L’anthropisation médicamenteuse des eaux usées favorise l’émergence et la diffusion dans l’environnement de microorganismes résistants aux antibiotiques. Les effluents hospitaliers pourraient être doublement impliqués en véhiculant antibiotiques et bactéries multirésistantes. L’objectif de ce travail est de caractériser les effluents hospitaliers d’un Centre Hospitalo-Universitaire en évaluant simultanément les concentrations d’antibiotiques (fluoroquinolones et imipénème) et la diversité des bactéries résistantes à ces antibiotiques au sein de biofilms constitués in situ. Les concentrations en antibiotiques mesurées par chromatographie en phase liquide - spectrométrie de masse après collecte via un échantillonnage passif pendant 15 jours sont égales à 2,08±0,88μg/L (ciprofloxacine), 101,06±18.47 μg/L (ofloxacine), 6,43±0.56 μg/L (norfloxacine) et indétectable pour l’imipénème. Comparées aux données de consommation à l’hôpital pendant cette même période, les concentrations estimées sont 5,84±1,78μg/L (ciprofloxacine), 11.22±1.09μg/L (ofloxacine), 7.68±3,7μg/L (norfloxacine) et 3,61±0,24ug/L (imipénème). La mesure du risque potentiel écotoxicologique s’est avérée positive pour la ciprofloxacine et la norfloxacine (hazard quotient >1). En parallèle, des bactéries résistantes aux fluoroquinolones (n=115) ou aux carbapénèmes (n=38) ont été isolées de biofilms formés dans les effluents hospitaliers. 60 % des isolats, constitués majoritairement de bacilles à Gram négatif, notamment Aeromonas spp et Klebsiella spp, sont résistants à plusieurs familles d’antibiotiques dont certains sont exclusivement utilisés à l’hôpital. La majorité des souches hébergent des éléments génétiques mobiles dont des plasmides conjugatifs porteurs de la résistance à l’imipénème ou aux fluoroquinolones. La présence combinée de bactéries résistantes aux antibiotiques hébergeant des éléments génétiques mobiles en lien avec ces résistances et de faibles concentrations en antibiotiques permet de qualifier l’interface hôpital-environnement comme un lieu propice au transfert des résistances. / The presence of pharmaceutical compounds in waste water favors the emergence and the spreading of antibiotic resistant microorganisms. The hospital effluents could be involved gathering antibiotics and multiresistant bacteria. The aim of this work is to characterize the hospital effluents of a teaching hospital measuring simultaneously the concentrations of antibiotics (fluoroquinolones and imipenem) and the diversity of the bacteria resistant to these antibiotics within hospital effluent biofilms.The antibiotics concentrations were measured by liquid-phase chromatography - mass spectrometry via a passive sampling during 15 days. The measured environmental concentrations were 2.08 ± 0.88μg/L (ciprofloxacin), 101.06 ± 18.47 μg/L (ofloxacine), 6.43 ± 0.56 μg/L (norfloxacine). Imipenem was not detected. Compared with the data of hospital consumption during the same period, the predicted estimated concentrations are 5.84±1.78µg/L(ciprofloxacin), 11.22 ± 1.09µg/L (ofloxacin), 7.68 ± 3.7µg/L, 7.68 ± 3.7μg/L (norfloxacin) and 3.61 ± 0.24ug/L (imipenem). The ecotoxicological risk was confirmed for the ciprofloxacin and the ofloxacin (hazard quotient > 1).In parallel, fluoroquinolones (n=115) and carbapenem (n=38) resistant bacteria were isolated from hospital effluent biofilm. Sixty % of isolates, mainly composed by Gram negative bacilli in particular Aeromona spp and Klebsiella spp, are resistant to several antibiotics among which some are exclusively used at the hospital. The majority of these strains have mobile genetic elements such as conjugative plasmids harboring imipenem or fluoroquinolones resistances.The presences of both antibiotics resistant bacteria harboring mobile genetic elements in connection with these resistances and low antibiotics concentrations make the hospital effluent a convenient place for the transfer of resistance between the hospital and the environment.

