RESUMEN
In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.
Asunto(s)
Antibacterianos , Infecciones por Bacteroides , Humanos , Antibacterianos/farmacología , Bacteroides , Bacteroides fragilis/genética , Ecuador , beta-Lactamasas/genética , Infecciones por Bacteroides/tratamiento farmacológico , Infecciones por Bacteroides/microbiología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana/genética , Proteínas Bacterianas/genéticaRESUMEN
The gut microbiota is a complex community of microorganisms that inhabit the digestive tracts of humans, living in symbiosis with the host. Dysbiosis, characterized by an imbalance between the beneficial and opportunistic gut microbiota, is associated with several gastrointestinal disorders, such as irritable bowel syndrome (IBS); inflammatory bowel disease (IBD), represented by ulcerative colitis and Crohn's disease; and colorectal cancer (CRC). Dysbiosis can disrupt the mucosal barrier, resulting in perpetuation of inflammation and carcinogenesis. The increase in some specific groups of harmful bacteria, such as Escherichia coli (E. coli) and enterotoxigenic Bacteroides fragilis (ETBF), has been associated with chronic tissue inflammation and the release of pro-inflammatory and carcinogenic mediators, increasing the chance of developing CRC, following the inflammation-dysplasia-cancer sequence in IBD patients. Therefore, the aim of the present review was to analyze the correlation between changes in the gut microbiota and the development and maintenance of IBD, CRC, and IBD-associated CRC. Patients with IBD and CRC have shown reduced bacterial diversity and abundance compared to healthy individuals, with enrichment of Firmicute sand Bacteroidetes. Specific bacteria are also associated with the onset and progression of CRC, such as Fusobacterium nucleatum, E. coli, Enterococcus faecalis, Streptococcus gallolyticus, and ETBF. Future research can evaluate the advantages of modulating the gut microbiota as preventive measures in CRC high-risk patients, directly affecting the prognosis of the disease and the quality of life of patients.
Asunto(s)
Neoplasias Colorrectales , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Bacterias , Bacteroides fragilis , Neoplasias Colorrectales/microbiología , Disbiosis/complicaciones , Disbiosis/microbiología , Escherichia coli , Humanos , Inflamación/complicaciones , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/microbiología , Calidad de Vida , ArenaRESUMEN
OBJECTIVE: An experimental infection based on a tissue cage model was reproduced to evaluate the interference subinhibitory concentration (SIC) of metronidazole in Bacteroides fragilis OMV production patterns and immunological and histological characteristics of the host facing the experimental challenge. METHODS: A tissue cage model was reproduced for B. fragilis experimental challenge in three Wistar rats groups: negative control group (NC) without bacterial inoculation; positive control group (PC) infected with parental strain; and experimental group (EG) infected with the parental strain and treated with metronidazole SIC. Tissue cage sections and histological preparations were evaluated under optical and transmission electron microscope. Observations included OMV identification and count and cellular envelope evaluation. Transcriptional analyses were performed to evaluate cytokines expression levels. RESULTS: Total counts of leukocytes and neutrophils were higher for EG, and slight increase in PC group. It was observed an exacerbated inflammatory infiltrate after 8 days on infection. The expression of TNF-α was increased during the experiments, along with IL-1α and IL-6. MCP-1 levels were suppressed in almost every evaluated time-point. The IL-10 was exacerbated in EG group. A massive production and release of OMV and cell wall thickening were observed especially the EG group. CONCLUSIONS: Despite literature data suggest positive association between OMV and antimicrobial stress for Gram negatives, no correlations are made for B. fragilis and drug-response during experimental model of infection. Results corroborate observations in which OMV may be involved in bacterial pathogenicity once the phenomenon was observed along histological evidence of exacerbated inflammation and cytokines modulation.
Asunto(s)
Infecciones Bacterianas , Bacteroides fragilis , Animales , Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Metronidazol/farmacología , Ratas , Ratas WistarRESUMEN
Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.
