Effets des fluorures sur l'activité antimicrobienne des antibiotiques contre Staphylococcus aureus et Pseudomonas aeruginosa

L’émergence des souches bactériennes résistantes aux antibiotiques est un phénomène inquiétant, qui se répand à travers le monde. Staphylococcus aureus et Pseudomonas aeruginosa sont des bactéries pathogènes opportunistes multi résistantes qui peuvent causer plusieurs maladies. Cependant, ces bactéries deviennent difficiles à traiter avec des antibiotiques sans occasionner de toxicité. Alors pour trouver des solutions, c’est nécessaire de développer de nouvelles molécules afin de combattre les agents pathogènes résistants. Grâce à leur action pharmacologique, les fluorures exercent un certain effet antibactérien au niveau de l'émail des dents; donc, leur association aux antibiotiques pourrait bien a méliorer l’activité antimicrobienne. De ce fait, nous nous sommes proposés d’étudier les activités in vitro de la vancomycine (VAN), l’oxacilline (OXA), la ceftazidime (CFT) et la méropenème (MER) libre ou associée au fluorure de sodium (NaF) et fluorure de lithium (LiF) qui ont été évaluées sur des souches S.aureus et P.aeruginosa sensibles et résistantes, par la méthode de la microdilution en bouillon, déterminant leur concentration minimale inhibitrice (CMI), leur concentration minimale bactéricide (CMB), leur courbe cinétique (Time-Kill). Leur cytotoxicité sur les globules rouges humains, et leur stabilité à la température de 4°C et 22°C ont été étudiées. Les associations des antimicrobiens aux dérivés des fluorures ont montré une amélioration de l’effet des antibiotiques par la réduction des leurs concentrations et toxicité pour traiter correctement ces pathogènes résistants. Par conséquent, des antibiotiques associés aux dérivés de fluorure pourraient devenir une option de traitement contre des souches résistantes afin de diminuer la toxicité causée par de fortes doses des antibiotiques conventionnels.

Detection and quantification of staphylococcus aureus enterotoxin B in food product using isotopic dilution techniques and mass spectrometry

L’entérotoxine B staphylococcique (SEB) est une toxine entérique hautement résistante à la chaleur et est responsable de plus de 50 % des cas d’intoxication d’origine alimentaire par une entérotoxine. L’objectif principal de ce projet de maîtrise est de développer et valider une méthode basée sur des nouvelles stratégies analytiques permettant la détection et la quantification de SEB dans les matrices alimentaires. Une carte de peptides tryptiques a été produite et 3 peptides tryptiques spécifiques ont été sélectionnés pour servir de peptides témoins à partir des 9 fragments protéolytiques identifiés (couverture de 35 % de la séquence). L’anhydride acétique et la forme deutérée furent utilisés afin de synthétiser des peptides standards marqués avec un isotope léger et lourd. La combinaison de mélanges des deux isotopes à des concentrations molaires différentes fut utilisée afin d’établir la linéarité et les résultats ont démontré que les mesures faites par dilution isotopique combinée au CL-SM/SM respectaient les critères généralement reconnus d’épreuves biologiques avec des valeurs de pente près de 1, des valeurs de R2 supérieure à 0,98 et des coefficients de variation (CV%) inférieurs à 8 %. La précision et l’exactitude de la méthode ont été évaluées à l’aide d’échantillons d’homogénat de viande de poulet dans lesquels SEB a été introduite. SEB a été enrichie à 0,2, 1 et 2 pmol/g. Les résultats analytiques révèlent que la méthode procure une plage d’exactitude de 84,9 à 91,1 %. Dans l’ensemble, les résultats présentés dans ce mémoire démontrent que les méthodes protéomiques peuvent être utilisées efficacement pour détecter et quantifier SEB dans les matrices alimentaires. Mots clés : spectrométrie de masse; marquage isotopique; protéomique quantitative; entérotoxines

Biofilm Forming Multi Drug Resistant Staphylococcus spp. Among Patients with Conjunctivitis

Biofilm forming multidrug resistant Staphylococcus spp. are major reservoirs for transmission of ophthalmic infections. They were isolated from ocular patients suffering from conjunctivitis. In this study we analyzed biofilm forming ability, antibiotic resistance profile of the Staphylococcus spp. isolated from clinical ocular patients, and their phylogenetic relationship with other community MRSA. Sixty Staphylococcus spp. strains isolated from clinical subjects were evaluated for their ability to form biofilm and express biofilm encoding ica gene. Among them 93% were slime producers and 87% were slime positive. Staphylococcus aureus and S. epidermidis were dominant strains among the isolates obtained from ocular patients. The strains also exhibited a differential biofilm formation quantitatively. Antibiotic susceptibility of the strains tested with Penicillin G, Ciprofloxacin, Ofloxacin, Methicillin, Amikacin, and Gentamicin indicated that they were resistant to more than one antibiotic. The amplicon of ica gene of strong biofilm producing S. aureus strains, obtained by polymerase chain reaction, was sequenced and their close genetic relationship with community acquired MRSA was analyzed based on phylogenetic tree. Our results indicate that they are genetically close to other community acquired MRSA

