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International audience; Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria, such as Gardnerella vaginalis, and low levels of "healthy" lactobacilli leads to bacterial vaginosis (BV), usually associated with a low-grade inflammatory process. Despite appropriate antibiotic treatment, G. vaginalis-associated BV is characterized by significant recurrence. The use of probiotics could be an interesting alternative therapy due to their ability to rebalance vaginal microbiota. In this study, we investigated the effects of a wellcharacterized probiotic strain, Lacticaseibadllus rhamnosus Lcr35, on epithelial vaginal and dendritic cell (DC) immune responses after G. vaginalis infection. In an in vitro coculture model with human monocyte-derived dendritic cells and a vaginal epithelial cell NEC) monolayer, the Lcr35 strain induced DC activation, as evidenced by the induction of maturation and synthesis of interleukin-8 (IL-8) and CCL-20 chemokines upon apical challenge of the VECs by G. vaginas. Analysis of the vaginal epithelial response showed that the presence of Lcr35 significantly increased the production of the proinflammatory cytokines IL-8 and IL-1 beta and human beta-defensin 2 (HBD-2), whereas the concentration of secretory leukocyte protease inhibitor (SLPI) was decreased in G. vagina/is-infected vaginal epithelial cells. Treatment with recombinant SLPI was associated with upregulation of Lcr35-stimulated IL-8 and HBD-2 production. These results suggest that inhibition of SLPI by Lcr35 in vaginal epithelial cells contributes to the host defense response against G. vaginalis infection.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

International audience; The treatment of osteomyelitis, a destructive inflammatory process caused by bacterial infections to bone tissue, is one of the most critical challenges of orthopedics and bone regenerative medicine. The standard treatment consists of intense antibiotic therapies combined with tissue surgical debridement and the application of a bone defect filler material. Unfortunately, commercially available candidates, such as gentamicin-impregnated polymethylmethacrylate cements, possess very poor pharmacokinetics (i.e., 24 hours burst release) and little to no regenerative potential. Fostered by the intrinsic limitations associated with conventional treatments, alternative osteostimulative biomaterials with local drug delivery have recently started to emerge. In this study, we propose the use of a polycaprolactone-silica sol–gel hybrid material as carrier for the delivery of rifampicin, an RNA-polymerase blocker often used to treat bone infections, and of osteostimulative silicate ions. The release of therapeutic agents from the material is dual, offering two separate and simultaneous effects, and decoupled, meaning that the kinetics of rifampicin and silicate releases are independent from each other. A series of hybrid formulations with increasing amounts of rifampicin was prepared. The antibiotic loading efficacy, as well as the release profiles of rifampicin and silicates were measured. The characterization of cell viability and differentiation of rat primary osteoblasts and antibacterial performance were also performed. Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Escherichia coli were selected due to their high occurrence in bone infections. Results confirmed that rifampicin can be successfully loaded within the hybrids without significant degradation and that it is possible to tailor the antibiotic release according to need. Once in a physiological environment, the rapid release of silicates was associated with optimal cell proliferation and the overexpression of osteoblastic differentiation. Simultaneously, rifampicin is delivered over the course of several weeks with significant inhibition of all tested strains. In particular, the materials caused a growth reduction of 7–10 orders of magnitude in Staphylococcus aureus, the major strain responsible for osteomyelitis worldwide. Our data strongly suggest that PCL/silica hybrids are a very promising candidate to develop bone fillers with superior biological performance compared to currently available options. Thanks to their unique synthesis route and their dual tailored release they can promote bone regeneration while reducing the risk of infection for several weeks upon implantation.

