RESUMO
Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.
Assuntos
Corynebacterium/patogenicidade , Células Epiteliais/microbiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Corynebacterium/genética , Corynebacterium/isolamento & purificação , Infecções por Corynebacterium/microbiologia , Impedância Elétrica , Proteínas de Fluorescência Verde/genética , Células HeLa/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Larva/microbiologia , Lepidópteros/microbiologia , Receptor 2 Toll-Like/metabolismo , Células Vero/microbiologia , VirulênciaRESUMO
Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS- and Stx-mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS-mediated cytotoxicity in an experiment mimicking "natural infection," (c) the effect of antibiotics to decrease Stx2e-mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS- and Stx2e-induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS- and Stx2e-mediated cytotoxicity.
Assuntos
Amoxicilina/farmacologia , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Colistina/farmacologia , Enrofloxacina/farmacologia , Toxina Shiga/antagonistas & inibidores , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Células A549/microbiologia , Animais , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Células Vero/microbiologiaRESUMO
Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli, which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.
Assuntos
Infecções por Escherichia coli/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genética , Animais , China , Chlorocebus aethiops , Infecções por Escherichia coli/microbiologia , Genótipo , Humanos , Marmota/microbiologia , Toxina Shiga II/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero/microbiologia , Fatores de VirulênciaRESUMO
This study aimed to evaluate, by means of artificial feeding, the interaction between a pathogenic rickettsia and the hard tick R. microplus. We used partially engorged females fed on calves free of Rickettsia spp. Group 1 (G1), containing 20 ticks, was fed bovine blood only. Group 2 (G2), containing 20 ticks, was fed blood containing uninfected VERO cells, and group 3 (G3), containing 40 ticks, was fed blood containing VERO cells infected with Rickettsia parkeri. Biological parameters of the non-parasitic phase and a possible bacterial transmission to the tick eggs and to guinea pigs were evaluated. At the end of oviposition, all G3 females were PCR-positive for genes specific for the genus Rickettsia. Although no guinea pigs were infected, the experimental infection of R. microplus by R. parkeri caused a deleterious effect on the oviposition and provided the first report of transovarian transmission of rickettsia in this tick.
Assuntos
Oviposição , Rhipicephalus/microbiologia , Rhipicephalus/fisiologia , Rickettsia/fisiologia , Animais , Chlorocebus aethiops , Feminino , Cobaias , Reação em Cadeia da Polimerase , Rickettsia/genética , Infecções por Rickettsia/transmissão , Células Vero/microbiologiaRESUMO
BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.
Assuntos
Antibacterianos/farmacologia , Coagulase/metabolismo , Farmacorresistência Bacteriana/genética , Carne Vermelha/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Virulência/genética , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Bovinos , Chlorocebus aethiops , DNA Girase/genética , Egito , Microbiologia de Alimentos , Genes Bacterianos/genética , Proteínas Hemolisinas/metabolismo , Meticilina/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Resistência a Meticilina/genética , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Fenótipo , RNA Ribossômico 16S/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus lugdunensis/efeitos dos fármacos , Staphylococcus lugdunensis/genética , Staphylococcus lugdunensis/isolamento & purificação , Vancomicina/farmacologia , Células Vero/microbiologiaRESUMO
The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoebae and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of three Acanthamoeba castellanii strains with Fusarium conidia and the possible implications on keratitis. F. conidia were internalized by A. castellanii strains and were able to germinate inside the amoebae. The co-culture with the live amoebae, as well as the amoebal culture supernatant and lysate, increased the fungal growth significantly. Moreover, live F. solani and its culture supernatant enhanced the survival of amoebae, but in a different way in each amoebal strain. The encystment of the A. castellanii strain re-isolated from rat lung was increased by the fungus. These results show that A. castellanii and F. solani interaction may have an important influence on survival of both, and specially indicate a possible effect on virulence characteristics of these microorganisms. These data suggest that the A. castellanii-F. solani interaction may cause severe impacts on keratitis.
Assuntos
Acanthamoeba castellanii/crescimento & desenvolvimento , Coinfecção/etiologia , Lentes de Contato/efeitos adversos , Fusarium/crescimento & desenvolvimento , Ceratite/etiologia , Ceratite/microbiologia , Células Vero/microbiologia , Animais , Chlorocebus aethiops/microbiologia , Técnicas de Cocultura , Coinfecção/microbiologia , Lentes de Contato/microbiologia , Ratos , Esporos Fúngicos/crescimento & desenvolvimento , VirulênciaRESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.
Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/efeitos adversos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/microbiologia , Toxina Shiga II/efeitos adversos , Subtilisinas/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Células Vero/efeitos dos fármacos , Células Vero/microbiologiaRESUMO
The genus Aeromonas includes some species that have now been identified as human pathogens of significant medical importance. We investigated the ability of 13 selected Aeromonas strains belonging to nine species isolated from clinical cases (n = 5), environmental waters (n = 5), and fish (n = 3) to adhere to and translocate Caco-2 cells in the absence and presence of two lactic acid bacteria (LAB), i.e., Lactobacillus acidophilus and Bifidobacterium breve. Aeromonas isolates were also assessed for their cytotoxicity, the presence of virulence genes, and hemolysin production. Among the clinical isolates, one strain of Aeromonas veronii biovar veronii and two strains of Aeromonas hydrophila carried cytotoxin (act), heat-labile toxin (alt), hemolysin (hlyA), and aerolysin (aerA) genes, were cytotoxic to Vero cells, produced hemolysin, and showed higher adherence to Caco-2 cells. In contrast, this was seen in only one environmental strain, a strain of A. veronii biovar sobria. When Aeromonas strains were coinoculated with LAB onto Caco-2 cells, their level of adhesion was reduced. However, their rate of translocation in the presence of LAB increased and was significantly (P < 0.05) higher among fish strains. We suggest that either the interaction between Aeromonas and LAB strains could have a detrimental effect on the Caco-2 cells, allowing the Aeromonas to translocate more readily, or the presence of the LAB stimulated the Aeromonas strains to produce more toxins and/or increase their translocation rate.
Assuntos
Aeromonas/fisiologia , Bifidobacterium/fisiologia , Células CACO-2/microbiologia , Lactobacillus acidophilus/fisiologia , Aeromonas/genética , Aeromonas/patogenicidade , Animais , Aderência Bacteriana , Toxinas Bacterianas/genética , Chlorocebus aethiops , Doenças dos Peixes/microbiologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Ácido Láctico/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Células Vero/microbiologia , Virulência/genéticaRESUMO
Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3, which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent antimycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Proteínas de Transporte de Ânions/genética , Antituberculosos/farmacocinética , Proteínas de Bactérias/genética , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Indóis/química , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Mutação , Polimorfismo de Nucleotídeo Único , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Células Vero/efeitos dos fármacos , Células Vero/microbiologiaRESUMO
Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.
Assuntos
Bacteriocinas/metabolismo , Rim/citologia , Lactobacillus plantarum/metabolismo , Fígado/citologia , Animais , Células Cultivadas , Chlorocebus aethiops , Citofotometria , Eletrofisiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Lactobacillus plantarum/crescimento & desenvolvimento , Masculino , Ratos , Ratos Wistar , Células Vero/metabolismo , Células Vero/microbiologiaRESUMO
Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strainsare the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and highmortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeuticanti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2 AB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ inthe expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. Thevaccine strains expressed Stx2 AB, either cell bound or secreted into the extracellular environment, andshowed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions.Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B(IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) andconferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2 AB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.
Assuntos
Humanos , Camundongos , Células Vero/microbiologia , Escherichia coli Enteropatogênica/imunologia , Vacinas , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Salmonella entericaRESUMO
Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment.
Assuntos
Dermacentor/microbiologia , Ixodes/microbiologia , Rickettsia/crescimento & desenvolvimento , Células Vero/microbiologia , Animais , Linhagem Celular/microbiologia , Chlorocebus aethiops , Estágios do Ciclo de Vida/fisiologia , Camundongos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Rickettsia/fisiologiaRESUMO
OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.
OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.
Assuntos
Animais , Proteínas de Bactérias/toxicidade , Cólera/virologia , Meios de Cultivo Condicionados/toxicidade , Proteínas Hemolisinas/toxicidade , Células Vero/microbiologia , Vibrio cholerae O1/patogenicidade , Austrália/epidemiologia , Proteínas de Bactérias/genética , Chlorocebus aethiops , Cólera/epidemiologia , DNA Bacteriano/genética , Proteínas Hemolisinas/genética , Hemólise , América Latina/epidemiologia , Fenótipo , Ribotipagem , Romênia/epidemiologia , Estados Unidos/epidemiologia , Vacúolos , Células Vero/ultraestrutura , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Virulência/genéticaRESUMO
OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.
