RESUMEN
Objectives: Capnocytophaga spp. are often reported to cause bacteraemia and extra-oral infections and are characterized by their significant contribution to resistance to ß-lactam and macrolide-lincosamide-streptogramin antibiotics in the human oral microbiota. The implication of mutations in the quinolone resistance-determining region (QRDR) of DNA gyrase A and B ( gyrA and gyrB ) and topoisomerase IV ( parC and parE ) of fluoroquinolone (FQ)-resistant Capnocytophaga spp., hitherto unknown, was explored in this study. Methods: Two reference strains ( Capnocytophaga gingivalis ATCC 33624 and Capnocytophaga sputigena ATCC 33612) and four Capnocytophaga spp. isolated from clinical samples were studied. Nine in vitro FQ-resistant mutants, derived from two reference strains and one FQ-susceptible clinical isolate, were selected by successive inoculations onto medium containing levofloxacin. MICs of ofloxacin, norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin were determined. The presumed QRDRs of GyrA, GyrB, ParC and ParE from Capnocytophaga spp. were determined by sequence homology to Bacteroides fragilis and Escherichia coli . PCR primers were designed to amplify the presumed QRDR genetic region of Capnocytophaga spp. and sequence analyses were performed using the BLAST program at the National Center for Biotechnology Information. Results and conclusions: gyrA mutations leading to a substitution from amino acid position 80 to 86 were systematically detected in Capnocytophaga spp. with ciprofloxacin MIC >1 mg/L and considered as the primary target of FQs. No mutational alteration in the QRDR of gyrB was detected. Other mutations in parC and parE led to spontaneous amino acid substitutions of DNA topoisomerase IV subunit B with no alteration in FQ susceptibility.
Asunto(s)
Antibacterianos/farmacología , Capnocytophaga/efectos de los fármacos , Capnocytophaga/enzimología , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Fluoroquinolonas/farmacología , Mutación Missense , Sustitución de Aminoácidos , Capnocytophaga/genética , Capnocytophaga/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Capnocytophga canimorsus and Capnocytophga cynodegmi can be transmitted from cats and dogs to humans, and can cause a wide range of infections including wound infections, sepsis, or endocarditis. We and others recently discovered two new Capnocytophaga species, C. canis and C. stomatis, mainly associated with wound infections. The first-line treatment of animal bite related infections is penicillin, and in case of allergy, doxycycline and trimethoprim/sulfamethoxazole. However, there is a lack of antibiotic susceptibility patterns for animal bite associated Capnocytophaga species. Thus, we set out to study the antibiotic profiles against animal bite associated Capnocytophaga species isolated from wound and blood cultures after cat and dog bites and coupled the findings to whole genome sequencing data. A total of 24 strains were included in the study. Phenotypic analysis of antibiotic resistance was performed with E-tests. The web-based tool 'Resfinder' was used to identify resistance genes in the whole genome dataset. Two strains of C. cynodegmi and two strains of the recently discovered C. stomatis were resistant to penicillin (MIC > 24 mg/L) and cephalosporins (MIC > 24 mg/L), and three out of these strains also exhibited resistance to imipenem (MIC = 32 mg/L). Genomic analysis revealed that these strains carried a class D beta-lactamase gene, which has not previously been found in Capnocytophaga spp. A class D beta lactamase with broad substrate specificity was found in animal bite associated Capnocytophaga species, which could have important implications when treating wound infections after cat and dog bites. It also suggests that pet animal bacteria can harbour resistance genes with relevance for human infections.
