliminary characterisation of the S. aureus containing vacuole (SACV) was performed and it was found to be positive for the Rab7 late-endosomal GTPase and for trans-Golgi markers. This suggests that SACVs converge at the Golgi apparatus. Interestingly, a USA300 mutant lacking the global regulatory system agr was unable to proliferate intracellularly and did not acquire Rab7 or Golgi markers. Since the Δagr mutation did not cause any impairment in carbon source dependent growth, these preliminary data suggest that modification of the SACV by Agr-regulated effectors may play a key role in modulating cellular processes that control staphylococcal intracellular survival and/or replication. Evidence presented in this thesis provides a platform for further exploration of S. aureus host cell nutrient dependence and the mechanisms that drive replication.

Infection is one of the factors that may contribute to non-healing of chronic wounds; the presence of antibiotic resistant bacteria serves to exacerbate the problem due to limited treatment options. Bacteria utilise several mechanisms to survive exposure to antibiotics, including synthesis of deactivating enzymes, target modification or substitution, changes to membrane permeability, upregulation of efflux pumps and the formation of a biofilm. Quorum sensing is a density-dependent mechanism of bacterial cell to cell communication that can be instrumental in co-ordinating biofilm initiation. Hyperbaric oxygen therapy (HBOT) is an option offered to some patients with chronic wounds, including diabetic foot ulcers. Evidence suggests that HBOT can reduce the incidence of major amputation in these patients. As well as the direct toxicity of increased tissue oxygenation on anaerobic bacteria HBOT may also increase levels of reactive oxygen and nitrogen species in the wound environment. This study aimed to investigate the effects of hyperoxia and oxidative damage on three specific mechanisms of antibiotic resistance: the activity of penicillinase, an antibiotic deactivating enzyme synthesised by bacteria; the activity of quorum sensing signalling molecules (AHLs); and biofilms and their associated bacteria. It also analysed the population dynamics of, primarily, bacteria in diabetic foot ulcers during HBOT, by the use of molecular analysis tools such as PCRDGGE. The presence of fungal species was investigated in wounds prior to HBOT and in two wounds at two points during HBOT. This study found that hydrogen peroxide, hypochlorous acid and peroxynitrite reduced the activity of penicillinase in vitro. Hypochlorous acid reduced the activity of a range of AHLs in vitro but not in vivo. Oxygen concentration did not have any impact on biofilm mass, nor did it significantly affect the ability of an oxidant-generating enzyme to reduce live bacterial cells within a biofilm. The population dynamics of bacterial species identified in all the wounds were complex and did not undergo identifiable changes during HBOT. Fungal species were identified in all wounds prior to HBOT, though different profiles were observed in the two wounds investigated during HBOT. These results suggest that oxidants could play a role in the attenuation of antibiotic resistance in chronic wound bacteria. It is unclear whether HBOT alters the population dynamics of non-healing wound microflora

Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics, however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here we report that mutations in the Arabidopsis locus Multiple Antibiotic Resistance (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Resistance is highly specific for aminoglycosides and does not extend to antibiotics of other classes, including the aminocyclitol, spectinomycin. Yeast expressing MAR1 are hypersensitive to the aminoglycoside, G418, but not to chloramphenicol or cycloheximide. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:YFP fusion protein localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside, gentamicin, while mar1-1 mutant chloroplasts accumulate less than wild type. MAR1 overexpression lines are slightly chlorotic, and this chlorosis is rescued by application of exogenous iron. MAR1 expression is also downregulated by low iron. Taken together, these data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis, and allows opportunistic entry of multiple antibiotics into the chloroplast. mar1 mutants represent an interesting example of plant antibiotic resistance that is based on the restriction of antibiotic entry into a subcellular compartment. Knowledge about this process – and other processes of antibiotic entry – could enable the production of crop plants that are incapable of antibiotic accumulation, aid in development of phytoremediation strategies for decontamination of water and soils polluted with antibiotics, and further the development of new plant-based molecular markers. The work described here also contributes to our understanding of how plants interact with the antibiotics they encounter, both in the laboratory (where aminoglycosides such as kanamycin are used heavily to select for transgenics) and in the natural environment. / text

<p>The study evaluated the Mutant Prevention Concentration (MPC) of Pradofloxacin on three Escherichia coli (E.coli) strains, 2 wildtypes and one first-step gyrA resistant mutant. We also measured the value of AUC (Under the Concentration)/MPC that prevents growth of resistant mutants. It is of importance to reach a concentration above MPC that prevent E.coli from developing resistance against the antibiotic.</p><p>We used an in vitro kinetic model where we added bacteria? and antibiotic. The culture flask was attached to a pump with an adjustable pump-speed. This made it possible to dilute the antibiotics in a satisfying elimination half-life (t1/2= 7 hours) pace. Samples were removed with a syringe at different times in the study. The samples where then cultured on agar- plates to enable counting of the viable colonies after incubation.</p><p>The optimal concentration to completely eradicate both E.coli wildtypes Nu14 and MG1655 with Pradofloxacin was Cmax ≥8 times MPC and AUC/MPC then became73. Additional experiments needs to be done on the resistant mutant LM378 before we can determine the optimal concentration. But results so far indicate that the concentration of Cmax would be about 8-12 timesMPC to completely eradicate that mutant.</p>

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.

