Comparison of the hemolytic activity between C. albicans and non-albicans Candida species

The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

Mecanismo de acción de la toxina alfa hemolisina de Escherichia coli / Action mechanism of Escherichia coli alpha-hemolysin toxin / Mecanismo de ação da toxina alfa-hemolisina de Escherichia coli

Escherichia coli es una de las bacterias anaerobias facultativas más predominantes en el intestino, siendo, en la mayoría de los casos, inocua para el huésped. Existen cepas que traslocan al torrente sanguíneo causando enfermedades extraintestinales como infecciones urinarias, septicemia y meningitis. Dentro de éstas se encuentran las cepas uropatogénicas (Uropathogenic Escherichia coli: UPEC), que secretan varios factores de virulencia. Estos últimos incluyen: toxinas, sistemas de adquisición de hierro, adhesinas y antígenos capsulares. Las principales toxinas secretadas son: alfa-hemolisina (HlyA) y el factor necrotizante citotóxico 1 (CNF-1). En esta revisión se presenta una descripción exhaustiva de HlyA, incluyendo su síntesis, maduración y exportación desde la bacteria. La acilación de la proteína en dos residuos internos de lisina la convierte en una toxina muy virulenta al exponer regiones intrínsecamente desordenadas que son esenciales en diferentes pasos del mecanismo de acción de la misma. Específicamente, la exposición de estas regiones está involucrada en interacciones proteína-proteína dentro del proceso de oligomerización. La formación del oligómero es responsable de la permeabilidad inducida en las células blanco. Finalmente, basado en los conocimientos acerca de las características estructurales y funcionales de HlyA, se presentan potenciales usos de HlyA en terapias basadas en toxinas.

Escherichia coli is one of the predominant species of facultative anaerobes in the human gut, and in the majority of the cases it is harmless to the host. Some strains of this species can translocate to blood and cause infection such as urinary infection, septicemia and meningitis. These are the uropathogenic E. coli strains (UPEC) that secrete a number of virulence factors. The latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens. Secreted toxins include HlyA, the cytotoxic necrotizing factor-1 (CNF-1). In this review an exhaustive description of the toxin has been delineated, including its synthesis, maturation, and export from the bacteria. The acylation of the protein at two internal lysine residues gives the toxin its virulence, by exposing intrinsic disordered regions that are essential in different steps of the toxin's mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsG-ible for the permeability induced in all the target cells. Based on the already known structural and functional characteristics of HlyA, the potential use in toxin-based therapy is presented.

Escherichia coli é uma das bactérias anaérobias facultativas mais predominantes no intestino, sendo na maioria dos casos inócua para o hóspede. Há cepas que passam ao torrente sanguíneo causando doenças extraintestinais como infecção urinária, septicemia e meningite. Dentro destas se encontram as cepas uropatogênicas (Uropathogenic Escherichia coli: UPEC) que secretam varios fatores de virulência. Estos últimos incluem: toxinas, sistemas de aquisição de ferro, adesinas e antígenos capsulares. As principais toxinas secretadas são: alfa hemolisina (HlyA) e o fator necrotizante citotóxico 1 (CNF-1). Nesta revisão apresenta-se uma descrição exaustiva de HlyA incluindo sua sintese, seu amadurecimento e exportação a partir da bactéria. A acilação da proteína em dois residuos internos de lisina a transforma numa toxina muito virulenta ao expor regiões intrinsecamente desordenadas que são essenciais em diferentes passos do mecanismo de ação da mesma. Especificamente, a exposição destas regiões esta envolvida em interações proteína-proteína dentro do processo de oligomerização. A formação do oligômero é responsável pela permeabilidade induzida nas células alvo. Finalmente, com base nos conhecimentos acerca das características estruturais e funcionais de HlyA, apresentam-se potenciais usos de HlyA em terapias baseadas em toxinas.

