Roseburia intestinalis Modulates PYY Expression in a New a Multicellular Model including Enteroendocrine Cells.

The gut microbiota contributes to human health and disease; however, the mechanisms by which commensal bacteria interact with the host are still unclear. To date, a number of in vitro systems have been designed to investigate the host-microbe interactions. In most of the intestinal models, the enteroendocrine cells, considered as a potential link between gut bacteria and several human diseases, were missing. In the present study, we have generated a new model by adding enteroendocrine cells (ECC) of L-type (NCI-H716) to the one that we have previously described including enterocytes, mucus, and M cells. After 21 days of culture with the other cells, enteroendocrine-differentiated NCI-H716 cells showed neuropods at their basolateral side and expressed their specific genes encoding proglucagon (GCG) and chromogranin A (CHGA). We showed that this model could be stimulated by commensal bacteria playing a key role in health, Roseburia intestinalis and Bacteroides fragilis, but also by a pathogenic strain such as Salmonella Heidelberg. Moreover, using cell-free supernatants of B. fragilis and R. intestinalis, we have shown that R. intestinalis supernatant induced a significant increase in IL-8 and PYY but not in GCG gene expression, while B. fragilis had no impact. Our data indicated that R. intestinalis produced short chain fatty acids (SCFAs) such as butyrate whereas B. fragilis produced more propionate. However, these SCFAs were probably not the only metabolites implicated in PYY expression since butyrate alone had no effect. In conclusion, our new quadricellular model of gut epithelium could be an effective tool to highlight potential beneficial effects of bacteria or their metabolites, in order to develop new classes of probiotics.

SARS-CoV-2 and Prevotella spp.: friend or foe? A systematic literature review.

During this global pandemic of the COVID-19 disease, a lot of information has arisen in the media and online without scientific validation, and among these is the possibility that this disease could be aggravated by a secondary bacterial infection such as Prevotella, as well as the interest or not in using azithromycin, a potentially active antimicrobial agent. The aim of this study was to carry out a systematic literature review, to prove or disprove these allegations by scientific arguments. The search included Medline, PubMed, and Pubtator Central databases for English-language articles published 1999-2021. After removing duplicates, a total of final eligible studies (n=149) were selected. There were more articles showing an increase of Prevotella abundance in the presence of viral infection like that related to Human Immunodeficiency Virus (HIV), Papillomavirus (HPV), Herpesviridae and respiratory virus, highlighting differences according to methodologies and patient groups. The arguments for or against the use of azithromycin are stated in light of the results of the literature, showing the role of intercurrent factors, such as age, drug consumption, the presence of cancer or periodontal diseases. However, clinical trials are lacking to prove the direct link between the presence of Prevotella spp. and a worsening of COVID-19, mainly those using azithromycin alone in this indication.

Next-Generation Probiotics and Their Metabolites in COVID-19.

Since December 2019, a global pandemic has been observed, caused by the emergence of a new coronavirus, SARS CoV-2. The latter is responsible for the respiratory disease, COVID-19. The infection is also characterized by renal, hepatic, and gastrointestinal dysfunctions suggesting the spread of the virus to other organs. A dysregulated immune response was also reported. To date, there is no measure to treat or prevent SARS CoV-2 infection. Additionally, as gut microbiota composition is altered in patients with COVID-19, alternative therapies using probiotics can be considered to fight SARS CoV-2 infection. This review aims at summarizing the current knowledge about next-generation probiotics (NGPs) and their benefits in viral respiratory tract infections and in COVID-19. We describe these bacteria, highlighted by studies using metagenomic approaches. In addition, these bacteria generate metabolites such as butyrate, desaminotyrosine, and secondary bile acid, suggested to prevent viral respiratory infections. Gut microbial metabolites transported via the circulation to the lungs could inhibit viral replication or improve the immune response against viruses. The use of probiotics and/or their metabolites may target either the virus itself and/or the immunologic process. However, this review showed that more studies are needed to determine the benefits of probiotics and metabolite products in COVID-19.

Lichen butyrolactone derivatives disrupt oral bacterial membrane.