Pseudomonas aeruginosa est une bactérie à Gram négatif à caractère ubiquitaire que l’on retrouve dans une grande diversité d’environnements. C’est un pathogène opportuniste qui est responsable chez l’homme d’infections chroniques ou aigües qui peuvent être mortelles pour des patients immuno-déficients. L’établissement d’une infection chronique est généralement associé à la capacité de la bactérie à former un biofilm, qui se définit comme une population bactérienne attachée sur une surface et englobée par une matrice extracellulaire formée entre autre depolysaccharides. La formation du biofilm est un processus bien défini dans le temps et dans l’espace et qui implique la mise en jeu de nombreuses structures de surfaces dont l’assemblage est strictement contrôlé. Une des voies de régulation contrôlant cet assemblage est le système à 2composants Roc1 (« regulation of cup genes »). Les gènes cup codent des composants de la voie « chaperone-usher » qui permet le transport de sous-unités pilines et leur assemblage à la surface bactérienne sous forme de pili. Ces pili Cup sont important dans l’établissement du biofilm. Le système Roc1 est aussi impliqué dans la mise en place du système de sécrétion de type III, qui est communément associé aux infections aigues. De fait le système Roc1 peut être considéré comme un «interrupteur» décidant du mode d’infection associé à P. aeruginosa. Le système Roc1 est constitué d’un senseur non-orthodoxe (RocS1) et de deux régulateurs de réponse, RocA1 et RocR, dont le domaine effecteur est un domaine de liaison à l’ADN ou un domaine EAL à activité phosphodiesterase, respectivement. Il existe également d’autres gènes paralogues de Roc1 qui sont le système Roc2 avec RocS2 et RocA2 très similaire à RocS1 et RocA1, ainsi que RocS3 similaire à RocS1. Le travail réalisé au cours de ma thèse a montré qu’il existe une régulation croisée entre Roc1 etRoc2. Cependant, chacune des branches du réseau de régulation contrôle l’expression d’une série de gènes bien spécifiques. Nous avons montré que la signalisation via RocS2 et RocS1 lorsqu’elle converge sur RocA1 contrôle l’expression des gènes cupC et ce contrôle est totalement indépendantde RocA2. Par contre lorsque la signalisation RocS1 et RocS2 converge vers RocA2 alors ce sont les gènes mexAB-oprM, qui codent une pompe d’efflux impliquée dans la résistance aux antibiotiques, dont l’expression est alors réprimée.En conclusion, nous avons mis en évidence un modèle unique de régulation croisée qui résulte dans un effet antagoniste entre formation du biofilm et résistance aux antibiotiques. Si cela peut paraître inattendu, quelques données cliniques sont en faveur d’une telle balance. En effet, l’analyse de souches de P. aeruginosa, isolées à partir de patients atteints de mucoviscidose, révèle que dans ces isolats la pompe MexAB-OprM est inactive. La raison de cette adaptation n’est pas élucidée, mais l’absence de pompe fonctionnelle pourrait procurer un avantage, une meilleure aptitude à la souche à persister dans cet environnement. Il est également reconnu que dans les poumons de ces patients le mode préféré de développement pour P. aeruginosa est le biofilm. Mises bout à bout ces observations suggèrent donc que le système Roc pourrait être un système de régulation important pour percevoir l’environnement du poumon chez le patient mucoviscidosique et déclencher une réponse adaptée. / The opportunistic pathogen Pseudomonas aeruginosa is responsible for diverse chronic and acute infections in human. Chronic infections are associated with the capacity of P. aeruginosa to form biofilms. One of the pathways controlling biofilm formation is the Roc1 two-component system, involved in the regulation of cup genes allow the assembly of thin fimbriae at the surface of the bacterium. Cup fimbiae are important in biofilm formation. There exist paralogues of the Roc1 system - the Roc2 and Roc3 system. The work in this thesis has shown that cross-regulation occurs between Roc1 and Roc2. However, each branch in this network appears to control the expression of a specific subset of genes whose role and functions are striking in the context of an infection process. We characterized here a unique model of cross-regulation which results in the antagonistic regulation of biofilm formation and antibiotic resistance

L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni. / Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain.