Eremoxylarins D-J, Antibacterial Eremophilane Sesquiterpenes Discovered from an Endolichenic Strain of Xylaria hypoxylon.

An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.

Design, synthesis and biological evaluation of potential antibacterial butyrolactones.

Novel butyrolactone analogues were designed and synthesized based on the known lichen antibacterial compounds, lichesterinic acids (B-10 and B-11), by substituting different functional groups on the butyrolactone ring trying to enhance its activity. All synthesized butyrolactone analogues were evaluated for their in vitro antibacterial activity against Streptococcus gordonii. Among the derivatives, B-12 and B-13 had the lowest MIC of 9.38µg/mL where they have shown to be stronger bactericidals, by 2-3 times, than the reference antibiotic, doxycycline. These two compounds were then checked for their cytotoxicity against human gingival epithelial cell lines, Ca9-22, and macrophages, THP-1, by MTT and LDH assays which confirmed their safety against the tested cell lines. A preliminary study of the structure-activity relationships unveiled that the functional groups at the C4 position had an important influence on the antibacterial activity. An optimum length of the alkyl chain at the C5 position registered the best antibacterial inhibitory activity however as its length increased the bactericidal effect increased as well. This efficiency was attained by a carboxyl group substitution at the C4 position indicating the important dual role contributed by these two substituents which might be involved in their mechanism of action.

Strong mutator phenotype drives faster adaptation from growth on glucose to growth on acetate in Salmonella.

The metabolic adaptation of strong mutator strains was studied to better understand the link between the strong mutator phenotype and virulence. Analysis of the growth curves of isogenic strains of Salmonella, which were previously grown in M63 glucose media, revealed that the exponential phase of growth was reached earlier in an M63 acetate medium with strong mutator strains (mutated in mutS or in mutL) than with normomutator strains (P<0.05). Complemented strains confirmed the direct role of the strong mutator phenotype in this faster metabolic adaptation to the assimilation of acetate. In a mixed cell population, proliferation of strong mutators over normomutators was observed when the carbon source was switched from glucose to acetate. These results add to the sparse body of knowledge about strong mutators and highlight the selective advantage conferred by the strong mutator phenotype to adapt to a switch of carbon source in the environment. This work may provide clinically useful information given that there is a high prevalence of strong mutators among pathogenic strains of Salmonella and that acetate is the principal short chain fatty acid of the human terminal ileum and colon where Salmonella infection is localized.

Two human milk-like synthetic bacterial communities displayed contrasted impacts on barrier and immune responses in an intestinal quadricellular model.

The human milk (HM) microbiota, a highly diverse microbial ecosystem, is thought to contribute to the health benefits associated with breast-feeding, notably through its impact on infant gut microbiota. Our objective was to further explore the role of HM bacteria on gut homeostasis through a "disassembly/reassembly" strategy. HM strains covering the diversity of HM cultivable microbiota were first characterized individually and then assembled in synthetic bacterial communities (SynComs) using two human cellular models, peripheral blood mononuclear cells and a quadricellular model mimicking intestinal epithelium. Selected HM bacteria displayed a large range of immunomodulatory properties and had variable effects on epithelial barrier, allowing their classification in functional groups. This multispecies characterization of HM bacteria showed no clear association between taxonomy and HM bacteria impacts on epithelial immune and barrier functions, revealing the entirety and complexity of HM bacteria potential. More importantly, the assembly of HM strains into two SynComs of similar taxonomic composition but with strains exhibiting distinct individual properties, resulted in contrasting impacts on the epithelium. These impacts of SynComs partially diverged from the predicted ones based on individual bacteria. Overall, our results indicate that the functional properties of the HM bacterial community rather than the taxonomic composition itself could play a crucial role in intestinal homeostasis of infants.

Complete genome sequence of the strong mutator Salmonella enterica subsp. enterica serotype Heidelberg strain B182.

In bacteria, normal mutation frequencies are mostly around 10(-10) per base pair. However, there exists natural isolates, called "mutators," that exhibit permanent mutation occurrences up to 1,000-fold greater than usual. As mutations play essential roles, particularly in the evolution of antibiotic resistance, bacteria showing elevated mutation rates could have an important responsibility in the emergence of antibiotic resistance, especially in the clinical background. In this announcement, we report the first complete genome sequence of the Salmonella enterica subsp. enterica serotype Heidelberg B182 mutator strain, isolated from bovine feces (France), which consists of a 4,750,465-bp circular chromosome (cB182_4750; GC, 52.2%) and one circular plasmid of 37,581 bp (pB182_37; GC, 42.8%).

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.

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.

Bacteroides fragilis derived metabolites, identified by molecular networking, decrease Salmonella virulence in mice model.

