Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (p. 134-135). Also available via World Wide Web.

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 216-244). Also available via World Wide Web.

Thesis (MTech (Medical Technology))--Cape Technikon, 1998. / This study was undertaken to investigate resistance to aminoglycoside antibiotics and the transfer of resistance in selected Gram negative bacilli in hospitals in the Greater Durban area in order to determine whether the development of resistance in this region was similar to that found in other countries and whether it was the same in the hospitals in the region. It was intended that the study might expose the existence of nosocomial pathogens of a particular strain or endemic plasmids responsible for aminoglycoside antibiotic resistance. Strains of Klebsiella, Enterobacter and Serratia species and Escherichia coli resistant to gentamicin, tobramycin, netilmicin or amikacin were obtained. Resistance of the isolates obtained to the above aminoglycoside antibiotics was confirmed using a disc diffusion technique. Resistance mechanisms were initially assigned on the basis of resistance to these four aminoglycoside antibiotics. In approximately 50% of the isolates, including donor isolates and their respective transconjugants, resistance mechanisms were confirmed or revised on the basis of a changed resistance profile to a range of 12 aminoglycoside antibiotics in conjunction with DNA/DNA hybridization tests. Bacterial conjugation studies were performed on selected isolates to investigate the transfer of aminoglycoside resistance from Klebsiella pneumoniae isolates to recipient Escherichia coli. Plasmid profiles of all isolates and Escherichia colitransconjugants were compared to establish similarities. Isolates in three of the four genera of bacteria and all isolates collectively, demonstrated the greatest incidence of resistance to tobramycin. Amikacin resistance was, in all groups of isolates, the least frequently encountered. Collectively, the most frequent mechanisms of resistance were the AAC(3)-V and AAC(6')-1 enzymes One large hospital showed a high frequency of the AAC(3)-V modifying enzyme while in other hospitals a wider range of enzyme resistance mechanisms were evident. Plasmid profiles were generally dissimilar within and between different genera and the different hospitals. / Mangosuthu Technikon Research Fund

Etant donné leur implication dans de nombreux processus biochimiques, les ARN sont maintenant considérés comme des cibles thérapeutiques très prometteuses. Cependant, nos connaissances limitées concernant les phénomènes d'interaction entre les ARN et de petites molécules, compliquent l'élaboration de nouveaux ligands (ou médicaments), capables de reconnaître sélectivement une structure complexe d'ARN. En absence de toute approche rationnelle, une stratégie de criblage pourrait permettre de mieux comprendre ces phénomènes de reconnaissance. Cette thèse porte sur la mise au point d'une méthode électrochimique, simple, adaptée pour du criblage haut-débit et permettant de détecter et de quantifier les interactions ARN/ligands. Le principe de la méthode repose sur la différence de coefficient de diffusion qui existe entre la forme libre d'un ligand possédant des propriétés redox et sa forme complexée à l'ARN. Cette stratégie de détection par voie électrochimique présente comme avantages d'être peu coûteuse, rapide, simple d'utilisation, adaptée pour du criblage haut-débit de molécules et utilisable dans de faibles volumes. Cette méthodologie a été utilisée pour caractériser la formation d'un complexe entre un analogue d'aminoglycoside porteur d'un groupe ferocene et une séquence d'ARNr 16S23. De plus, des expériences de compétition entre le complexe ARN/ligand redox et des aminoglycosides non modifiées permettent d'étendre la méthode à la détermination de constantes de dissociation (KD) pour des molécules non marquées en phase homogène. Ces expériences de compétition pourront être généralisées pour mesurer le KD de librairies de molécules, permettant ainsi de trouver de meilleurs ligands d'ARN. / RNA molecules play a major role in various biochemical processes and they are now considered as an important drug target. However, our limited understanding of the interactions occurring between small molecules and RNA complicate the search for new ligands (or drugs) with improved specific interaction and binding to elaborated RNA structures. In the absence of any rational approach, a screening strategy could shed light on the ligand/RNA interactions. In this thesis, we describe a simple electrochemical approach allowing for high-throughput detection and quantification of small molecule/RNA interactions. The principle of the method relies on the difference of diffusion rates between a redoxmolecular probe free or bound to its RNA target and thus to the ability to more easily electrochemically detect the forme rover the latter in a homogenous solution. This electrochemical detection strategy has the advantages of being affordable,fast, easy to use, sensitive and well-adapted to a high-throughput screening strategy in small volume samples. This methodology was used to characterize the binding of an aminoglycoside analog bearing a ferocenyl group to the ribosomal RNA fragment (rRNA 16S23). Furthermore, competitive binding of unlabelled aminoglycosides on theRNA/electrochemical probe complex allowed us to evaluate their dissociation constants (KD). These competitive experiments could further be generalized to measure KD values for libraries of molecules, which could help to find better RNA ligands.