International audience; Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

Cytotoxicity and antibacterial properties associated with the dopant release of Cu-doped Biphasic Calcium Phosphate (BCP) powders, mainly composed of hydroxyapatite mixed with β-tricalcium phosphate powders, were investigated. Twelve BCP ceramics were synthesized at three different sintering temperatures (600 °C, 900 °C and 1200 °C) and four copper doping rates (x = 0.0, 0.05, 0.10 and 0.20, corresponding to the stoichiometric amount of copper in Ca10Cux(PO4)6(OH)2-2xO2x). Cytotoxicity assessments of Cu-doped BCP powders, using MTT assay with human-Mesenchymal Stem Cells (h-MSCs), indicated no cytotoxicity and the release of less than 12 ppm of copper into the biological medium. The antibacterial activity of the powders was determined against both Gram-positive (methicillin-sensitive (MS) and methicillin resistant (MR) Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The Cu-doped biomaterials exhibited a strong antibacterial activity against MSSA, MRSA and E. coli, releasing approximatively 2.5 ppm after 24 h, whereas 10 ppm were required to induce an antibacterial effect against P. aeruginosa. This study also demonstrated that the culture medium used during experiments can directly impact the antibacterial effect observed, only 4 ppm of Cu2+ were effective for killing all the bacteria in a 1:500 diluted TS medium, whereas 20 ppm were necessary to achieve the same result in a rich, non-diluted standard marrow cell culture medium.

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International audience; Copper is an essential trace element required for human life, and is involved in several physiological mechanisms. Today researchers have found and confirmed that Cu has biological properties which are particularly useful for orthopedic biomaterials applications such as implant coatings or biodegradable filler bone substitutes. Indeed, Cu exhibits antibacterial functions, provides angiogenic ability and favors osteogenesis; these represent major key points for ideal biomaterial integration and the healing process that follows. The antibacterial performances of copper-doped biomaterials present an interesting alternative to the massive use of prophylactic antibiotics and help to limit the development of antibiotic resistance. By stimulating blood vessel growth and new bone formation, copper contributes to the improved bio-integration of biomaterials. This review describes the bio-functional advantages offered by Cu and focuses on the antibacterial, angiogenic and osteogenic properties of Cu-doped biomaterials with potential for orthopedic applications.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Copper is an essential trace element required for human life, and is involved in several physiological mechanisms. Today researchers have found and confirmed that Cu has biological properties which are particularly useful for biomaterials applications. Indeed, Cu exhibits antibacterial functions, provides angiogenic ability and favors osteogenesis; these represent major key points for ideal biomaterial integration and the healing process that follows. Copper-doped biomaterials thus present an interesting alternative to the massive use of prophylactic antibiotics to prevent infection complications following a surgical act for bone tissue reconstruction. This review describes the bio-functional advantages offered by Cu and focuses on the antibacterial, angiogenic and osteogenic properties of Cu-doped biomaterials used in orthopedic applications. The chemical role of doping - used in all families of biomaterials (ceramics, composites and metals) - is the main concern of this article. Other aspects, independent of doping, which may be important for optimizing biomaterial efficiency (in particular surface structuration), are also mentioned here, although they are not the focus of this review.

Persisters form sub-populations of stress-tolerant cells that play a major role in the capacity of biofilms to survive and recover from disturbances such as antibiotic treatments. The mechanisms of persistence are diverse and influenced by environmental conditions, and persister populations are more heterogeneous than formerly suspected. We used computational modeling to assess the impact of three switching strategies between susceptible and persister cells on the capacity of bacterial biofilms to grow, survive and recover from antibiotic treatments. The strategies tested were: (1) constant switches, (2) substrate-dependent switches and (3) antibiotic-dependent switches. We implemented these strategies in an individual-based biofilm model and simulated antibiotic shocks on virtual biofilms. Because of limited available data on switching rates in the literature, nine parameter sets were assessed for each strategy. Substrate and antibiotic-dependent switches allowed high switching rates without affecting the growth of the biofilms. Compared to substrate-dependent switches, constant and antibiotic-dependent switches were associated with higher proportions of persisters in the top of the biofilms, close to the substrate source, which probably confers a competitive advantage within multi-species biofilms. The constant and substrate-dependent strategies need a compromise between limiting the wake-up and death of persisters during treatments and leaving the persister state fast enough to recover quickly after antibiotic-removal. Overall, the simulations gave new insights into the relationships between the dynamics of persister populations in biofilms and their dynamics of growth, survival and recovery when faced with disturbances., Biofilm persistence: Strategies for switching Computer simulations yield useful insights into strategies bacteria in biofilms use to switch into antibiotic-tolerant “persister” states. A diverse range of mechanisms are available to promote the transition into persister forms. The populations of cells in persister states are proving to be more varied than previously thought. Researchers in France, led by Gabriel Carvalho at the Complex Systems Engineering Laboratory in Aubière, modelled different switching strategies that allow biofilms to survive, recover and grow despite antibiotic treatments. Their procedure examines the effect on switching rates of a greater range of variable factors than previous simulation methods. These variables include the concentration and flow patterns of relevant chemical compounds, changing environmental conditions, and the challenge presented by different antibiotics. The results will help research toward understanding and treating a variety of medically significant biofilm infections.

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self-produced extracellular matrix. Although a biofilm is commonly described as a "cozy house" where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm-dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.

Background: Klebsiella pneumoniae is a major cause of community-acquired and nosocomial infections. This germ is responsible for acute and chronic infections, most of which are due to its ability to adhere to medical implants and form a biofilm. The objective of this work is to study the interaction between clinical isolates of K. pneumoniae and abiotic surfaces (medical devices) and some factors influencing biofilm formation. Methods: Over a period of 2 years, 115 strains of K. pneumoniae were isolated from medical devices CHU Tlemcen, most of which had a high level of resistance to cephalosporins 1st, 2nd, and 3rd generation. Their capacity to form biofilm was assessed using two 3 techniques: TCP, TP, and RCA. We determined in vitro the effects of three antimicrobial agents against planktonic and biofilm forms of K. pneumoniae. The presence of MrkD genes was detected by polymerase chain reaction (PCR). Results: According to the studied (TCP, TP, RCA) strains of K .pneumoniae isolated from urinary catheters have proved very good, forming the biofilm to those isolated from other medical devices. 24 of 115 isolated strains showed a clear difference in antibiotic susceptibility between planktonic populations and biofilm populations. They were 10-20 times higher. All strains presented a highly hydrophilic character and adhesion 2-10 times greater in PVC with respect to glass support. The MrkD gene (detected by PCR) responsible for biofilm formation was found in 22 strains of K. pneumoniae, which may explain their adhesion and therefore their pathogenicity. Conclusion: Our results show the great ability of K.pneumoniae strains to form a biofilm on medical devices, and the isolates were at least 10 times more resistant than their planktonic counterparts. In addition, we showed that the presence of type 3-encoding gene mrkD was associated with high adhesion indexes.

International audience; Azo dyes are widely used in industries and their release in the environment contributes to the pollution of effluents. The authors aim to develop a new eco-friendly water treatment method for the degradation of azo dyes based on in situ magnetic separation and immobilisation of bacterial cells. The immobilisation was achieved using superparamagnetic Fe3O4 nanoparticles and offers the possibility of reusing bacteria by magnetic separation for several degradation cycles. The iron–oxide nanoparticles were synthesised by reverse co-precipitation. The Gram-positive bacteria Bacillus subtilis were immobilised using iron–oxide nanoparticles by adsorption and then separated with an external magnetic field. Transmission electron microscopy observation showed that the particles' diameter was ∼20 nm with a narrow size distribution. Moreover, the iron–oxide nanoparticles were adsorbed onto the surface in order to coat the cells. B. subtilis has proved its ability to decolorise and degrade several azo dyes at different values of pH, with the highest decolorisation rate for Congo red. Furthermore, immobilised cells have a degradation activity similar to that of free cells. The system provided a degradation rate up to 80% and could be reused for seven batch cycles.

International audience; Resilience is the capacity of systems to recover their initial state or functions after a disturbance. The concepts of resilience and resistance are complementary in ecology and both represent different aspects of the stability of ecosystems. However, antibiotic resilience is not used in clinical bacteriology whereas antibiotic resistance is a recognized major problem. To join the fields of ecology and clinical bacteriology, we first review the resilience concept from ecology, socio-ecological systems and microbiology where it is widely developed. We then review resilience-related concepts in microbiology, including bacterial tolerance and persistence, phenotypic heterogeneity and collective tolerance and resistance. We discuss how antibiotic resilience could be defined and argue that the use of this concept largely relies on its experimental measure and its clinical relevance. We review indicators in microbiology which could be used to reflect antibiotic resilience and used as valuable indicators to anticipate the capacity of bacteria to recover from antibiotic treatments.

Some respiratory infections have been associated with dysbiosis of the intestinal microbiota. The underlying mechanism is incompletely understood, but cross talk between the intestinal microbiota and local immune cells could influence the immune response at distal mucosal sites. This has led to the concept of enhancing respiratory defenses by modulating the intestinal microbiota with exogenous supplementation of beneficial strains. In this study, we examined the effect of Lactobacillus plantarum CIRM653 on the inflammatory response induced by the pathogen Klebsiella pneumoniae. Oral administration of L. plantarum CIRM653 to mice subsequently infected by K. pneumoniae via the nasal route (i) reduced the pulmonary inflammation response, with decreased numbers of lung innate immune cells (macrophages and neutrophils) and cytokines (mouse keratinocyte-derived chemokine [KC], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) in the bronchoalveolar fluid, and (ii) induced an immunosuppressive Treg response in lungs. In vitro coincubation of L. plantarum CIRM653 and K. pneumoniae with human dendritic cells and peripheral blood mononuclear cells resulted in decreased Th1 (IL-12p70 and interferon gamma [IFN-γ]) and Th17 (IL-23 and IL-17) and increased Treg (IL-10) cytokine levels compared to those observed for K. pneumoniae-infected cells. Neither K. pneumoniae nor L. plantarum CIRM653 had any effect on cytokine production by intestinal epithelial cells in vitro, but the induction of the NF-κB pathway and IL-8 and IL-6 production by K. pneumoniae in airway epithelial cells was significantly reduced when the pathogen was coincubated with L. plantarum CIRM653. The remote IL-10-mediated modulation of the K. pneumoniae inflammatory response by L. plantarum CIRM653 supports the concept of immunomodulation by beneficial bacteria through the gut-lung axis.

International audience; Bacterial resistance to carbapenem, which is mainly due to the successful dissemination of carbapenemase-encoding genes, has become a major health problem. Few studies have aimed to characterize the level of resistance in the environment, notably in hospital wastewater, which is a likely hotspot for exchange of antibiotic resistance genes.In this work, we looked for the presence of imipenem-resistant bacteria and imipenem in the effluent of the teaching hospital of Clermont-Ferrand, France. Selective growth of bacteria from 14-day old biofilms formed in the pipe sewer showed that 22.1% of the isolates were imipenem-resistant and identified as Aeromonas (n = 23), Pseudomonas (n = 10), Stenotrophomonas (n = 4) and Acinetobacter (n = 1). Fifteen of these strains harbored acquired carbapenemase-encoding genes blaVIM (n = 11), blaOXA-48 (n = 2), blaGES (n = 1), blaNDM (n = 1). All isolates also harbored associated resistances to aminoglycosides, fluoroquinolones and/or tetracyclin. S1-nuclease pulsed-field gel electrophoresis analysis of eight selected isolates showed that four of them harbored one to two plasmids of molecular weight between 48.5 Kb and 194 Kb. In vitro transformation assays evidenced the presence of blaVIM and blaNDM on plasmids with the blaVIM harboring 80 Kb plasmid having conjugative capacity.The predicted environmental concentration of imipenem in the hospital effluent was 3.16 μg/L, suggesting that biofilm bacteria are subjected to sub-MICs of imipenem within the effluent. However, no imipenem molecule was detected in the hospital effluent, probably owing to its instability: in vitro assays indicated that imipenem's biological activity was no longer detectable after 45 h of storage. However, the predictive value of the hazard quotient relative to the development of resistance was >1.0 (HQr = 28.9 ± 1.9), which indicates a possible risk.The presence of carbapenemase-encoding genes in hospital effluent biofilm strains and their ability to transfer are therefore a potential hazard that should not be neglected and points to the need for monitoring antibiotic resistance in hospital wastewater.

Biofilm-dispersal is a key determinant for further dissemination of biofilm-embedded bacteria. Recent evidence indicates that biofilm-dispersed bacteria have transcriptional features different from those of both biofilm and planktonic bacteria. In this study, the in vitro and in vivo phenotypic properties of Klebsiella pneumoniae cells spontaneously dispersed from biofilm were compared with those of planktonic and sessile cells. Biofilm-dispersed cells, whose growth rate was the same as that of exponential planktonic bacteria but significantly higher than those of sessile and stationary planktonic forms, colonized both abiotic and biotic surfaces more efficiently than their planktonic counterparts regardless of their initial adhesion capabilities. Microscopy studies suggested that dispersed bacteria initiate formation of microcolonies more rapidly than planktonic bacteria. In addition, dispersed cells have both a higher engulfment rate and better survival/multiplication inside macrophages than planktonic cells and sessile cells. In an in vivo murine pneumonia model, the bacterial load in mice lungs infected with biofilm-dispersed bacteria was similar at 6, 24 and 48 h after infection to that of mice lungs infected with planktonic or sessile bacteria. However, biofilm-dispersed and sessile bacteria trend to elicit innate immune response in lungs to a lesser extent than planktonic bacteria. Collectively, the findings from this study suggest that the greater ability of K. pneumoniae biofilm-dispersed cells to efficiently achieve surface colonization and to subvert the host immune response confers them substantial advantages in the first steps of the infection process over planktonic bacteria.

