Distribución de los genotipos de fimA en cepas de Porphyromonas gingivalis aisladas de placas subgingivales y de sangre durante bacteriemias

Introduction. Porphyromonas gingivalis is considered as a major etiological agent in the onset and progression of chronic destructive periodontitis. Porphyromonus gingivalis fimA type has been correlated to the virulence potential of the strain; therefore this gene could be involved in the ability of P. gingivalis to reach blood stream.Objective. The classifications of P. gingivalis fimA types will be compared in subgingival plaque and blood samples collected after scaling and root root planing of periodontitis patients.Materials and methods. Fifteen periodontitis patients requiring scaling and root planing were enrolled. P. gingivalis isolates were classed to genotype with fimA type-specific PCR assay. fimA gene was sequenced if the isolate was listed as unclassifiable after PCR technique.Results. Six patients showed positive P. gingivalis bacteremia. The most frequent fimA was fimA type II, followed by Ib, III and IV. In blood strains, type II was followed by IV, Ib and III.Conclusion. Type II was the most frequent genotype in blood samples and in subgingival plaque samples. However, no correlation was found between the frequency of any fimA type with SRP induced bacteremia. P. gingivalis fimA type appears to be conserved within individual patients throughout the times of sample collection. fimA gene sequence results were not in agreement with results of fimA genotyping by PCR.Introducción. Porphyromonas gingivalis es el principal agente etiológico de la periodontitis. El gen fimA ha sido relacionado con la virulencia del microorganismo, lo cual sugiere la participación de dicho gen en la capacidad del microorganismo para alcanzar el torrente sanguíneo.Objetivo. Estudiar la distribución de los tipos de fimA de P. gingivalis en muestras de placa subgingival y de sangre obtenidas durante bacteriemias después de raspaje y alisado radicular.Materiales y métodos. Se practicó un alisado radicular a 15 pacientes con periodontitis. Se obtuvieron aislamientos clínicos de P. gingivalis de la placa subgingival y durante la bacteriemia inducida por el procedimiento. Para la genotipificación se utilizó la técnica de reacción en cadena de la polimerasa (PCR) específica para fimA. En los aislamientos no clasificables por PCR se realizó secuenciación del gen fimA.Resultados. Seis pacientes fueron positivos para bacteriemia por P. gingivalis. La distribución de fimA evaluada en 30 aislamientos de placa subgingival y de sangre mostró una mayor frecuencia del fimA tipo II de P. gingivalis. En los aislamientos de placa subgingival, la detección de fimA tipo II fue seguida por Ib, III y IV; sin embargo, en los aislamientos de sangre el tipo II fue seguido por los tipos IV, Ib y III.Conclusión. En los aislamientos de sangre y de placa subgingival de pacientes con periodontitis el fimA más frecuente fue el tipo II; no fue posible correlacionar el tipo de fimA con la bacteriemia inducida por el alisado radicular. Los resultados de la secuenciación del gen fimA no concuerdan con los obtenidos por PCR

Prevalence of oropharyngeal beta-lactamase-producing Capnocytophaga spp. in pediatric oncology patients over a ten-year period

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

Antibiothérapie probabiliste en médecine bucco-dentaire et capnocytophaga spp.

La résistance bactérienne aux antibiotiques représente aujourd hui un véritable problème de santé publique. En médecine bucco-dentaire, le risque de sélection de souches résistantes est plus grand du fait du caractère poly-microbien des infections et de l antibiothérapie probabiliste, notamment via la prescription des béta-lactamines et du métronidazole. Parmi les bactéries de la cavité buccale, les Capnocytophaga sont des espèces ubiquitaires au sein des biofilms oraux. La détection concomitante de gènes de résistance aux béta-lactamines et de gènes de mobilité chez les Capnocytopahaga cliniques, par l équipe EA-1254, caractérise ces souches comme des réservoirs de gènes de résistance, capables de se mouvoir d une espèce bactérienne à l autre. Le présent travail a mis en évidence un caractère supplémentaire, la résistance au métronidazole, chez un certain nombre de ces souches. Il s est intéressé également à leur mécanisme moléculaire. Par ailleurs, il montre que la capacité de formation de biofilm chez ces souches est en grande partie espèce-dépendante.Today, antibiotics bacterial resistance represents a real public health problem. In oral medicine, there is a higher risk of resistant strains selection due to the particularity of poly-microbial infections and probabilistic antibacterial treatments, particularly in the prescription of beta-lactam antibiotics and metronidazole. Among oral bacterial strains, Capnocytophaga species are described as ubiquitous inside oral biofilms. The concomitant presence of beta-lactam resistance genes and mobility genes in the clinical Capnocytopahaga strains described by the EA-1254 team characterizes these bacteria as resistance gene reservoirs capable to move the resistance from a bacterial species to the other one. The present work highlights an additional character, the metronidazole resistance, in a number of these Capnocytophaga strains. This work equally focuses on the molecular mechanism of this resistance. Furthermore, it shows that the biofilm formation capacity of these bacteria is mainly species-dependent.RENNES1-BU Santé (352382103) / SudocSudocFranceF