In the gut microbiota, resident bacteria prevent pathogens infection by producing specific metabolites. Among bacteria belonging to phylum Bacteroidota, we have previously shown that Bacteroides fragilis or its cell-free supernatant inhibited in vitro Salmonella Heidelberg translocation. In the present study, we have analyzed this supernatant to identify bioactive molecules after extraction and subsequent fractionation using a semi-preparative reversed-phase Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS). The results indicated that only two fractions (F3 and F4) strongly inhibited S. Heidelberg translocation in a model mimicking the intestinal epithelium. The efficiency of the bioactive fractions was evaluated in BALB/c mice, and the results showed a decrease of S. Heidelberg in Peyer's patches and spleen, associated with a decrease in inflammatory cytokines and neutrophils infiltration. The reduction of the genus Alistipes in mice receiving the fractions could be related to the anti-inflammatory effects of bioactive fractions. Furthermore, these bioactive fractions did not alter the gut microbiota diversity in mice. To further characterize the compounds present in these bioactive fractions, Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS) data were analyzed through molecular networking, highlighting cholic acid (CA) and deoxycholic acid. In vitro, CA had inhibitory activity against the translocation of S. Heidelberg by significantly decreasing the expression of Salmonella virulence genes such as sipA. The bioactive fractions also significantly downregulated the flagellar gene fliC, suggesting the involvement of other active molecules. This study showed the interest to characterize better the metabolites produced by B. fragilis to make them means of fighting pathogenic bacteria by targeting their virulence factor without modifying the gut microbiota.

The L1 major capsid protein of HPV16 differentially modulates APC trafficking according to the vaccination or natural infection context.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. The aim of this study was to determine whether HPV16 viral particles can influence the trafficking of human DC/Langerhans cells (LC), either by direct interactions with DC or following incubation with human normal keratinocytes that are in close contact with LC in the squamous epithelium. We first demonstrated that HPV16 L1 major capsid protein, when self-assembled into virus-like particles (VLP), is able to induce in DC an over-expression of CXC receptor 4 (CXCR4) via the activation of the NF-κB signaling pathway and to enhance DC motility in the presence of CXCL12, suggesting an ability to migrate towards lymph nodes. We also showed that conditioned media of HPV16 VLP-treated keratinocytes induce a lower LC migration than those from untreated keratinocytes and that prostaglandin E2 (PGE(2)), detected in HPV16 VLP-treated keratinocyte supernatants, may reduce LC recruitment into the squamous epithelium. Taken together, our data demonstrate that HPV16 VLP may differentially regulate the immune protective response according to their target cells.

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-associated bacteria transform antibacterial usnic acid to products of lower antibiotic activity.

Lichens are specific symbiotic organisms harboring various microorganisms in addition to the two classic partners (algae or cyanobacterium and fungus). Although lichens produce many antibiotic compounds such as (+)-usnic acid, their associated microorganisms possess the ability to colonize an environment where antibiosis exists. Here, we have studied the behavior of several lichen-associated bacterial strains in the presence of (+)-usnic acid, a known antibiotic lichen compound. The effect of this compound was firstly evaluated on the growth and metabolism of three bacteria, thus showing its ability to inhibit Gram-positive bacteria. This inhibition was not thwarted with the usnic acid producer strain Streptomyces cyaneofuscatus. The biotransformation of this lichen metabolite was also studied. An ethanolamine derivative of (+)-usnic acid with low antibiotic activity was highlighted with chemical profiling, using HPLC-UV combined with low resolution mass spectrometry. These findings highlight the way in which some strains develop resistance mechanisms. A methylated derivative of (+)-usnic acid was annotated using the molecular networking method, thus showing the interest of this computer-based approach in biotransformation studies.

Inhibition of cervical cancer cell growth by human papillomavirus virus-like particles packaged with human papillomavirus oncoprotein short hairpin RNAs.

Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth.

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.

Antibacterial activities of natural lichen compounds against Streptococcus gordonii and Porphyromonas gingivalis.

The oral bacteria not only infect the mouth and reside there, but also travel through the blood and reach distant body organs. If left untreated, the dental biofilm that can cause destructive inflammation in the oral cavity may result in serious medical complications. In dental biofilm, Streptococcus gordonii, a primary oral colonizer, constitutes the platform on which late pathogenic colonizers like Porphyromonas gingivalis, the causative agent of periodontal diseases, will bind. The aim of this study was to determine the antibacterial activity of eleven natural lichen compounds belonging to different chemical families and spanning from linear into cyclic and aromatic structures to uncover new antibiotics which can fight against the oral bacteria. The compounds were screened by broth microdilution assay. Three compounds were shown to have promising antibacterial activities where the depsidone core with certain functional groups constituted the best compound, psoromic acid, with the lowest MICs=11.72 and 5.86µg/mL against S. gordonii and P. gingivalis, respectively. The compounds screened had promising antibacterial activity which might be attributed to some important functional groups as discussed in our study. The best compounds did not induce the death of gingival epithelial carcinoma cells (Ca9-22). These results introduce new compounds having potent antibacterial activities against oral pathogens causing serious medical complications.

