Clostridioides difficile Biofilm.

Clostridioides difficile infection (CDI), previously Clostridium difficile infection, is a symptomatic infection of the large intestine caused by the spore-forming anaerobic, gram-positive bacterium Clostridioides difficile. CDI is an important healthcare-associated disease worldwide, characterized by high levels of recurrence, morbidity, and mortality. CDI is observed at a higher rate in immunocompromised patients after antimicrobial therapy, with antibiotics disrupting the commensal microbiota and promoting C. difficile colonization of the gastrointestinal tract.A rise in clinical isolates resistant to multiple antibiotics and the reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of CDI more complicated, allowing the persistence of C. difficile in the intestinal environment.Gut colonization and biofilm formation have been suggested to contribute to the pathogenesis and persistence of C. difficile. In fact, biofilm growth is considered as a serious threat because of the related antimicrobial tolerance that makes antibiotic therapy often ineffective. This is the reason why the involvement of C. difficile biofilm in the pathogenesis and recurrence of CDI is attracting more and more interest, and the mechanisms underlying biofilm formation of C. difficile as well as the role of biofilm in CDI are increasingly being studied by researchers in the field.Findings on C. difficile biofilm, possible implications in CDI pathogenesis and treatment, efficacy of currently available antibiotics in treating biofilm-forming C. difficile strains, and some antimicrobial alternatives under investigation will be discussed here.

An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory.

The common view is that T lymphocytes activate telomerase to delay senescence. Here we show that some T cells (primarily naïve and central memory cells) elongated telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs) independently of telomerase action. Upon contact with these T cells, APCs degraded shelterin to donate telomeres, which were cleaved by the telomere trimming factor TZAP, and then transferred in extracellular vesicles at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled telomere fusion with T-cell chromosome ends lengthening them by an average of ~3,000 base pairs. Thus, there are antigen-specific populations of T cells whose ageing fate decisions are based on telomere vesicle transfer upon initial contact with APCs. These telomere-acquiring T cells are protected from senescence before clonal division begins, conferring long-lasting immune protection.

The Multifunctional Role of Poloxamer P338 as a Biofilm Disrupter and Antibiotic Enhancer: A Small Step forward against the Big Trouble of Catheter-Associated Escherichia coli Urinary Tract Infections.

Poloxamer 338 (P338), a nonionic surfactant amphiphilic copolymer, is herein proposed as an anti-biofilm compound for the management of catheter-associated urinary tract infections (CAUTIs). P338's ability to disrupt Escherichia coli biofilms on silicone urinary catheters and to serve as antibiotic enhancer was evaluated for biofilm-producing E. coli Ec5FSL and Ec9FSL clinical strains, isolated from urinary catheters. In static conditions, quantitative biofilm formation assay allowed us to determine the active P338 concentration. In dynamic conditions, the BioFlux system, combined with confocal laser scanning microscopy, allowed us to investigate the P338 solution's ability to detach biofilm, alone or in combination with sub-MIC concentrations of cefoxitin (FOX). The 0.5% P338 solution was able to destroy the structure of E. coli biofilms, to reduce the volume and area fraction covered by adherent cells (41.42 ± 4.79% and 56.20 ± 9.22% reduction for the Ec5FSL and Ec9FSL biofilms, respectively), and to potentiate the activity of 1\2 MIC FOX in disaggregating biofilms (19.41 ± 7.41% and 34.66 ± 3.75% reduction in the area fraction covered by biofilm for Ec5FSL and Ec9FSL, respectively) and killing cells (36.85 ± 7.13% and 32.33 ± 4.65% increase in the biofilm area covered by dead Ec5FSL and Ec9FSL cells, respectively).

Synthesis of Novel Hyaluronic Acid Sulfonated Hydrogels Using Safe Reactants: A Chemical and Biological Characterization.

