RESUMEN

Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

Asunto(s)

Bacteroides fragilis/genética , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Transcriptoma/genética , Animales , Bacteroides fragilis/patogenicidad , Regulación Bacteriana de la Expresión Génica/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ARN

RESUMEN

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Asunto(s)

Antibacterianos/farmacología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/crecimiento & desarrollo , Animales , Infecciones por Bacteroides/genética , Infecciones por Bacteroides/metabolismo , Bacteroides fragilis/patogenicidad , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/efectos de los fármacos

RESUMEN

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

Asunto(s)

Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Bacteroides fragilis/fisiología , Adhesinas Bacterianas/genética , Animales , Bacteroides fragilis/patogenicidad , Western Blotting , Técnicas de Inactivación de Genes , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Viabilidad Microbiana , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36 degrees C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P < 0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.

Asunto(s)

Bacteroides fragilis/patogenicidad , Enterotoxinas/análisis , Vaginosis Bacteriana/microbiología , Adolescente , Adulto , Toxinas Bacterianas/análisis , Bacteroides fragilis/aislamiento & purificación , Bacteroides fragilis/metabolismo , Candida albicans/aislamiento & purificación , Candidiasis Vulvovaginal/microbiología , Coinfección , Exudados y Transudados/microbiología , Femenino , Gardnerella vaginalis/aislamiento & purificación , Humanos , Metaloendopeptidasas/análisis , Persona de Mediana Edad , Infecciones por Mycoplasma/microbiología , Mycoplasma hominis/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Vagina/microbiología , Adulto Joven

RESUMEN

La vaginitis es un trastorno ginecológico frecuente producido por distintas causas, algunas de las cuales permanecen desconocidas. Bacteroides fragilis es el anaerobio más importante en bacteriología clínica. Algunas cepas son enterotoxigénicas y se asocian con síndromes intestinales y extraintestinales. Recientemente han sido aisladas de pacientes con vaginitis. En este trabajo se planteó investigar la posible asociación de B. fragilis enterotoxigénico con la vaginitis infecciosa. Fueron procesadas 265 muestras de exudado vaginal. 202 de mujeres sintomáticas y 63 mujeres sanas. La identificación de los microorganismos se realizó por métodos convencionales. En 31,2% de las pacientes sintomáticas se identificaron: Gardnerella vaginalis, Candida albicans, Mobiluncus, Mycoplasma hominis, Ureaplasma urealyticum y Streptococcus agalactiae. En 27 pacientes sintomáticas y en 5 mujeres sanas se identificó B. fragilis. Estas cepas fueron cultivadas en medio líquido e incubadas durante 48 h a 36° C en anaerobiosis. La toxicidad en los sobrenadantes se ensayó en células HT-29. 18 cepas de B. fragilis aisladas de pacientes sintomáticas fueron enterotoxigénicas, ya que indujeron alteraciones en la monocapa celular y en las células. No se identificó en mujeres sanas (P<0,05). 77,7% de las cepas de B. fragilis enterotoxigénicas no se encontraron asociadas con otros patógenos específicos. Este hecho sugiere que pudiera ser un agente causante de vaginitis, ya que el efecto de la enterotoxina sobre la E-cadherina del epitelio vaginal podría facilitar la invasión y su posible papel patógeno en la vagina. Esta es la primera investigación que asocia a Bacteroides fragilis enterotoxigénico como posible causa de vaginitis infecciosa.

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Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36°C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P<0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.

Asunto(s)

Adolescente , Adulto , Femenino , Humanos , Persona de Mediana Edad , Adulto Joven , Bacteroides fragilis/patogenicidad , Enterotoxinas/análisis , Vaginosis Bacteriana/microbiología , Toxinas Bacterianas/análisis , Bacteroides fragilis/aislamiento & purificación , Bacteroides fragilis/metabolismo , Coinfección , Candida albicans/aislamiento & purificación , Candidiasis Vulvovaginal/microbiología , Exudados y Transudados/microbiología , Gardnerella vaginalis/aislamiento & purificación , Metaloendopeptidasas/análisis , Infecciones por Mycoplasma/microbiología , Mycoplasma hominis/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Vagina/microbiología

RESUMEN

Ertapenem and piperacillin/tazobactam are beta-lactam antibiotics with a broad spectrum of activity, used for the treatment of mixed infections, in which Bacteroides fragilis plays an important etiological role. The aim of this study was to select strains of B. fragilis resistant to these drugs and correlate the phenotype profiles of these lineages with changes in the virulence of the original bacterium. B. fragilis ATCC 25285, sensitive to the drugs listed, was used in this study. Strains resistant to these drugs were obtained by multi-step method and this condition was confirmed by comparing the time-kill curve of the original strain with those curves obtained from derived-resistant strains. To assess the virulence, germ-free mice were challenged intragastrically with the original strain or those derived-resistant. The mouse infection by the piperacillin/tazobactam-resistant B. fragilis strain produced increased levels of C-reactive protein, alkaline phosphatase and white blood cells and reduced platelet counts, what may indicate that acquisition of piperacillin/tazobactam resistance may enhance the pathogenic properties of these B. fragilis strains.