Après la mise en évidence de la forte activité antibactérienne à large spectre contre des bactéries Gram (+) et Gram (-) sauvages et résistantes aux antibiotiques des dérivés 3',4',6-tri-naphtylméthyl (2NM) et -trihexyl d'un petit aminoglycoside, la néamine, et de l'activité, contre les bactéries Gram (+) sauvages et résistantes, des 3',4'- et 3',6-di2NM néamines, les travaux réalisés ont cherché à obtenir des dérivés antibactériens plus actifs et moins toxiques que les dérivés trialkylés. Deux approches en séries aminoglycosides amphiphiles ont été développées dans ce but : (1) la synthèse à partir de la néomycine B de nouveaux dérivés de la néamine portant des groupements alkyles de différentes lipophilies de façon à diminuer globalement celle-ci et (2) après les premiers résultats en série néamine, la synthèse à partir de la N-acétyl glucosamine de dérivés amphiphiles d'un des constituants de la néamine, la néosamine.Dans la première approche, après la mise au point de méthodes d'alkylation de la néamine, il a été possible de passer de dérivés trialkylés de la néamine à des dérivés dialkylés, en particulier 3',6- dialkylés, plus actifs et moins cytotoxiques ceci en diminuant la lipophilie globale des composés. Dans la deuxième approche, à partir de la 1-allyl 3,4-dinonyl néosamine, des dérivés amphiphiles antibactériens à large spectre portant en position anomérique des groupements polaires ont été préparés après époxydation de la double liaison. Le dérivé tri2NM de la néamine s'était avéré avoir une forte affinité pour les lipopolysaccharides de la membrane externe de P. aeruginosa et agir à travers son caractère amphiphiles pour dépolariser la membrane bactérienne. L'étude du mode d'action des dérivés de la néamine préparés les plus actifs a suggéré un mécanisme d'action similaire. / In regard to the antibacterial activity of amphiphilic aminoglycosides which are (i) 3',4',6-trinaphtylmethyl (2NM) et -trihexyl neamine derivatives active against wild-type and resistant to antibiotic drugs Gram (+) et Gram (–) bacteria, and (ii) 3',4'- and 3',6-di2NM derivatives active against bacteria Gram (+) bacteria, the works were focused on the search for more active and less toxic derivatives than the trialkyl derivatives. With this aim, two approaches were developed for obtaining antibacterial amphiphilic aminoglycosides: (1) the synthesis from neomycin B of new neamine derivatives carrying alkyl groups of various lipophily in order to decrease the global lipophily and (2) after the first results in the neamine series, the synthesis from N-acetylglucosamine of amphiphilic derivatives of the aminosugar part of the neamine core named neosamine.In the first approach, after optimisation of alkylation methods of neamine, we shifted from active 3',4',6-trialkylated derivatives to less lipophilic more active and less cytotoxic dialkylated derivatives, especially 3',6 derivatives. In the second approach, from 1-allyl 3,4-dinonyl neosamine, large spectrum antibacterial derivatives carrying at the anomeric position polar groups were obtained through epoxidation of the allylic double bond. Previously, the tri2NM neamine derivative has appeared to be able to strongly bind to the lipopolysaccharides of the outer membrane of P. aeruginosa and to destabilize membranes. The study of the mechanism of action of the most active derivatives prepared suggested a similar mode of action.