Here, we present a one-pot procedure for the preparation of hyaluronic acid (HA) sulfonated hydrogels in aqueous alkaline medium. The HA hydrogels were crosslinked using 1,4-butanedioldiglycidyl ether (BDDE) alone, or together with N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (Bes), as a safe sulfonating agent. Conditions for the simultaneous reaction of HA with BDDE and Bes were optimized and the resulting hydrogels were characterized under different reaction times (24, 72, and 96 h). The incorporation of sulfonic groups into the HA network was proven by elemental analysis and FTIR spectroscopy and its effect on water uptake was evaluated. Compared with the non-sulfonated sample, sulfonated gels showed improved mechanical properties, with their compressive modulus increased from 15 to 70 kPa, higher stability towards hyaluronidase, and better biocompatibility to 10T1/2 fibroblasts, especially after the absorption of collagen. As main advantages, the procedure described represents an easy and reproducible methodology for the fabrication of sulfonated hydrogels, which does not require toxic chemicals and/or solvents.

Proinflammatory mucosal-associated invariant CD8+ T cells react to gut flora yeasts and infiltrate multiple sclerosis brain.

The composition of the intestinal microbiota plays a critical role in shaping the immune system. Modern lifestyle, the inappropriate use of antibiotics, and exposure to pollution have significantly affected the composition of commensal microorganisms. The intestinal microbiota has been shown to sustain inappropriate autoimmune responses at distant sites in animal models of disease, and may also have a role in immune-mediated central nervous system (CNS) diseases such as multiple sclerosis (MS). We studied the composition of the gut mycobiota in fecal samples from 27 persons with MS (pwMS) and in 18 healthy donors (HD), including 5 pairs of homozygous twins discordant for MS. We found a tendency towards higher fungal abundance and richness in the MS group, and we observed that MS twins showed a higher rate of food-associated strains, such as Saccharomyces cerevisiae. We then found that in pwMS, a distinct population of cells with antibacterial and antifungal activity is expanded during the remitting phase and markedly decreases during clinically and/or radiologically active disease. These cells, named MAIT (mucosal-associated invariant T cells) lymphocytes, were significantly more activated in pwMS compared to HD in response to S. cerevisiae and Candida albicans strains isolated from fecal samples. This activation was also mediated by fungal-induced IL-23 secretion by innate immune cells. Finally, immunofluorescent stainings of MS post-mortem brain tissues from persons with the secondary progressive form of the disease showed that MAIT cells cross the blood-brain barrier (BBB) and produce pro-inflammatory cytokines in the brain. These results were in agreement with the hypothesis that dysbiosis of the gut microbiota might determine the inappropriate response of a subset of pathogenic mucosal T cells and favor the development of systemic inflammatory and autoimmune diseases.

Poloxamer 338 Affects Cell Adhesion and Biofilm Formation in Escherichia coli: Potential Applications in the Management of Catheter-Associated Urinary Tract Infections.

Poloxamers are nontoxic, amphiphilic copolymers used in different formulations. Due to its surfactant properties, Poloxamer 338 (P388) is herein proposed as a strategy to avoid biofilm formation often causing recalcitrant catheter-associated urinary tract infections (CAUTI). The aim is to evaluate the ability of P388 coatings to affect the adhesion of Ec5FSL and Ec9FSL Escherichia coli strains on silicone urinary catheters. Attenuated total reflection infrared spectroscopy, atomic force microscopy, and static water contact angle measurement were employed to characterize the P388-coated silicone catheter in terms of amount of P388 layered, coating thickness, homogeneity, and hydrophilicity. In static conditions, the antifouling power of P388 was defined by comparing the E. coli cells adherent on a hydrophilic P388-adsorbed catheter segment with those on an uncoated one. A P388-coated catheter, having a homogeneous coverage of 35 nm in thickness, reduced of 0.83 log10 and 0.51 log10 the biofilm of Ec5FSL and Ec9FSL, respectively. In dynamic conditions, the percentage of cell adhesion on P388-adsorbed silicone channels was investigated by a microfluidic system, simulating the in vivo conditions of catheterized patients. As a result, both E. coli isolates were undetected. The strong and stable antifouling property against E. coli biofilm lead us to consider P388 as a promising anti-biofilm agent for CAUTIs control.