A new device for rapid evaluation of antibiofilm formation

Objectives: This study describes the implementation of a new assay: Biofilm Ring Test ® to evaluate the efficacy of decontamination solutions to inhibit biofilm formation. This method is based on the immobilization of magnetic microbeads by bacterial cells forming biofilm in a modified polystyrene microtiter plate. Proteinase K (PK) was used as biofilm inhibitor in two models of biofilm formation in anaerobic conditions; Porphyromonas gingivalis ATCC 33277 mono specie and P. gingivalis/Streptococcus gordonii DL1 bi-species biofilms. Methods: Two hundred µl from P. gingivalis or P. gingivalis/S. gordonii culture were loaded in each well in triplicate. The PK solution was applied directly at a final concentration of 0.2%. After incubation at 37°C in an anaerobic incubator, plates were scanned prior and after magnetization and a BioFilm Index (BFI) was measured with the Biofilm Control®Software. A BFI ≤ 2 means a full immobilization of beads by a completely formed biofilm whereas a BFI ≥12 indicates no biofilm formation. Results: The capacity of different bacterial strains to form biofilm was validated using BioFilm Ring Test®. The BFI reached a value of 1.5 after 7 h for P. gingivalis alone and 3 h for the mixture of strains. Under the same conditions, the BFI increased when the 0.2% PK solution was added and reached a value of 4.70 ± 0.2 and 9.40 ± 0.1 respectively. The increased BFI compared to those observed in the absence of biofilm inhibitor showed the capacity of this new device to evaluate bacterial biofilm formation. Conclusion: In comparison with laborious methods, the Biofilm Ring Test ® may be used as a rapid, reproducible and easy-handling device to determine the effectiveness of anti-biofilm molecules. Considering these properties, this assay can be implemented for screening of biofilm formation by high numbers of bacterial strains with various molecules in the same assay

High prevalence of β-lactam and macrolide resistance genes in human oral Capnocytophaga species.

International audienceOBJECTIVES: To determine macrolide-lincosamide-streptogramin (MLS) resistance determinants in the Capnocytophaga genus and to describe the prevalence of β-lactam resistance genes in human oral Capnocytophaga species. METHODS: Forty-eight Capnocytophaga isolates identified by analysis of 16S rRNA sequences were isolated from subgingival samples from 14 haematology patients (HPs), 11 periodontitis patients (PPs) and 17 healthy volunteers (HVs). MICs of β-lactam and MLS antibiotics were obtained for all isolates. blaCfxA, blaCSP-1 (encoding a new class A β-lactamase) and MLS resistance genes [erm(F), erm(B), erm(Q), erm(D), erm(C) and erm(A)] were evaluated using specific PCR and sequencing. RESULTS: In HVs, which had the lowest prevalence of β-lactamase-producing isolates in comparison with the other groups (16%; P < 0.001), Capnocytophaga ochracea was the prominent species (68%; P < 0.03). In PPs, which had a high prevalence of β-lactamase-positive isolates (82%; P < 0.001), Capnocytophaga sputigena was more frequently identified (64%; P < 0.03). In HPs, 50% of isolates were β-lactamase-positive. The more rarely identified species (15%) Capnocytophaga gingivalis, Capnocytophaga granulosa and Capnocytophaga leadbetteri were isolated only from PPs and/or HPs. All β-lactam-resistant isolates (44%) were PCR-positive for blaCfxA (31%) or blaCSP-1 (12.5%). Interestingly, blaCSP-1 was identified only in a subgroup of the C. sputigena species. Twenty-nine percent of isolates were MLS resistant independently of species identification, β-lactamase production or patient group. The MLS-resistant isolates carried the erm(F) or erm(C) gene (93% and 7%, respectively), previously unknown in the Capnocytophaga genus. CONCLUSIONS: Our findings illustrate that Capnocytophaga species are important contributors to the β-lactam and MLS resistance gene reservoir in the oral microbiome

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of human oral Capnocytophaga species

International audienceMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for rapid identification of cfxA PCR positive and negative Capnocytophaga strains. Colonies were grown on blood agar, incubated anaerobically at 37 °C for 48 h, and were then evaluated by MALDI-TOF MS and 16S rRNA gene sequencing. Both methods identified all colonies to the genus level. The MALDI-TOF MS method gave the same result, at the species level, as 16S rRNA gene sequencing for 41/53 Capnocytophaga sp. strains (77.4%), but the limit of this technique was the absence of some species (C. leadbetteri, C. AHN) in the Biotyper-Bruker® database used in this study. Distinction between the cefotaxime resistant and susceptible strains was unsuccessful using the MALDI-TOF MS method. This technique had low discriminatory power to rapidly detect beta-lactamase-producing Capnocytophaga strains in clinical samples. However, the results from a score-oriented dendrogram confirmed MALDI-TOF MS is a rapid, inexpensive, and reliable method for Capnocytophaga species identification. Enrichment of the reference database used (Biotyper®) will improve future results

Silver-zeolite combined to polyphenol-rich extracts of Ascophyllum nodosum: potential active role in prevention of periodontal diseases.

International audienceThe purpose of this study was to evaluate various biological effects of silver-zeolite and a polyphenol-rich extract of A. nodosum (ASCOP) to prevent and/or treat biofilm-related oral diseases. Porphyromonas gingivalis and Streptococcus gordonii contribute to the biofilm formation associated with chronic periodontitis. In this study, we evaluated in vitro antibacterial and anti-biofilm effects of silver-zeolite (Ag-zeolite) combined to ASCOP on P. gingivalis and S. gordonii growth and biofilm formation capacity. We also studied the anti-inflammatory and antioxidant capacities of ASCOP in cell culture models. While Ag-zeolite combined with ASCOP was ineffective against the growth of S. gordonii, it showed a strong bactericidal effect on P. gingivalis growth. Ag-zeolite combined with ASCOP was able to completely inhibit S. gordonii monospecies biofilm formation as well as to reduce the formation of a bi-species S. gordonii/P. gingivalis biofilm. ASCOP alone was ineffective towards the growth and/or biofilm formation of S. gordonii and P. gingivalis while it significantly reduced the secretion of inflammatory cytokines (TNFα and IL-6) by LPS-stimulated human like-macrophages. It also exhibited antioxidant properties and decreased LPS induced lipid peroxidation in gingival epithelial cells. These findings support promising use of these products in future preventive or therapeutic strategies against periodontal diseases