Genes de virulencia y bacteriocinas en cepas de Enterococcus faecalis aisladas desde diferentes muestras clínicas en la Región del Maule, Chile / Virulence genes and bacteriocins in Enterococcus faecalis strains isolated from different clinical samples in Maule Region, Chile

The presence of virulence genes (VG) and bacteriocins from different clinical samples was studied in Enterococcus faecalis isolated from urinary tract infections (UTI), bacteremia and endodontitis and was correlated with haemolysin and gelatinase activity. We evaluated the presence of VG by PCR in 150 strains of E. faecalis including cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA andentl071. Haemolysin and gelatinase activity was studied. gelE and cylA genes expressed hemolysin and gelatinase, respectively. This activity was observed in some strains of bacteremia, UTI and endodontitis. The highest number of VG was detected in bacteremic strains, being aggA and entA genes the most frequent. efaA, esp, entA, entL50A/B were associated with their clinical origin (p < 0.05). The most common genetic profile was aggA-eep-enlA-entL50A/B. E. faecalis from UTI, bacteremia and endodontitis presented different gene combinations. Some of the genes studied were related to their clinical origin. The results obtained in this study are similar to those reported in other countries.

Desde diferentes muestras clínicas se determinó la presencia de genes codificantes de factores de virulencia (FV) y bacteriocinas en Enterococcus faecalis aislados desde infecciones del tracto urinario (ITU), bacteriemias y endodontitis, correlacionándose con la actividad hemolisina y gelatinasa. En 150 cepas de E. faecalis fue evaluada mediante RPC la presencia de cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA, y ent1071 determinándose actividad hemolisina y gelatinasa. Los genes cylA y gelE expresaron hemolisina y gelatinasa, respectivamente. Esta actividad fue observada en algunas de las cepas causantes de bacteriemia, ITU y endodontitis. El mayor número de genes estudiados se detectó en cepas bacteriémicas. Los genes aggA y entA, fueron los más frecuentes. Los genes efaA, esp, entL50/AB y entA se asociaron a su origen clínico (p < 0,05). El perfil genético más recurrente fue aggA-eep-enlA-entL50A/B. Enterococcusfaecalis de ITU, bacteriemias y endodontitis presentaron distintas combinaciones génicas. AAlgunos de los genes estudiados se relacionaron con su origen clínico. Los resultados obtenidos son similares a los reportados en otros países.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29 percent of the strains. Enterohaemolysin production was detected in 29 percent of the strains. Most of the bacterial strains (95 percent) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50 percent of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections

A total of 120 strains of Pseudomonas aeruginosa, isolated from cystic fibrosis (CF) patients (n = 80) and from patients having extra-pulmonary infections (n = 40) were studied regarding the presence of some virulence factors (hemolysin, gelatinase and elastase production) and presence of the algD and algU genes as detected by polymerase chain reaction-PCR. There was not a significant difference for the production of gelatinase and hemolysin between non-mucoid strains from CF patients and other isolates from extra-pulmonary infections and mucoid strains. The production of elastase was found to be significant among these strains. The algD gene was detected by PCR in all studied strains but the algU gene was detected only in 25 percent of the mucoid strains. Conclusion withdrawn from the results were: (i) hemolysin and gelatinase production although present in many strains of P aeruginosa should not be considered as general virulence factors for the mucoid phenotype but could help in the pathogenic process; (ii) elastase production could be a necessary virulence factor for the initial pathogenesis process; (iii) mucoid and non-mucoid phenotypes could also be expressed according to the host's tissues or environment, and finally, (iv) more than one regulator system for alginate production is probably present in each strain.

Effect of exposure to hydrogen peroxide on the virulence of Escherichia coli.

PURPOSE: To eliminate pathogenic bacteria, the host presents conditions that are stressful for bacteria. Oxidative stress arises when the concentration of pro-oxidants like hydrogen peroxide (H2O2 ) and superoxide anion increases to a level over the basal defence capacity of the cell. In the present study, we studied the effect of oxidative stress on the production of certain virulence factors by Escherichia coli . METHODS: E. coli was exposed to oxidative stress by growing in the presence of different concentrations of H2O2 . The effect of oxidative stress on the expression of surface hydrophobicity, adherence, haemolysin production, serum resistance and phagocytosis was studied. RESULTS: Oxidative stress caused a significant decrease in the expression of all the virulence factors of E. coli . CONCLUSIONS: Synthesis of virulence factors can be significantly altered by oxidative stress and such changes may affect the pathogenicity of E. coli.