International audience; Amphipathic cationic peptoids (N‐substituted glycine oligomers) represent a promising class of antimicrobial peptide mimics. The aim of this study is to explore the potential of the triazolium group as a cationic moiety and helix inducer to develop potent antimicrobial helical peptoids. Herein we report the first solid‐phase synthesis of peptoid oligomers incorporating 1,2,3‐triazolium‐type side chains and their evaluation against Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Several triazolium‐based oligomers, even of short length, selectively kill bacteria over mammalian cells. SEM visualization of S. aureus cells treated with a dodecamer and a hexamer reveals severe cell membrane damage and suggests that the longer oligomer acts by pore formation.

International audience; Beneficial bacteria represent potential sources of therapy, particularly in the battle against antibiotic-resistant pathogens. The Gram-negative bacillus Klebsiella pneumoniae is not only a paradigm of multi-resistant opportunistic pathogen, but it is also able to colonise the human intestine and displays a high capacity to form biofilm. In this study, the anti-biofilm activity of 140 neutralised Lactobacillus supernatants was assessed against K. pneumoniae. Among the 13 strains whose supernatant significantly impaired biofilm formation, Lactobacillus plantarum CIRM653 was selected because it was also able to impair K. pneumoniae preformed biofilm, independently of a bactericidal effect. Mixed K. pneumoniae/L. plantarum CIRM653 biofilms had reduced tridimensional structures associated with a significant decrease in K. pneumoniae biomass. Further investigation showed that L. plantarum CIRM653 supernatant induced transcriptional modifications of K. pneumoniae biofilm-related genes, including down-regulation of the quorum sensing-related lsr operons and over-expression of type 3 pili structure genes. Increased production of type 3 pili was validated by Western-blot, hemagglutination and adhesion assays. L. plantarum CIRM653 activity against K. pneumoniae was also assessed in a murine intestinal colonisation model: a constant faecal pathogen burden was observed, as against a gradual decrease in the control group. These results reveal that an in vitro a priori attracting anti-biofilm activity of Lactobacillus might be counterbalanced by an in vivo behaviour in a complex microbiota environment with potential deleterious dispersal of highly adherent K. pneumoniae cells, raising the question of the accuracy of in vitro assays in screening of beneficial microbes.

This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2 M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self-produced extracellular matrix. Although a biofilm is commonly described as a "cozy house" where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm-dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.

Isolates of Klebsiella pneumoniae are responsible for opportunistic infections in humans, particularly of the urinary respiratory tracts. These bacteria express type 3 fimbriae that have been implicated in binding to eukaryotic cells and matrix proteins. Twenty four (24) K. pneumoniae strains isolated from medical devices were studied. Their capacity to form biofilm was assessed using two types of materials, polyvinylchloride (PVC) and glass of microfermenter in static or kinetic conditions. Strains with adherence to PVC also cling strongly to glass slides. We determined the in vitro effects of three antimicrobial agents against planktonic and biofilm forms of K. pneumoniae and we demonstrated that isolates of the biofilm form were at least 10-25 times more resistant than the planktonic form. Most strains of K. pneumoniae harbored the mrkD gene and exhibited a strong ability to adhere to inert surfaces. Key words: Biofilm, Fimbriae, Klebsiella pneumoniae, polyvinylchloride (PVC), microfermenter.

International audience; Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria-sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non-capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild-type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA-independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.