Asunto(s)
Mordeduras y Picaduras/complicaciones , Capnocytophaga/enzimología , Infecciones por Bacterias Gramnegativas/microbiología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamas/metabolismo , Animales , Capnocytophaga/genética , Capnocytophaga/aislamiento & purificación , Gatos , Biología Computacional , Pruebas Antimicrobianas de Difusión por Disco , Perros , Genoma Bacteriano , Humanos , Especificidad por Sustrato , beta-Lactamasas/clasificaciónRESUMEN
Essentials Capnocytophaga canimorsus causes severe dog bite related blood stream infections. We investigated if C. canimorsus contributes to bleeding abnormalities during infection. The C. canimorsus protease CcDPP7 causes factor X dysfunction by N-terminal cleavage. CcDPP7 inhibits coagulation in vivo, which could promote immune evasion and trigger hemorrhage. SUMMARY: Background Capnocytophaga canimorsus is a Gram-negative bacterium that is present in the oral flora of dogs and causes fulminant sepsis in humans who have been bitten, licked, or scratched. In patients, bleeding abnormalities, such as petechiae, purpura fulminans, or disseminated intravascular coagulation (DIC), occur frequently. Objective To investigate whether C. canimorsus could actively contribute to these bleeding abnormalities. Methods Calibrated automated thrombogram and clotting time assays were performed to assess the anticoagulant activity of C. canimorsus 5 (Cc5), a strain isolated from a fatal human infection. Clotting factor activities were measured with factor-deficient plasma. Factor X cleavage was monitored with the radiolabeled zymogen and western blotting. Mutagenesis of Cc5 genes encoding putative serine proteases was performed to identify the protease that cleaves FX. Protein purification was performed with affinity chromatography. Edman degradation allowed the detection of N-terminal cleavage of FX. Tail bleeding times were measured in mice. Results We found that Cc5 inhibited thrombin generation and increased the prothrombin time and the activated partial thromboplastin time of human plasma via FX cleavage. A mutant that was unable to synthesize a type 7 dipeptidyl peptidase (DPP7) of the S46 serine protease family failed to proteolyse FX. The purified protease (CcDPP7) cleaved FX heavy and light chains from the N-terminus, and was active in vivo after intravenous injection. Conclusions This is, to our knowledge, the first study demonstrating a detailed mechanism for FX inactivation by a bacterial protease, and it is the first functional study associating DPP7 proteases with a potentially pathogenic outcome.
Asunto(s)
Mordeduras y Picaduras/microbiología , Capnocytophaga/enzimología , Coagulación Intravascular Diseminada/microbiología , Factor X/antagonistas & inhibidores , Péptido Hidrolasas/química , Animales , Catálisis , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Tiempo de Tromboplastina Parcial , Plásmidos/metabolismo , Dominios Proteicos , Sepsis/microbiología , Análisis de Secuencia de ADNRESUMEN
Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.
Asunto(s)
Capnocytophaga/genética , Capnocytophaga/metabolismo , Lípido A/biosíntesis , Fosfatos/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Capnocytophaga/efectos de los fármacos , Capnocytophaga/enzimología , Perros , Genes Bacterianos/genética , Prueba de Complementación Genética , Genoma Bacteriano , Humanos , Lípido A/química , Lípido A/genética , Espectrometría de Masas , Operón , Eliminación de SecuenciaRESUMEN
Capnocytophaga canimorsus are gram-negative bacteria living as commensals in the mouth of dogs and cats. C. canimorsus cause rare but life-threatening generalized infections in humans that have been in contact with a dog or a cat. Over the last years we collected 105 C. canimorsus strains from different geographical origins and from severe human infections or healthy dogs. All these strains were analyzed by 16S rDNA sequencing and a phylogenetic tree revealed two main groups of bacteria instead of one with no relation to the geographical origin. This branching was confirmed by the whole-genome sequencing of 10 strains, supporting the evidence of a new Capnocytophaga species in dogs. Interestingly, 19 out of 19 C. canimorsus strains isolated from human infections belonged to the same species. Furthermore, most strains from this species could grow in heat-inactivated human serum (HIHS) (40/46 tested), deglycosylate IgM (48/66) and were cytochrome-oxidase positive (60/66) while most strains from the other species could not grow in HIHS (22/23 tested), could not deglycosylate IgM (33/34) and were cytochrome-oxidase negative (33/34). Here, we propose to call Capnocytophaga canis (Latin: dog) the novel, presumably less virulent dog-hosted Capnocytophaga species and to keep the name C. canimorsus for the species including human pathogens.