Enzymatic and hemolytic activities of Candida dubliniensis strains

Candida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl2; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g percent CaCl2, the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).

C. dubliniensis é uma levedura oportunista que, embora já tenha sido isolada de vários sítios anatômicos é, com maior frequência, encontrada na boca de pacientes infectados pelo HIV. Embora tenham sido realizados numerosos estudos sobre a epidemiologia e filogenia, seus fatores de virulência como atividade exoenzimática e atividade hemolítica, são, ainda, pouco conhecidos. Neste estudo comparou-se a atividade in vitro de proteinase, hialuronidase, condroitin sulfatase e atividade hemolítica de 18 cultivos de C. dubliniensis com 30 cultivos de C. albicans, todos isolados de pacientes com SIDA. Foi evidenciada maior atividade de proteinase em C. albicans em relação a C. dubliniensis (p < 0,05). Todos os isolados de C. dubliniensis evidenciaram atividade de hialuronidase e condroitin-sulfatase de forma similar ao observado com C. albicans (p > 0,05). Constatou-se que a atividade hemolítica foi influenciada pelo CaCl2; em sua ausência não foram observadas diferenças na atividade hemolítica das duas espécies; todavia, ao se agregar 2,5 por cento de CaCl2, a atividade hemolítica de C. dubliniensis foi reduzida enquanto a de C. albicans, estimulada (p < 0,05).

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil / Fatores de virulência de Escherichia coli uropatogênicas provenientes de um Hospital Universitário em Ribeirão Preto, São Paulo, Brasil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0 percent, 76.0 percent, 24.0 percent, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0 percent, 19.0 percent and 11.0 percent for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

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 por cento, 76,0 por cento e 24,0 por cento para hemolisina, aerobactina e colicina respectivamente, e a prevalência dos genes para os sistemas de adesinas fimbriais foi de 32,0 por cento, 19,0 por cento e 11,0 por cento 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.

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum.

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity

Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil. Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals. Although rare, infections with C. violaceum are characterized by rapid dissemination and high mortality. With the complete genome sequence of C. violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed. Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged. The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis. We described a number of genes involved in infectious processes, such as host cell adhesion, [quot ]contact-dependent secretion[quot ] of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins. We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response

Serum resistance of Escherichia coli strains causing urinary tract infection and diarrhoea in relation to alpha haemolysin production and O type.

A total of 46 alpha-hemolytic and 40 non-hemolytic clinical isolates of Escherichia coli were collected from pediatric patients with urinary tract infection and diarrhoea. Of 39 (84.7%) alpha-hemolytic strains and 27 (67.5%) non-hemolytic strains were resistant to 10% serum and there was no significant difference between urinary and stool isolates. On the contrary when 100% serum was used, 22 (47.8%) of the alpha-hemolytic and 7 (17.5%) of the non-hemolytic strains were resistant (p<0.01). and significantly greater resistance was found in urinary tract infection than from the stool samples (47% versus 24%, p<0.01). Serum resistance was higher in serogroups O6, O18 and O75. Production of alpha-hemolysin was more frequent in serogrops O2, O6, O8, O18 and O75. Thus, the resistance to human serum can determine clinical significance of Escherichia coli from different sources and alpha-hemolysin contributes to the virulence of Escherichia coli in initiation and perpetuation of clinical infection.

Uropathogenic Escherichia coli--a preliminary study.

Urinary isolates of Escherichia coli were studied for presence of haemolysin, adhesins, serum resistance and O serotype prevalence. Of the 144 isolates studied, 72 exhibited hemolysin, 7 were resistant to bactericidal effect of serum and 50 strains showed Mannose resistant Haemagglutination (MRHA). O101,O68,O04 and O25 were the commonest serotypes in this study.