Variación del estado de portador de Staphylococcus aureus en una población de estudiantes de medicina

Para evaluar en estudiantes de medicina la variación del estado de portador de Staphylococcus aureus y su resistencia antimicrobiana, antes y después de la práctica clínica, se realizó un estudio longitudinal en una cohorte de 159 estudiantes de cuarto y noveno semestre universitario. Se tomaron muestras de las zonas periamigdalianas y/o pared posterior de orofaringe, de las fosas nasales y las manos, se cultivaron en agar sangre de cordero al 5% y se incubaron en aerobiosis a 37°C, durante 48 horas. La identificación de Staphylococcus aureus se realizó según las características macroscópicas y pruebas bioquímicas. La susceptibilidad a los antimicrobianos se evaluó mediante el método de difusión de disco, por la técnica de Kirby-Bauer, siguiendo las normas internacionales del Clinical and Laboratory Standards Institute (CLSI), con los siguientes antimicrobianos: ciprofloxacina, vancomicina, oxacilina, cefalotina,clindamicina y rifampicina. La edad promedio de los alumnos de cuarto semestre fue 19,1±1,2 años y el género femenino fue 2/1 más frecuente que el masculino. Se analizaron la presencia de antecedentes como: infecciones, alergias, estado de fumador, otras patologías no infecciosas, uso de antibióticos en los últimos tres meses y procedimientos quirúrgicos u hospitalizaciones seis meses previos a la toma de las muestras. No hubo relación significativa entre la incidencia del estado de portador y los antecedentes estudiados. Se observó un aumento significativo del 15,1%, con respecto al grupo de estudiantes de cuarto semestre, en el estado de portador de S. aureus en el grupo de estudiantes de noveno semestre, después de haber estado expuestos durante tres años al ambiente hospitalario, (p=0,001 Test exacto de Mc Nemar). De los portadores, el 16,4% presentó la bacteria en manos (p<0,001), el 13,8% en fosas nasales (p=0,0015) y el 3,2% en faringe. Por otra parte,el 35,8% de los portadores presentó persistencia, de los cuales el 25,2% fue en fosas nasales; el 4,4%, en faringe y el 3,8% en manos. En cuanto a la resistencia a los antimicrobianos, el 1,9% de las cepas aisladas de los estudiantes de cuarto semestre presentó resistencia: una a ciprofloxacina y dos a clindamicina (tres estudiantes). Por su parte, el 2,5% de las cepas aisladas de estudiantes de noveno semestre fue resistente: una a cefalotina, ciprofloxacina, oxacilina y clindamicina, una a cefalotina y oxacilina y dos a clindamicina (cuatro estudiantes). En el 1,3% del grupo estudiado se aislaron cepas de Staphylococcus aureus Resistentes a la Meticilina (MRSA, por sus siglas en inglés). Estos resultados no muestran diferencias significativas (p=1.000). 

The staphylococcus aureus surface protein IsdA mediates resistance to the innate defenses of human skin

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Staphylococcus aureus MnhF mediates cholate efflux and facilitates survival under human colonic conditions

Resistance to the innate defences of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common coloniser of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent anti-microbial activity. The mechanisms by which S. aureus is able to resist such defences in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates efflux of radiolabelled cholic acid in both S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated survival of S. aureus in an anaerobic three stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine.

Variabilidades fenotípica e genotípica de estirpes de Staphylococcus aureus isoladas em casos de mastite subclínica bovina

Foram submetidas a PCR-Ribotipagem e aos testes de sensibilidade in vitro frente a 12 antimicrobianos 77 estirpes de Staphylococcus aureus isoladas em amostras de leite procedentes de 40 vacas da raça holandesa que apresentaram mastite subclínica, em uma propriedade rural localizada no Estado de São Paulo, Brasil. Os resultados obtidos revelaram quatro diferentes padrões de resistência a antimicrobianos, sendo observada a predominância de resistência à lincomicina entre 19 (24,7%) estirpes de S.aureus. As 58 (75,3%) estirpes restantes foram sensíveis aos 12 antimicrobianos testados. A PCR-ribotipagem revelou a ocorrência de nove padrões genotípicos distintos, além de apresentar uma capacidade discriminatória maior (D = 0,82) que a obtida nos antibiogramas (D = 0,42). Entre as 19 estirpes resistentes aos antimicrobianos, 14 (73,7%) foram agrupadas em três padrões de ribotipagem e, destas, 13 (92,9%) apresentaram resistência à eritromicina e à lincomicina, isoladamente ou em associação. O grande número de ribotipos e de padrões de resistência a antimicrobianos observados nesta propriedade demonstrou que há grande heterogeneidade genética em populações naturais de S. aureus, fato este que deve ser levado em consideração em programas de controle da mastite bovina.