International audience; Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (

BACKGROUND AND OBJECTIVES:Interdialytic lock solutions should maintain catheter patency and prevent catheter infections. We aimed to determine in which conditions injectable anticoagulant agents (IAAs) combined with ethanol are compatible and to assess the antibiofilm activity of the selected combination and its effects on dialysis catheters (DC). METHODS:The solubility and compatibility of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), heparinoids and fondaparinux (50 to 2,500 U/mL) in 30 to 70% ethanol were determined by visual observation. The stability of enoxaparin in ethanol and the ethanol content were assessed by high performance liquid chromatography (HPLC) and titrimetric control, respectively. The bactericidal effect was determined on 24h-old biofilms embedded in silicone-DC. The integrity of polyurethane-DC immersed in anticoagulant-ethanol was assessed by gas chromatography-mass spectrometry (GC-MS) and compared with previously published results. RESULTS:The compatibility of IAAs and ethanol varied according to IAA type and concentration, and ethanol content. UFH in 40% ethanol was not compatible, whatever the UFH concentration used. Established limits of compatibility of enoxaparin, nadroparin, dalteparin and tinzaparin in 40% ethanol were 1350, 575, 307 and 207 U/ml, respectively, and up to 300 U/ml for danaparoid and 1 mg/mL for fondaparinux. Enoxaparin 400 U/mL in 40% ethanol (Enox/Eth) eradicated biofilm after 4 hours of exposure for Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans and after 24 hours for Klebsiella pneumoniae and S. aureus. Aliphatic carbonate and alcohol compounds were released by polyurethane-DC after Enox/Eth exposure, as after 40% ethanol or saline exposure. There was no significant difference between the amounts released after 30 minutes of exposure to Enox/Eth and 15 days to saline. CONCLUSIONS:A 40% ethanol solution can be combined with all IAAs but UFH. Enox/Eth was effective as an anti-biofilm agent with minor impacts on DC integrity and could be a useful interdialytic lock solution.

International audience; Discharge of antimicrobial residues and resistant bacteria in hospital effluents is supposed to have strong impacts on the spread of antibiotic resistant bacteria in the environment. This study aimed to characterize the effluents of the Gabriel Montpied teaching hospital, Clermont-Ferrand, France, by simultaneously measuring the concentration of ciprofloxacin and of biological indicators resistant to this molecule in biofilms formed in the hospital effluent and by comparing these data to ciprofloxacin consumption and resistant bacterial isolates of the hospital. Determination of the measured environmental concentration of ciprofloxacin by spot sampling and polar organic chemical integrative (POCIS) sampling over 2 weeks, and comparison with predicted environmental concentrations produced a hazard quotient >1, indicating a potential ecotoxicological risk. A negative impact was also observed with whole hospital effluent samples using the Tetrahymena pyriformis biological model.During the same period, biofilms were formed within the hospital effluent, and analysis of ciprofloxacin-resistant isolates indicated that Gamma-Proteobacteria were numerous, predominantly Aeromonadaceae (69.56%) and Enterobacteriaceae (22.61%). Among the 115 isolates collected, plasmid-mediated fluoroquinolone-resistant genes were detected, with mostly aac(6′)-lb-cr and qnrS. In addition, 60% of the isolates were resistant to up to six antibiotics, including molecules mostly used in the hospital (aminosides and third-generation cephalosporins).In parallel, 1247 bacteria isolated from hospitalized patients and resistant to at least one of the fluoroquinolones were collected. Only 5 of the 14 species identified in the effluent biofilm were also found in the clinical isolates, but PFGE typing of the Gram-negative isolates found in both compartments showed there was no clonality among the strains.Altogether, these data confirm the role of hospital loads as sources of pollution for wastewater and question the role of environmental biofilms communities as efficient shelters for hospital-released resistance genes.