Gut Microbiota and Disorders of the Central Nervous System.

The gut microbiota, consisting of bacteria, fungi, archaea, viruses, and protozoa, together with their collective genomes (microbiome), plays a key role in immune system development and maturation, gut morphology, and in performing essential metabolic functions. Several factors, including lifestyle, body mass index, diet, antibiotic use, and the environment, influence the balance of the intestinal microbiota, whose alterations (the so-called dysbiosis) in recent years have been associated with the onset and/or progression of neurological and neuropsychiatric disorders. The purpose of this narrative review is to provide an overview of the possible involvement of the microbiota-gut-brain axis in the pathogenesis of diseases of the central nervous system, with a special focus on key issues and common misjudgments on the potential contribution of specific microorganisms.

Novel Treatment Strategies for Biofilm-Based Infections.

Biofilm-growing cells show an enhanced antimicrobial tolerance with respect to the same cells growing in a free-floating way. This is due to physical or chemical diffusion barriers and increased transfer of resistance markers. Thus, tissue- and medical device-related biofilms can be considered among the leading sources of antibiotic treatment failure, causing many of the deadliest chronic infections afflicting humans nowadays. To find a satisfying way to counteract this major health threat, a great effort has been made in recent years to develop safe, effective and fast-acting anti-biofilm strategies. In this review, we summarise and evaluate the most promising tools and molecules that have demonstrated their ability to modulate steps involved in biofilm formation or to disperse pre-formed biofilms, without conferring evolutionary pressure to microorganisms.

Clostridium difficile Biofilm.

Clostridium difficile infection (CDI) is an important healthcare-associated disease worldwide, mainly occurring after antimicrobial therapy. Antibiotics administered to treat a number of infections can promote C. difficile colonization of the gastrointestinal tract and, thus, CDI. A rise in multidrug resistant clinical isolates to multiple antibiotics and their reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of CDI more complicated, allowing the persistence of C. difficile in the intestinal environment.Gut colonization and biofilm formation have been suggested to contribute to the pathogenesis and persistence of C. difficile. In fact, biofilm growth is considered as a serious threat because of the related increase in bacterial resistance that makes antibiotic therapy often ineffective. However, although the involvement of the C. difficile biofilm in the pathogenesis and recurrence of CDI is attracting more and more interest, the mechanisms underlying biofilm formation of C. difficile as well as the role of biofilm in CDI have not been extensively described.Findings on C. difficile biofilm, possible implications in CDI pathogenesis and treatment, efficacy of currently available antibiotics in treating biofilm-forming C. difficile strains, and some antimicrobial alternatives under investigation will be discussed here.

Biofilm-Forming Ability and Clonality in Acinetobacter baumannii Strains Isolated from Urine Samples and Urinary Catheters in Different European Hospitals.

OBJECTIVE: Biofilm formation has been associated with the persistence of Acinetobacter baumannii in hospital settings and its propensity to cause infection. We investigated the adhesion ability and clonality of 128 A. baumannii isolates recovered from urine and urinary catheters of patients admitted to 5 European hospitals during 1991-2013. METHODS: Isolates identification was confirmed by rpoB sequencing and by the presence of blaOXA-51. The presence of carbapenemases was detected by PCR. Clonality was determined by Sequence Group (SG) identification, Pulsed field gel electrophoresis (PFGE) and Multilocus sequence typing. Adhesion ability was defined by quantitative biofilm production assay and biofilms were characterized by Confocal Laser Microscopy and Scanning Electron Microscopy. RESULTS: The 128 isolates, either resistant (85.9%) or susceptible (14.1%) to carbapenems, and belonging to 50 different PFGE types and 24 different STs, were distributed among SG1 (67.2%), SG2 (10.2%) and other allelic profiles (22.7%). ST218 was the most frequent ST, corresponding to 54,5% of the isolates collected between 2011 and 2013. Among the 109 isolates showing resistance to at least 1 carbapenem, 55% revealed the presence of an acquired carbapenem-hydrolyzing class D - lactamases (CHDL): blaOXA-23 were the most frequent gene detected from 2008 onwards (75%). Among all the clinical isolates, 42.2% were strong biofilm producers, with the older isolates having the highest adhesion ability. Most isolates recovered later, belonging to ST218 and harbouring blaOXA-23, were homogeneously less adhesive. CONCLUSIONS: An evolution towards a decrease in adhesion ability and a CHDL content change was observed along the years in several European countries.