Background: Antibiotic overuse/misuse has been documented in several reports to increase the risk of Clostridioides difficile (C.diff) infection and antibiotic resistance. The older adult population is more prone to use antibiotic medications than any other age group due to decreased immune function, use of urinary catheters, ventilation during hospitalization and other factors. Antibiotic resistance and C.diff are major public health problems. However, studies examining the trends of antibiotic use and the association between the antibiotic use and negative health outcomes among older adults in the outpatient and emergency department settings are limited. Objectives: The main objectives of this dissertation were to: 1) calculate the national antibiotic trends among community-dwelling older adults in the United State; 2) evaluate the antibiotic trends and antibiotic appropriateness among older adult patients visiting the geriatrics clinic and adult internal ambulatory care clinic at VCU Health; and 3) examine the antibiotic trends and antibiotic appropriateness among older adult patients and middle-aged patients visiting the emergency department at VCU Health. Methods: For the first objective, data were obtained from Medical Expenditure Panel survey (MEPS) a nationally representative dataset (2011-2015). Descriptive analyses were conducted and multiple logistic regression was performed to assess the association between the antibiotic use and demographic and sociodemographic characteristics. In the second objective, data were obtained from VCU Health outpatient clinics (geriatrics, and Internal medicine ambulatory care clinic only). Descriptive statistics were calculated and multiple logistic regression was performed to assess the association between antibiotic appropriateness and type of clinics and other demographic characteristics. In the third objective, the emergency department electronic medical records at VCU were used. Trend analysis was performed across the dissertation studies using the Cochran–Armitage test. All variables were considered statistically significant at an α level of 0.05. All the statistical analyses were conducted using the Statistical Analysis Software Version 9.4 (SAS v.9.4), (SAS Institute Inc, Cary, NC). Results: There were 105,762,134 prescriptions dispensed to older adults in the outpatient setting in the US from 2011 to 2015. Antibiotic prescriptions were more common among women (18%) compared to men (12%). White participants received more antibiotics (27%) than African Americans (1.77%) and others (1.4%). Among the 3,515 patients who visited either Geriatrics or Internal Medicine ambulatory clinic at VCU Health from 2012-2017, 1,534 antibiotics were prescribed. Potentially inappropriate antibiotic prescriptions were similar between the two clinics (30% in Geriatrics clinic and 28% in Internal Medicine ambulatory clinic) with p-value of 0.08. In addition, 6,343 middle-aged or older adult patients were dispensed and prescribed an antibiotic in the ED at VCU Health from (2012 to 2017). Eighteen percent of the antibiotic prescriptions received by middle age group were considered potentially inappropriate, compared to 9% among the older adult patient (p < 0.0001). Conclusions: The rate of antibiotic use overall remains unchanged despite the national and international efforts to reduce antibiotic prescriptions and eventually to reduce antibiotic resistance. The changes in the patterns of use in some of the antibiotic categories appear to be driven more by the safety concerns rather than reducing overall use. Future research is needed to strengthen antibiotic stewardship programs for older adults in outpatient settings.

Urinary tract infections are diagnosed in female populations primarily in ambulatory care settings in the United States. Yet, published evidence documents that many of the antibiotics prescribed in these settings are unnecessary, erroneous, or, inappropriately prescribed. Improper management of uncomplicated urinary tract infections in nonpregnant women has resulted in higher morbidity rates due to antibiotic resistance. The purpose of this retrospective observational cohort study was to explore a current national database for associations between nonpregnant American female patients who were exposed to poverty and at risk for urinary tract infection antibiotic resistance in an ambulatory care setting. Krieger's ecosocial theory was utilized as the study's theoretical foundation to complement current public health social change priorities. Data from the National Ambulatory Medical Care Survey were analyzed to explore potential associations with urinary tract infections and antibiotic resistance. The sample consisted of ambulatory patients with urinary tract infection symptoms (n=45). The independent variables selected were antibiotics prescribed initially in 3 months or less after the onset of urinary tract infection symptoms, the continuation of antibiotics prescribed in 12 months or less after recurrence, and three classes of antibiotics prescribed for urinary tract infection symptoms known as broad-spectrum, narrow-spectrum, and combined broad- and narrow-spectrum antibiotics, while the dependent variable was urinary tract infection antibiotic resistance. Relationships between the variables were analyzed using binary logistic regression, however, there were no statistically significant outcomes. Promoting antibiotic stewardship programs in all health care settings in the U.S. can effect positive social changes that will prevent further antibiotic resistance.