Asunto(s)
Infecciones por Bacteroides/inmunología , Bacteroides fragilis/inmunología , Fibras de la Dieta/efectos adversos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Peritonitis/inmunología , Animales , Infecciones por Bacteroides/microbiología , Dieta , Fibras de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Peritonitis/microbiología , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
Several factors affect the composition of species that inhabit our intestinal tract, including mode of delivery, genetics and nutrition. Antimicrobial peptides and proteins secreted in the gastrointestinal tract are powerful tools against bacteria. Lactoferrin (LF) inhibits the growth of several bacterial species, such as Enterobacteriaceae, but may stimulate probiotic bacteria. Activity of LF against gut symbiotic species of the Bacteroides genus could give us insights on how these species colonize the gut. We investigated the effects of the antimicrobial protein lactoferrin and its derived peptide, lactoferricin B on two species of strict anaerobes, opportunistic pathogens that cause diseases in both adults and children, commonly found in the microbiota of the human gastrointestinal tract, Bacteroides fragilis and B. thetaiotaomicron., In vitro biofilm formation and binding to laminin were strongly inhibited by a low concentration of lactoferrin (12.5⯵g/ml). Conversely, the growth of the strains in a micro-dilution assay in minimal media with different iron sources was not affected by physiological concentrations (2â¯mg/ml) of apo-lactoferrin or holo-lactoferrin. The combination of lactoferrin with antibiotics in synergism assays was also negative. The lactoferricin B fragment was also unable to inhibit growth in a similar test with concentrations of up to 32⯵g/ml. Resistance to lactoferrin could confer an advantage to these species, even when high amount of this protein is present in the gastrointestinal tract. However, colonization is hampered by the binding and biofilm inhibitiory effect of lactoferrin, which may explain the low prevalence of Bacteroides in healthy babies. Resistance to this antimicrobial protein may help understand the success of these opportunistic pathogens during infection in the peritoneum.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Bacteroides/efectos de los fármacos , Bacteroides/fisiología , Biopelículas/efectos de los fármacos , Lactoferrina/farmacología , Antibacterianos/farmacología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/fisiología , Bacteroides thetaiotaomicron/efectos de los fármacos , Bacteroides thetaiotaomicron/fisiología , Tracto Gastrointestinal/microbiología , HumanosRESUMEN
The most commonly identified pathogens related to bacterial meningitis are group B streptococcus and gram-negative enteric flora; anaerobic sepsis and meningitis are very rare. We report a case on a preterm and extremely low-birth weight infant who developed meningitis caused by Bacteroides fragilis and his mother who had postpartum sepsis also caused by the same agent.
Asunto(s)
Bacterias Anaerobias , Bacteroides fragilis , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Adulto , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacteroides fragilis/efectos de los fármacos , Biomarcadores , Femenino , Humanos , Recién Nacido , Meningitis Bacterianas/complicaciones , Meningitis Bacterianas/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Evaluación de Síntomas , Resultado del TratamientoRESUMEN
Bacteroides genus are commonly found on mucous membranes, including the female genital tract, acting as agents for several site infections. Anaerobic infections are usually polymicrobial and endogenous. Trichomonas vaginalis, the trichomoniasis etiologic agent, is a facultative anaerobic flagellated parasite spread worldwide. The purpose of this study was to explore the association between vaginal bacteria and T. vaginalis, as well as to understand factors that may favour the infection of T. vaginalis. We have, therefore, used T. vaginalis trophozoites and the species Bacteroides fragilis, which is considered the most important in its genus, once it is the most commonly isolated bacteria from endogenous infections. The parasite-bacteria interaction was performed in different proportions in periods varying from 1 to 12 hours applying viability tests. The data were analyzed to compare the parasite viability in vitro in the presence and absence of B. fragilis. The results indicate that in the 1:100 proportion postinteraction analysis, ultrastructural alterations were noticeable after 6 hours. After 8 hours, T. vaginalis viability decreased, and after 12 hours of interaction no viable trophozoites were found. These data suggest that the parasite can deal with B. fragilis in short interaction periods. However, in longer interaction periods the trophozoites collapse, indicating that B. fragilis may produce toxic metabolites against T. vaginalis activity.