Background: Intra-amniotic infections (IAIs) are common complications of labor and delivery. If inadequately treated, these infections can lead to significant morbidity and mortality in the mother and the fetus. Intrapartum aminoglycoside (AG) administration is recommended for the management of IAIs. AGs are known to cross the placenta and achieve bactericidal concentrations in fetal serum. However, the highest and most persistent fetal levels are achieved in renal tissue. So, the fetus may be vulnerable to the nephrotoxic effects of AGs. Megalin, a 600 kDaendocytic receptor, is responsible for the uptake of AGs into renal proximal tubular epithelial cells. This receptor is also expressed in human term placenta and it is reasonable to speculate that it is similarly involved in the placental transport of AGs. However, the mechanisms responsible for placental AG uptake and transport have not yet been characterized. Objective: To evaluate the role of megalin in the transport of AGs across human placenta. Specific aims: (1) To assess and compare megalin expression in term and preterm placental villous tissue, and (2) assess the functional activity of megalin in in vitro placental models. Methods: (1) Following IRB approval, placental tissue samples were collected from pregnant women undergoing term or preterm deliveries. Placental villous tissueswere used to quantify megalin expression by western blotting and q-PCR (2) The human choriocarcinoma cell line (BeWo cells) were grown on Transwell plates, and then megalin expression and function were assessed. Results: Megalin protein and mRNA expression were confirmed in samples of human placental villous tissues. Megalin mRNA expression declined steeply with gestational age till week 31 of gestation then it plateaued thereafter. Also, the expression in the early preterm (n=2) was six fold higher than that of both late preterm (n=3) and term placenta (n=10) (p<0.05). The uptake of 3H-gentamicin by the BeWo cells was time-dependent, saturable (Vmax=42.9 ± 4.9 nmol/mg protein/min; Km=2.93±0.68mM) and partially inhibited by megalin inhibitors. Conclusion: Megalin is expressed in human placental villous tissues as well as the BeWo cells. When grown on Transwell® plates, the BeWo cells appear to be the most appropriate model to study the in vitro transport of AGs across the apical membrane. Time, temperature and concentration dependence of gentamicin uptake in the BeWo cells indicate protein-mediated transport. The inhibition data are consistent with megalin-mediated endocytosis of AGs.

Agentes causadores de infecções urinárias, endocardites, meningites e septicemias os membros do gênero Enterococcus ganharam grande importância epidemiológica nos últimos anos, já que possuem resistência, tanto intrínseca quanto adquirida, a uma ampla gama de antibióticos. Entre as trinta e seis espécies descritas atualmente, duas recebem maior destaque,E. fecalis e E. faecium devido à alta frequência de multirresistência a antimicrobianos e a sua maior participação nos casos de infecções humanas. Vários estudos tem associado o uso de facilitadores de crescimento em animais de produção com o aumento da frequência de multirresistência em várias espécies de Enterococcus. Diante do exposto, no presente estudo foram avaliadas 245 cepas de Enterococcus faecalis isoladas de 171 suínos comercias quanto ao perfil de resistência a antimicrobianos através da determinação da concentração inibitória mínima e quanto ao perfil genotípico através da eletroforese em campo pulsado. As maiores taxas de resistência observadas foram contra a tilosina (98,7%) e lincomicina (98,7%), seguidas pela tetraciclina (97,1%), eritromicina (96,7%), estreptomicina (96,3%), combinação quinupristinadalfopristina (95,5%), kanamicina (93,8%), gentamicina (85,3%), ciprofloxacina (76,7%) e cloranfenicol (71,8%). Não foram identificadas cepas resistentes a vancomicina e a taxa de resistência a princípios como daptomicina (0,4,%), nitrofurantoína (1,2%) e tigeciclina (1,6%) foi baixa. Através da eletroforese em campo pulsado as cepas foram agrupadas em 109 pulsotipos, não havendo grupamentos diretamente relacionados ao perfil de resistência. As cepas foram agrupadas em maior frequência, de acordo com o animal e a granja de origem. No Brasil, o uso de avoparcina em suinocultura não foi muito intensivo, o que provavelmente não contribuiu para a seleção de cepas resistentes a vancomicina, no entanto, a resistência a altos níveis de gentamicina e estreptomicina é alarmante, e devido à importância destes antimicrobianos no tratamento das infecções humanas por Enterococcus, estes níveis de resistência