Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on other sensitive yeast genera but are immune to their own toxins. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer phenotypes are classified into 11 typical types (K1-K11). The toxic actions of yeast killer proteins on sensitive cells show differences and one of the most important toxic actions involves the selective functional damage by hydrolyzing major cell wall components. Because mammalian cells lack a cell wall, novel highly selective antifungals tend to be harmless to people by targeting important cell wall components specific to fungi. We have previously characterized the K9 type yeast killer protein isolated from Hansenula mrakii. This protein is stable at pH and temperature values appropriate for its medical usage. Antifungal activity of this protein was tested against 23 human pathogenic yeast and 9 dermathophyte strains. Pathogenic yeast strains found to be susceptible and both the MIC and MFC values ranged from 0.25 to 8 &micro / g/ml except C. parapsilosis and C guilliermondii isolates. 9 dermatophyte strains were not susceptible to this protein and MICs were &gt / 64 &micro / g/ml. According to the cell killing analysis toxin activity starts within the first 4 hours and complete cell death was observed for the 4, 8 and 16 times the MIC concentrations at 24 hour. The results obtained from this study might make the potential use of this protein possible as a selective antimycotic agent.

The mucus-dwelling bacteria previously isolated from the surface of a freshwater fish species (Alburnus alburnus from Lake Mogan, Ankara), were studied to discover their cobalt resistance. The minimum inhibitory concentrations (MIC) were determined for a total of thirty six bacterial isolates. The results of the resistance studies led us to design experiments on adaptation to cobalt and subsequent memory retention. Three selected isolates were exposed to an inhibitory cobalt concentration as a mixed culture and individually. The delayed formation of colonies along with competitive exclusion of one of the isolates in the mixed culture were recorded. The delay for colony formation was followed up for liquid culture conditions. After some of our isolates acclimated to cobalt and started to exhibit constant time of growth period, it is assumed that they were adapted. We regarded adaptation as a result of memory formation. Next, we did a further study to find out how long this memory could be retained via serial multiple passages in cobalt free medium. We expressed our observations quantitatively by measuring the growth by using spectrophotometer and by performing viable counts. Interestingly, where there was a high CFU, the photometric values were very low. We interpreted the finding such that the presence of cobalt above tolerance limits were causing size reduction in the cells. So that their presence was underestimated by optic devices in visible range. Our study hinted that freshwater bacteria was adapting cobalt in a memory based mechanism and able to retain this memory for some time.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &amp / #946 / -1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &amp / #946 / -1,3-glucanase which is stable at pH&rsquo / s and temperatures appropriate for its medical usage. &amp / #946 / -1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 &micro / g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

[pt] Este trabalho investiga a formação de biofilmes sobre ligas de aço inoxidável S32750, conhecido como superduplex, por cepas de bactérias redutoras de sulfato (BRS) em circuito de teste, submetido a condições de vazão e temperatura constantes, utilizando como fluido de processo a água do mar sintética. As ligas de superduplex são geralmente empregadas em serviços cuja concentração de íons cloretos é alta. As BRS desempenham papel significativo nos processos corrosivos por pites em aços inoxidáveis. Foram elaborados corpos de prova em S32750 e em AISI 316, a título de comparação com a literatura, que foram expostos ao fluido de processo por 15 e 35 dias. Também foi avaliada a influência das características físicas da superfície e para isso foram testados três diferentes tratamentos superficiais nos corpos de prova antes de serem inseridos no sistema: jateamento com microsfera de vidro e polimento com pasta de diamante com grãos de 6 μm e 1μm. Foram realizados a quantificação das BRS sésseis e planctônicas, o teor de sulfetos totais produzidos, a quantificação