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.

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles.

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCß), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCß values in poultry muscle were found: penicillin G ≤ 12.5 µg kg(-1), total sulfonamides ≤ 75 µg kg(-1), erythromycin 75 µg kg(-1) and lincomycin 50 µg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 µg kg(-1)) and the method did not detect gentamicin (1000 µg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening ß-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.

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.

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.