deveriam ser monitorados em isolados de origem animal e ambiental. / Causative agents of urinary tract infections, endocarditis, meningitis and septicemia members of the genus Enterococcus gained great epidemiological importance in the last years, due to resistance, both intrinsic and acquired a wide range of antibiotics. Among the thirty-six species currently described two receive greater emphasis, E. faecalis and E. faecium due to the high frequency of multidrug resistance to antimicrobial agents and their greater involvement in cases of human infections. Several studies have associated the use of growth promoters in production animals to increase the frequency of multidrug resistance in various species of Enterococci. On the exposed, in the present study, 245 strains of E. faecalis isolated from 171 commercial pigs were evaluated for the antimicrobial resistance profile by determining the minimum inhibitory concentration and for the genotypic profile by pulsed field gel electrophoresis. The highest resistance rates were observed against tylosin (98.7 %) and lincomycin (98.7 %), followed by tetracycline (97.1 %), erythromycin (96.7%), streptomycin (96.3%), a combination quinupristin - dalfopristin (95.5 %), kanamycin (93.8 %), gentamicin (85.3 %), ciprofloxacin (76.7 %) and chloramphenicol (71.8%). Strains resistant to vancomycin were not found, and the rate of resistance to daptomycin (0.4 %), nitrofurantoin (1.2%) and tigecycline (1.6%) was low. By pulsed field gel electrophoresis the strains were grouped into 109 pulsotypes, with no groups directly related to the resistance profile. The strains were grouped into higher frequency, according to the animal and farm of origin. In Brazil, the use of avoparcin in swine production was not very intensive, which probably contribute to the low selection of vancomycin-resistant strains, however, resistance to high levels of gentamicin and streptomycin is alarming, and because of the importance of these antimicrobials in treatment of human infections caused by Enterococcus, these resistance levels should be monitored in isolates of animal and environmental origin.

Antibióticos antimicrobianos são moléculas capazes de inibir o crescimento de microrganismos, na maioria, produtos naturais derivados de fungos ou bactérias que bloqueiam processos cruciais para a sobrevivência de microrganismos. A gentamicina é um aminoglicosídeo 4,6 bisubstituído que exerce papel importante no tratamento de infecções graves causadas por bactérias gram-negativas; apresenta uma complexa e pouco entendida rota de biossíntese. Dessa via, a desidrogenase GenD2 e a aminotransferase GenS2 atuam sobre a posição C3 da gentamicina A2 produzindo gentamicina X2, e a GenB2 atua na posição C6 epimerizando a molécula de gentamicina C2a. As enzimas foram purificadas por cromatografia de afinidade e por exclusão de tamanho, no tampão composto por 50 mM de Tris HCl, 100 mM de NaCl pH 7,7. Com a intenção de compreender a rota de biossíntese da gentamicina, o objetivo deste trabalho foi estudar estruturalmente as enzimas GenD2, GenS2 e GenB2 em complexo com seus cofatores e ligantes. Cristais da enzima GenD2 difrataram, porém não foi possível determinar sua estrutura, análises biofísicas através de calorimetria de titulação isotérmica e por gel filtração analítica, puderam confirmar seu cofator, o NAD+, e um estado oligomérico compatível à um tetrâmero em solução. A enzima GenB2 em complexo com o cofator PMP e gentamicina X2 difratou a uma resolução de 1,7 Å e foi resolvida por substituição molecular, na qual foi identificada uma molécula na unidade assimétrica pertencente ao grupo espacial C 2 2 21. Essa enzima é um homodímero, seu sítio de ligação está na interface entre os protômeros com contribuições de resíduos de ambas as moléculas. A região onde se encontra o anel pirimidínico do PMP é carregada positivamente, favorecendo a interação com o cofator. A cavidade onde se encontra posicionada a gentamicina X2, é bastante ampla e eletronegativa, por seu substrato ser um aminoglicosídeo. A lisina 227 é o resíduo que realiza a clássica base de Schiff, presente em enzimas PLP/PMP dependentes, entre o anel pirimidínico do PMP e a tirosina 124. Os avanços promissores sobre a GenD2 e a compreensão estrutural da GenB2 auxiliam no entendimento da via de biossíntese de gentamicina bem como contribuem para o crescente entendimento sobre o enovelamento de enzimas PLP dependentes. / Antimicrobial antibiotics are molecules capable of inhibiting the growth of microorganisms, most of them are natural products derived from fungi or bacteria that block some crucial processes to the survival