dos ácidos orgânicos e sulfato depletado, a morfologia dos biofilmes, a rugosidade superficial, além da avaliação dos pites formados. Foram realizados ensaios abióticos (sem a presença de bactérias) a fim de avaliar a ação corrosiva do meio. / [en] The goal of this work is to determine the biofilm colonization on the surface of superduplex stainless steel S32750 by Sulfate Reducing Bacteria (SRB) in a flow loop at constant flow rate and room temperature, exposed to artificial sea water. The duplex stainless steel has been widely used as material for offshore applications due to their excellent corrosion resistance in chloride environments, compared with austenitic stainless steel. At high concentrations these sulfides are known to adsorb and catalyze anodic dissolution and hence facilitate pitting. The samples of S32750 were exposed to flowing conditions for 15 and 35 days. Other 316 stainless steel coupons were exposed under the same time in order to compare with superduplex. The physical and chemical characteristics of a solid surface affect biofilm formation in flowing systems. Surface roughness affects bacterial adhesion. The degree of surface roughness may well be a critical factor in biofilm development. Some coupons were ground and others were polished (6 μm e 1 μm - diamond paste). The bacterial cells were quantified using most probable number (NMP) method. It was also quantified the total sulfide, the organics acids and sulfate depleted. Biofilm morphology, surface roughness and pitting formation were explored.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pythiosis is a chronic disease that affects humans, other mammals and birds. It is caused by the aquatic oomycete Pythium insidiosum. Pythiosis progresses rapidly and can become life threatening if not treated in the early stages. The absence of ergosterol in the cell wall of this oomycete prevents the treatment of pythiosis with antifungal therapy because most antifungal drugs act by inhibiting the synthesis of ergosterol. Members of the genus Pythium are known to be susceptible to some antimicrobial of the tetracycline, macrolide, aminoglycoside and chloramphenicol classes. This study aimed to evaluate the in vitro susceptibility of isolates of P. insidiosum to the aminoglycosides gentamicin, neomycin, paromomycin and streptomycin and to the minocycline derivative tigecycline. The susceptibility tests were carried out with 24 P. insidiosum isolates using the broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) document M38-A2. The minimum inhibitory concentration (MIC) values for gentamicin, neomycin, paromomycin and streptomycin ranged from 32 to 64 mg/L, and the minimal fungicidal concentration (MFC) values ranged from 32 to 128 mg/L. Tigecycline had a good inhibitory activity, with values of mic MIC (0.25-2 mg/L) and MFC (1-8 mg/L) values. The observed in vitro susceptibility to tigecycline makes this drug a good option for future in vivo assays investigating options of treatment of pythiosis. / Pitiose é uma doença crônica que afeta humanos, outros mamíferos e pássaros. O seu agente etiológico é um oomiceto aquático denominado Pythium insidiosum. A Pitiose evolui rapidamente e pode se tornar fatal se não tratada nos primeiros estágios. A ausência de ergosterol na parede da célula deste oomiceto impede o tratamento de pitiose com terapia antifúngica, pois a maioria das drogas antifúngicas atua sobre a síntese de ergosterol. Os membros do gênero Pythium são conhecidos por serem susceptíveis a alguns antimicrobianos do grupo das tetraciclinas, macrolídeos, aminoglicosídeos e cloranfenicol. Este estudo teve como objetivo avaliar a susceptibilidade in vitro de isolados de P. insidiosum frente aos aminoglicosídeos gentamicina, neomicina, paromomicina e estreptomicina e ao derivado da minociclina denominado de tigeciclina. Os testes de susceptibilidade foram realizados com 24 isolados de P. insidiosum, utilizando o método de microdiluição em caldo, de acordo com o documento M38- A2 do Clinical and Laboratory Standards Institute (CLSI). Os valores de Concentração Inibitória Mínima (CIM) de gentamicina, neomicina, paromomicina e estreptomicina variaram de 32 a 64 mg/L, e os valores de concentração fungicida mínima (CFM) variaram de 32 a 128 mg/l. Tigeciclina apresentou boa atividade inibitória, com valores de CIM (0,25-2 mg/L) e CFM (1-8 mg/L). A susceptibilidade à tigeciclina observada in vitro faz deste fármaco uma boa opção em futuros ensaios e in vivo, investigando o tratamento da pitiose.

