WGS permet d'analyser et de déchiffrer l'étude de résistances de bactéries multirésistantes(MDR), en comprenant les différents mécanismes de résistance, les annuaires génétiques. Au cours de ma thèse de doctorat, j'ai réalisé: 1 revue de la littérature sur l'utilisation de nouveaux outils de diagnostic contemporains et les capacités dans la détection des foyers dans les maladies infectieuses causées par MDR. L'identification et l'analyse de résistances de bactéries multirésistantes Comme étant des Shewanellalgae, normalement de l'environnement marin, dans notre cas une souche clinique isolée du lavage bronchoalvéolaire d'un patient hospitalis avec pneumonie et Chryseobacteriumin dologenes, isolé d'une fibrose kystique du patient. Dans cette analyse, nous pouvons montrer que les bactéries environnementales telles que les S.algae peuvent être un réservoir de gènes de résistance aux antibiotiques. L'analyse exhaustive de ces bactéries a montré leur capacité à s'adapter à leurs écosystèmes, y compris l'acquisition de nouveaux éléments génétiques par transfert latéral de gènes. La détection des gènes impliqués dans la synthèse de peptides synthetasenon ribosomale et de polycétide synthétase peut avoir un rôle dans leur capacité à survivre dans des environnements hostiles tels que le tractus respiratoire des patients atteints de fibrose kystique ou leur présence chez des patients ayant subi plusieurs antibiotiques. Nous avons réalisé une analyse standardisée «insilico» afin de déterminer la résistance de ces bactéries et la présence de métabolites secondaires associés aux bactériocines et aux NRPS/PKS. L'application du NTS pour le séquençage du génome bactérien de nouvelles espèces bactériennes isolées dans le microbiome humain nous a permis de développer une plateforme capable d'analyser ces nouvelles espèces dans les 48heures. Ce travail permet de mieux comprendre la biodiversité des bactéries isolées dans le microbiome humain. / Theuse of WG Sallows to analyze and to decipherthe study of resistome of Multi Drug Resistant bacteria (MDR), understanding the different resistance mechanisms, genetic directories and their dissemination mechanisms at global level. During them y thesis I have achieved: 1. A literature review on the use of new contemporary diagnostic tools and capabilities in detecting out breaksin infectious diseases caused by MDR. 2: The identification and the analysis of resistome of multidrug resistant bacteria from clinical isolates suchasShewanellaalgae, normally marine environmental, in our case clinical strain isolated from the broncho alveolar lavage of a hospitalized patient with pneumonia and Chryseobacteriumin dologenes, isolated from a patient cysticfibrosis. In this analysis, we can show that environment albacteria suchas S.algae can be a reservoir of antibiotic resistance genes. The exhaustive analysis of these bacteria showed their ability to a dapttotheirecosystemsincludingtheacquisitionofnewgeneticelementsbylateralgenetransfer. The detection of genes in volved in the synthesis of nonribosomal peptide synthetase and polyketide synthases may have a role in their ability to survive in hostile environments suchas therespiratorytractofCFpatients or their presence inpatients having suffered multipleofantibiotic. 3:In this work,through theuse of the NTS onnew bacterial species isolated from human microbiome,we have a chieveda standardized analysis"insilico"to determine there sistome of these bacteria and the presence of secondary metabolites associated bacteriocins and the NRPS/PKS. The application of the NTS for sequenc in go bacterial genome of new bacterial species isolated in the human microbiome, allowe dus to develop a platform capable of analyzing the senew species within 48

No description available.