of microorganisms. Gentamicin is a 4,6 disubstituted aminoglycoside that plays an important role in the treatment of severe infections caused by gram-negative bacteria. This aminoglycoside presents a complex and poorly understood biosynthesis route. From this route, GenD2 dehydrogenase and GenS2 aminotransferase have been identified to act at C3 position of gentamicin A2 producing gentamicin X2, and the enzyme GenB2 acts at the C6 position, epimerizing the gentamicin C2a molecule. These enzymes were purified by immobilized metal ion affinity and size exclusion chromatography in a buffer composed of 50 mM Tris HCl, 100 mM NaCl pH 7,7. In order to understand the route of gentamicin biosynthesis, this work aims to study the enzymes GenD2, GenS2 and GenB2 in complex with their cofactors and ligands. Some crystals of GenD2 diffracted, however it was not possible to determine its structure. Biophysical analysis by isothermal titration calorimetry and analytical gel filtration; confirmed NAD+ as its cofactor, and an oligomeric state compatible with a tetramer in solution. GenB2 crystals in complex with PMP cofactor and gentamicin X2 diffracted at a resolution of 1.7 Å and its structure was resolved by molecular replacement, which presented one molecule in the asymmetric unit belonging to the space group C 2 2 21. This enzyme is a homodimer, the binding site is located at the interface between protomers and residues from both molecules contribute toward the formation of the active site. The region where the PMP pyrimidine ring is located is positively charged, favoring the interaction with the cofactor. The active site cavity, where gentamicin X2 is positioned, is a large groove and predominantly electronegative, compatible with the substrate of the enzyme, which is an aminoglycoside. Lysine 227 is the key residue that performs the classical Schiff base, commonly present in PLP/PMP dependent enzymes, between the PMP pyrimidine ring and tyrosine 124. Promising advances on GenD2 and the structural understanding of GenB2 aids in the understanding of the gentamicin biosynthetic pathway as well as contribute towards the knowledge about the folding of PLP dependent enzymes.

Agentes causadores de infecções urinárias, endocardites, meningites e septicemias os membros do gênero Enterococcus ganharam grande importância epidemiológica nos últimos anos, já que possuem resistência, tanto intrínseca quanto adquirida, a uma ampla gama de antibióticos. Entre as trinta e seis espécies descritas atualmente, duas recebem maior destaque,E. fecalis e E. faecium devido à alta frequência de multirresistência a antimicrobianos e a sua maior participação nos casos de infecções humanas. Vários estudos tem associado o uso de facilitadores de crescimento em animais de produção com o aumento da frequência de multirresistência em várias espécies de Enterococcus. Diante do exposto, no presente estudo foram avaliadas 245 cepas de Enterococcus faecalis isoladas de 171 suínos comercias quanto ao perfil de resistência a antimicrobianos através da determinação da concentração inibitória mínima e quanto ao perfil genotípico através da eletroforese em campo pulsado. As maiores taxas de resistência observadas foram contra a tilosina (98,7%) e lincomicina (98,7%), seguidas pela tetraciclina (97,1%), eritromicina (96,7%), estreptomicina (96,3%), combinação quinupristinadalfopristina (95,5%), kanamicina (93,8%), gentamicina (85,3%), ciprofloxacina (76,7%) e cloranfenicol (71,8%). Não foram identificadas cepas resistentes a vancomicina e a taxa de resistência a princípios como daptomicina (0,4,%), nitrofurantoína (1,2%) e tigeciclina (1,6%) foi baixa. Através da eletroforese em campo pulsado as cepas foram agrupadas em 109 pulsotipos, não havendo grupamentos diretamente relacionados ao perfil de resistência. As cepas foram agrupadas em maior frequência, de acordo com o animal e a granja de origem. No Brasil, o uso de avoparcina em suinocultura não foi muito intensivo, o que provavelmente não contribuiu para a seleção de cepas resistentes a vancomicina, no entanto, a resistência a altos níveis de gentamicina e estreptomicina é alarmante, e devido à importância destes antimicrobianos no tratamento das infecções humanas por Enterococcus, estes níveis de resistência deveriam ser monitorados em isolados de origem animal e ambiental. / Causative agents of urinary tract infections, endocarditis, meningitis and septicemia members of the genus Enterococcus gained great epidemiological importance in the last years, due to resistance, both intrinsic and acquired a wide range of antibiotics. Among the thirty-six species currently