Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

The antimicrobial susceptibility, biofilm formation and genotypic profiles of Staphylococcus haemolyticus from bloodstream infections

We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Biofilm production by multiresistant Corynebacterium striatumassociated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatumbound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Parâmetros microbiológicos indicadores do efeito do diuron sobre a microflora do solo

A biomassa microbiana (Cmic), a respiração básica (RB), as razões Cmic:Corg e o qCO2 foram usados para avaliar o efeito do herbicida diuron, nas dosagens de 0, 2 e 20 mig g-1 , na microflora do solo. O estudo foi conduzido em dois solos com texturas diferentes, e as avaliações, realizadas aos 0, 3, 10, 20 e 33 dias após a aplicação do pesticida. No solo mais argiloso, o Cmic e a razão Cmic:Corg forneceram resultados semelhantes quanto ao efeito prejudicial do diuron. A RB não foi afetada pelo diuron. O qCO2 foi o único parâmetro capaz de demonstrar uma diferenciação no efeito prejudicial entre doses. No solo mais arenoso não se detectou efeito do diuron.

Isolamento de fungos degradadores de carbendazim

Os fungicidas sistêmicos do grupo dos benzimidazóis são amplamente utilizados no controle de muitas doenças de plantas, tanto no solo como na parte aérea, e isso tem sido causa de contaminação ambiental. A ação de alguns microorganismos contribui para degradação e perda da atividade biológica desses fungicidas, podendo reduzir, dessa forma, o risco de impactos negativos. O objetivo deste trabalho foi selecionar fungos com potencial para degradar benomil e seu produto de hidrólise, carbendazim, e quantificar o potencial de degradação. Fungos foram isolados de três diferentes solos da região de agricultura irrigada do município de Guaíra, SP, com histórico de aplicação intensiva de fungicidas benzimidazóis. Dentre os fungos isolados, Alternaria alternata foi a linhagem menos afetada pelo aumento na concentração de benomil no meio de cultura. Na concentração máxima (100 mig mL-1) a inibição da taxa de crescimento de A. alternata foi 22%, enquanto nas demais linhagens foi superior a 45%. Em meio de cultura líquido suplementado com carbendazim, a taxa de crescimento em biomassa de A. alternata foi 43% maior do que a no meio sem carbendazim, o que indica o consumo do fungicida como fonte de carbono. A. alternata degradou rapidamente o fungicida, chegando a 66,21% de desaparecimento do produto em dois dias. A meia-vida de carbendazim nessas condições foi de 1,16 dias.

Evolvimento de dióxido de carbono e mineralização de nitrogênio em Latossolo Vermelho-Escuro com diferentes manejos

Este trabalho foi elaborado com o objetivo de verificar o efeito das palhas de milho e lab-lab, dos tipos de manejos do solo e da presença de uma fonte externa de N, no evolvimento de CO2 e na mineralização de N. Usou-se um Latossolo Vermelho-Escuro distrófico textura argilosa fase cerrado, da região de Sete Lagoas (MG), com histórico de uso envolvendo cinco anos sob plantio direto e plantio convencional com arado de disco e arado de aiveca. Amostras de solo (0-20 cm) foram incubadas a 25ºC durante 55 dias, na presença e na ausência de palhada residual e de fonte externa de N aplicada no início e 25 dias após. De modo geral, a palhada de milho apresentou maior taxa de CO2 evolvido do que o lab-lab (935 e 764 mig CO2 g-1 solo, respectivamente). O tipo de palhada residual influenciou mais o evolvimento de CO2 do que os manejos de solo. A adição de N aumenta a taxa de CO2 evolvida, apenas quando N é aplicado no início do período de incubação. A disponibilidade de N e o tempo de imobilização são alterados pela relação C/N da palhada incorporada e pelo preparo do solo.

Avaliação de programas hormonais para a indução e sincronização do estro em caprinos

O objetivo deste trabalho foi estabelecer alternativas para indução e sincronização do estro em cabras leiteiras manejadas semi-intensivamente. Foram conduzidos quatro experimentos com 411 cabras na Embrapa-Centro Nacional de Pesquisa deCaprinos, Sobral, CE. No protocolo básico, utilizaram-se esponjas intra-vaginais com 50 mg de acetato de medroxiprogesterona (MAP) por dez dias e aplicação intra-muscular de 100 mig de cloprostenol e 200 UI de gonadotropina coriônica eqüina (eCG) no 8º dia; a inseminação artificial (IA), com sêmen congelado foi feita 38 horas após remoção da esponja. No experimento1 substituiu-se a e CG pelo "efeito macho"; no experimento 2 substituiu-se a dose de MAP para 60 mg; no experimento3 compararam-se diferentes momentos de IA: 38, 44 e 50 horas e no experimento 4 substituiu-se a eCG pela gonadotropina humana (hCG). Nenhuma das alternativas testadas modificou (P>0,05) a prolificidade. A IA em cio natural gerou maior (P<0,05) índice de parição no experimento2(67,7%) e no experimento 4 (73,3%). A dose de 60 mg de MAP permitiu realizar a IA mais tarde (44 horas apósretirar a esponja) sem detrimento da fertilidade. A hCG equivaleu a eCG, se aplicada 48 horas antes de retirar a esponja.

