RESUMO
Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a "vacuolating factor" in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae.
Assuntos
Citotoxinas/toxicidade , Haemophilus influenzae/metabolismo , Vacúolos/microbiologia , Células Cultivadas , Citotoxinas/biossíntese , Haemophilus influenzae/ultraestrutura , Microscopia Eletrônica de Transmissão , Vacúolos/ultraestruturaRESUMO
In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.
Com o objetivo de selecionar rizobactérias produtoras de fitotoxinas para uso no controle de plantas daninhas, vinte e cinco isolados bacterianos previamente obtidos das rizosferas de diferentes plantas foram cultivados em meio líquido e, após remoção das células por centrifugação, as fases líquidas foram liofilizadas e os resíduos obtidos foram submetidos à extração com acetato de etila/metanol. Os extratos foram concentrados sob vácuo até secura e dissolvidos em água e solução de sacarose para serem submetidos a testes in vitro de germinação de sementes de alface (Lactuca sativa L.) e de crescimento de coleóptilos de trigo (Triticum aestivum L.). Embora a maior parte das amostras tenha desfavorecido o crescimento dos coleóptilos de trigo, somente as provenientes de quatro isolados reduziram a germinação das sementes de alface. Dois isolados de Bacillus cereus, um isolado de B. pumilus e um de Stenotrophomonas maltophilia foram os microrganismos mais promissores para a produção de fitotoxinas, com possibilidade de uso no desenvolvimento de novos produtos para o controle de plantas daninhas.
Assuntos
Toxinas Bacterianas/farmacologia , Citotoxinas/farmacologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Lactuca/efeitos dos fármacos , Rizosfera , Triticum/efeitos dos fármacos , Toxinas Bacterianas/biossíntese , Citotoxinas/biossíntese , Bacilos e Cocos Aeróbios Gram-Negativos/química , Bacilos e Cocos Aeróbios Gram-Negativos/metabolismo , Lactuca/crescimento & desenvolvimento , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimentoRESUMO
In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.
Assuntos
Toxinas Bacterianas/farmacologia , Citotoxinas/farmacologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Lactuca/efeitos dos fármacos , Rizosfera , Triticum/efeitos dos fármacos , Toxinas Bacterianas/biossíntese , Citotoxinas/biossíntese , Bacilos e Cocos Aeróbios Gram-Negativos/química , Bacilos e Cocos Aeróbios Gram-Negativos/metabolismo , Lactuca/crescimento & desenvolvimento , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimentoRESUMO
Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90 percent sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica, Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.
Assuntos
Bactérias/citologia , Bactérias/patogenicidade , Citotoxinas/biossíntese , Citotoxinas , Cytophaga/citologia , Cytophaga/patogenicidade , Flavobacterium/citologia , Flavobacterium/patogenicidade , Citotoxinas/análise , Reação em Cadeia da Polimerase , TailândiaRESUMO
The vacA and cagA genotypes of Helicobacter pylori exhibited distinct geographic distribution and correlation with severity of disease. In the above genotypes (obtained from 150 H. pylori-positive patients--139 with gastritis, 10 with ulcer and 1 patient with gastric cancer) combinations vacA s1/m1 and s2/m2 were detected using PCR in 75 and 25% of isolates, respectively, in patients with chronic gastritis. The of s1/m1 and s2/m2 combinations were also detected from ulcers (60 and 40%, respectively). The cagA was detected in 30% of isolates. Concentrated culture supernatants of 7 (64%) out of 11 H. pylori strains induced vacuolization in Vero cells in titers ranging from 1:5 to 1:40. The vacA s1 genotype was significantly associated with, but not predictive of the presence of vacuolating cytotoxin activity and the cagA gene.
Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Gastropatias/microbiologia , Adulto , Idoso , Antígenos de Bactérias/genética , Argentina/epidemiologia , Proteínas de Bactérias/genética , Citotoxinas/biossíntese , Gastroscopia , Genótipo , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Gastropatias/epidemiologiaRESUMO
AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.
Assuntos
Citotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Stenotrophomonas maltophilia/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citotoxinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Ferro/farmacologia , Ovinos , Células Vero , Zinco/farmacologiaRESUMO
The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.
Assuntos
Toxinas Bacterianas/biossíntese , Citotoxinas/biossíntese , Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Doença Aguda , Toxinas Bacterianas/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Citotoxinas/genética , Sondas de DNA/genética , DNA Bacteriano/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Fezes/microbiologia , Humanos , LactenteRESUMO
AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.
Assuntos
Plesiomonas/patogenicidade , Microbiologia da Água , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/toxicidade , Citotoxinas/biossíntese , Citotoxinas/toxicidade , Proteínas Hemolisinas/biossíntese , Hemólise , Humanos , Vacúolos/efeitos dos fármacos , VirulênciaRESUMO
The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.
Assuntos
Toxinas Bacterianas/biossíntese , Citotoxinas/biossíntese , Infecções por Escherichia coli/veterinária , Escherichia coli/metabolismo , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas de Bactérias/farmacologia , Células CHO , Linhagem Celular , Celulite (Flegmão)/microbiologia , Celulite (Flegmão)/veterinária , Embrião de Galinha , Galinhas , Chlorocebus aethiops , Cricetinae , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Células HeLa , Helicobacter pylori , Temperatura Alta , Humanos , Peso Molecular , Sepse/microbiologia , Sepse/veterinária , Células Tumorais Cultivadas , Células VeroRESUMO
In this work, culture filtrates of entomopathogenic and phytopathogenic Serratia marcescens strains induced cytotoxic effects on CHO, Vero and HEp-2 cell lines. Morphological changes on sensitive cells were characterized by cell rounding and detachment as soon as 30 min of incubation, culminating in cell death after 24 h. The cytotoxic effect was completely neutralized by specific antiserum indicating that occur antigenic similarity among cytotoxins produced by these strains. The toxicity assays on plants showed that the culture supernatants did not provoke any visible morphological change and did not affect their growth. By contrast, the plants treated with bacterial suspension showed disease symptom, such as shriveling and decay of stores bulbus in onion and lettuce plantlets. In conclusion, this study show that phytopathogenic and entomopathogenic S. marcescens may produce a cytototoxin similar to that produced by clinical isolates and it is toxic to different mammalian cell lines. These results are especially important for studies involving this bacterium as biological control agent.