We have previously demonstrated that out of the butyrolactones series synthesized based on the natural lichen metabolite lichesterinic acid, compound (B-13) was the most effective against oral bacteria. However, its antibacterial mechanism is still unknown. In this study, we have investigated its bacterial localization by synthesizing a fluorescently labeled B-13 with NBD while maintaining its antibacterial activity. We showed that this compound binds to Streptococcus gordonii cell surface, as demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. A Gram-negative counterpart, Porphyromanas gingivalis, showed also cracked and ruptured cells in the presence of B-13. Besides, we also demonstrated that the analog of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. This study revealed that butyrolactones can be considered as potent antibacterial compounds against oral pathogens causing medical complications.

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

Matrix-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.

A new mathematical model of bacterial interactions in two-species oral biofilms.

Periodontitis are bacterial inflammatory diseases, where the bacterial biofilms present on the tooth-supporting tissues switch from a healthy state towards a pathogenic state. Among bacterial species involved in the disease, Porphyromonas gingivalis has been shown to induce dysbiosis, and to induce virulence of otherwise healthy bacteria like Streptococcus gordonii. During biofilm development, primary colonizers such as S. gordonii first attach to the surface and allow the subsequent adhesion of periodontal pathogens such as P. gingivalis. Interactions between those two bacteria have been extensively studied during the adhesion step of the biofilm. The aim of the study was to understand interactions of both species during the growing phase of the biofilm, for which little knowledge is available, using a mathematical model. This two-species biofilm model was based on a substrate-dependent growth, implemented with damage parameters, and validated thanks to data obtained on experimental biofilms. Three different hypothesis of interactions were proposed and assayed using this model: independence, competition between both bacteria species, or induction of toxicity by one species for the other species. Adequacy between experimental and simulated biofilms were found with the last hypothetic mathematical model. This new mathematical model of two species bacteria biofilms, dependent on different substrates for growing, can be applied to any bacteria species, environmental conditions, or steps of biofilm development. It will be of great interest for exploring bacterial interactions in biofilm conditions.

Roseburia spp.: a marker of health?

The genus Roseburia consists of obligate Gram-positive anaerobic bacteria that are slightly curved, rod-shaped and motile by means of multiple subterminal flagella. It includes five species: Roseburia intestinalis, R. hominis, R. inulinivorans, R. faecis and R. cecicola. Gut Roseburia spp. metabolize dietary components that stimulate their proliferation and metabolic activities. They are part of commensal bacteria producing short-chain fatty acids, especially butyrate, affecting colonic motility, immunity maintenance and anti-inflammatory properties. Modification in Roseburia spp. representation may affect various metabolic pathways and is associated with several diseases (including irritable bowel syndrome, obesity, Type 2 diabetes, nervous system conditions and allergies). Roseburia spp. could also serve as biomarkers for symptomatic pathologies (e.g., gallstone formation) or as probiotics for restoration of beneficial flora.

cfxA expression in oral clinical Capnocytophaga isolates.

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.

Oral Gram-negative anaerobic bacilli as a reservoir of ß-lactam resistance genes facilitating infections with multiresistant bacteria.

Many ß-lactamases have been described in various Gram-negative bacilli (Capnocytophaga, Prevotella, Fusobacterium, etc.) of the oral cavity, belonging to class A of the Ambler classification (CepA, CblA, CfxA, CSP-1 and TEM), class B (CfiA) or class D in Fusobacterium nucleatum (FUS-1). The minimum inhibitory concentrations of ß-lactams are variable and this variation is often related to the presence of plasmids or other mobile genetic elements (MGEs) that modulate the expression of resistance genes. DNA persistence and bacterial promiscuity in oral biofilms also contribute to genetic transformation and conjugation in this particular microcosm. Overexpression of efflux pumps is facilitated because the encoding genes are located on MGEs, in some multidrug-resistant clinical isolates, similar to conjugative transposons harbouring genes encoding ß-lactamases. All these facts lead us to consider the oral cavity as an important reservoir of ß-lactam resistance genes and a privileged place for genetic exchange, especially in commensal strictly anaerobic Gram-negative bacilli.

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

The 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.