Antifouling and antimicrobial biomaterials: an overview.

The use of implantable medical devices is a common and indispensable part of medical care for both diagnostic and therapeutic purposes. However, as side effect, the implant of medical devices quite often leads to the occurrence of difficult-to-treat infections, as a consequence of the colonization of their abiotic surfaces by biofilm-growing microorganisms increasingly resistant to antimicrobial therapies. A promising strategy to combat device-related infections is based on anti-infective biomaterials that either repel microbes, so they cannot attach to the device surfaces, or kill them in the surrounding areas. In general, such biomaterials are characterized by antifouling coatings, exhibiting low adhesion or even repellent properties towards microorganisms, or antimicrobial coatings, able to kill microbes approaching the surface. In this light, the present overview will address the development in the last two decades of antifouling and antimicrobial biomaterials designed to potentially limit the initial stages of microbial adhesion, as well as the microbial growth and biofilm formation on medical device surfaces.

Antioxidant Hydroxytyrosol-Based Polyacrylate with Antimicrobial and Antiadhesive Activity Versus Staphylococcus Epidermidis.

The accumulation of reactive oxygen species (ROS) in microbial biofilms has been recently recognized to play a role in promoting antibiotic resistance in biofilm-growing bacteria. ROS are also over-produced when a medical device is implanted and they can promote device susceptibility to infection or aseptic loosening. High levels of ROS seem also to be responsible for the establishment of chronic wounds.In this study, a novel antioxidant polyacrylate was synthesized and investigated in terms of antimicrobial and antibiofilm activity. The polymer possesses in side-chain hydroxytyrosol (HTy), that is a polyphenolic compound extracted from olive oil wastewaters.The obtained 60 nm in size polymer nanoparticles showed good scavenging and antibacterial activity versus a strain of Staphylococcus epidermidis. Microbial adherence assays evidenced that the hydroxytyrosol-containing polymer was able to significantly reduce bacterial adhesion compared to the control. These findings open novel perspective for a successful use of this antioxidant polymer for the prevention or treatment of biofilm-based infections as those related to medical devices or chronic wounds.

Antiseptics for treating infected wounds: Efficacy on biofilms and effect of pH.

Biofilm recalcitrance is a persistent problem when managing difficult to heal and infected chronic wounds. The wound biofilm is a fundamental factor in the re-occurrence and delayed healing commonly observed in non-healing and infected chronic wounds. However, there is presently no single antimicrobial agent that is completely efficacious against both the planktonic and sessile polymicrobial communities evident in at risk or infected wounds. We will review currently available antimicrobials, with particular emphasis on silver and iodine, employed to help suppress biofilms in wounds. In addition, we will also review the effect of pH on antimicrobial efficacy. Available evidence suggests that it is best to take a multifactorial approach towards controlling biofilm in chronic, "at risk" and infected wounds. This highlights the growing importance of avoiding indiscriminate or inappropriate use of antimicrobials in the treatment of chronic wounds.

Subinhibitory concentrations of metronidazole increase biofilm formation in Clostridium difficile strains.