In the United States, influenza causes approximately 36,000 deaths and over 200,000 hospitalizations each year with elderly most often affected. Clostridium difficile infection (CDI) is another major health care challenge and pressing public health issue associated with 14,000 deaths and over 335,000 hospitalizations annually. The use of antibiotics has been implicated in the development of CDI. This study's purpose was to test the relationship of seasonal influenza incidence and antiviral/antibiotic use in CDI development among hospitalized patients. Grounded in the epidemiologic wheel model of man-environment interactions, this retrospective observational study described and analyzed data from a proprietary, laboratory, and pharmacy-based system from a cohort of hospitals. The association between 147 patients with a diagnosis and/or positive test for influenza, the independent variables of delivery of antivirals/antibiotics (n = 130) during the patient's hospitalization, and the dependent variable of positive test or diagnosis of CDI (n = 17) was tested using multiple logistic regressions. The study results did not prove to be significant for the 3 research questions, suggesting no impact of antiviral use (R2 = .05, p = .336), antibiotic use (R2 = .05, p = .290), or antiviral and/or antibiotic use (R2 = .04, p = .382) on development of CDI within 60 days of discharge. However, findings indicated that recommended antiviral medication was inconsistently administered to influenza positive patients and that inappropriate prescribing patterns for antimicrobial agents coincided with seasonal influenza. Implications for positive social change include confirming the importance of antibiotic stewardship as an essential aspect of quality healthcare.

Methicillin-Resistant Staphylococcus aureus (MRSA) began as a nosocomial infection due to overuse of antibiotics. Several previous studies have reported an increase in this infection in adult patients who have not been hospitalized. It has also been reported that there is an increase in MRSA in children. Some of these children became infected even though they were not at high risk for the infection. After approval from the All Children's Hospital Institutional Review Board (IRB), a cross sectional study was conducted with pediatric admissions and pediatric emergency room visits to determine the characteristics of Methicillin-Sensitive Staphylococcus aureus and MRSA. During this study, a review of 672 medical charts was conducted. The study participants ranged in age from newborns to 18 years of age. In order to be enrolled in the study, the subjects' cultures were collected either as outpatients or within 72 hours of admission. The data that was collected from each chart included age, race/ethnicity, gender, type of infection, preexisting medical conditions, and risk factors for infection. The potential risk factors include antibiotic use, previous surgery or outpatient procedure, previous MRSA infection, immunotherapy, community worn device, and residence in a facility. Statistical analysis was conducted using Epi Info and SAS software packages. In regards to demographic characteristics, black children are 2.98 times more likely to have an MRSA infection than white children. Gender and age were not risk factors for the development of the infection. The risk factors that were significant in whites were home health care (OR= 6.12, CI= 5.16, 7.08), community worn device (OR= 2.28, CI= 1.67, 2.89), previous hospitalization (OR= 2.43, CI= 1.95, 2.91), previous MRSA infection (OR= 3.69, CI= 2.90, 4.48), and previous surgery (OR= 2.02, CI= 1.51, 2.53). In blacks, females were more likely to have MRSA (OR= 2.57, CI= 1.73, 3.41). This finding may be due to the small sample size of black children in the study. Of the analyzed risk factors, home health care (OR= 2.95, CI= 1.11, 4.79), community worn device (OR= 2.85, CI= 1.71, 4.01), previous hospitalization (OR= 1.98, CI= 1.13, 2.83), previous surgery (OR= 2.79, CI= 1.79, 3.79), and previous antibiotic (OR= 5.60, CI= 4.66, 6.54) use were all significant risk factors in blacks. Effect modification was tested between race and all risk factors. Race was an effect modifier only for the risk factor of previous antibiotic use (pvalue =.02). Adjustment of confounding was performed for each race due to the presence of effect modification. After the adjustment for confounding in whites, only home health care (OR=4.37 CI= 1.55, 12.32), previous MRSA infection (OR= 2.86 CI= 1.16, 7.05), and previous hospitalization (OR= 2.00 CI= 1.14, 3.50) remained statistically significant. In blacks, after adjustment of confounding, only previous antibiotic use (OR= 5.13 CI= 1.75, 15.08) remained significant. Adjustment for confounding was also preformed on the total risk factors model. A dose response relationship was present with increasing risk factors present.