Adsorção de boro em solos de várzea do Sul de Minas Gerais

O objetivo deste trabalho foi avaliar a adsorção de B em quatro solos de várzea da região de Lavras, MG, por meio dos parâmetros das isotermas de Langmuir e Freundlich, no período de outubro a novembro de 1998. Amostraram-se solos Aluvial, Glei Pouco Húmico, Glei Húmico, e Orgânico artificialmente drenado, coletados na camada de 0-20 cm e peneirados para 2 mm. Amostras com e sem calagem foram incubadas durante 30 dias. Duplicatas de 4,0 g de solo de cada classe foram acondicionadas em tubos de polietileno com oito diferentes doses de B (0, 2, 4, 8, 12, 16, 24 e 32 mig mL-1) preparadas em CaCl2.2H2O 0,01 mol L-1, com ácido bórico como fonte. Os teores de B na solução de equilíbrio foram determinados pelo método da Azometina-H. Os resultados mostraram que alto teor de matéria orgânica confere ao solo Glei Húmico maior capacidade de adsorver boro. A matéria orgânica, a superfície específica, caulinita e alumínio trocável foram os atributos dos solos que se correlacionaram diretamente com a capacidade máxima de adsorção de B (CMAB). A calagem proporcionou diminuição da CMAB em todos os solos.

Solubilização de fosfatos naturais por microrganismos isolados de cultivos de Pinus e Eucalyptus de Santa Catarina

A utilização de microrganismos solubilizadores tem sido sugerida como alternativa ao uso de fertilizantes fosfáticos. Para serem utilizados num programa de inoculação controlada, os microrganismos devem apresentar grande capacidade e potencial de solubilização. O objetivo deste trabalho foi avaliar a capacidade e o potencial de solubilização de 56 isolados inoculados em meio GES contendo fosfato (Anitápolis, Araxá ou Catalão), usando-se um fatorial 57x3 (56 isolados + testemunha e três fosfatos) conduzido em delineamento completamente casualizado com três repetições. Após 15 dias de incubação, determinaram-se as quantidades de P e o pH do meio. Foram verificados efeitos dos fosfatos, dos isolados e da interação. Baixos valores de pH foram obtidos por isolados que apresentaram médio a alto potencial. O teor médio de P foi superior no fosfato de Anitápolis, seguido do Araxá. Trinta e um isolados solubilizaram quantidades significativas. O 310 apresentou o mais alto potencial (média de 263 mig mL-1 de P). Quatro isolados (177, 262, 251 e 269) apresentaram alto potencial (120 a 150 mig), e doze (201, 309, 199, 195, 249, 202, 198, 305, 253, 196, 203 e 307), mostraram valores médios (80 a 120 mig). O comportamento dos isolados foi diferente entre os fosfatos. Apenas quatro isolados solubilizaram os três fosfatos (310, 251, 199 e 249). As características apresentadas pelos isolados 310, 251, 199 e 249 os qualificam para um programa de seleção visando à inoculação controlada.

A técnica da reflectância difusa aplicada ao estudo da fluorescência de 2,3-naftalimidas n-substituídas com grupos alquila incluídas em b-ciclodextrina e adsorvidas em celulose microcristalina

The solution fluorescence of N-alkyl-2,3-naphthalimides (1-4) in polar protic and aprotic solvents was compared to the emission from solid samples resulting from the imide complexation with b-cyclodextrin or adsorption on the surface of microcrystalline cellulose. Solid samples of the inclusion complex 2,3-naphthalimides/b-cyclodextrin show maximum for fluorescence emission significantly different to the observed in methanolic solution. Beside this, a clear effect on the alkyl chain length could be observed for these samples which is probably due to differences in probe location inside the cyclodextrin cavity. The constancy for fluorescence quantum yield and fluorescence lifetime for the imides 1 - 4 adsorbed on microcrystalline cellulose suggests that, independently of the polarity of the solvent used for sample preparation, the probe is preferentially located on the cellulose surface. An increase of fluorescence quantum yield and fluorescence lifetime for solid samples, when compared to the values obtained in solution for the different solvents employed in this study (acetonitrile, methanol and water), is fully in accordance with a decrease of the probe mobility due to inclusion in b-cyclodextrin or to adsorption on cellulose.

Injetor multicanal com válvulas de estrangulamento para análise em fluxo

An important component for the automation of flow injection analysis (FIA) systems is the sample injection valve. A simple and inexpensive commutator with 16 pinch valves (8 normally open and 8 closed) was developed and configured as a multichannel injection valve. It is activated by a single solenoid of 3 Kgf, powered by a pulsed driver circuit, controlled by a microcomputer or a switch. FIA with spectrophometric detection of potassium dichromate solution was used for the evaluation of the new injection valve and its comparison with other valves, for sample loops of 50, 100, 200, 300 and 500 muL. The repeatability was favorable (RSD 1.0% for 15 injections at each loop volume) compared to a manual injector, an electropneumatic injector and an injector configured with three mini solenoid valves (RSD 1.1, 1.3 and 1.0%, respectively, for15 injections at each loop volume).

Aplicação da espectroscopia de ressonância magnética nuclear para estudos de difusão molecular em líquidos: a técnica DOSY

Diffusion coefficients provide uniquely detailed and easily interpreted information on molecular organization and phase structure. They are quite sensitive to structural changes, and to binding and association phenomena, in particular for liquid colloidal or macromolecular systems. This paper describes the principles of diffusion measurements in liquids by pulsed magnetic field gradient spin-echo (PFG-SE) NMR spectroscopy. The important PFG-SE technique known as DOSY is presented and discussed. This is a noninvasive technique that can provide individual multicomponent translational diffusion coefficients with good precision in a few minutes, without the need for radioactive isotopic labelling.