Asunto(s)
Capnocytophaga/clasificación , ADN Ribosómico/química , Enfermedades de los Perros/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , ARN Ribosómico 16S/genética , Animales , Capnocytophaga/enzimología , Capnocytophaga/genética , Capnocytophaga/patogenicidad , Gatos , Secuencia de Consenso , ADN Ribosómico/aislamiento & purificación , Enfermedades de los Perros/transmisión , Perros , Complejo IV de Transporte de Electrones/metabolismo , Genoma Bacteriano/genética , Estudio de Asociación del Genoma Completo , Infecciones por Bacterias Gramnegativas/transmisión , Humanos , Inmunoglobulina M/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Polisacáridos/metabolismo , Saliva/microbiología , Especificidad de la EspecieAsunto(s)
Capnocytophaga/enzimología , Capnocytophaga/genética , Genes Bacterianos , beta-Lactamasas/genética , Capnocytophaga/clasificación , Capnocytophaga/aislamiento & purificación , Perfilación de la Expresión Génica , Variación Genética , Infecciones por Bacterias Gramnegativas/microbiología , HumanosRESUMEN
Capnocytophaga spp. are commensal bacteria involved in oral and systemic diseases, with a variable susceptibility to beta-lactams. The cfxA gene expression level was assessed using quantitative RT-PCR, and reasons of the observed misexpression were discussed, as insertion of foreign genetic material, contributing to dissemination and evolution of antibiotic resistance genes.
Asunto(s)
Capnocytophaga/enzimología , Infecciones por Bacterias Gramnegativas/microbiología , Mucosa Bucal/microbiología , beta-Lactamasas/biosíntesis , Antibacterianos/farmacología , Capnocytophaga/efectos de los fármacos , Capnocytophaga/genética , Capnocytophaga/aislamiento & purificación , Perfilación de la Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Mutagénesis Insercional , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinación Genética , beta-Lactamasas/genética , beta-Lactamas/farmacologíaRESUMEN
Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.
Asunto(s)
Ingeniería de Proteínas , Eliminación de Secuencia , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo , Secuencia de Aminoácidos , Capnocytophaga/enzimología , Capnocytophaga/genética , Clonación Molecular , Estabilidad de Enzimas , Biblioteca de Genes , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Thermotoga maritima/enzimología , Thermotoga maritima/genética , beta-Fructofuranosidasa/químicaRESUMEN
Coeliac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in patients with coeliac disease. We found that the oral microbiome is a novel and rich source of gluten-degrading organisms. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on the immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: (i) hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; (ii) gliadin degradation in-gel (gliadin zymography); (iii) gliadin degradation in solution; (iv) proteolysis of the highly immunogenic α-gliadin-derived 33-mer peptide. For selected strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastrointestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for coeliac disease.
Asunto(s)
Bacterias/enzimología , Bacterias/aislamiento & purificación , Placa Dental/microbiología , Gliadina/metabolismo , Microbiota , Saliva/microbiología , Actinomyces/enzimología , Actinomyces/aislamiento & purificación , Capnocytophaga/enzimología , Capnocytophaga/aislamiento & purificación , Enfermedad Celíaca/tratamiento farmacológico , Enfermedad Celíaca/enzimología , Gliadina/química , Glútenes/inmunología , Glútenes/metabolismo , Humanos , Neisseria mucosa/enzimología , Neisseria mucosa/aislamiento & purificación , Streptococcus/enzimología , Streptococcus/aislamiento & purificaciónRESUMEN
INTRODUCTION: Antimicrobial resistance is on the increase in 'medical and dental fields. Beta-lactam antibiotics are the most widely used antimicrobials in dental patients. OBJECTIVES: This study investigated the prevalence of beta-lactamase-producing anaerobic oral bacteria in patients with chronic periodontitis. METHODS: Pooled subgingival samples from two sites in 42 patients with chronic periodontitis were cultured anaerobically on blood agar plates containing amoxycillin or/and Augmentin. Colonies that grew on amoxycillin but not Augmentin were identified and tested for beta-lactamase production. RESULTS: Sixty-nine percent of patients carried beta-lactamase-producing anaerobes, with a mean of one to two strains per patient. Seventy isolates of the beta-lactamase- producing strains formed 4% of the total cultivable anaerobic flora. Prevotella was the most prevalent beta-lactamase-producing species, followed by Capnocytophaga, Veillonella and Bacteroides. CONCLUSIONS: A high prevalence of beta-lactamase-producing oral anaerobes was detected in this preliminary study. However, the percentage of beta-lactamase producers in the total number of bacteria per patient was low. Therefore, beta-lactam antibiotics still remain the drug of choice in infections with odontogenic origin.