Asunto(s)
Bacteroides fragilis , Trichomonas vaginalis , Técnicas In Vitro , Vaginosis Bacteriana , Enfermedades de los Genitales FemeninosRESUMEN
Abstract Endodontic infections result from oral pathogenic bacteria which reach and infect dental pulp, as well as surrounding tissues, through cracks, unrepaired caries and failed caries restorations. This study aims to determine the chemical composition of essential oil from Psidium cattleianum leaves (PC-EO) and to assess its antibacterial activity against endodontic bacteria. Antibacterial activity of PC-EO was evaluated in terms of its minimum inhibitory concentration (MIC) values by the broth microdilution method on 96-well microplates. Bacteria Porphyromonas gingivalis (MIC = 20 µg/mL), Prevotella nigrescens (MIC = 62.5 µg/mL), Fusobacterium nucleatum (MIC = 12.5 µg/mL), Actinomyces naeslundii (MIC = 50 µg/mL), Bacteroides fragilis (MIC = 12.5 µg/mL), Aggregatibacter actinomycetemcomitans (MIC = 6.25 µg/mL) and Peptostreptococcus anaerobius (MIC = 62.5 µg/mL) were evaluated and compared to chlorhexidine dihydrochloride (CDH), the positive control. PC-EO was obtained by hydrodistillation with the use of a Clevenger-type apparatus whereas its chemical composition was analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Viridiflorol (17.9%), β-caryophyllene (11.8%), 1,8-cineole (10.8%) and β-selinene (8.6%) were the major constituents found in PC-EO, which exhibited high antibacterial activity against all endodontic pathogens under investigation. Therefore, PC-EO, a promising source of bioactive compounds, may provide therapeutic solutions for the field of endodontics.
Asunto(s)
Aceites Volátiles/farmacología , Clorhexidina/farmacología , Psidium/química , Antibacterianos/farmacología , Peptostreptococcus/efectos de los fármacos , Bacteroides fragilis/efectos de los fármacos , Actinomyces/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fusobacterium nucleatum/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Prevotella nigrescens/efectos de los fármacos , Cromatografía de Gases y Espectrometría de MasasRESUMEN
Introducción. La apendicitis aguda es la primera causa de abdomen agudo; sin embargo, poco se conoce sobre las bacterias asociadas y su perfil de sensibilidad. Objetivo. Identificar y determinar el patrón de resistencia de las bacterias aerobias y anaerobias aisladas en cultivo de líquido periapendicular tomado de los pacientes con apendicitis aguda, y establecer la proporción de bacterias según la fase clínica. Materiales y métodos. Se llevó a cabo un estudio descriptivo y prospectivo en el Hospital Universitario de San José de Bogotá (Colombia), en pacientes mayores de 16 años sometidos a apendicectomía abierta. Se tomaron muestras de líquido periapendicular, las cuales se sembraron directamente en botellas de hemocultivos para aerobios y anaerobios. Resultados. Se incluyeron 154 pacientes. Del total de cultivos, el 87 % (n=134) fueron positivos: 77 % (n=118) para aerobios y 51 % (n=79) para anaerobios. La proporción de cultivos positivos fue inferior en los casos de apendicitis no complicada, en comparación con aquellos de apendicitis complicada (80 % (66/83) Vs. 95 % (67/71); p=0,003). Los microorganismos aislados con mayor frecuencia fueron: Escherichia coli (53 %) (n=84), Bacteroides sp. (25 %) (n=25), Propionibacterium acnes (21 %) (n=21), Staphylococci coagulasa negativo (17 %) (n=27), Enterococcus sp. (10 %) (n=15) y Fusobacterium sp. (11 %) (n=11). La sensibilidad de E. coli a la amplicilina sulbactam fue de 30 %. La sensibilidad de Bacteroides spp. a la clindamicina y la ampicilina sulbactam fue de 91 %. El 100 % de los anaerobios fueron sensibles a piperacilina tazobactam, ertapenem, meropenem y metronidazol. Conclusiones. Los cultivos intraoperatorios son pertinentes en la apendicitis para determinar el patrón epidemiológico local, y establecer los antibióticos profilácticos y terapéuticos para esta enfermedad. Su siembra directa en botellas de hemocultivo permite una gran recuperación de microorganismos.