Assuntos
Proteínas de Bactérias/toxicidade , Cólera/virologia , Meios de Cultivo Condicionados/toxicidade , Proteínas Hemolisinas/toxicidade , Células Vero/microbiologia , Vibrio cholerae O1/patogenicidade , Animais , Austrália/epidemiologia , Proteínas de Bactérias/genética , Chlorocebus aethiops , Cólera/epidemiologia , DNA Bacteriano/genética , Proteínas Hemolisinas/genética , Hemólise , América Latina/epidemiologia , Fenótipo , Ribotipagem , Romênia/epidemiologia , Estados Unidos/epidemiologia , Vacúolos , Células Vero/ultraestrutura , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Virulência/genéticaRESUMO
Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.
Assuntos
Antígenos Nucleares/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Rickettsia conorii/patogenicidade , Actinas/metabolismo , Animais , Caveolina 2/metabolismo , Chlorocebus aethiops , Clatrina/metabolismo , Células HeLa/microbiologia , Humanos , Autoantígeno Ku , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Rickettsia conorii/metabolismo , Rickettsia conorii/fisiologia , Células Vero/microbiologiaRESUMO
Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6%), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60% of strains vs 11.7% of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75% for fimH, fyuA, kpsMTII and iucD, and between 35-65% for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.
Assuntos
Escherichia coli/patogenicidade , Sepse/microbiologia , Fatores de Virulência/genética , Adulto , Animais , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Humanos , Reação em Cadeia da Polimerase , Células Vero/microbiologia , Virulência/genéticaRESUMO
Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6 percent), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60 percent of strains vs 11.7 percent of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75 percent for fimH, fyuA, kpsMTII and iucD, and between 35-65 percent for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.
Assuntos
Adulto , Animais , Humanos , Escherichia coli/patogenicidade , Sepse/microbiologia , Fatores de Virulência/genética , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Reação em Cadeia da Polimerase , Células Vero/microbiologia , Virulência/genéticaRESUMO
Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.
Assuntos
Antígenos Glicosídicos Associados a Tumores/farmacologia , Escherichia coli/química , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Toxinas Shiga/antagonistas & inibidores , Animais , Antígenos Glicosídicos Associados a Tumores/fisiologia , Chlorocebus aethiops , Cromatografia em Camada Fina , Receptores de Superfície Celular/análise , Toxinas Shiga/metabolismo , Toxinas Shiga/toxicidade , Triexosilceramidas , Células Vero/microbiologiaRESUMO
Shiga toxin producing Escherichia coli (STEC) harbouring the stx(2d-activatable) gene and expressing the mucus- and elastase-activatable phenotype have been associated with severe outcomes of human disease. However, there is limited data available on the occurrence of such strains in livestock reservoirs. In this study, we analyzed 11 STEC strains isolated from healthy cattle and sheep at slaughter that were originally detected to contain the stx(2c) allele, for the presence of the stx(2d-activatable) genotype. Ten of the eleven strains displayed the stx(2d-activatable) genotype as determine by PstI restriction fragment length polymorphism (RFLP) of 890-bp fragments of their stx genes. However, only in 6 of the 10 strains whose stx genes were sequenced, the presence of stx(2d-activatable) could be confirmed based on the predicted amino acid sequence of their StxA subunits; the remaining four strains contained Stx2c A subunit. Five of the six strains which contained stx(2d-activatable) displayed the activatable phenotype on Vero cells. Genes for adhesins such as the outer membrane protein intimin (eae), which is essential for the intimate attachment and the formation of attaching-and-effacing lesions on intestinal epithelial cells, or the STEC autoagglutinating adhesin (saa), potentially important in eae-negative STEC, were not detected. Moreover, all the strains tested negative for EHEC-hlyA encoding enterohaemorrhagic E. coli (EHEC) hemolysin. To our knowledge, this is the first study that reports the presence of STEC harbouring stx(2d-activatable) and producing the activatable Stx2d in fecal samples of sheep. Therefore both cattle and sheep are reservoirs of such strains and potential sources of human infections. This is of particular importance, because in contrast to other eae-negative STEC, strains producing Stx2d(activatable) may cause severe diseases such as bloody diarrhoea and haemolytic uremic syndrome in humans.
Assuntos
Adesinas Bacterianas , Bovinos/microbiologia , Ovinos/microbiologia , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Matadouros , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , DNA Bacteriano/química , DNA Bacteriano/genética , Reservatórios de Doenças/veterinária , Fezes/microbiologia , Genótipo , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Toxina Shiga II/biossíntese , Toxina Shiga II/isolamento & purificação , Escherichia coli Shiga Toxigênica/metabolismo , Células Vero/microbiologiaRESUMO
Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective antifungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.