Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

Isolation of extraintestinal pathogenic Escherichia coli from diarrheic dogs and their antimicrobial resistance profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Incidence of non-01 Vibrio cholerae and Aeromonas spp. in fresh water in Araraquara, Brazil

The occurrence of Aeromonas spp., Vibrio cholerae, and Plesiomonas shigelloides in fresh water from various sources in Araraquara, State of São Paulo, Brazil was determined. Samples from ten distinct irrigation systems used in vegetable cultivation, from five distinct streams, from two reservoirs, from one artificial lake, and from three distinct springs were analyzed. All isolates were serotyped and tested for hemolysin, cytotoxin, heat-stable (ST) and heat-labile (LT) enterotoxins production; presence of plasmid; autoagglutination and drug resistance. V. cholerae isolates were also tested for cholera enterotoxin (CT) production, and Aeromonas isolates for suicide phenomenon. No P. shigelloides was found. V. cholerae non 01 was found in five irrigation water samples and in three stream samples. Aeromonas sp. were isolated in two samples of irrigation water, in three streams, and in one reservoir. All the V. cholerae and Aeromonas isolates were positive for P-hemolysin production, and all Aeromonas isolates were positive for suicide phenomenon; cytotoxic activities were observed in two Aeromonas strains. Cholera enterotoxin was not found in eight V. cholerae non-01 isolates tested by the Y-1 mouse adrenal cell. All isolates were also negative for the other virulence markers. Ii cholelerae isolates were found to be sensitive to the majority of drugs tested, while Aeromonas strains presented multiple drug resistance..

Freqüência de hemolisinas anti-A e anti-B em doadores de sangue do Hemocentro de Botucatu

O Sistema ABO foi descoberto em 1900 e permanece até hoje como sendo o sistema mais importante dentro da prática transfusional. A transfusão ABO incorreta pode resultar na morte do paciente, com uma reação hemolítica intravascular, seguida de alterações imunológicas e bioquímicas. Os anticorpos ABO estão presentes nos soros dos indivíduos, dirigidos contra os antígenos A e/ou B ausentes nas hemácias. Embora as transfusões com pequenas quantias de plasmas incompatíveis sejam geralmente consideradas uma prática segura, alguns casos de reações hemolíticas por plasma incompatível são encontrados na literatura. Tendo em vista a pequena quantidade de estudos sobre as hemolisinas anti-A e anti-B e a importância desses anticorpos na prática transfusional, objetivamos neste trabalho verificar a freqüência dessas hemolisinas em doadores de sangue do Hemocentro da Unesp de Botucatu. Foram analisadas 600 amostras de soros de doadores do grupo O para presença ou ausência das hemolisinas anti-A e anti-B. Desses doadores, 77 (12,8%) foram classificados como perigosos por apresentarem em seu soro altos títulos de hemolisinas e 523 (87,2%) como não perigosos por apresentarem baixos títulos. No grupo dos doadores perigosos, 45 (58,4%) foram reativos para hemolisina anti-A, 11 (14,2%) reativos para hemolisina anti-B e 21 (27,2%) reativos para ambas. O título de aglutininas superior a 1/100 já considera o doador O como perigoso. Assim, o teste realizado em nossa rotina é suficiente para detecção de altos títulos fazendo com que os pacientes dos outros grupos sangüíneos não corram o risco de reação transfusional se necessitarem de transfusão sangüínea não-isogrupo.

The role of virulence factors in the outcome of staphylococcal peritonitis in CAPD patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peritoneal Dialysis-Related Peritonitis Due to Staphylococcus aureus: A Single-Center Experience over 15 Years

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prevalence of virulence factors in Escherichia coli strains isolated from the genital tract of healthy cows

Determinou-se a prevalência dos genes de virulência expressando fimbrias, produção de hemolisina, colicina e aerobactina em cepas de Escherichia coli obtidas do trato genital de vacas saudáveis que não apresentam sinais clínicos indicativos de infecção. A presença dos genes responsáveis pela expressão de fimbrias (pap, sfa, afa) foi avaliada através de reação em cadeia da polimerase utilizando primers especificos para cada um dos genes, nenhum deles foi detectado em qualquer uma das cepas isoladas. A prevalência dos fatores de virulência foi de 90,4%, 69,8%, 28,5% para colicina, hemolisina e aerobactina, respectivamente. A análise da patogenicidade das cepas do trato genital pode contribuir para o entendimento do comportamento das cepas de E. coli.