Resistance mechanism to metronidazole is still poorly understood, even if the number of reports on Clostridium difficile strains with reduced susceptibility to this antibiotic is increasing. In this study, we investigated the ability of the C. difficile strains 7032994, 7032985 and 7032989, showing different susceptibility profiles to metronidazole but all belonging to the PCR ribotype 010, to form biofilm in vitro in presence and absence of subinhibitory concentrations of metronidazole. The quantitative biofilm production assay performed in presence of metronidazole revealed a significant increase in biofilm formation in both the susceptible strain 7032994 and the strain 7032985 exhibiting a reduced susceptibility to this antibiotic, while antibiotic pressure did not affect the biofilm-forming ability of the stable-resistant strain 7032989. Moreover, confocal microscopy analysis showed an abundant biofilm matrix production by the strains 7032994 and 7032885, when grown in presence of metronidazole, but not in the stable-resistant one. These results seem to demonstrate that subinhibitory concentrations of metronidazole are able to enhance the in vitro biofilm production of the above-mentioned PCR ribotype 010 C. difficile strains, susceptible or with reduced susceptibility to this antibiotic, suggesting a possible role of biofilm formation in the multifactorial mechanism of metronidazole resistance developed by C. difficile.

Biofilms and Wounds: An Identification Algorithm and Potential Treatment Options.

Significance: The presence of a "pathogenic" or "highly virulent" biofilm is a fundamental risk factor that prevents a chronic wound from healing and increases the risk of the wound becoming clinically infected. There is presently no unequivocal gold standard method available for clinicians to confirm the presence of biofilms in a wound. Thus, to help support clinician practice, we devised an algorithm intended to demonstrate evidence of the presence of a biofilm in a wound to assist with wound management. Recent Advances: A variety of histological and microscopic methods applied to tissue biopsies are currently the most informative techniques available for demonstrating the presence of generic (not classified as pathogenic or commensal) biofilms and the effect they are having in promoting inflammation and downregulating cellular functions. Critical Issues: Even as we rely on microscopic techniques to visualize biofilms, they are entities which are patchy and dispersed rather than confluent, particularly on biotic surfaces. Consequently, detection of biofilms by microscopic techniques alone can lead to frequent false-negative results. Furthermore, visual identification using the naked eye of a pathogenic biofilm on a macroscopic level on the wound will not be possible, unlike with biofilms on abiotic surfaces. Future Direction: Lacking specific biomarkers to demonstrate microscopic, nonconfluent, virulent biofilms in wounds, the present focus on biofilm research should be placed on changing clinical practice. This is best done by utilizing an anti-biofilm toolbox approach, rather than speculating on unscientific approaches to identifying biofilms, with or without staining, in wounds with the naked eye. The approach to controlling biofilm should include initial wound cleansing, periodic debridement, followed by the application of appropriate antimicrobial wound dressings. This approach appears to be effective in removing pathogenic biofilms.

Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control.

Biofilms are of great importance in infection control and healthcare-associated infections owing to their inherent tolerance and 'resistance' to antimicrobial therapies. Biofilms have been shown to develop on medical device surfaces, and dispersal of single and clustered cells implies a significant risk of microbial dissemination within the host and increased risk of infection. Although routine microbiological testing assists with the diagnosis of a clinical infection, there is no 'gold standard' available to reveal the presence of microbial biofilm from samples collected within clinical settings. Furthermore, such limiting factors as viable but non-culturable micro-organisms and small-colony variants often prevent successful detection. In order to increase the chances of detection and provide a more accurate diagnosis, a combination of microbiological culture techniques and molecular methods should be employed. Measures such as antimicrobial coating and surface alterations of medical devices provide promising opportunities in the prevention of biofilm formation on medical devices.

Anaerobes in biofilm-based healthcare-associated infections.