Mycoplasma (M.) bovis est une bactérie pathogène des bovins, à l'origine de signes cliniques divers, comme des mammites, des arthrites, des otites et des bronchopneumonies, ces dernières étant majoritaires en France. Les mycoplasmoses à M. bovis ont un fort coût économique et leur contrôle impose une importante mobilisation sanitaire et un recours très fréquent à l'antibiothérapie. Peu de données étaient disponibles jusque récemment concernant le typage moléculaire et l'antibiosensibilité des souches françaises de M. bovis. Deux études antérieures à ce travail et réalisées au sein de l'UMR « Mycoplasmoses des ruminants » ont montré que les isolats cliniques de M. bovis collectés en France après 2000 appartiennent à un sous-type moléculaire majoritaire (ST2), très homogène et sont par ailleurs multirésistants à la plupart des familles antibiotiques à l'exception des fluoroquinolones. Ces résultats suggèrent la diffusion sur le territoire national d'un clone unique multirésistant. Le premier objectif de cette étude était de déterminer les mécanismes à la base de la perte de sensibilité aux antibiotiques des isolats français. Dans un deuxième temps, les liens entre les différents sous-types moléculaires, les profils d'antibiosensibilité, les maladies associées et le polymorphisme des gènes cibles des antibiotiques ont été investigués. Cette approche a été déployée pour trois familles d'antibiotiques utilisées en pratique vétérinaire: les macrolides, les tétracyclines et également les fluoroquinolones, quoique récemment classées comme molécules critiques. De façon générale, les mutations identifiées dans les cibles des antibiotiques expliquent à elles seules les phénotypes de résistance observés. Des mutations dans les ARNs ribosomaux, cibles des macrolides et des tétracyclines, ont été observées sur des isolats cliniques dès 1978 et sont devenues systématiques sur tous les isolats collectés après 2000 et appartenant au sous-type ST2 majoritaire. En ce qui concerne les fluoroquinolones, la faible augmentation des CMI (concentrations minimales inhibitrices) mesurée chez la plupart des isolats cliniques récents n'a pas été associée à des mutations des QRDR (« Quinolones Resistance-Determining Regions »). Par contre, des altérations cumulées de façon séquentielle dans ces QRDR, associées à une hausse des CMI, ont été mises en évidence lors d'expériences de sélection in vitro et majoritairement pour des souches appartenant à un sous-type récent minoritaire, ST3, apparemment plus variable et plus apte à fixer les mutations. En 2013, le premier isolat clinique présentant une CMI augmentée aux fluoroquinolones a été isolé: il appartient à ce sous-type ST3. L'ensemble des résultats obtenus montrent que les différents sous-types de M. bovis n'évoluent pas de la même façon vers la résistance. Ce constat ajouté à celui de la multirésistance des isolats récents (ST2 ou ST3) met en exergue l'intérêt de la surveillance (sous-typage et antibiosensibilité) et le suivi de l'évolution des isolats de M. bovis circulant en France. Ce suivi permettrait notamment d'anticiper une éventuelle émergence de la résistance aux fluoroquinolones / Mycoplasma (M.) bovis is a bacterial pathogen for cattle, responsible for various clinical signs, like mastitis, arthritis, otitis and respiratory diseases, the latter being the main syndrome present in France. Mycoplasmoses have a great economic impact and their control entails drastic sanitary measures and a frequent use of antibiotherapy. Few data was available until recently on the molecular subtyping and the antimicrobial susceptibility of the French strains of M. bovis. Two previous studies done in the UMR « Mycoplasmoses des ruminants » proved that clinical isolates collected in France after the year 2000 belonged to one major subtype (ST2), which is very homogeneous, and that they were multiresistant to the main antimicrobial families except fluoroquinolones. These results suggested the diffusion of one unique multiresistant clone on the national territory. The first aim of the present study was to decipher the molecular mechanisms underlying the loss of susceptibility to antimicrobials of the French strains. Secondly the links between the molecular subtypes, the antibiotics susceptibility profiles, the clinical origins and the polymorphisms of the target genes were assessed. This approach was used for 3 antimicrobial families currently used in veterinary medicine: macrolides, tetracyclines and fluoroquinolones, although recently classified as critical. Actually, the point mutations observed in the target