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea

Escherichia coli Shiga toxigênica (STEC) e E. coli Attaching- effacing (AEEC) têm sido associadas à doença diarréica em cachorros. Entre janeiro e dezembro de 2006, 92 cepas de E. coli isoladas de 25 cachorros diarréicos foram examinadas. As cepas foram analisadas para a detecção dos genes produtores de Shiga toxina (stx 1 e stx 2) e da intimina (eae). Por meio de PCR foi observado que sete cepas (7,6%) portavam o gene stx 1, cinco cepas (5,4%) carregavam o gene stx 2 e nenhum cepa apresentou ambos os genes associados. Nove cepas de E. coli (9,8%) apresentaram o gene eae isoladamente. Treze das cepas (62,0%) que apresentaram os genes stx ou eae também apresentaram a produção de a hemolisina. As cepas que apresentaram genes de virulência foram também examinadas em relação à resistência a 12 agentes antimicrobianos. As resistências mais comuns foram para cefalotina (85,7%), estreptomicina (81,0%), amoxicilina (71,4%) e gentamicina (71,4%).

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

O objetivo do trabalho foi determinar a ocorrência de fatores de virulência, tais como, a expressão de fímbrias, produção de hemolisina, colicina e aerobactina em 100 cepas de Escherichia coli isoladas de pacientes ambulatoriais e hospitalizados de um hospital universitário de nível de atendimento terciário, entre os meses de julho e agosto de 2000, que apresentavam sinais clínicos e laboratoriais de infecção do trato urinário (ITU). Foram pesquisados os genes pap, afa e sfa responsáveis pela expressão de fímbrias através da técnica de PCR. A freqüência dos fatores de virulência entre as cepas estudadas foi de 96,0%, 76,0% e 24,0% para hemolisina, aerobactina e colicina respectivamente, e a prevalência dos genes para os sistemas de adesinas fimbriais foi de 32,0%, 19,0% e 11,0% para os genes pap, sfa e afa respectivamente. As cepas isoladas dos pacientes ambulatoriais exibiram um número maior de fatores de virulência quando comparadas com aquelas provenientes de indivíduos hospitalizados.

Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Influence of antimicrobial subinhibitory concentrations on hemolytic activity and bacteriocin-like substances in oral: Fusobacterium nucleatum

Fusobacterium nucleatum is considered for its role in colonization of initial and late microorganisms in dental plaque and for its coaggregation with other bacterial species. It is known that action of different antimicrobial substances may interfere with either virulence factors or with host-bacteria interaction. The goal of this study was to examine the influence of subinhibitory concentrations of chlorhexidine, triclosan , penicillin G and metronidazole on hemolytic activity and bacteriocin-like substance production of oral F. nucleatum. A high resistance to penicillin G was observed and 63% of the isolates were β-lactamase positive. All the tested isolates were susceptible to metronidazole. F. nucleatum isolates grown with or without antimicrobials were alpha-hemolytics. Bacteriocin-like substance production was increased in isolates grown with penicillin G. Impaired production of hemolytic or antagonic substances can suggest a role in the regulation of oral microbiota.

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cryl genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.

Presence of extraintestinal pathogenic Escherichia coli in butcheries in Taquaritinga, SP, Brazil

The study was conducted in twenty-three butcheries in the city of Taquaritinga, State of São Paulo, Brazil, surveyed during a 10 months period. Among two hundred and eighty-seven Escherichia coli strains isolated from samples of ground beef, meat-grinding-machines and the hands of manipulators, five were recognized as extraintestinal pathogenic E. coli (ExPEC), showing virulence factors (P and S fimbriae, hemolysin and aerobactin) and presenting multidrug resistance. Retail-sold food may constitute an important vehicle for the dissemination of ExPEC in communities, giving rise to reasons for concern.