Bakgrund: Sjukdom kan innebära en påverkan på något mer än det fysiska, sjukdom kan även innebära något annat än ohälsa. Sjuksköterskans kunskap samt relationen mellan sjuksköterska och patient är en viktigdel i skapandet av trygghet i hälsoprocesserna. För somligakan sjukdom ha en positiv effekt på hälsan. Penicillinet kom med stora möjligheter att behandla infektioner, men snabbt började bakterier utveckla resistens. Idag är de vanligaste, anmälningspliktiga multiresistenta bakterierna i Sverige MRSA, ESBL och VRE. Det innebär förhållningsregler för patienter och gör att hälso-och sjukvården behöver genomföra smittskyddsåtgärder för att minska spridning. Sjuksköterskan kan känna osäkerhet och rädsla i omvårdnaden av dessa patienter vilket kan ha en inverkan på den jämlika vård som enligt lagen ska ges. Syfte: Syftet var att belysa patienters upplevelser av att leva med multiresistenta bakterier (MRSA, ESBL eller VRE). Metod: En litteraturöversikt där informationssökning har gjorts i PubMed, CINAHL och SwePub. Artiklar inkluderades om de svarade på syftet, var orginalartiklar, publicerade mellan 2010-2020, skrivna på engelska, hade kvalitativ eller mixad metod, var granskade av referenter och hade ett etiskt godkännande. Den insamlade datan analyserades av författarna och sammanställdes i tre teman och tillhörande underteman. Resultat: Tre teman vilka rubricerades som känslomässig påverkan, upplevelser i mötet med hälso-och sjukvård och förändringar i relationen till andra. Diskussion: Litteraturöversiktens resultat diskuteras gentemot Livsvärldsteorin, bakgrunden, andra referenser och författarnas egna tankar. / Background: Disease can have impact on something more than the physical, disease can be something else than illness. The nurse’s knowledge and the relationship between nurse and patient is an importantpart in creating safety in the health processes. For some, disease can have a positive effecton the health. The penicillin came with great possibilities in treating infections, but the bacteria quickly began to develop resistance. Today, the most common notifiable multidrug-resistant bacteria in Sweden are MRSA, ESBL and VRE. It induces instructions for patients and the health care has to conduct disease control to reduce spread. The nurse can feel uncertainty and fear in the caring of these patients, which can have an impact on the equal care which is to be given according to the law. Aim: The aim was to illustrate patients’ experiences of living with multidrug-resistant bacteria (MRSA, ESBL or VRE). Method: A literature review where the information search has been conducted in PubMed, CINAHL and SwePub. Articles were included if they answered to the aim, was original articles, published between 2010-2020, written in English, had qualitative or mixed method, were peer-reviewed and had an ethical approval. The collected data were analysed by the authors and compiled in to three themes and belonging sub-themes. Results: Three themes who were labelled emotional impact, experiences in the encounter with health care and change in the relationship with others. Discussion: The result of the literaturereview was discussed towards Life World Theory, the background, other references and the authors’ own thoughts.

Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

Thesis ( MPH.) -- University of Limpopo, 2022 / Background Multidrug-resistant tuberculosis (MDR-TB) has recently resulted to be in an emergence state globally and this of constitute a big challenge for TB control and the goals of the World Health Organization’s End TB Strategy. Aminoglycosides (AG) were often used as part of treatment of life-threatening illnesses such as MDR-TB for decades, however their adverse effects are widely described and hearing loss is one of the major side effects. The risk factors for hearing loss in patients treated with AG include the dose and duration of AG, infection with human immunodeficiency virus (HIV), older age and persons exposed to a high level of noise while the damage can be total and permanent. Severe hearing impairment has been reported to occur among patients treated for MDR-TB with injectable drugs, especially among the elderly and patients infected with human immunodeficiency virus, however, Bedaquiline containing regimens have demonstrated improved outcomes over injectable containing regimens in the long-term treatment of MDR-TB. Methods The objective of the current study was to investigate the burden of hearing loss amongst MDR-TB patients on bedaquiline at Zithulele Hospital in Eastern Cape Province. Therefore, the current study followed a quantitative retrospective approach using simple random sampling to select MDR-TB patients treated with bedaquiline and having a baseline audiogram be the initiation of treatment. The data was captured in a Microsoft Excel spreadsheet and then transferred