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.

This project aimed to find a method for producing potential probe targets for identification of ESBL (Extended Spectrum Beta Lactamase) genes in bacteria. ESBLs are a type of enzymes responsible for antibiotic resistance in many bacteria. The result we developed was a semi-automated pipeline that utilises several Perl scripts to download gene sequences, identify sequence subgroups based on sequence similarity, find common target sequences among them and screen the target sequences against a background database. These target sequences should work with padlock probes and therefore had specific requirements regarding length and highest number of allowed mismatches. This report includes descriptions of the scripts and ideas for future improvements, as well as an ethical analysis about aspects relevant to research on antibiotic resistance.

Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.

This study characterized Salmonella enterica serovar Heidelberg from human, abattoir poultry and retail poultry isolates to examine the molecular relationships of cefoxitin resistance between these groups. A total of 147 S. Heidelberg (70 cefoxitin-resistant and 77 cefoxitin-susceptible) isolates were studied. All cefoxitin-resistant isolates were also resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur and ceftriaxone, and all contained the CMY-2 gene. Pulsed-field gel electrophoresis typing illustrated that 93.9% isolates clustered together with ≥ 90% similarity. Core genome analysis using whole genome sequencing identified 12 clusters of isolates with zero to four single nucleotide variations. These clusters consisted of cefoxitin-resistant and susceptible human, abattoir poultry and retail poultry isolates. Analysis of CMY-2 plasmids from cefoxitin-resistant isolates revealed all belonged to incompatibility group I1. Analysis of plasmid sequences using WGS revealed high identity (95-99%) to a previously described plasmid (pCVM29188_101) found in Salmonella Kentucky. When compared to pCVM29188_101, all sequenced cefoxitin-resistant isolates were found to carry one of ten possible variant plasmids. The discovery of several clusters of isolates from different sources with zero to four SNVs suggests that transmission between human, abattoir poultry and retail poultry sources may be occurring. The classification of newly sequenced plasmids into one of ten sequence variant types suggests transmission of a common CMY-2 plasmid amongst S. Heidelberg with variable genetic backgrounds. / October 2016

The transferrin iron acquisition system of Neisseria consists of two dissimilar proteins, transferrin binding protein A and B (TbpA and TbpB). TbpA and TbpB both specifically and independently bind human transferrin (Tf). TbpA is a TonB-dependent transporter, expression of which is necessary for Tf iron acquisition. In contrast, the lipoprotein TbpB is not necessary for iron internalization; however it makes this process more efficient. The role of TbpB in the transferrin iron acquisition system has not been completely elucidated. It has been suggested that TbpB is entirely surface exposed and tethered to the outer membrane by its lipid moiety. We inserted the hemagluttinin antigen (HA) epitope into TbpB in an effort to examine surface accessible and functional domains of the lipoprotein. We determined that TbpB was entirely surface exposed from just beyond the mature N-terminus. It was previously reported that the N- and C-terminus of TbpB independently bind Tf. HA epitope analysis defined both the N-terminal and C-terminal binding domains. TbpB was previously reported to play an important role in the release of Tf from the receptor. We established that TbpB exhibited a biphasic dissociation pattern; a C-terminal rapid release followed by a slower N-terminal release. These results suggested that the C-terminus plays a role in ligand turnover of the wild-type receptor. Little is known about the transport of TbpB to the outer membrane. In an attempt to identify the signals/mechanisms required for TbpB localization, the signal sequence of the protein was altered. In the absence of lipid modification, TbpB remained associated with the cell, localized to the periplasm. We also noted that internal cysteine residues were not critical for TbpB localization. Our results suggested that TbpB was transported by a lipoprotein-specific mechanism. Additionally, we demonstrated the major outer membrane secretin, PilQ, was not necessary for proper localization of TbpB. The mechanism responsible for this process remains elusive. This body of work represents the first comprehensive study of TbpB topology and function, utilizing the lipoprotein expressed in its native membrane. These results may translate to other, similar lipoprotein receptors of the pathogenic Neisseria, helping to shed light on these poorly understood proteins.