Introduction: Acute appendicitis is the first cause of acute abdomen, however, there is a little information about the associated bacteria and its sensibility profile. Objetive: To identify and to determine the resistance pattern of aerobic and anaerobic bacteria isolated in periapendicular fluid cultures taken in patients with acute appendicitis and to establish the proportions of isolates according to the clinical phase. Materials and methods: A descriptive and prospective study was undertaken at the Hospital Universitario de San José (Bogotá, Colombia) of patients older than sixteen years of age, undergoing an open appendectomy. A sample of periappendiceal fluid was taken, which was deposited directly into aerobic and anaerobic blood culture bottles. Results: One hundred and fifty-four patients were included. The overall positivity of cultures was 87% (n=1344); 77% (n=118) for aerobes and 51% (n=79) for anaerobes. The proportion of positive cultures was lower in the uncomplicated appendicitis cases as compared to the complicated ones (80% (66/83) vs. 95%(67/71), p = 0.003). The microorganisms isolated most frequently were: Escherichia coli (53%) (n=84); Bacteroides spp. (25%) (n=25); Propionibacterium acnes (21%) (n=21); coagulase negative Staphylococci (17%) (n=27); Enterococcus spp. (11%) (n=15), and Fusobacterium spp. (11%) (n=11). The sensitivity of E.coli to ampicillin/sulbactam was 30%. The sensitivity of Bacteroides spp. to clindamycin and ampicillin/sulbactam was 91%. All anaerobe isolates were sensitive to piperacillin/tazobactam, ertapenem, meropenem and metronidazole. Conclusions: Intraoperative cultures in acute appendicits are relevant in order to determine the local epidemiological pattern and to establish prophylactic and therapeutic antibiotics for this pathology; direct inoculation in blood culture bottles allows a high recovery of microorganisms.
Asunto(s)
Apendicitis , Bacterias Anaerobias , Bacterias Aerobias , Apendicectomía , Bacteroides fragilis , Líquido Ascítico , Pruebas de Sensibilidad MicrobianaRESUMEN
Bacteroides fragilis, an opportunistic pathogen and commensal bacterium in the gut, is one the most aerotolerant species among strict anaerobes. However, the mechanisms that control gene regulation in response to oxidative stress are not completely understood. In this study, we show that the MarR type regulator, BmoR, regulates the expression of genes involved in the homeostasis of intracellular redox state. Transcriptome analysis showed that absence of BmoR leads to altered expression in total of 167 genes. Sixteen of these genes had a 2-fold or greater change in their expression. Most of these genes are related to LPS biosynthesis and carbohydrates metabolism, but there was a significant increase in the expression of genes related to the redox balance inside the cell. A pyridine nucleotide-disulfide oxidoreductase located directly upstream of bmoR was shown to be repressed by direct binding of BmoR to the promoter region. The expression of two other genes, coding for a thiosulphate:quinone-oxidoreductase and a thioredoxin, are indirectly affected by bmoR mutation during oxygen exposure. Phenotypic assays showed that BmoR is important to maintain the thiol/disulfide balance in the cell, confirming its relevance to B. fragilis response to oxidative stress.