Asunto(s)
Bacterias Anaerobias/enzimología , Periodontitis Crónica/microbiología , beta-Lactamasas/biosíntesis , Adulto , Anciano , Amoxicilina/farmacología , Combinación Amoxicilina-Clavulanato de Potasio/farmacología , Antibacterianos/farmacología , Bacterias Anaerobias/clasificación , Carga Bacteriana , Técnicas Bacteriológicas , Bacteroides/enzimología , Bacteroides/aislamiento & purificación , Capnocytophaga/enzimología , Capnocytophaga/aislamiento & purificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Bolsa Periodontal/microbiología , Prevotella/enzimología , Prevotella/aislamiento & purificación , Sudáfrica , Veillonella/enzimología , Veillonella/aislamiento & purificación , Adulto JovenRESUMEN
Capnocytophaga canimorsus, a commensal bacterium of the canine oral flora, has been repeatedly isolated since 1976 from severe human infections transmitted by dog bites. Here, we show that C. canimorsus exhibits robust growth when it is in direct contact with mammalian cells, including phagocytes. This property was found to be dependent on a surface-exposed sialidase allowing C. canimorsus to utilize internal aminosugars of glycan chains from host cell glycoproteins. Although sialidase probably evolved to sustain commensalism, by releasing carbohydrates from mucosal surfaces, it also contributed to bacterial persistence in a murine infection model: the wild type, but not the sialidase-deficient mutant, grew and persisted, both when infected singly or in competition. This study reveals an example of pathogenic bacteria feeding on mammalian cells, including phagocytes by deglycosylation of host glycans, and it illustrates how the adaptation of a commensal to its ecological niche in the host, here the dog's oral cavity, contributes to being a potential pathogen.
Asunto(s)
Capnocytophaga/metabolismo , Células Epiteliales/microbiología , Fagocitos/microbiología , Animales , Capnocytophaga/enzimología , Capnocytophaga/aislamiento & purificación , Células Cultivadas , Infecciones por Bacterias Gramnegativas , Humanos , Ratones , Neuraminidasa/metabolismo , Polisacáridos/metabolismoRESUMEN
AIM: In order to assess the prevalence of beta-lactamase producing oral bacteria in childhood, the influence of different parameters on the oral carriage of Capnocytophaga was studied in a specific population of children with cancer. The examined parameters included clinical observation of oropharyngeal mucosa, type of malignant disease and intake of chemotherapy and antimicrobial treatment. METHODS: The gingival and mucosal status of the patients was recorded before each sampling procedure. Samples were collected by oropharyngeal swabbing in children with leukaemia or other oncological diseases for isolation of Capnocytophaga strains. RESULTS: Capnocytophaga strains were more often isolated in samples from children with oncological diseases (71%) other than leukaemia (57%). Concomitant chemotherapy had no influence on oral Capnocytophaga carriage. A significant decrease of the prevalence of Capnocytophaga strains isolated was observed in children who received antimicrobial treatment within 8 days before the sampling procedure (15.5% vs. 28%). But, the incidence of beta-lactamase-producing strains was not linked to previous antimicrobial treatments. CONCLUSION: Oral carriage of Capnocytophaga strains can be linked to haematological disease and previous antibiotherapy, but results did not confirm that beta-lactamase treatments exert a selective pressure. Other factors might be involved in emerging for oral beta-lactamase-producing Capnocytophaga strains.