FbpA is the periplasmic binding protein of the transferrin and lactoferrin-iron transport systems. FbpA is conserved among neisserial species and is required for Neisseria gonorrhoeae to sustain growth on transferrin and lactoferrin. The identification of other putative TonB-dependent outer membrane transporters suggests that gonococci may employ other uncharacterized iron uptake systems that do not require FbpA. Previous work in our lab demonstrated that gonococcal strain FA19 utilizes iron from a number of xenosiderophores of the catecholate and hydroxamate classes. In this study we created conditional FbpA mutants to evaluate whether FbpA plays a role in the ability of gonococci to utilize iron from xenosiderophores. Strain FA19 was able to acquire iron from the xenosiderophores enterobactin and salmochelin in an FbpA-dependent and TonB-independent manner. We were also able to detect an extracellular population of FbpA indicating that FbpA may play a novel role in the internalization of iron in the absence of a dedicated transporter.

The gene encoding resistance to β-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated Staphylococcal Chromosome Cassette mec or SCCmec. In addition to the resistance gene, mecA, SCCmec also contains site specific recombinase genes, ccrAB, that are capable of catalyzing the chromosomal excision and integration of SCCmec. The increasing prevalence of methicillin-resistant Staphylococcus aureus infections may be due, in part, to the transfer of SCCmec into successful methicillin-sensitive S. aureus lineages. In this work we attempt to better characterize the factors associated with SCCmec transfer, beginning with CcrAB-mediated SCCmec excision in a collection of MRSA containing type IV SCCmec. CcrAB-mediated excision of type IV SCCmec was not demonstrated for all strains tested, as a subset of S. aureus strains with type IV SCCmed did not excise their element. These strains are all highly related and represent a lineage of successful community associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the presence of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately outside of SCCmec on the chromosome. Staphylococcus aureus strain USA300 contains SCCmec type IV and a genomic island containing an arginine deiminase pathway, known as ACME, inserted adjacent to one another in the SCCmec chromosomal attachment site. Each element was site-specifically excised from the chromosome by CcrAB, resulting in two independent, extra-chromosomal, circularized elements. Therefore the presence of ACME did not disrupt SCCmec excision.Next, we describe three MRSA strains that become resistant to vancomyein during passage on increasing concentrations of the drug. In each case, mecA was lost during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec while the other two strains (3130VR and VP32) deleted portions of their SCCmec elements in a manner that appears to involve IS431. Conversion to vancomycin resistance caused a decrease in growth rate that was partially compensated for by deletion of mecA. In mixed culture competition experiements, vancomycin resistant strains that lacked mecA readily out-competed their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.Finally, we examined the genetic structure surrounding the SCCmec attachment site in a diverse collection of methicillin-sensitive S. aureus isolates. This region of the chromosome varies greatly from strain to strain and appaers to be prone to recomination. Open reading frames found in this region were homologous to enterotoxins, restriction-modification enzymes, and transposases. Several open reading frames that have not been previously reported in staphylococci were also present in this region. 28 out of the 42 isolates examined did not contain the attachment site sequence found in S. aureus isolates known to be capable of CcrAB-mediated SCCmec integration or excision. This may indicate that these strains do not contain a functional attachment site and therefore may not have the potential to acquire SCCmec by CcrAB-mediated recombination.