Bacterial source tracking (BST) methods often involve the use of phenotypic or genotypic fingerprinting techniques to compare indicator bacteria such as Escherichia coli isolated from unknown sources against a library of fingerprints from indicator bacteria found in the feces of various known source animals. The predictive capability of a library is based in part on how well the library isolates reflect the true population diversity of indicator bacteria that can potentially impact a water body. The purpose of this study was to compare the behavior of E. coli population structures in the feces of humans, beef cattle and horses across different parameters. Ribotyping and antibiotic resistance analysis were used to "fingerprint", or subtype E. coli isolates. Significantly greater diversity was observed in the E. coli population of horses compared to the human or beef cattle sampled. Subtype sharing between individuals from all host categories was infrequent, therefore the majority of E. coli subtypes were sampled from a single individual. The dominant E. coli populations of nine individuals (three per host source category) were monitored over time, which demonstrated that E. coli subtypes within a host individual vary on a monthly time frame, and an increase in the frequency of subtype sharing was noted between individuals within the same source group over time. The E. coli population of a single human that had just finished antibiotic treatment was studied on a daily basis for one month. The loss of an E. coli subtype with high antibiotic resistance was observed over time, however there was a single dominant E. coli subtype that was present at every sampling event during the entire month. Geographic distinctiveness of E. coli populations was investigated by sampling four herds located in different geographical regions. We observed that E. coli populations are not geographically distinct, but are somewhat individual-specific, as most E. coli isolates had a subtype that was found in a single individual. This study defines factors that should be considered when constructing a successful BST library, and suggests that E. coli may not be the appropriate indicator organism for BST.

N-Methylthio beta-lactams represent a promising new family of antibacterial agents whose in vitro activity is confined largely to Staphylococcus species, including multidrug-resistant forms of S. aureus. Originally developed in the 1980's for use as synthetic intermediates, N-methylthio beta-lactams have recently been shown in these laboratories to possess intriguing biological properties which are addressed in Chapters I-IV. In terms of the antibacterial activities, the structural features and species specificities exhibited by these compounds are unlike those of any existing family of beta-lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active Beta-lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes. The most active members of this Beta-lactam class appear to be those bearing an aryl (Ar) substituent at C4 of the ring. The synthesis and structure-activity relationship of these analogues is discussed in Chapter III. Moreover, microscopy and 3H pulse-labeling studies, which are described in Chapter IV, demonstrate that N-methylthio beta-lactams appear to be inhibitors of protein biosynthesis.

>Magister Scientiae - MSc / The continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.