Asunto(s)

Antibacterianos/farmacología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/patogenicidad , Resistencia betalactámica , Animales , Infecciones por Bacteroides/metabolismo , Ertapenem , Vida Libre de Gérmenes , Técnicas In Vitro , Ratones , Pruebas de Sensibilidad Microbiana , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/farmacología , Fenotipo , Piperacilina/farmacología , Tazobactam , Virulencia/efectos de los fármacos , beta-Lactamas/farmacología

RESUMEN

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Asunto(s)

Infecciones por Bacteroides/microbiología , Infecciones por Bacteroides/patología , Bacteroides fragilis/patogenicidad , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/patología , Animales , Femenino , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Necrosis/microbiología , Necrosis/patología , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/metabolismo , Bacteroides fragilis/patogenicidad , Activadores Plasminogénicos/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Infecciones por Bacteroides/metabolismo , Bacteroides fragilis/genética , Cromatografía de Afinidad , Clonación Molecular , ADN Bacteriano/análisis , Fibrinolisina/metabolismo , Humanos , Laminina/metabolismo , Espectrometría de Masas , Plasminógeno/metabolismo , Activadores Plasminogénicos/química , Activadores Plasminogénicos/genética , Análisis de Secuencia de ADN , Virulencia

RESUMEN

The presence of enterotoxigenic Bacteroides fragilis and nontoxigenic B. fragilis (NTBF) among 109 strains isolated from 1980-2008 in Brazil were investigated by PCR. One strain, representing 0.9% of the total analyzed strains, harbored the bft gene which was identified as bft-1 isoform based on PCR-RFLP and sequencing. Forty-nine strains (44.9%) exhibited the NTBF pattern III which possesses the flanking region required for pathogenicity island acquisition in which the bft gene is codified. These data reinforce the potential of B. fragilis as an emerging enteropathogen in our country.

Asunto(s)

Bacteroides fragilis/genética , Enterotoxinas/biosíntesis , Genes Bacterianos/genética , Bacteroides fragilis/clasificación , Bacteroides fragilis/patogenicidad , Brasil , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción

RESUMEN

The presence of enterotoxigenic Bacteroides fragilis and nontoxigenic B. fragilis (NTBF) among 109 strains isolated from 1980-2008 in Brazil were investigated by PCR. One strain, representing 0.9 percent of the total analyzed strains, harbored the bft gene which was identified as bft-1 isoform based on PCR-RFLP and sequencing. Forty-nine strains (44.9 percent) exhibited the NTBF pattern III which possesses the flanking region required for pathogenicity island acquisition in which the bftgene is codified. These data reinforce the potential of B. fragilis as an emerging enteropathogen in our country.

Asunto(s)

Humanos , Bacteroides fragilis/genética , Enterotoxinas/biosíntesis , Genes Bacterianos/genética , Brasil , Bacteroides fragilis/clasificación , Bacteroides fragilis/patogenicidad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción

RESUMEN

In this study, a novel, simple and rapid hemagglutination assay by using a peanut lectin to detect a neuraminidase activity in strains of the Bacteroides fragilis group was developed. One hundred and fourteen species of the B. fragilis group isolated from children with and without diarrhea and 15 reference strains were evaluated. Neuraminidase production was determined by using the method above described and its inhibition was observed by using galactose. The neuraminidase production was observed in 54 (84.37%) diarrhea and in 43 (86%) non-diarrhea strains. HA titers were ranged from 2 to 32. This neuraminidase assays based on PNA hemagglutination is highly sensitive, reproducible and could be used as a tool to detect the sialidase activity in anaerobic bacteria, particularly, in species of the B. fragilis group.

Asunto(s)

Bacteroides fragilis/enzimología , Pruebas de Hemaglutinación/métodos , Neuraminidasa/metabolismo , Bacteroides fragilis/clasificación , Bacteroides fragilis/patogenicidad , Niño , Diarrea/microbiología , Heces/microbiología , Humanos , Aglutinina de Mani/farmacología

RESUMEN

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.