to Statistical Package for Social Sciences (SPSS) Version 20 for data analysis in which categorical variables were presented as percentages and frequencies, while continuous variables was presented as mean, median and standard deviation lastly, comparison of categorical variables was done using a Chi-Squared test, whereas continuous variables were compared using a t-test. P-value of <0.05 will be considered significant. Results The mean age for the participants was 39.2 years with standard deviation of 11.8 and there was no statistical significance difference between the age groups (p value = 0.178). There no was a statistical significance difference between the employment status (p value = 0.794), previous use of injectables (p value = 0.360) and type of hearing of loss (p value = 0.536). Majority of the MDR-TB patients on bedaquiline did not have hearing loss at 67% while those who had gradual hearing loss and sudden hearing loss were 26.8% and 6.2% respectively. There was no statistical significance difference between males and females in both the right and left ears, however, the right ear results appeared to be slightly worse than the left ear results. It was found that both males and females had a high frequency hearing loss in the left ears of 26.8% and 22.2% respectively as compared to the right ears with of 25.9% and 1.6% respectively. The was a statistical significance difference between the age groups in both ears for hearing loss at p-value <0.001. The overall prevalence of hearing loss was found to be 32.9% and hearing loss at 20dB or more loss at any frequency was low at 11.9% while hearing loss at 10B or more loss at any frequency was the highest at 32.9% followed by loss response at 3 consecutive frequencies at 26.2%. Hearing loss was increasing with increasing age from 8.3% in age group and age was significantly associated with hearing loss as older patients were 2.2 times more likely to have a hearing loss at a degree of 20dB and 4.4 times more likely to have a hearing loss at a degree of 10dB. Previous use of injectables was also significantly associated with hearing loss as patients who used injectables previously were 11.5 times more likely to have a hearing loss at degree of 10dB, 5.6 and 11.3 times more likely to have a hearing loss at loss response at 3 consecutive frequencies and overall hearing loss respectively. Conclusion South Africa has a high burden of drug-resistant tuberculosis (DRTB) and until recently, ototoxic aminoglycosides were predominant in treatment regimens. Drug resistant TB treatment with bedaquilines caused clinically and statistically significant deterioration of hearing loss in patients, most prominently at high frequencies. Although public health interventions to prevent hearing loss have been deemed cost effective and have meaningful individual and economic implications, hearing loss and its prevention consistently receive inadequate attention as a global public health priority. Despite the serious impacts of hearing loss, little is known regarding prevalence of ototoxic hearing loss after treatment for DR-TB. Therefore, when the use of injectable ototoxic medications is unavoidable, audiological ototoxicity monitoring is essential to optimise hearing-related outcomes.

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is permanent hearing loss and this is thought to have a significant genetic component. To date, at least six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced hearing loss. It is imperative that we investigate the frequency of these mutations in our populations and determine whether certain sub-groups are at increased risk. The aim of the present study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in the South African population. A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen for six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C. A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who were receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA sequencing, respectively. A total of 97 family members from a South African family known to harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition, mitochondrial functioning in the presence of different streptomycin drug concentrations, in transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software. We successfully developed a robust and cost-effective method that detects the presence of all six mutations simultaneously. The method worked equally well on both blood (from adults) and buccal swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high frequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5% of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation was III detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since they were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls. Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eight MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3 and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved across five species from human to frog, and the four variants in the protein coding genes resulted in missense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive (on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversible hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin were (compared to wild type) observed but this was not statistically significant (p-value: 0.615- 0.999). The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. However, future studies with larger numbers of samples are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. Our data suggests that the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the high frequencies observed in controls (>1%). The identification of the first novel variants within protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss holds great possibilities with regards to the identification of a second gene involved in drug induced hearing loss. Future studies where the possible effect of these variants on the normal functioning of these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive members of the family were given genetic reports and counseled about their risk and that of their children for developing hearing loss due to aminoglycoside use. The development of a rapid and cost-effective genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is of critical important in a low-resource country like South Africa where, despite their adverse sideeffects, aminoglycosides will be continue to be used routinely and are accompanied with very limited or no audiological monitoring. Future studies and greater public awareness is therefore needed to address this serious problem. / AFRIKAANSE OPSOMMING: Suid Afrika beleef tans „n grootskaalse tuberculose epidemie (veral weerstandige vorme van tuberculose) (MDR-TB), met een van die hoogste voorkomssyfers in die wêreld. Aminoglikosied antibiotikums word baie algemeen gebruik in Suid Afrika vir die behandeling van MDR-TB. ‟n Bekende newe effek van die middels is permanente gehoor verlies en dit is van mening dat dit gekoppel is aan „n genetiese component. Daar is tans ses mutasies in die mitochondriale genoom wat vatbaarheid tot aminoglikosied-geinduseerde gehoor verlies veroorsaak. Daarom is dit van uiterse belang dat die frekwensie van die mutasies in ons populasies bepaal word sodat daar vasgestel kan word watter groepe „n hoë risiko het om gehoor verlies te kan ontwikkel. Die ABI Prism® SNaPshotTM Multipleks sisteem is gebruik en geoptimiseer om te toets vir die ses mutasies in die MT-RNR1: C1494T, T1095C, 961delT+C(n), A827G and T1291C. „n Totaal van 115 MDR-TB pasiente van die Brooklyn Chest Hospital in Kaap Stad is gewerf vir die studie. Hierdie pasiente ontvang daaglikse hoë dosese van een van die volgende aminoglikosiede: streptomycin, kanamycin of capreomycin. Verder is „n totaal van 439 kontrole DNA monsters gewerf vanuit die volgende etniese groepe: 93 Afrikaner, 104 Blank, 112 Swart and 130 Kleurling. Hierdie monsters is ook getoets vir die ses mutatsies. Hoë Resolusie Smelt analise (HRS) is gebruik om nuwe DNS volgorde veranderinge in die MT-RNR geen te identifiseer. Die hele mitochondriale genoom is blootgestel aan DNA volgorde bepaling in „n poging om nuwe DNS volgorde verandering in die genoom te identifiseer wat moontlik betrokke kan wees by aminoglikosied-geinduseerde gehoor verlies. „n Total van 97 lede van „n Suid Afrikaanse familie waar die A1555G mutasie teenwoordig is, is deur middle van die SNaPshot metode gegenotipeer. Verder is die normale funcitoneering van die mitochondrion in getransformeerde witbloed selle, getoets in die teenwoordigheid van verskillende konsentrasies streptomycin met behulp van die MTT kleurmetrie toets. Deteksie van heteroplasmiese mutasies is gedoen deur middle van die PCR-RFLP tegniek en alle analises is gedoen op die UN-SCAN-IT program. Ons was suksesvol in die ontwikkeling van „n vinnige, koste effektiewe en kragtige tegniek wat al ses die mutasies in MT-RNR1 in een reaksie kan optel. Hierdie tegniek het goed gewerk met DNA monsters van bloed en van selle verkry vanuit die wangholte (geneem van kinders jonger as 12 jaar). Die C1494T, T1095C en T1291C mutasies is glad nie waargeneem in enige van ons MDR-TB patiente of kontroles nie. Skrikwekkend is die hoë frekwensie (0.9%) waarby die A1555G mutasie in die Swart kontrole groep waargeneem is. Hierdie mutasie is ook in 1.1% van die Afrikaner kontrole groep opgemerk in heteroplasmie van 25%. Die A827G mutasie was teenwoordig in 0.9% en 1.1% van die MDR-TB patiente en Afrikaner kontrole monsters, onerskeidelik. Die 961delT + insC(n) mutasie is opgemerk in baie hoë frekwensies in beide die MDR-TB (3.5%) en kontrole groepe (1.1% van die Afrikaner, 1.5% van die