Minimum inhibitory concentrations of Staphylococcus aureus recovered from clinical and subclinical cases of bovine mastitis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ocorrência e determinação do perfil de sensibilidade às drogas das cepas de Staphylococcus aureus isoladas de fossas nasais e orofaringe dos servidores hospitalares

Staphylococcus aureus is a very important hospital and community pathogen. This species is related to supurative disease, systemic and widespread metastatic lesions. The ability of S. aureus to develop resistance to antibiotics, more recently to methicillin, associates this bacteria with epidemic outbreaks of severe nosocomial infection. The source of staphylococcal infection is a patient with a staphylococcal lesion or a career member of the hospital staff. We aimed to detect the frequency of S. aureus isolated from anterior nares and oral cavities among the hospital staff in Bauru - SP, and to determine the antibiotic susceptibility of the isolates. Within 213 of the staff members analyzed. S. aureus was found in 94 (44.13%) of careers, with 47 (50%) of nasal carriers, 23 (24.4%) of oral carriers and 24 (25.5%) of both carriers. The biochemical characteristics analyzed for the species identification were similar to S. aureus ATCC 29213. All strains identified as S. aureus showed varied sensibility to the antimicrobial agents tested. No vancomicin resistant strains and only 8 (8.5%) strains with oxacilin resistance were found.

Risk of peritonitis during peritoneal dialysis in carriers of Staphylococcus aureus and coagulase-negative staphylococci

The presence of Staphylococcus aureus in the nasal cavities and pericatheter skin of peritoneal dialysis patients put them at high risk of developing peritonitis. However, it is not clear whether the presence of coagulase-negative staphylococci (CNS) in the nasal passages and skin of patients is related to subsequent occurrence of peritoneal infection. The aim of the present study was to verify the relationship between endogenous sources of S. aureus and CNS and occurrence of peritonitis in patients undergoing peritoneal dialysis. Thirty-two patients on peritoneal hemodialysis were observed for 18 months. Staphylococcus species present in their nasal passage, pericatheter skin and peritoneal effluent were identified and compared based on drug susceptibility tests and dendrograms, which were drawn to better visualize the similarity among strains from extraperitoneal sites as well as their involvement in the causes of infection. Out of 288 Staphylococcus strains isolated, 155 (53.8%) were detected in the nasal cavity, 122 (42.4%) on the skin, and 11 (3.8%) in the peritoneal effluent of patients who developed peritonitis during the study. The most frequent Staphylococcus species were CNS (78.1%), compared with S. aureus (21.9%). Among CNS, S. epidermidis was predominant (64.4%), followed by S. warneri (15.1%), S. haemolyticus (10.7%), and other species (9.8%). Seven (64%) out of 11 cases of peritonitis analyzed presented similar strains. The same strain was isolated from different sites in two (66%) out of three S. aureus infection cases. In the six cases of S. epidermidis peritonitis, the species that caused infection was also found in the normal flora. From these, two cases (33%) presented highly similar strains and in three cases (50%), it was difficult to group strains as to similarity. Patients colonized with multidrug-resistant S. epidermidis strains were more predisposed to infection. Results demonstrated that an endogenous source of S. epidermidis could cause peritonitis in peritoneal dialysis patients, similarly to what has been observed with S. aureus.

Perfil de resistência a antimicrobianos de Staphylococcus aureus isolados de granjas e frigoríficos de suínos

Staphylococcus aureus are involved in a wide range of clinical problems to swine industry as son in humans. Epidemiological researchs prove his potential to acquire resistantence to antibiotics. Nowadays, methicillin-resistant S. aureus (MRSA) are responsabilized for nosocomial infections and many studies are done because MRSA are spread to extra hospitalar enrivonment and frequentely isolated from domestic animals including pigs. The aim of this study was to determine the presence o S. aureus at swine farms and identify the mecA, icaA and icaD genes and the resistant proflife to antibiotics. Overal, 458 swabs were taked from five pigeris and two slautherhouses. All the samples were placed on Braid - Parker and blood agar follow by biochemical analyses. The suspect colonies were submitted to PCR to confirm the S. aureus species, by the detection of the coa gene, mecA to avaible meticillin-resistant as son to the virulence gens icaA and icaD that can determine slime production. Antibiogram were done to evaluate the response to 11 antibiotics. All pigeris and slautherhouse were positive and 81 (79%) samples were S. aureus positive including four isolates from pigs employeers. The mecA gene was not detected. The icaD gene was most frequent and 41% were positive to both genes. The antibiogram show a lot of samples penicillin and tetraciclin resistant. Most of the samples were multirestant.

Condições higiênico-sanitárias de alimentos prontos para o consumo servidos em escolas públicas de um município da região noroeste do Estado de São Paulo e perfil de sensibilidade das cepas de Staphylococcus aureus e Escherichia coli isoladas frente a agentes antimicrobianos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de cepas de Staphylococcus aureus isoladas de amostras nasais e linguais de portadores voluntários adultos saudáveis

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Caracterização da resistência à oxacilina em Staphylococcus aureus isolados de feridas de pacientes atendidos em Unidades Básicas de Saúde da cidade de Botucatu

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)