Asunto(s)
Bacteroides fragilis , Disulfuros/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo/genética , Proteínas Represoras , Compuestos de Sulfhidrilo/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Perfilación de la Expresión Génica , Oxidación-Reducción , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.
Asunto(s)
Bacteroides fragilis/enzimología , Bacteroides fragilis/ultraestructura , Electroforesis en Gel de Poliacrilamida , Fosfopiruvato Hidratasa , Plasminógeno , Vesículas ExtracelularesRESUMEN
Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.(AU)
Asunto(s)
Bacteroides fragilis , Antiinfecciosos , Resistencia a Múltiples Medicamentos , Estrés Oxidativo , Bacterias Anaerobias , Silenciador del GenRESUMEN
ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.
Asunto(s)
Humanos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/genética , Infecciones por Bacteroides/microbiología , Proteínas Represoras/genética , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/aislamiento & purificación , Bacteroides fragilis/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Pruebas de Sensibilidad Microbiana , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.
Asunto(s)
Bacteroides fragilis/enzimología , Vesículas Extracelulares/enzimología , Fosfopiruvato Hidratasa/análisis , Bacteroides fragilis/ultraestructura , Electroforesis en Gel de Poliacrilamida , Vesículas Extracelulares/ultraestructura , Humanos , Laminina , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Fosfopiruvato Hidratasa/metabolismo , PlasminógenoRESUMEN
Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/genética , Proteínas Represoras/genética , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/aislamiento & purificación , Bacteroides fragilis/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Represoras/metabolismoRESUMEN
CfiA (CcrA) metallo-ß-lactamase is the main carbapenem resistance mechanism in B. fragilis. From cfiA positive isolates detected in a previous surveillance study, 3 displayed resistance to imipenem while the remaining were susceptible. The aim of this study was to identify the cfiA alleles and to analyze the presence of IS elements in their upstream regions. CfiA-1, CfiA-4, CfiA-13, CfiA-19 and CfiA-22 were detected. IS elements belonging to IS21 family and IS942 group were identified upstream to cfiA in the 3 imipenem resistant isolates. We present an exhaustive analysis of cfiA/CfiA registers in databases, illustrating the inconsistencies in both organization and nomenclature. According to this analysis CfiA family comprises nowadays 15 different CfiA variants coded by 24 cfiA sequences. Curation of CfiA database is mandatory, if not new cfiA admission at GenBank will contribute to make this classification more complex.
Asunto(s)
Proteínas Bacterianas/genética , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/clasificación , Bacteroides fragilis/genética , Sistemas de Lectura Abierta , beta-Lactamasas/genética , Alelos , Antibacterianos/farmacología , Argentina/epidemiología , Infecciones por Bacteroides/epidemiología , Bacteroides fragilis/efectos de los fármacos , Bacteroides fragilis/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Filogenia , Vigilancia en Salud PúblicaRESUMEN
Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Bacteroides fragilis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Proteínas Portadoras/aislamiento & purificación , Membrana Celular/efectos de los fármacos , Estrés Fisiológico/genética , Adaptación Fisiológica , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/química , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/química , Medios de Cultivo/química , Expresión Génica , Ontología de Genes , Anotación de Secuencia Molecular , Proteómica/métodosRESUMEN
Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.
Asunto(s)
Toxinas Bacterianas/genética , Infecciones por Bacteroides/microbiología , Bacteroides fragilis/genética , Bacteroides fragilis/aislamiento & purificación , Heces/microbiología , Genotipo , Metaloendopeptidasas/genética , Animales , Infecciones por Bacteroides/epidemiología , Bacteroides fragilis/clasificación , Brasil/epidemiología , Niño , Preescolar , ADN Bacteriano/genética , Femenino , Humanos , Incidencia , Masculino , Tipificación Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.(AU)
Asunto(s)
Humanos , Niño , Bacteroides fragilis , Heces , Firmicutes , Reacción en Cadena de la PolimerasaRESUMEN
Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.