Kleurling en 7.1% van die Swart monsters). Die T961G mutasie is ook in hoë frekwensies in slegs die Blanke (2.9%) en die Afrikaner (3.2%) kontrole groepe waargeneem. Nuwe DNS volgorde veranderinge in MT-RNR1 is gesoek in „n groep MDR-TB patiente wat gehoor verlies ondervind. Slegs twee nuwe verandering is ontdek (G719A en T1040C). Dit is onwaarskynlik dat hierdie veranderinge patogenies is siende dat hulle teen frekwensies van 20.7% en 1.8% waargeneem is in die Kleurling en Swart kontrole groepe onderskeidelik. Tydens die soeke na nuwe DNS volgorde veranderinge wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies in die mitochondriale genoom is 50 onbekende veranderinge ontdek (een in die MT-TH, ND3, COX3 en twee in die CYTB gene). Die veranderinge is verder ondersoek vir evolusionêre konservasie op beide die nukliotied en amino suur vlak van mens to padda. Dit is bevind dat 76 uit die 97 familie lede positief is vir die A1555G mutasie en het dus „n hoë risiko om aminoglikosied-geinduseerde gehoor verlies te ontwikkel as hul bloot gestel word aan hierdie antibiotikums. Verder is gevind dat hierdie familie op die L0d mitochondriale haplogroep lê. Geen van die sogenaamde genetiese modifiseerde gene of DNS volgorde veranderinge in hierdie gene (tRNASer(UCN), A10S in TRMU in homosigotiese vorm en die 35delG in GJB2) is gevind in die familie nie. Die MTT toets het „n afname in die mitochondriale funksioneering van selle waar die A1555G mutasie teenwoordig was getoon, alhoewel die verskil tussen selle wat nie die A1555G mutasie het nie, nie statisties betekenisvol was nie (p-waarde: 0.615-0.999). Die hoë frekwensie van die A1555G mutasie (0.9%) in die Swart populasie van Suid Afrika is skrikwekkend siende dat die voorkomssyfer van MDR-TB in hierdie groep baie hoog is. Toekomstige studies met grooter getalle is nodig om die ware frekwensie van die mutasies geassosieer met aminoglikosied-geinduseerde gehoor verlies in die algemende Suid Afrikaanse populasie te bepaal. Ons data dui aan dat die 961delT + insC(n) en die T961G mutasies slegs algemene nie-patogeniese polimorphismis is siende dat dit in sulke hoë frekwensies (>1%) in kontroles opgemerk is. Die identifiseering van die eerste DNS volgorde veranderinge in proteïen kodeerende gene wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies hou groot en belowende moontlikehede in, interme van die identifiseering van „n tweede geen. Toekomstige studies waarin die effek van hierdie veranderinge op die normale funktioneering van hierdie gene ondersoek word sal „n besondere groot bydrae lewer tot hierdie veld van navorsing. Al 76 van die A1555G positiewe familie lede is voorsien van genetiese verslae en het berading ontvang in verband met hul risiko en die risiko van hul kinders om aminoglikosied-geinduseerde gehoor verlies te ontwikkel. Die ontwikkeling van „n kragtige, vinnige en koste-effektiewe genetiese metode vergemaklik die vinnige identifiseering van hoë risiko individue vir die ontwikkeling van gehoor verlies voordat hulle met hul aminoglikosiede behandeling begin. Dit is veral noodsaaklik in „n derde wêreld land soos Suid Afrika waar, ten spyte van hul gevaarlike newe effekte, aminoglikosied antibiotikums steeds gebruik sal word. Daarom is grooter publieke bewusmaking nodig om hierdie problem te probeer oplos en te verhoed.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered. / AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante. / The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University / DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust / Harry Crossley Foundation

As infecções relacionadas à assistência à saúde (IRAS) são um grave problema de saúde pública cujo prognóstico tem sido desfavorecido pela emergência e endemicidade de bactérias multirresistentes (MRs). Neste cenário, seguindo uma tendência mundial, no Brasil, infecções por cepas de Acinetobacter baumannii MRs produtoras de carbapenemases do tipo OXA são atualmente consideradas uma emergência clínica e epidemiológica. Na falta de alternativas terapêuticas efetivas para infecções relacionadas, este trabalho objetivou avaliar efeitos sinérgicos (utilizando checkerboard e time-kill) decorrentes da combinação de 10 antimicrobianos de diferentes classes, contra 8 cepas MRs de A. baumannii, clonalmente não relacionadas, produtoras de carbapenemases do tipo OXA-23, OXA-72, OXA-58 e OXA-143, representativas de diferentes centros hospitalares do Brasil. Como resultado, a combinação amicacina/tigeciclina apresentou atividade sinérgica (S= &#931;CIF &#8804; 0,5) e parcialmente sinérgica (PS= &#931CIF ;0,5-0,75) contra 4 (50%) cepas produtoras de OXA-143 ou OXA-72, e 2 cepas (25%) produtoras de OXA-23, respectivamente. Por outro lado, a combinação polimixina B/imipenem apresentou atividade S e PS contra 3 (37,5%) isolados OXA-143, OXA-23 ou OXA-72 positivos, e 1 (12,5%) isolado produtor de OXA-58, respectivamente. Já, a combinação amicacina/ampicilina-sulbactam foi S contra 2 (25%) A. baumannii OXA-143 ou OXA-23 positivos, sendo PS contra dois (25%) A. baumannii OXA-58 ou OXA-143/23 positivos. De interesse, foi o efeito S da combinação polimixina B/vancomicina, contra 2 cepas (25%) produtoras de OXA-72 ou OXA-23. Por outro lado, a combinação ampicilina-sulbactam/rifampicina apresentou atividade PS contra 6 (75%) cepas produtoras das variantes OXA-23, OXA-143, OXA-72 ou OXA-58. Da mesma forma, rifampicina combinada com polimixina B foi sinérgica para uma cepa OXA-23 (12,5%) e PS para 5/8 (62,5%) cepas produtoras de OXA-72, OXA-58, OXA-23/-OXA143 ou OXA-143. O efeito sinérgico da combinação polimixina B/imipenem foi confirmado, in vivo, no modelo murino de infecção, tanto por avaliação histopatológica como por redução das UFC/g pulmão ou baço (p &#8804; 0,05). Finalmente, foi avaliada a atividade, in vitro, do lípide catiônico brometo de dioctadecildimetilamônio (DDA), individualmente e em combinação com tigeciclina. DDA possui efeito bactericida, e potencializou sinergicamente a tigeciclina contra 2 (25%) cepas OXA-143 ou OXA-23 positivas. A atividade do DDA, assim como a atividade da sua combinação com tigeciclina foram efetivas já na segunda hora de interação, como avaliado pelas curvas de morte. Em resumo, o efeito sinérgico decorrente do uso combinado de amicacina, tigeciclina, polimixina B, imipenem, rifampicina ou ampicilina/sulbactam, pode constituir uma alternativa terapêutica para o tratamento de infecções produzidas por cepas de A. baumannii MRs produtoras de oxacilinases, sendo que nanofragmentos catiônicos de bicamada do lipídeo sintético de DDA tem potencial para consolidar um produto de aplicação clínica. / Healthcare-associated infections (HAIs) are a serious public health issue, which have been related with an unfavorable prognosis due to the emergence and endemicity of multidrug-resistant (MDR) bacteria. In this scenario, following a worldwide trend, in Brazil, infections produced by MDR OXA-type carbapenemase-producing Acinetobacter baumannii are currently considered a clinical and epidemiological urgency. In the absence of effective therapeutic alternatives for related infections, this study aimed to evaluate synergistic effects (by using time-kill and checkerboard assays) achieved by the combination of 10 different classes of antimicrobial against 8 strains of MDR, clonally unrelated, A. baumannii strains producing OXA-23, OXA-72, OXA-58 and OXA-143 carbapenemases, being representatives of different medical centers in Brazil. As a result, the combination of amikacin / tigecycline showed synergistic (S = &#931;FIC &#8804; 0.5) and partially synergistic (PS = 0.5 to 0.75 &#931;FIC) activity against 4 (50%) OXA-72 or OXA-143 producing A. baumannii strains, and two strains (25%) producing OXA-23, respectively. Moreover, the combination of polymyxin B / imipenem showed S and PS activity against 3 (37.5%) OXA-143, OXA-23 and OXA-72 positive isolates, and 1 (12.5%) OXA-58 producer, respectively. On the other hand, the combination amikacin / ampicillin-sulbactam was S against 2 (25%) OXA-143 and OXA-23 positive strains, being PS against two (25%) OXA-58- and OXA-143/23-producing A. baumannii. Of interest was the synergistic effect achieved by polymyxin B plus vancomycin against two strains (25%) producing OXA-72 and OXA-23, respectively. Furthermore, the ampicilina-sulbactam / rifampicin combination displayed a PS activity against six (75%) strains producing OXA-23, OXA-143, OXA-72 or OXA-58-type enzymes. Likewise, rifampicin combined with polymyxin B was S against 1 (25%) OXA-23-positive A. baumannii being PS to 5/8 (62.5%) strains producing OXA-72, OXA-58, OXA-23/-OXA143 or OXA-143. The synergistic effect of the combination polymyxin B / imipenem was confirmed, in vivo, in the murine model of infection, by using both histopathological studies and bacterial clearance from the lungs and spleen (CFU/g, p&#8804; 0.05). Finally, we evaluated the in vitro activity of the cationic lipid dioctadecyldimethylammonium bromide (DDA), alone and in combination with tigecycline. DDA display a bactericidal effect, enhancing synergistically the activity of tigecycline against 2 (25%) OXA-143 and OXA-23 positive strains, respectively. DDA activity alone and in combination with tigecycline was effective on the second hour of interaction, as evaluated by time-kill assays. In summary, the synergistic effect resulting from the combined use of amikacin, tigecycline, polymyxin B, imipenem, rifampicin or ampicillin / sulbactam, could be an alternative therapy for the treatment of infections caused by MDR A. baumannii strains producing oxacilinases. On the other hand, cationic bilayer nanofragments of DDA has potential for consolidating a product for medical application.