Asunto(s)
Antibacterianos/uso terapéutico , Capnocytophaga/aislamiento & purificación , Neoplasias/microbiología , Orofaringe/microbiología , beta-Lactamasas/biosíntesis , Adolescente , Capnocytophaga/enzimología , Niño , Preescolar , Femenino , Humanos , Lactante , MasculinoRESUMEN
Bacteremia due to Capnocytophaga sputigena occurred in a 4-year and 9-month-old Japanese girl patient with acute erythroblastic leukemia in Shinshu University Hospital, Japan. On her admission to the hospital, she had a temperature of 38.2 degrees C with canker sore. Prior to the commencement of chemotherapy, peripheral blood culture was carried out with the BacT/Alert 3D System ver. 4.00D (bioMerieux Japan Ltd., Tokyo, Japan) using both the PF and the SN bottles. At 48 hrs of incubation, the System showed the positive sign only in the anaerobic SN bottle for bacterial growth. The strain isolated from the SN bottle was morphologically, biochemically, and genetically characterized, and finally identified as Capnocytophaga sputigena. The causative Capnocytophaga sputigena isolate was found to be a beta-lactamase-producer demonstrating to possess cfxA3 gene. The gene responsible for the production of CfxA3-beta-lactamase was proved to be chromosome-encoded, by means of southern hybridization analysis. This was the first case of bacteremia caused by chromosome-encoded CfxA3-beta-lactamase-producing Capnocytophaga sputigena.
Asunto(s)
Bacteriemia/microbiología , Capnocytophaga/aislamiento & purificación , Cromosomas Bacterianos , Leucemia Eritroblástica Aguda/complicaciones , beta-Lactamasas/biosíntesis , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacteriemia/complicaciones , Bacteriemia/diagnóstico , Bacteriemia/tratamiento farmacológico , Capnocytophaga/efectos de los fármacos , Capnocytophaga/enzimología , Preescolar , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
Chromosome- and plasmid-encoded CfxA2 and CfxA3 beta-lactamases were detected in Capnocytophaga spp. from oral sources in France, Norway, and the United States. Unidentified chromosome-encoded beta-lactamases were present in Capnocytophaga sputigena. Nucleotide sequence analysis of the CfxA3-encoding plasmid from C. ochracea revealed an unreported insertion sequence (ISCoc1) upstream of the cfxA gene.
Asunto(s)
Capnocytophaga/genética , Cromosomas Bacterianos/genética , Plásmidos/genética , beta-Lactamasas/genética , Secuencia de Bases , Capnocytophaga/enzimología , Cartilla de ADN , Elementos Transponibles de ADN , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Infecciones por Bacterias Gramnegativas/microbiología , Focalización Isoeléctrica , Datos de Secuencia MolecularRESUMEN
BACKGROUND: The aim of this study was to evaluate the prevalence of beta-lactamase-producing Capnocytophaga isolates in young children hospitalized in the Pediatric Oncology Department of Hôpital Sud (Rennes, France) over a ten-year period (1993-2002). METHODS: In neutropenic children, a periodic survey of the oral cavity allows a predictive evaluation of the risk of systemic infections by Capnocytophaga spp. In 449 children with cancer, 3,053 samples were collected by oral swabbing and plated on TBBP agar. The susceptibility of Capnocytophaga isolates to five beta-lactams was determined. RESULTS: A total of 440 strains of Capnocytophaga spp. were isolated, 309 (70%) of which were beta-lactamase producers. The beta-lactamase-producing strains were all resistant to cefazolin, 86% to amoxicillin, and 63% to ceftazidime. The proportion of strains resistant to third-generation cephalosporins remained high throughout the ten-year study, while susceptibility to imipenem and amoxicillin combined with clavulanic acid was always conserved. CONCLUSION: These results highlight the risk of antibiotic failure in Capnocytophaga infections and the importance of monitoring immunosuppressed patients and testing for antibiotic susceptibility and beta-lactamase production.