genes of the antimicrobials accounted for the observed resistance phenotypes. Some mutations in the ribosomal RNAs, targets of the macrolides and the tetracyclines, were observed in clinical isolates as soon as 1978 and they were generalized in all isolates collected after 2000 and belonging to the major subtype ST2. Concerning the fluoroquinolones, the slight increase in MIC (Minimum Inhibitory Concentration) observed in most of the recent isolates was not associated with mutations in the QRDR (Quinolone Resistance-Determining Regions). However alterations that were associated with increased MICs were highlighted and proved to be sequentially cumulated during experiments of in vitro selection under antimicrobials pressure. This was mainly true for strains belonging to a recent and uncommon subtype, ST3, which is apparently more variable and more able to fix the mutations. In 2013 the first clinical strain showing an increased MIC to fluoroquinolones was isolated and proved to belong to ST3. The whole results of this study showed that the different subtypes did not evolve with the same speed towards resistance. This fact, associated with the multiresistant phenotype of the recent isolates (ST2 or ST3), highlights the urge to monitor (subtyping and antimicrobial susceptibility profiles) and to follow-up the evolution of the isolates of M. bovis circulating in France in order to anticipate a potential emergence of the resistance to fluoroquinolones

The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli are found as commensals in the intestinal tract of healthy mammals and birds. Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans which is predominantly contracted from poultry products. Although the vast majority of these infections are mild, life-threatening complications should be treated with antimicrobials. Patients are usually treated with either macrolides of fluoroquinolones. However, globally there is an increased trend in the development of resistance to these antibiotics. This trend has also been observed in infection of poultry and pigs. The aim of this investigation was to determine antimicrobial sensitivity of thermophilic Campylobacters isolated from pigs and poultry by broth microdilution minimum inhibitory concentration testing. A total of 482 samples of the small intestinal content from poultry and pigs from the Western Cape and Gauteng Provinces were collected and analysed. Thirty-eight Campylobacter isolates were obtained. Statistical analyses included percentage resistance, minimum inhibitory concentrations (MIC50 and MIC90) as well as the distribution percentages of the MICs. The non-parametric Mann-Whitney U test was used to establish any significant differences at an interspecies, interhost and interprovincial level. Analyses of the data obtained revealed indications of decreasing susceptibility to several antibiotic groups including the tetracyclines, macrolides, erythromycin and tylosin, as well as the lincoasamides, and fluoroquinolones. It was found that isolates from the Western Cape were more likely to be resistant to the fluoroquinolones (p = 0.0392), macrolides (p = 0.0262), and lincoasmides (p = 0.0001) and, as well as to a certain extent the pleuromutulins (p = 0.0985), whereas isolates from Gauteng were more resistant to the tetracycyclines (p = <.0001). Poultry Campylobacter spp. were more prone to be resistant to enrofloxacin (p = 0.0021). Campylobacter jejuni, mainly isolated from poultry, was more liable to be resistant to the tetracyclines (chlrotetracycline p= 0.0307), whereas C. coli, predominatly isolated from pigs was more likely to be resistant to the macrolides (tylosin p= 0.063). Four of the bacteria isolated from the Western Cape were resistant to three or more antibiotic classes, namely; tetracyclines, macrolides, lincosamides, pleuromutulins and fluoroquinolones. No multi-resistant Campylobacter spp. were isolated from the flocks in Gauteng. With the exception of tiamulin, the bacterial populations could clearly be divided into resistant and susceptible populations. As consequence of the increased resistance to the antimicrobial classes used for human therapy and the geographical differences in antimicrobial susceptibility, it is recommended that an antimicrobial resistance monitoring system for the thermophilic Campylobacter spp. be initiated in the South Africa National Veterinary Surveillance and Monitoring Programme for Resistance to Antimicorbial Drugs (SANVAD) Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

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