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

OBJECTIVES: To determine macrolide-lincosamide-streptogramin (MLS) resistance determinants in the Capnocytophaga genus and to describe the prevalence of ß-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 ß-lactam and MLS antibiotics were obtained for all isolates. blaCfxA, blaCSP-1 (encoding a new class A ß-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 ß-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 ß-lactamase-positive isolates (82%; P < 0.001), Capnocytophaga sputigena was more frequently identified (64%; P < 0.03). In HPs, 50% of isolates were ß-lactamase-positive. The more rarely identified species (15%) Capnocytophaga gingivalis, Capnocytophaga granulosa and Capnocytophaga leadbetteri were isolated only from PPs and/or HPs. All ß-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, ß-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 ß-lactam and MLS resistance gene reservoir in the oral microbiome.

Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis.

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Distribution of Porphyromonas gingivalis fimA genotypes in isolates from subgingival plaque and blood sample during bacteremia

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.

Distribution of Porphyromonas gingivalis fimA genotypes in isolates from subgingival plaque and blood sample during bacteremia.

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.

Influence of previous antimicrobial therapy on oral carriage of beta-lactamase producing Capnocytophaga isolates.

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.

The antibacterial activity of tramadol against bacteria associated with infectious complications after local or regional anesthesia.

BACKGROUND: Tramadol is a synthetic analog of codeine with opioid and local anesthetic properties. It is used as a central-acting analgesic, and recently, in subcutaneous or intradermal injections, as a local anesthetic. We investigated in vitro the antibacterial activity of tramadol in the absence of any local anesthetics against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa pathogens that can cause infectious complications after local or regional anesthesia. METHODS: Bacterial cultures were grown for 18 h, diluted in sterile physiological saline, and incubated for 6 or 24 h at 37 degrees C with 6.25, 12.5, or 25 mg/mL tramadol. The mixtures were then plated onto blood agar and colony counts were recorded after 24 h incubation at 37 degrees C. RESULTS: Tramadol had bactericidal activity against E. coli and S. epidermidis compared with controls: at 25 mg/mL for 6 h or at 12.5 mg/mL for 24 h, tramadol decreased by approximately 7 log(10) (P < 0.001) the colony counts of E. coli (100% kill). Similar results were obtained with S. epidermidis, with approximately 6 log(10) reduction (100% kill) when tramadol was used at 25 mg/mL for 24 h (P < 0.001). The antibacterial effect of 25 mg/mL tramadol was lower against S. aureus and P. aeruginosa, reducing the growth of these strains by approximately 3log(10) after 24 h (P < 0.001). CONCLUSIONS: Tramadol has dose- and time-dependent bactericidal activity against E. coli and S. epidermidis, as well as antibacterial activity against S. aureus and P. aeruginosa. The antibacterial properties of tramadol may be useful for reducing the risk of bacterial infection after local or regional anesthesia.

Antimicrobial treatment of Capnocytophaga infections.

Capnocytophaga spp. are normal inhabitants of the oropharyngeal flora. They are also involved in periodontal diseases or animal bites, complicated by septicaemia with dissemination to a great variety of sites, both in immunocompetent and immunocompromised hosts. This review will focus on their pathogenesis, spectrum of clinical infections and susceptibility to disinfectants and antibiotics. The spread of beta-lactamase-producing strains limits the use of beta-lactams as first-line treatments, underlying the necessity to test the in vitro susceptibility of clinical strains. Many antimicrobial treatments have been used, despite an absence of randomised studies and guidelines regarding the duration of treatment according to infected sites. Imipenem/cilastatin, clindamycin or beta-lactamase inhibitor combinations are always effective and their use can be recommended in all infections.

Peracetic acid stress-induced genetic rearrangements in Escherichia coli H10407 detected by RAPD and RFLP analyses.

The discriminatory powers of random amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP) were assessed for the detection and comparison of DNA modifications caused by an oxidative stress. DNA extracted from peracetic acid (PAA)-treated Escherichia coli H10407 was randomly amplified with the 10-mer primer OPZ14, which generated one stress-induced fragment. RFLP and RAPD profiles were hybridized by Southern blotting with the digoxigenin-labelled RAPD product. Untreated and PAA-treated cells had difference band profiles. The results indicate that RAPD analysis could be used as a discriminatory tool for investigating genetic rearrangements in E. coli caused by oxidative stress and that RFLP analysis could be used to confirm the rearrangements.

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.