Biofilms are a crucial adaptation for bacterial survival against stresses from external environments. Biofilms are adherent colonies of sessile bacteria embedded within a self-produced matrix. Bacterial control over formation, maintenance, and response to external stresses are strictly regulated. However, complexities of intracellular signaling for biofilm regulation are still not fully understood. In this thesis, I report on two distinct regulatory systems important for biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. The first regulatory system I report on is the two-component system TctD-TctE. This system is involved in regulating the uptake of tricarboxylic acids such as citric acid and is involved in biofilm-specific susceptibility to aminoglycoside antibiotics. Here I describe work I performed characterizing the involvement of TctD-TctE in biofilm development when citric acid is present as a carbon source in nutrient media. In further characterizing a previously observed aminoglycoside susceptibility, I found that a strain with a deletion of TctD-TctE (ΔtctED) has a heightened accumulation of tobramycin in its biofilms when grown in the presence of citric acid. In ΔtctED, I determined that there was an inhibition of overall cell growth when citric acid was present in nutrient media. Additionally, in the presence of citric acid, ΔtctED displayed high levels of biofilm formation. This contrasted with normal biofilm development observed in the PA14 wild type strain where biofilm mass was reduced in the presence citric acid. The second project of this thesis reports on a novel regulatory small RNA, the Small RNA Regulator of Biofilms (SrbA). SrbA was found to be unique to P. aeruginosa and displayed no homology with any other sequenced bacterial species. I found that loss of SrbA resulted in a significant reduction in biofilm mass. Subsequently, loss of SrbA also leads to attenuation of P. aeruginosa pathogenicity in Caenorhabditis elegans nematodes. Bacterial biofilms possess specific regulatory programs that are still just being appreciated for their complexity. This thesis work adds to our understanding of biofilm regulation by studying roles of the two-component system TctD-TctE and the small RNA SrbA in P. aeruginosa.

Le développement de nouveaux antibiotiques est un enjeu majeur de santé publique. Etant donné, le fort potentiel des aminoglycosides en tant qu’antibiotique, ces composés ont attisé l’intérêt de plusieurs groupes de recherche. Cependant, leur usage est encore très limité, malgré leur ancienneté, du fait de leur toxicité et du développement toujours croissant des mécanismes de résistances aux aminoglycosides. Afin de mieux appréhender les problèmes inhérents à leur utilisation, il est crucial de mieux comprendre leur action sur les différentes cibles cellulaires, et d’étudier leur interaction avec leur cible moléculaire (ARN et protéine). En plus de leur pouvoir antibiotique, les aminoglycosides sont également des ligands universels pour des ARN, capables d’interagir spécifiquement avec notamment les ARN du VIH-1 suivants : DIS, TAR, RRE. L’élaboration d’aminoglycosides modifiés présente un énorme avantage car le domaine d’application, et en conséquence les retombées, sont grandes. Néanmoins, la complexité structurale de ces molécules est un frein majeur, la fonctionnalisation chimiosélective est indispensable mais malheureusement peu décrite dans la littérature. Dans le cadre de ce travail, nous avons développé deux types d’approches pour cibler le DIS et/ou le site A du ribosome bactérien. La première originale, mais risquée se base sur le concept de click in situ. La seconde approche est traditionnelle et est basée sur la fonctionnalisation sélective de certaines positions clés des aminoglycosides. / The development of new antibiotics is a major public health issue. Given the high potential of aminoglycosides as antibiotics, these compounds have aroused great interest in many research groups. However, despite their maturity, their use is still limited because of their toxicity and the increasing development of resistance mechanisms to aminoglycosides. To better understand the problems inherent to their use, it is crucial to understand their action a cellular level, and to study the interactions with their molecular targets (RNA and protein). In addition to their antibiotic power, aminoglycosides are also universal ligands for several RNAs, capable of specific interactions with RNAs of HIV-1: DIS, TAR and RRE. The elaboration of modified aminoglycosides presents a huge advantage because the domain of application, and therefore the benefits, are important. Nevertheless, the structural complexity of these molecules is a major constraint, chemoselective functionalization is essential but unfortunately poorly described in the literature.In this work, we developed two approaches to target the DIS and/or the A site of the bacterialribosome. The first one, unique but challenging is based on the concept of in situ click chemistry. The second approach is conventional and is based on the selective functionalization of some keypositions of aminoglycosides.