Antibiotic resistance threatens to throw mankind back into an era when infectious disease was the predominant cause of death. In an effort to mitigate this danger, we targeted ErmC’ and KsgA. Both methylate N6-adenosine of ribosomal RNA, though each serve different roles in their bacterial host. ErmC’ dimethylates A2058 on 23S rRNA, conferring resistance to MLSB antibiotics (macrolides, lincosamides, streptogramin B). KsgA aids in ribosome assembly, binding inactive 30S until dimethylating A1518/A1519 of 16S rRNA. Like most methyltransferases, ErmC’ and KsgA use cofactor S-adenosylmethionine (SAM) as their methyl source, which binds adjacent to their specific adenosine substrate. ErmC’ inhibitors could restore MLSB antibiotics against infections with this resistance mechanism. KsgA inhibitors could form novel antibiotics that stall 30S assembly. Previous studies reported a potent ErmC’ inhibitor, N6-cyclopentyl adenosine (1), binding to the substrate pocket with cyclopentyl bridging into the SAM pocket. We expanded this study by synthesizing 1 and 22 other N6-substituted analogs to explore more favorable interactions within the SAM pocket. When these compounds (1mM) were screened against ErmC’ and KsgA, some showed greater inhibition than 1. Two of these inhibitors that were crystallized with ErmC’, N6-8-octylamine adenosine (2.60Å) and N6-phenethyl adenosine (2.40Å), unexpectedly docked into the SAM pocket with their 5’-C pointing towards the substrate pocket. New compounds were made to exploit this orientation by adding substituents off the 5’-C to probe the substrate pocket. Through a five step synthesis, the 5’-OH of 1 was substituted with an amine linked to benzyl (30), phenethyl (31), propylphenyl (32) or butylphenyl (33). When 30-33 were screened using 20μM SAM, ErmC’ showed greater inhibition (relative to 1), while KsgA showed virtually none. However, when ErmC’ was tested using 0.5μM SAM, inhibition from 30-33 was nearly unchanged, whereas 1 became significantly more potent than 30-33, suggesting 30-33 were not binding to the SAM pocket. Preliminary data showed that raising 23S concentrations lowered inhibition from 32-33, while inhibition from 1, 30 and 31 was nearly unchanged, suggesting that at least 32-33 bound within the substrate pocket.

The purpose of this study was to determine the changes in prescribing habits of active members of American Association of Endodontics (AAE) with regards to antibiotics in comparison to the findings reported by Yingling et al. in 1999. The invitations to take the online survey were sent via email to 2593 active members. A response rate of 37.75% was obtained. It was determined to be adequate for analysis and for comparison to the results obtained by Yingling et al. Comparisons between the percentages shown in this survey and the previous survey were tested using a z-test. An ANOVA model was used to determine the relationships between predictive factors and the number of prescriptions written. The change in distribution of respondents was notable with an increase in younger clinicians (25% in 1999 to 36% at present). They were more likely to be in private practice and much less in part-time academic and private practice setting. The number of patients being seen per week and the number of prescriptions written per week also decreased in comparison (p<0.001). For all the considered factors, it was also noted that board certified endodontists were prescribing less antibiotics per week. A positive correlation was noted for number of years in practice (p=0.0006), type of practice (p<0.001) and number of prescriptions written per week. Changes in choice of antibiotics were also noted. There was a decrease in use of Penicillin (61.48% to 43%), an increase in the use of Amoxicillin (27.5% to 37.6%), and an increase in use clindamycin (45.3% to 64%) for patients with no medical allergies. As for patients with medical allergies, there was a steep incline in the use of clindamycin (56.03% to 90.3%) as first choice to an increase in azithromycin (7.4% to 38%) as a second choice. An improved trend was noted with a significant decrease in use of antibiotics in managing most of the endodontic scenarios given. Antibiotic use in cases of irreversible pulpitis significantly dropped from 16.76% to 12% (p<0.05); in necrotic pulps with acute apical periodontitis with no swelling, a significant decline from 53.9% to 28.3% (p<0.001); significant decreases were also noted for necrotic pulp with chronic apical periodontitis with no/mild symptoms, 18.8% to 16.1% (p=0.029), and necrotic pulp with acute apical periodontitis with swelling and mod/severe symptoms, 99.2% to 92.4% (p<0.001). An exception was noted for necrotic pulp with chronic apical periodontitis with a sinus tract where there was a significant increase in antibiotic use from 11.9% to 29.1% (p<0.001). Many clinicians (19%) were still giving antibiotics due to soliciting of patients and referring general dentists in fear of losing referrals. A disturbing find is that 50% of the respondents were using antibiotics to manage post treatment flare-ups and pain, while 13% were using antibiotics for inter-appointment pain. As for prophylactic antibiotics, most clinicians were aware of the new AHA/ADA guidelines and were abiding by them. Most of the clinicians responding to survey were choosing the appropriate antibiotics and regimen (i.e. dosage, loading dose, and duration). Although there is an improvement in trends, it has to be noted that there is still an indiscriminate and overuse of antibiotics at large. There needs to be greater improvement in the use of antibiotics in endodontics, and a group effort as a specialty is needed in halting this alarming problem of antibiotic resistance globally.