described two receive greater emphasis, E. faecalis and E. faecium due to the high frequency of multidrug resistance to antimicrobial agents and their greater involvement in cases of human infections. Several studies have associated the use of growth promoters in production animals to increase the frequency of multidrug resistance in various species of Enterococci. On the exposed, in the present study, 245 strains of E. faecalis isolated from 171 commercial pigs were evaluated for the antimicrobial resistance profile by determining the minimum inhibitory concentration and for the genotypic profile by pulsed field gel electrophoresis. The highest resistance rates were observed against tylosin (98.7 %) and lincomycin (98.7 %), followed by tetracycline (97.1 %), erythromycin (96.7%), streptomycin (96.3%), a combination quinupristin - dalfopristin (95.5 %), kanamycin (93.8 %), gentamicin (85.3 %), ciprofloxacin (76.7 %) and chloramphenicol (71.8%). Strains resistant to vancomycin were not found, and the rate of resistance to daptomycin (0.4 %), nitrofurantoin (1.2%) and tigecycline (1.6%) was low. By pulsed field gel electrophoresis the strains were grouped into 109 pulsotypes, with no groups directly related to the resistance profile. The strains were grouped into higher frequency, according to the animal and farm of origin. In Brazil, the use of avoparcin in swine production was not very intensive, which probably contribute to the low selection of vancomycin-resistant strains, however, resistance to high levels of gentamicin and streptomycin is alarming, and because of the importance of these antimicrobials in treatment of human infections caused by Enterococcus, these resistance levels should be monitored in isolates of animal and environmental origin.

Introdução: O tratamento dos pacientes com fibrose cística envolve o uso de medicamentos ototóxicos, sendo que os mais frequentemente utilizados são os antibióticos aminoglicosídeos. Devido ao uso frequente deste tipo de medicamento, os pacientes com fibrose cística apresentam risco de desenvolver perda auditiva. Objetivo: o objetivo deste estudo foi avaliar a audição dos pacientes com fibrose cística pela audiometria de altas frequências (AAF) e emissões otoacústicas por produto de distorção (EOAPD). Pacientes e métodos: estudo transversal retrospectivo e prospectivo, incluindo 75 indivíduos, sendo 39 do grupo de estudo e 36 do grupo controle. Foram realizados os exames de AAF (de 250 a 16.000 Hz) e EOAPD. Resultados: o grupo de estudo apresentou limiares na AAF significativamente mais elevados em 250, 1.000, 8.000, 9.000, 10.000, 12.500 e 16.000 Hz. (p=0,004) e maior prevalência de alterações nas EOAPD em 1.000 e 6.000 Hz (p=0,001), com amplitudes significativamente mais baixas em 1.000, 1.400 e 6.000 Hz. Houve associação significativa entre as alterações dos limiares auditivos na AAF com o número de cursos de aminoglicosídeos realizados (p=0,005). Oitenta e três por cento dos pacientes que realizaram mais de 10 cursos de aminoglicosídeos apresentaram perda auditiva na AAF. Conclusão: Um número expressivo de pacientes com fibrose cística que receberam repetidos cursos de aminoglicosídeos apresentou alterações na AAF e EOAPD. realização de 10 ou mais cursos de aminoglicosídeos esteve associada às alterações na AAF. / Introduction: the treatment of patients with cystic fibrosis involves the use of ototoxic drugs, and the most frequently used are the aminoglycoside antibiotics. Due to the frequent use of this drug, cystic fibrosis patients are at risk to develop hearing loss. Objective: the aim of this study was to evaluate the hearing of patients with cystic fibrosis by high frequency audiometry (HFA) and distortion product otoacoustic emissions (DPOAE). Patients and methods: retrospective and prospective crosssectional study including 75 individuals, 39 of the study group and 36 in the control group. HFA (250 – 16,000 Hz) and DPOAE tests were conducted. Results: the study group had thresholds significantly higher in the HFA in 250, 1,000, 8,000, 9,000, 10,000, 12,500 and 16,000 Hz (p=0.004) and higher prevalence of abnormal DPOAE at 1,000 and 6,000 Hz (p=0.001), with significantly lower amplitudes of 1,000, 1,400 and 6,000 Hz. There was a significant association between changes in hearing thresholds in HFA with the number of courses of aminoglycosides performed (p=0.005). Eighty-three percent of patients who completed more than 10 courses of aminoglycosides had hearing loss in HFA. Conclusion: a significant number of patients with cystic fibrosis who received repeated courses of aminoglycosides showed alterations in HFA and DPOAE.