Asunto(s)

Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/patogenicidad , Metaloendopeptidasas/genética , Metaloendopeptidasas/toxicidad , Animales , Bacteroides fragilis/aislamiento & purificación , Niño , Preescolar , Diarrea/microbiología , Edema/microbiología , Enterotoxinas/genética , Enterotoxinas/toxicidad , Vida Libre de Gérmenes , Humanos , Ratones

RESUMEN

Bacteroides fragilis is the anaerobe most commonly recoverable from clinical specimens. The wide genetic diversity of this bacterium related with virulence potential is still an open question. In this study, we analyzed the morphological aspects and microbicide action of MØ during interactions with B. fragilis. A filamentous cytoplasm content release and a different actin organization colocalized with iNOS were detected. It was also possible to observe the reduction of NO production in the same conditions. The scanning electron microscopy showed the formation of pore-like structures in the surface of macrophages in the bacterial presence and by transmission electron microscopy we could observe the extrusion of cytoplasm contents as well as the condensation of chromatin in the nucleus periphery. These data suggest the existence of an inhibitory mechanism developed by B. fragilis strains for one of the macrophage microbicide actions.

Asunto(s)

Infecciones por Bacteroides/metabolismo , Bacteroides fragilis/metabolismo , Macrófagos Peritoneales/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Actinas/metabolismo , Animales , Bacteroides fragilis/patogenicidad , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/patología , Masculino , Ratones , Microscopía Electrónica de Rastreo , Microscopía de Contraste de Fase , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II

RESUMEN

Diarrhoeic stool samples from 334 0-5-year-old children were analysed with respect to the incidence of Bacteroides fragilis as well as other enteropathogens. B. fragilis was recovered in 9.3% (31/334) of the samples, and 79 strains were examined for the presence of the bft gene or the BfPAI flanking region using polymerase chain reaction assays. No enterotoxigenic B. fragilis strains were detected. In 29% (9/31) of the samples the coexistence of both II and III non-toxigenic B. fragilis (NTBF) patterns could be seen. In 51.6% (16/31) of the samples there existed a pattern II NTBF only, and in 19.4% (6/31) only pattern III could be detected. Strains from the same patient representing different patterns were submitted to pulsed-field gel electrophoresis assays. Fingerprints obtained by this technique showed that there was strong heterogeneity among strains from different individuals. However, different patterns from the same individual shared 100% similarity.

Asunto(s)

Infecciones por Bacteroides/microbiología , Bacteroides fragilis/patogenicidad , Enterotoxinas/biosíntesis , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos , Virulencia/genética , Bacteroides fragilis/clasificación , Bacteroides fragilis/genética , Bacteroides fragilis/fisiología , Preescolar , Diarrea/microbiología , Heces/microbiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Reacción en Cadena de la Polimerasa

RESUMEN

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60 masculine C, for 30 min, and among them, 12 (18.75%) were resistant at 60 masculine C, for 60 min. Also, none strain was resistant at 100 masculine C. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Asunto(s)

Antibacterianos/farmacología , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/aislamiento & purificación , Diarrea/microbiología , Factores de Virulencia , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/patogenicidad , Brasil , Niño , Preescolar , Frío , Farmacorresistencia Microbiana , Heces/microbiología , Humanos , Lactante , Pruebas de Sensibilidad Microbiana

RESUMEN

Different concentrations of metronidazole are used widely to treat protozoan and fungal infections. As an antibacterial drug, metronidazole is mainly used against anaerobes, of which the Bacteroides fragilis group is the most important in terms of the frequency of recovery and antimicrobial resistance patterns. The objective of this study was to investigate (1) in vivo metronidazole-induced modifications in the B. fragilis group reflected by altered virulence, and (2) the interference of metronidazole in cellular viability of these samples when subjected in vitro to human polymorphonuclear leukocytes (PMNs). Strains adapted to low metronidazole concentrations were observed to be more virulent, as demonstrated experimentally in mice by weight loss, quantitative evidence of tissue damage, hemorrhage and anatomopathology of spleen, liver and small intestine samples. A significant increase (P < 0.05) in mean bacterial viability rate of about 2.62-fold was observed for all the drug-adapted strains after contact with human PMNs. However, the level of this phenomenon was quite different among the tested species. These results draw attention to the risk that prolonged therapy, even with low concentrations of metronidazole, may affect the pathogenicity of Bacteroides strains, producing changes in host-bacteria relationships.

Asunto(s)

Antibacterianos/farmacología , Infecciones por Bacteroides/tratamiento farmacológico , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/patogenicidad , Metronidazol/farmacología , Animales , Antibacterianos/uso terapéutico , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/genética , Farmacorresistencia Microbiana , Heces/microbiología , Histocitoquímica/métodos , Humanos , Metronidazol/metabolismo , Metronidazol/uso terapéutico , Ratones , Neutrófilos/inmunología , Virulencia/efectos de los fármacos

