Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens.

AIMS: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. METHODS AND RESULTS: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin-producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6-8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at -70 degrees C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono-Q FPLC and C-18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N-terminus was: Met-Asn-Phe-Asp-Glu-Lys-Val-Gly-Lys-Val-X-Phe-Lys-Val-Gly-Asp. CONCLUSIONS: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. SIGNIFICANCE AND IMPACT OF THE STUDY: We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.

Phenotypic changes in a laboratory-derived ertapenem-resistant Escherichia coli strain.

The aim of this study was to identify phenotypic changes in a laboratory-derived strain of ertapenem-resistant Escherichia coli (Ec-ERT) when compared to its susceptible parent strain (Ec-WT). In both strains, we assessed both the effects of ertapenem via time-kill curves and the occurrence of cross resistance with other beta-lactams. The strains were compared based on growth pattern, biochemical-physiological profile and changes in the subproteome using 2D-DIGE followed by MALDI-TOF/TOF MS. To assess virulence, we employed a murine model of intraperitoneal infection in which we investigated the invasiveness of both strains. Growth persistence of the laboratory-derived resistant strain was observed via the time-kill curve assay, but cross resistance was not observed for other beta-lactams. We also observed a slower growth rate and changes in the biochemical and physiological characteristics of the drug-resistant bacteria. In the resistant strain, a total of 51 protein spots were increased in abundance relative to the wild-type strain, including an outer membrane protein A, which is related to bacterial virulence. The mouse infection assay showed a higher invasiveness of the Ec-ERT strain in relation to the Ec-WT strain. In conclusion, the alterations driven by ertapenem in E. coli reinforce the idea that antimicrobial agents may interfere in several aspects of bacterial cell biology, with possible implications for host-bacteria interactions.

TSST-1, enterotoxin and bacteriocin-like substance production by Staphylococcus aureus isolated from foods / TSST-1, enterotoxinas e substâncias tipo bacteriocina produzidas por Staphylococcus aureus isolados de alimentos

The production of Toxic Shock Syndrome Toxin-1 (TSST-1), enterotoxins and bacteriocin-like substances was evaluated in 95 strains of Staphylococcus aureus recovered from raw bovine milk (n=31) and from food samples involved in staphylococcal food poisoning (n=64). Enterotoxigenicity tests with the membrane over agar associated to optimal sensibility plate assays were performed and showed that 96.77% of strains recovered from milk and 95.31% from food samples produced enterotoxins A, B, C, D or TSST-1. Reference strains S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis and Bacteroides fragilis were used as indicator bacteria in the antagonistic assays, the first five being sensitive to antagonistic substances. Brain heart infusion agar, in pH values ranging from 5.0 to 7.0 in aerobic atmosphere showed to be the optimum condition for antagonistic activity as evaluated with the best producer strains against the most sensitive indicator bacterium, L. monocytogenes. Sensitivity to enzymes confirmed the proteinaceous nature of these substances. Neither bacteriophage activity nor fatty acids were detected and the antagonistic activity was not due to residual chloroform. Results did not establish a positive correlation between the bacteriocinogenic profile and toxigenicity in the tested S. aureus strains.

Avaliou-se a produção de toxina-1 da síndrome do choque tóxico (TSST-1), enterotoxinas e substâncias antagonistas tipo bacteriocina em 95 amostras de Staphylococcus aureus recuperadas de leite bovino in natura (n=31) e de alimentos envolvidos em surto de intoxicação (n=64). Testes de enterotoxigenicidade pelo método da membrana sobre ágar, associado à técnica da sensibilidade ótima em placa, revelaram que 96,77% das amostras do leite e 95,31% daquelas dos alimentos produziram enterotoxinas estafilocócicas tipos A, B, C, D ou TSST-1. Nos ensaios de antagonismo, foram utilizadas como reveladoras amostras de referência de S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella typhimurium, Escherichia coli, Enterococcus faecalis e Bacteroides fragilis, sendo as cinco primeiras sensíveis às substâncias produzidas. As condições ótimas para a atividade antagonista, avaliadas com as melhores produtoras contra a indicadora mais sensível, L. monocytogenes, foram observadas em aerobiose, em ágar infuso de cérebro-coração, nos valores de pH entre 5,0 e 7,0. A sensibilidade a enzimas confirmou a natureza proteica destas substâncias. Não foram detectadas atividades de bacteriófagos nem de ácidos graxos, e a atividade antagonista não foi devido ao clorofórmio residual. Os resultados não mostraram correlação entre o perfil bacteriocinogênico e a toxigenicidade nas amostras de Staphylococcus testadas.

Production of antagonistic substance by Eikenella corrodens isolated from the oral cavity of human beings with and without periodontal disease.

AIMS: Antagonistic abilities may confer ecological advantages for micro-organisms in competitive ecosystems. However, reports regarding this phenomenon in Eikenella corrodens are not available. METHODS AND RESULTS: Nineteen E. corrodens strains, isolated from the oral cavity of human beings without periodontal disease (n = 5) and with aggressive (n = 9) and chronic (n = 5) periodontitis, as well as a reference strain (E. corrodens ATCC23834), were evaluated for antagonistic activity. The following indicators were used: Porphyromonas gingivalis FDC381, Prevotella intermedia ATCC25611, Actinomyces israelii ATCC12102, Eubacterium lentum ATCC25559, Peptostreptococcus anaerobius ATCC27337, Actinobacillus actinomycetemcomitans FDCY4, Fusobacterium nucleatum ATCC10953, Streptococcus sanguinis ATCC10557, Streptococcus uberis ATCC9927, Streptococcus mutans IM/UFRJ, Staphylococcus aureus ATCC33591 and Candida albicans ATCC18804. All the strains showed antagonism against at least one of the indicator strains. This phenomenon was more frequently observed for strains isolated from patients with chronic periodontitis (36.4%), than those from healthy subjects (20.6%) and those with aggressive periodontitis (10.8%). CONCLUSIONS: The heterogeneous antagonistic spectrum exhibited by E. corrodens isolates suggests their ability to produce more than one antagonistic substance, whose ecological relevance is yet to be demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description of antagonistic compound production by E. corrodens and its relationships with the clinical status of the patients.

Assessment of the antimicrobial activity of Casearia sylvestris extract against oral pathogenic microorganisms

An ethanolic extract of leaves from the tree Casearia sylvestris, known as guaçatonga in Brazil, was tested for in vitro activity against oral pathogenic bacteria and fungi. The results showed susceptibility of all the microorganisms tested. This study suggests a potential use of ethanolic extract of C. sylvestris as a novel treatment of oral infectious conditions, such as denture stomatitis, periodontitis and dental caries.