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Towards in silico prediction of mutations related to antibiotic resistance / Vers la prédiction in silico des mutations liées à la résistance aux antibiotiquesElisée, Eddy 11 October 2019 (has links)
La résistance aux antibiotiques est une menace sérieuse pour la santé publique. En effet, si on ne change pas rapidement notre consommation excessive d'antibiotiques, la situation actuelle va se dégrader jusqu'à basculer dans une ère dite "post-antibiotique", dans laquelle plus aucun antibiotique ne sera efficace contre les infections microbiennes. Bien que ce phénomène de résistance apparaît naturellement, l'utilisation abusive d'antibiotiques accélère le processus. De plus, la présence de pathogènes multi-résistants neutralise l'effet des traitements existants et dans le cas de chirurgies courantes (césariennes, transplantations d'organe...), la situation peut rapidement s'aggraver voire devenir mortelle. C'est pourquoi des directives, émanant des autorités sanitaires, doivent être mises en place afin de contrôler l'utilisation des médicaments, et ce, à tous les niveaux de la société, des individus au secteur agricole en passant par les professionnels de santé et les industries pharmaceutiques. Le monde de la recherche scientifique, quant à elle, doit trouver des nouvelles stratégies pour enrayer la propagation de la résistance. Dans ce contexte, cette thèse a pour objectif le développement d'une méthode de prédiction, par calculs d'énergie libre, des mutations de β-lactamases favorables à l'hydrolyse des β-lactames. Ces travaux méthodologiques ont donc conduit au développement : (1) de nouveaux paramètres pour les enzymes à zinc, implémentés dans le champ de force OPLS-AA et validés par des simulations de dynamique moléculaire sur un panel de métalloenzymes représentatives, (2) d'un protocole de paramétrisation de ligands covalents pour étudier le comportement de certains β-lactames dans CMY-136, une nouvelle β-lactamase caractérisée au laboratoire, et (3) d'un protocole de calcul d'énergie libre évalué au moyen de compétitions internationales de prédiction. Ce dernier a ensuite été utilisé pour tenter d'expliquer pourquoi la carbamylation de la sérine catalytique n'a pas lieu dans certaines oxacillinases. Au travers de ces travaux, nous avons pu améliorer significativement notre approche computationnelle et désormais tout est en place pour une exploration exhaustive des mutations possibles dans les β-lactamases. / Antibiotic resistance is a global concern threatening worldwide health. Indeed, if we don't change our overconsumption of antibiotics, the current situation could worsen until a "post-antibiotic" era in which existing treatment would be ineffective against microbial infections. Despite the natural occurrence of antibiotic resistance, the misuse of antibiotics is speeding up the process. Furthermore, presence of multi-resistant pathogens negates the effect of modern treatments and usual surgeries (caesarean sections, organ transplantations...) might be riskier in the future, or even lethal. That's why, common guidelines have to be edicted by health authorities in order to control antibiotic use at every level of society, from individuals to healthcare industry including health professionals and agriculture sector. As for scientific research, new strategies have to be considered in order to limit the spread of antibiotic resistance. In that context, the presented thesis aimed at developing a protocol to predict, by free energy calculations, β-lactamase mutations which could promote the hydolysis of β-lactams antibiotics. In order to achieve that, we developed several methodological approaches including: (1) new parameters for zinc enzymes implemented in OPLS-AA force field and thereafter validated using molecular dynamics simulations of representative zinc-containing metalloenzymes, (2) a protocol to parameterize covalent ligands in order to analyze the dynamical behavior of some β-lactams in CMY-136, a novel β-lactamase recently characterized in our laboratory, and (3) a pmx-based free energy protocol. The latter was also assessed through several international blinded prediction challenges, and finally used to find out why carbamylation of the catalytic serine is not observed in certain OXA enzymes. Throughout this work, we made significant improvements in our protocol, and now everything is in place for an exhaustive prediction of possible mutations in β-lactamases.
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Detection of aeromonas species in relation to the occurrence of estrogens and testosterone in various water resources in Limpopo Province, South Africa and Lusaka, ZambiaManavhela, Murendeni 18 May 2019 (has links)
MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals
(EDCs) in water poses a serious concern due to their effects on humans, animals and
environment. In recent years, EDCs have been increasingly reported in rivers that
receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic
hormones are among those that are recognized for their potential to mimic or interfere
with normal hormonal functions of humans and animals. The present study aimed at
assessing the occurrence of these hormones in relation to the molecular diversity of
Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to
assess anti-bacterial activity of two selected traditional medicinal plants.
Methods: Wastewater, water and fish samples were collected from various sources
(rivers, wastewater treatment plants, taps, and dams) for the detection of hormones
and isolation of Aeromonas species. The analysis of hormones from various organs
of the fish and from water samples was conducted, after extraction using enzymelinked
immunosorbent assays (ELISA). Different types of hormones including Estriol,
Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations
determined. Aeromonas spp were isolated rom the samples using microbiological
methods and Conventional PCR was used for genotyping as well as for detection of
the beta-lactamase genes. Kirby-bauer method was used to determine the
susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was
used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia
milneana) extracts against Aeromonas species.
Results: A total of 144 samples were collected from 23 different locations in two
countries: South Africa and Zambia. These included wastewater and treated
wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected
in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65
ng/ml. The hormones were also detected from drinking water, with testosterone
detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment
plants were not able to remove the EE2 from the wastewater as the concentration of
this hormone in the final effluent was almost always higher than that in the influent.
These homones were also detected in drinking water at high concentrations of up to
53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of
Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent
species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were
completely resistant to cefuroxime accounting for 100% resistance. Aeromonas
isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila),
cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was
the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked
the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A.
veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic
resistance was also observed with the Aeromonas isolates being resistant to up to 11
antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the
presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were
the most predominant species in the presence of total estriol, A. veronii had a
prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila
and Aeromonas caviae had the lower prevalence in the presence of hormones with
the percentages of 26.1% and 27.8% respectively. The methanol extracts of both
Zornia milneana and Annona species showed good activity against the Aeromonas
spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective.
Conclusion: This study has shown high occurrence of steroid hormones in all types
of environmental samples tested. These included tap water, river water, wastewater
and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and
important health problem in the Southern African Sub-Region. The incapacity of the
wastewater treatment plants to remove EE2 is an important problem that needs to be
tackled immediately. The prevalence of Aeromonas species is very high in our
environmental water as well as in drinking water, with the highest prevalence observed
in fish and wastewater. It was also revealed that there is relationship between steroid
hormones and Aeromonas species, with the hormones supporting the growth of
Aeromonas species. The presence of beta-lactamase genes which causes
Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia
milneana and Annona spp were the most effective against Aeromonas spp and could
serve as primary sources for the isolation of lead compounds. / NRF
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