RESUMEN

Bacteroides fragilis isolates from intestinal and non-intestinal infections, normal flora and the environment were examined for properties linked with interactions among cells in vitro. Different adhesion molecules were detected in agglutination assays with human erythrocytes and tests for auto-agglutination and adherence to human colon carcinoma cells (HT29). There was no correlation between these properties, indicating that independent molecules are involved. Treatment with trypsin, heat or EDTA inhibited agglutination and adherence, suggesting that these molecules are proteins. The lack of correlation with the origin of the strains did not permit any of these activities to be recognised as virulence markers. The expression of fragilysin, a protease associated with damage to intestinal cells and bacterial translocation, was examined. Only those strains from patients with diarrhoea expressed this protease activity in assays with HT29 cells and this was confirmed by specific PCR for the bft gene. The activity of fragilysin as an enterotoxin was confirmed in the rabbit intestinal ligated loop assay. The association of this property only with strains from intestinal infections indicates that it is too early to suggest this protease as a determinant factor of B. fragilis invasiveness.

Asunto(s)

Infecciones por Bacteroides/microbiología , Bacteroides fragilis/patogenicidad , Pruebas de Aglutinación , Animales , Anticoagulantes/farmacología , Adhesión Bacteriana/efectos de los fármacos , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/genética , Ácido Edético/farmacología , Enterotoxinas/genética , Células HT29 , Pruebas de Hemaglutinación , Calor , Humanos , Íleon/microbiología , Enfermedades Intestinales/microbiología , Metaloendopeptidasas/genética , Reacción en Cadena de la Polimerasa , Conejos , Propiedades de Superficie , Tripsina/farmacología , Virulencia , Microbiología del Agua , Contaminación del Agua

RESUMEN

In order to investigate the relationship among virulent and avirulent Bacteroides fragilis strains, SDS-PAGE of whole-cell proteins (WP) and periplasmic proteins (PP) were used to establish a protein profile of strains isolated from human infections, fecal flora and environmental water. Despite different sources of the strains, no significant differences were observed as determined by the WP SDS-PAGE analysis. In contrast, the proteins obtained from the bacterial periplasm showed differences in the electrophoretic protein profile. Two distinct PP profile patterns were obtained. Pattern A included 6 out of the 8 virulent strains and pattern B, 6 out of 8 avirulent strains. Interestingly, an environmental strain that was capable of inducing abscesses in mice, had a PP profile highly similar to that of the virulent strains from human infections. These data indicate that PP from B. fragilis may be useful to characterize differences among virulent and avirulent strains. Moreover, strains isolated from environmental water may also be a source of exogenous infections by B. fragilis.

Asunto(s)

Proteínas Bacterianas/análisis , Bacteroides fragilis/química , Animales , Proteínas Bacterianas/aislamiento & purificación , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/clasificación , Bacteroides fragilis/patogenicidad , Electroforesis en Gel de Poliacrilamida , Heces/microbiología , Humanos , Ratones , Periplasma/química , Virulencia , Microbiología del Agua

RESUMEN

Thirteen strains of Bacteroides fragilis isolated from contaminated water or from cases of intestinal and non-intestinal infections were comparatively analyzed in order to detect a possible biological relationship among them. The observation of similar profiles among the two groups, regarding the capacity of inducing abscesses and some surface properties, may support the hypothesis that the species could also act as an exogenous pathogen.

Asunto(s)

Infecciones por Bacteroides/microbiología , Bacteroides fragilis/patogenicidad , Enfermedades Intestinales/microbiología , Microbiología del Agua , Absceso/microbiología , Aglutinación , Animales , Antígenos Bacterianos , Bacteroides fragilis/aislamiento & purificación , Bacteroides fragilis/metabolismo , Humanos , Ratones , Neuraminidasa/metabolismo , Propiedades de Superficie , Virulencia

RESUMEN

A Susceptibilidade de 104 cepas do grupo B. fragilis isolados de humanos (52) e de micos C. penicillata (52) para clindamicina, metronidazol, penicillina G e bicloreto de mercúrio, foi determinada em três diferentes meios de cultivos, pelos métodos de diluiçäo em ágar e diluiçäo em caldo. O ágar infuso cérebro coraçäo, suplementado com hemina ou sangue, foi usado para o primeiro método e, o caldo infuso cérebro coraçäo para o último. Nos meios sólidos, somente 10 (por cento) dos isolados humanose, nenhum dos isolados de micos foram resistentes para clindamicina. O metronidazol foi o antimicrobiano mais eficaz contra todos os microrganismos testados nos três meios apresentaram níveis elevados de resistência para penicilina G em todos os meios testados. O bicloreto de mecúrio apresentou menor atividade em ágar sangue, apresentando faixas de CIMs de 2 a 128 ug/ml.

Asunto(s)

Bacterias Anaerobias/patogenicidad , Bacteroides fragilis/patogenicidad , Medios de Cultivo Condicionados , Metronidazol/farmacología , Farmacorresistencia Microbiana