Haemolysin production by environmental isolates of Vibrio parahaemolyticus from Andamans.

Sixty marine water samples were collected from various coastal sites around Port Blair at different times during August 1996 to July 1997. The specimens were subjected to standard procedure for isolation and identification of V. parahaemolyticus. Forty four V. parahaemolyticus isolates were detected from these specimens and all showed clear haemolysis on Wagatsuma agar plates. The haemolytic activity was abolished by heating the culture supernatants at 60 degrees C for 10 min and enhanced when plates were kept at 4 degrees C. When isolates were subjected to PCR assay for tdh gene, only one showed the presence of the gene. The results indicate the existence of pathogenic V. parahaemolyticus in the marine environment of these islands.

Incidence of Aeromonas isolated from diarrhoeal children and study of some virulence factors in the isolates.

Stool samples from 305 children with diarrhoea and equal number of age and sex matched non-diarrhoeal control children, less than 5 years of age, were examined during the period from Sept 1988 to April 1989. Aeromonas spp. were isolated from 37 (12.1%) diarrhoeal and 05 (1.6%) control cases. Out of 37 diarrhoeal isolates 13 (35.1%) were A. hydrophila, 19 (51.1%) A. sobria and 05 (13.5%) A. caviae. All the isolated strains were tested for haem agglutination property and haemolysin production. Seventeen diarrhoeal and 05 control isolates were tested for cytotoxin production in He La cell line and enterotoxin production in rat ileal loop model and suckling mouse model. Chinese hamster ovary cell (CHO) assay and Gm-1 ELISA methods were also employed. Cytotoxin production was found in 82.5% of diarrhoeal and 40% of control isolates. Haemagglutination was found in 62.1% of Aeromonas isolated from diarrhoeal children and 20% from control children. Enterotoxin production was detected in 58.8% diarrhoeal and none of the control isolates by either of the methods. Of the virulence factors enterotoxin production was found to correlate well with enteropathogenicity but haemolysin, cytotoxin and haemagglutinin did not.

Virulence factors of Eschericha coli in urinary isolates

Escherichia coli strains isolated from 100 urine samples taken from patients with urinary tract infections (UTI) and from 20 normal fecal (NF) samples were examined for serum resistance, mannose-resistant hemagglutination of human erythrocytes (MRHA) and for production of aerobactin, hemolysis and colicin. Among the UTI E. coli strains, 79% produced aerobactin, 69% showed serum resistance, 44% produced MRHA, 32% were beta-hemolytic and 22% were colicinogenic. A greater proportion of UTI E. coli strains produced aerobactin, colicin V, beta-hemolysis and MRHA when compared to NF strains. Production of MR hemagglutins was significant correlated with that of aerobactin and hemolysin. These results suggest that the presence of aerobactin may be a significant etiological factor in UTI, and that the production of MR adhesins and of hemolysin also might contribute to the virulence of these strains

Antibiotic resistance, haemagglutination type & haemolysin production in relation to serogroups of uropathogenic Escherichia coli.

A total of 56 urinary isolates of Esch. coli were characterised according to serotype, haemagglutination (HA) type, production of beta haemolysin and antibiotic resistance pattern. Forty five strains were serotyped with prevalence of 057 followed by other serotypes. Eleven different Esch. coli serotypes were found to have mannose resistant haemagglutinating property (MRHA) and just five strains showed haemolysin production (Hly+). Multidrug resistance was common with preponderance of ampicillin, co-trimoxazole and tetracycline resistance. No correlation between serogroup, HA type, haemolysin production and antibiotic resistance was found.

Production of enterotoxin and cytotoxin by aeromonas veronii

Fourteen strains of Aeromonas veronii were isolated from salt and fresh water in Rio de Janeiro and investigated for the presence of birulence factors. Eleven (79%) A. veronii strains were positive for enterotoxin by the suckling-mouse test and thirteen (93%) produced hemolysin. Of the 13 A. veronii strains that produced hemolysin, seven were investigated for cytotoxin production using Vero cells as indicator cells and all of them were positive