Anaerobic bacteria can cause an infection when they encounter a permissive environment within the host. These opportunistic pathogens are seldom recovered as single isolates but more frequently are involved in polymicrobial infections, together with other anaerobes or aerobes. Nowadays it's known that some anaerobic bacteria are also able to grow as biofilm even if this feature and its role in the healthcare-associated infections (HAIs) are still poorly characterized. As consequence, the involvement of biofilm-forming anaerobic bacteria in infections related to healthcare procedures, including surgery and medical devices implantation, is underestimated.The current knowledge on the role of biofilm-growing anaerobes in HAIs has been here reviewed, with particular reference to respiratory, intestinal, intra-abdominal, wound, and urogenital tract infections. Even if the data are still scarce, the ability to form biofilm of opportunistic anaerobic species and their possible role as causative agents of HAIs should alert even more clinicians and microbiologists on the need to search for anaerobes in clinical samples when their presence can be reasonably assumed.

Antibiotic Resistance Related to Biofilm Formation in Klebsiella pneumoniae.

The Gram-negative opportunistic pathogen, Klebsiella pneumoniae, is responsible for causing a spectrum of community-acquired and nosocomial infections and typically infects patients with indwelling medical devices, especially urinary catheters, on which this microorganism is able to grow as a biofilm. The increasingly frequent acquisition of antibiotic resistance by K. pneumoniae strains has given rise to a global spread of this multidrug-resistant pathogen, mostly at the hospital level. This scenario is exacerbated when it is noted that intrinsic resistance to antimicrobial agents dramatically increases when K. pneumoniae strains grow as a biofilm. This review will summarize the findings about the antibiotic resistance related to biofilm formation in K. pneumoniae.

Role of daptomycin in the induction and persistence of the viable but non-culturable state of Staphylococcus aureus biofilms.

We have recently demonstrated that antibiotic pressure can induce the viable but non-culturable (VBNC) state in Staphylococcus aureus biofilms. Since dormant bacterial cells can undermine anti-infective therapy, a greater understanding of the role of antibiotics of last resort, including daptomycin, is crucial. Methicillin-resistant S. aureus 10850 biofilms were maintained on non-nutrient (NN) agar in the presence or absence of the MIC of daptomycin until loss of culturability. Viable cells were monitored by epifluorescence microscopy and flow cytometry for 150 days. All biofilms reached non-culturability at 40 days and showed a similar amount of viable cells; however, in biofilms exposed to daptomycin, their number remained unchanged throughout the experiment, whereas in those maintained on NN agar alone, no viable cells were detected after 150 days. Gene expression assays showed that after achievement of non-culturability, 16S rDNA and mecA were expressed by all biofilms, whereas glt expression was found only in daptomycin-exposed biofilms. Our findings suggest that low daptomycin concentrations, such as those that are likely to obtain within biofilms, can influence the viability and gene expression of non-culturable S. aureus cells. Resuscitation experiments are needed to establish the VBNC state of daptomycin-exposed biofilms.

Probiotics to counteract biofilm-associated infections: promising and conflicting data.

Altered bowel flora is currently thought to play a role in a variety of disease conditions, and the use of Bifidobacterium spp. and Lactobacillus spp. as probiotics has been demonstrated to be health-promoting, even if the success of their administration depends on the applied bacterial strain(s) and the targeted disease. In the last few decades, specific probiotics have been shown to be effective in the treatment or the prevention of acute viral gastroenteritis, pediatric post-antibiotic-associated diarrhea, some pediatric allergic disorders, necrotizing enterocolitis in preterm infants, inflammatory bowel diseases and postsurgical pouchitis. The potential application of probiotics is continuously widening, with new evidence accumulating to support their effect on the prevention and treatment of other disease conditions, including several oral diseases, such as dental caries, periodontal diseases and oral malodor, as well as genitourinary and wound infections. Considering the increasingly widespread ability of pathogens to generate persistent biofilm-related infections, an even more attractive proposal is to administer probiotics to prevent or counteract biofilm development. The response of biofilm-based oral, intestinal, vaginal and wound infections to probiotics treatment will be reviewed here in light of the most recent results obtained in this field.