Arranjos supramoleculares combinando o lípide catiônico brometo de dioctadecildimetilamônio (DOD) com polímeros, como carboximetilcelulose (CMC) e cloreto de poli(dialildimetilamônio) (PDDA), foram preparados na forma de nanopartículas (NPs), na ausência ou presença de antimicrobiano tradicional, como a claritromicina (CLA). NPs preparadas por atração eletrostática entre os fragmentos de bicamada (BF) de DOD, CMC e PDDA foram avaliadas, in vitro, quanto à atividade contra isolados clínicos de micro-organismos multirresistentes (MR) a antimicrobianos, como Pseudomonas aeruginosa MR, Klebsiella pneumoniae produtora da enzima carbapenemase do tipo KPC, Staphylococcus aureus resistente à meticilina/oxacilina (MRSA) e Candida albicans resistente ao fluconazol, através do método de plaqueamento e contagem de viáveis. As NPs de DOD BF/CMC/PDDA apresentam alta atividade de amplo espectro contra micro-organismos MR, em que o PDDA é o componente responsável pela excelente atividade biocida das NPs. O mecanismo de ação antimicrobiana indica a dissociação dessas NPs na presença dos micro-organismos, com a remoção de biopolímeros da parede celular microbiana pelo PDDA, conforme visualizado por microscopia eletrônica de varredura, ocorrendo lise da membrana microbiana e liberação de compostos fosforilados para o meio extracelular. Também foram desenvolvidas neste trabalho NPs carreadoras de CLA à base de DOD e polímeros. Solução etanólica contendo CLA/DOD foi injetada em solução aquosa de CMC, formando arranjos coloidalmente estáveis e aniônicos, que posteriormente foram adicionados de solução de PDDA, para a obtenção de arranjos estáveis e catiônicos. CLA/DOD/CMC e CLA/DOD/CMC/PDDA NPs incorporaram CLA em quantidade suficiente para inibir o crescimento de M. abscessus no interior de macrófagos bem como evitar a formação de biofilmes, sendo que altas doses de CLA foram tóxicas aos macrófagos, enquanto doses menores apresentaram baixa toxicidade e boa atividade antimicrobiana. NPs catiônicas carreando CLA foram tóxicas aos macrófagos nas concentrações de PDDA testadas. A natureza particulada das CLA NPs possivelmente aumenta a retenção intracelular de CLA em comparação com CLA livre, podendo prolongar atividade da CLA contra patógenos intracelulares. Desta maneira, arranjos supramoleculares combinando lípide e polímeros, com ou sem antimicrobianos tradicionais poderão encontrar diversas aplicações nas áreas farmacêutica, médica, alimentícia e biotecnológica. / Supramolecular assemblies combining cationic lipid dioctadecyldimethylammonium bromide (DOD) and polymers, such as sodium carboxymethylcellulose (CMC) and poly(diallyldimethylammonium chloride) (PDDA), were prepared as nanoparticles (NPs), in the absence or presence of traditional antibiotic, such as clarithromycin (CLA). NPs prepared by electrostatic attraction between DOD bilayer fragments (BF), CMC and PDDA were evaluated against clinical strains of multidrug resistant (MDR) microorganisms, such as Pseudomonas aeruginosa MDR, Klebsiella pneumoniae producer of KPC carbapenemase enzyme, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans fluconazole resistant, by plating and colony forming unities counting. DOD BF/CMC/PDDA NPs display high and broad-spectrum activity against MDR microrganisms, and PDDA is the excellent biocidal component in the NPs. The mechanism of antimicrobial action shows that NPs disassembly in the presence of microrganisms, with biopolymers withdrawn from the cell wall, as observed by scanning electron microscopy, consecutively lysing bacterial membrane as determined from the leakage of inner phosphorylated compounds. In this work there have also been developed NPs, based on lipid and polymers, as carriers for CLA. Ethanolic solution co-solubilizing CLA/DOD was injected in CMC aqueous solution, yielding colloidaly stable and anionic NPs, that were further added of PDDA solution, yielding stable and cationic NPs. CLA/DOD/CMC NPs and CLA/DOD/CMC/PDDA NPs incorporated CLA at doses high enough to inhibit M. abscessus growth inside macrophages or in biofilms. Larger CLA doses were toxic to macrophages while lower CLA doses reduced toxicity to macrophages despite their high antimicrobial activity. Cationic CLA NPs exhibited substantial toxicity against macrophages at the PDDA concentrations tested. The particulate nature of these CLA NPs possibly increases intracellular CLA retention in comparison to free CLA, probably extending CLA activity against intracellular pathogens. In conclusion, supramolecular assemblies combining cationic lipid and polymers, with or without traditional antibiotics, may find multiple possibilities of applications at pharmaceutical, medical, food and biotecnological fields.

La résistance bactérienne aux antibiotiques dans les établissements de santé complique la prise en charge thérapeutique et entraîne une surmortalité des patients infectés. L'objectif de ce travail était d'évaluer différentes approches ayant pour finalité la prévention et la maîtrise des infections nosocomiales. Ce travail s'articule autour de trois questions : (i) Existe-t-il une stratégie de promotion d'un juste usage des antibiotiques à privilégier ? (ii) Comment évaluer la performance en matière d'hygiène des mains ? (iii) Quelles mesures pouvons-nous proposer pour limiter la diffusion de P. aeruginosa ?Nous avons d'abord montré que la diffusion de recommandations sous la forme de guides régionaux ou de messages de pharmacovigilance était suivie d'une modification des prescriptions antibiotiques conformes aux recommandations.Ensuite, nous avons évalué le niveau de performance des établissements de santé en matière d'hygiène des mains en étudiant plus particulièrement l'indicateur de consommation de solution hydro-alcoolique (ICSHA). Nous avons ainsi pu montrer que le nombre minimal d'opportunité d'hygiène des mains servant à son calcul, était sous-estime. Dans une autre étude, nous avons été confrontés aux limites liées à sa construction, ces dernières étant en partie responsable de l'absence de relation observée entre cet indicateur et la prévalence des infections associées aux dispositifs invasifs.Enfin, nous avons montré que la charge en soins et la contamination des réseaux d'eau propre des établissements de santé étaient des facteurs de risque contextuels d'acquisition du bacille pyocyanique. Un autre travail a mis en évidence que les réseaux d'eau usée étaient impliqués dans la diffusion extra-hospitalière de souches résistantes. Une étude est actuellement en cours pour évaluer l'intérêt d'une approche globale associant dépistage et précautions complémentaires chez les patients porteurs de P. aeruginosa.En conclusion, si ce travail confirme l'efficacité de certaines actions de prévention tout en mettant en avant les limites d'autres approches, il ne permet pas de privilégier une stratégie particulière. Il apparaît ainsi nécessaire de mettre en place des stratégies globales et transversales allant au-delà des seuls établissements de santé / Bacterial resistance to antibiotics in health care facilities complicates the therapeutic burden and increased mortality of infected patients. The objective of this work was to evaluate different approaches which aim was to prevent and control hospital-acquired infections. This work focuses on three issues: (i) Is there a strategy already in place to promote the appropriate use of antibiotics? (ii) How can we evaluate performance in terms of hand hygiene? (iii) What measures can we implement to limit the spread of P. aeruginosa?We first showed that the distributions of regional guidelines or drug monitoring alerts were followed by a change in the uptake of antibiotic prescriptions in line with such recommandations.Then we evaluated the performance of health care facilities for hand hygiene, focusing especially on the index of consumption of alcohol-base hand-rub solution. We showed that the number of alcohol-based hand-rub is far higher than that defined by the French Ministry of Health. In another study, we were faced with limitations in its construction, the latter being partly responsible for the lack of a relationship between this indicator and the prevalence of invasive devices associated with infections.Finally, we have shown that the burden of care and the contamination of clean water networks of health facilities were contextual risk factors for acquisition of Pseudomonas aeruginosa. Another study showed that wastewater networks were involved in extra-hospital spread of resistant strains. A study is currently underway to assess the value of a global approach combining screening and additional precautions in patients with P. aeruginosa.In conclusion, this study confirms the effectiveness of some preventive measures while underlining the limitations of other approaches. However it does not promote a particular strategy. Because in terms of BMR, it is necessary to define global and cross-sectorial strategies which go beyond the health care facilities