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.

Hypermutator Salmonella Heidelberg induces an early cell death in epithelial cells.

We have previously described that a strain of Salmonella Heidelberg with a hypermutator phenotype, B182, adhered strongly to HeLa cells. In this work, we showed that this hypermutator Salmonella strain invaded HeLa epithelial cells and induced cytoskeleton alteration. Those changes lead to HeLa cell death which was characteristic of apoptosis. For the first time, we showed that this hypermutator strain induced apoptosis associated with the activation of caspases 2, 9 and 3. Complementation of B182 strain showed a decrease in cells death induction. In the presence of other Salmonella Heidelberg with a normomutator phenotype, such as WT and SL486, cell death and caspase 3 were undetectable. These results suggested that early apoptosis and caspase 3 activation were specific to B182. Besides, B182 induced LDH release and caspase 3 activation in CaCo-2 and HCT116 cells. Heat-treated B182 and diffusible products failed to induce this phenotype. Epithelial cells treatment with cytochalasin D caused the inhibition of B182 internalisation and caspase 3 activation. These results showed that this cell death required active S. Heidelberg B182 protein synthesis and bacterial internalisation. However sipB and sopB, usually involved in apoptosis induced by Salmonella were not overexpressed in B182, contrary to fimA and fliC. Comparative genome analysis showed numerous mutations as in rpoS which would be more investigated. The role of the hypermutator phenotype might be suspected to be implicated in these specific features. This result expands our knowledge about strong mutators frequently found in bacterial organisms isolated from clinical specimens.

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.

Role of a short tandem leucine/arginine repeat in strong mutator phenotype acquisition in a clinical isolate of Salmonella Typhimurium.

In this prospective study, a strong mutator strain of Salmonella Typhimurium was isolated from a collection of 130 human clinical strains of Salmonella. Sequence analysis of the mutS, mutL, and mutH genes, which encode three proteins that are essential for initiation of methyl-directed DNA mismatch repair, revealed insertion of a short tandem repeat (STR) of leucine/alanine in the histidine kinase-like ATPase domain of MutL. The role of this STR in the acquisition of the strong mutator phenotype was confirmed by the construction of an isogenic mutant (6bpinsmutL) from a normomutator strain of Salmonella Heidelberg. This result adds to the sparse body of knowledge about strong mutators and highlights the role of this STR as a hotspot for the acquisition of a strong mutator phenotype in Salmonella.

Impact of a mutator phenotype on motility and cell adherence in Salmonella Heidelberg.

In this study, we investigated adherence and motility of the hypermutator Salmonella enterica Heidelberg B182 bovine strain related to a 12bp deletion in mutS. This mutator phenotype was associated with increased adherence to epithelial cells and with high expression of fimA as shown by real-time RT-PCR. Motility studies showed that fliC were up-regulated in the B182 strain, while fljA and fljB were down-regulated. In order to determine if mutated mutS is implicated in this genes expression, isogenic strains, derived from a WT strain, containing the 12bp deletion in mutS (Δ12bpmutS) or an inactivated mutS (ΔmutS) were generated. Δ12bpmutS and ΔmutS strains showed a spontaneous mutation rate similar to the environmental strain B182, but exhibited lower adherence capacity and fimA expression. In contrast to the fimbriae genes, in Δ12bpmutS, fliC expression was up-regulated, but fljA and fljB expression were decreased, as in the B182 strain. Only fljB expression was increased in ΔmutS mutants. Taken together, our data suggest that mutS alteration does not influence fimbriae expression but can impact flagella genes.

Bacterial hypermutation: clinical implications.

Heritable hypermutation in bacteria is mainly due to alterations in the methyl-directed mismatch repair (MMR) system. MMR-deficient strains have been described from several bacterial species, and all of the strains exhibit increased mutation frequency and recombination, which are important mechanisms for acquired drug resistance in bacteria. Antibiotics select for drug-resistant strains and refine resistance determinants on plasmids, thus stimulating DNA recombination via the MMR system. Antibiotics can also act as indirect promoters of antibiotic resistance by inducing the SOS system and certain error-prone DNA polymerases. These alterations have clinical consequences in that efficacious treatment of bacterial infections requires high doses of antibiotics and/or a combination of different classes of antimicrobial agents. There are currently few new drugs with low endogenous resistance potential, and the development of such drugs merits further research.