Asunto(s)
Capnocytophaga/enzimología , Capnocytophaga/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/complicaciones , Infecciones por Bacterias Gramnegativas/microbiología , Neoplasias/complicaciones , Orofaringe/microbiología , beta-Lactamasas/metabolismo , Niño , Femenino , Infecciones por Bacterias Gramnegativas/epidemiología , Humanos , Masculino , Prevalencia , Factores de Tiempo , Resistencia betalactámicaRESUMEN
There is evidence that enzymes from the genus Capnocytophaga play a role in dental calculus formation. Although most of the species in the genus produce aminopeptidases, there is a paucity of data on the purification and characterization of the enzyme, except in the case of Capnocytophaga gingivalis. The aim of this study was to purify aminopeptidase from culture supernatant of Capnocytophaga granulosa ATCC 51502, a new species of the genus. Purification was performed using ammonium sulfate fractionation and two chromatographic steps. The aminopeptidase was purified 158,433-fold with a yield of 12.0%. The enzyme appeared to be a trimer with a molecular mass of 270 kDa. The optimal pH of the aminopeptidase was 6.5 and its activity was completely inhibited by incubation at 50 degrees C for 10 min. The enzyme showed maximum specificity for basic amino acids (Arg and Lys) and also hydrolyzed noncharged amino acids (Met, Leu and Ala). Ca(2+), Zn(2+) and Fe(3+) activated the enzyme, while EDTA, Ag(+), Hg(+) and Cu(2+) inhibited it. These results suggest that aminopeptidase of C. granulosa is different from that of C. gingivalis but similar to aminopeptidase B.
Asunto(s)
Aminopeptidasas/química , Aminopeptidasas/aislamiento & purificación , Capnocytophaga/enzimología , Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Fraccionamiento Químico , Cromatografía por Intercambio Iónico , Cálculos Dentales/microbiología , Electroforesis en Gel de Poliacrilamida , Punto Isoeléctrico , Peso Molecular , Inhibidores de Proteasas/metabolismo , Especificidad por SustratoAsunto(s)
Endopeptidasas/clasificación , Líquido del Surco Gingival/enzimología , Bacterias Gramnegativas/enzimología , Hidrolasas/clasificación , Enfermedades Periodontales/microbiología , Aggregatibacter actinomycetemcomitans/enzimología , Aggregatibacter actinomycetemcomitans/inmunología , Capnocytophaga/enzimología , Capnocytophaga/inmunología , Colagenasas/clasificación , Eikenella corrodens/enzimología , Eikenella corrodens/inmunología , Endopeptidasas/genética , Endopeptidasas/inmunología , Bacterias Gramnegativas/inmunología , Humanos , Hidrolasas/genética , Hidrolasas/inmunología , Isoenzimas/clasificación , Biología Molecular , Enfermedades Periodontales/inmunología , Periodontitis/inmunología , Periodontitis/microbiología , Porphyromonas gingivalis/enzimología , Prevotella/enzimología , Prevotella/inmunología , Prevotella intermedia/enzimología , Prevotella intermedia/inmunología , Treponema/enzimología , Treponema/inmunología , Tripsina/clasificación , Tripsina/genéticaRESUMEN
The susceptibilities of 43 pharyngeal isolates of Capnocytophaga to beta-lactam antibiotics, alone or in combination with beta-lactamase inhibitors, were tested by an agar dilution method. The 34 beta-lactamase-positive strains were highly resistant to beta-lactams, but the intrinsic activities of clavulanate, tazobactam, and sulbactam against Capnocytophaga, even beta-lactamase producers, indicates that these beta-lactamase inhibitors could be used for empirical treatment of neutropenic patients with oral sources of infection.
Asunto(s)
Antibacterianos/farmacología , Capnocytophaga/efectos de los fármacos , Ácido Penicilánico/análogos & derivados , Inhibidores de beta-Lactamasas , Antibacterianos/uso terapéutico , Capnocytophaga/enzimología , Ácido Clavulánico/farmacología , Ácido Clavulánico/uso terapéutico , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Ácido Penicilánico/farmacología , Ácido Penicilánico/uso terapéutico , Tazobactam , Resistencia betalactámicaRESUMEN
A plasmid-encoded extended-spectrum TEM beta-lactamase with a pI of 5.5 was detected in a Capnocytophaga ochracea clinical isolate. The bla gene was associated with a strong TEM-2 promoter and was derived from bla(TEM-1a) with a single-amino-acid substitution: Glu(104)-->Lys, previously assigned to TEM-17, which is thus the first TEM beta-lactamase to be reported in the phylum Flavobacter-Bacteroides.