The authors thank the CICS of Clermont-Ferrand and are very grateful to Christelle Blavignac, Claire Szczepaniak, and Lorraine Novais Gameiro for their generous cooperation. This work was partially supported by a grant of the regional council of Auvergne; Bacterial vaginosis (BV), the most common genital infection in reproductive-aged women, is associated with increased risk of sexually transmitted infections. Its etiology remains unclear, especially the role of Gardnerella (G.) vaginalis, an anaerobic bacterium characteristic of the BV-alteration of the vaginal ecosystem. In the genital mucosa, dendritic cells (DCs) sense bacteria of the microenvironment via receptors and then orchestrate the immune response by induction of different T cell subtypes. We investigated the interactions between G. vaginalis and human monocyte-derived DCs using a wide range of bacterial concentrations (multiplicity of infection from 0.01 to 100), and the effects of this pathogen on PHA-induced lymphocyte proliferation. As observed by electron microscopy and cytometry, G. vaginalis reduced the internalization ability of DCs by forming extracellular clusters and induced neither DC maturation, nor DC secretion of cytokines, except at the highest dose with a very early DC maturation state. The same profile was observed on lymphocytes with significant increases of proliferation and cytokine secretion only at the highest bacterial concentration. Our findings indicate that G. vaginalis possesses slight immune-stimulating activities against DCs and T cells, reflecting thus a defective inflammatory response and giving rise to the atypical, non-or low-grade, inflammatory clinical disease profile

Fungicides reaching stream ecosystems have different capacities to adsorb to benthic substrata and thus cause varying effects on benthic microbial communities. We evaluated the potential of a triazole fungicide, tebuconazole (TBZ), to adsorb to submerged leaves and sand and assessed TBZ effects on sand- and leaf-associated microbial communities. An indoor stream channel experiment was designed to test TBZ effects slightly above concentrations measured in agricultural streams (10.7 ± 0.3 μg L−1) on the microbial communities associated with either submerged leaves alone, sand alone or a mixed substratum of leaves + sand over 5 weeks. Weekly samples were taken to determine TBZ effects on the biomass and activity (litter breakdown rate, fungal sporulation rate and extracellular enzyme activities) of fungi and bacteria as well as the dissipation of TBZ in stream channels. TBZ significantly reduced fungal biomass (17–20% relative to controls) but increased bacterial biomass (34–50%) on leaves and sand incubated separately. TBZ also significantly inhibited phenol oxidase activity (36%) in sand but not in leaves. Differences in TBZ effects between leaves and sand communities were not explained by differences between substrata in TBZ adsorption but more likely were related to differences in biomass accrual and structure of the microbial communities. Mixing leaves+sand tended to attenuate TBZ effects on microbial communities, which was probably explained by a greater surface area available for TBZ adsorption to the substrata. This hypothesis is supported by the greater TBZ dissipation in stream channels receiving both leaves and sand. However, leaf and sand mixtures not only diluted but also modified TBZ effects on sand communities (14% increase in fungal biomass and 82% increase in the sporulation of aquatic hyphomycetes). This result suggests that sand could serve as a refuge for fungi during TBZ-contamination episodes. Our study points to the importance of substratum heterogeneity for the ecotoxicological effects of TBZ on agricultural streams and highlights the contrasting responses of fungi and bacteria to contamination by a widespread fungicide.

Here, we report the genome sequence of Staphylococcus aureus LYO-S2, an isolate with sequence type (ST) 45 that was isolated in 2001 from a prosthetic joint infection.

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(®)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(®) packet and capsule and FLOREA Lcr Lenio(®)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(®)). Lcr35(®) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(®)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(®) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(®) packet and Lcr Restituo(®) capsule vs Lcr35(®). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.

All accessible mucous membranes of the human body are colonized by an abundant and diversified microbial flora called microbiota. Recent studies have shown that these microorganisms, long regarded as purely commensal, have essential beneficial effects on human health. Thus, numerous human ailments are linked to dysbiosis; that is, imbalances in the microflora composition. The administration of probiotic microorganisms could, in some situations, provide substantial relief from such disorders. These live microorganisms, which, according to the definition, confer a health benefit to the host when administered in adequate amounts, are often derived from human flora and belong mostly to lactic acid bacteria, in particular to the genus Lactobacillus . The constant improvement of knowledge of the role of human microbiota and the growing popularity of probiotics are now opening the door to new prophylactic and therapeutic strategies in human health.

In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

Objectives. The ability of a probioticLactobacillus rhamnosusstrain (Lcr35) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis-associated pathogens,Prevotella bivia, Gardnerella vaginalis, as well asCandida albicanswas investigated.Methods. Adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix, ectocervix, and vagina. Coculture experiments were performed to count viable pathogens cells in the presence of Lcr35.Results. Lcr35 was able to specifically and rapidly adhere to the three cell lines. In coculture assays, a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation, mostly withP. bivia.Conclusion. The ability of Lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis-associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy.