Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Strong Activity and No Resistance Induction Exerted by Cell-Free Supernatants from Lacticaseibacillus rhamnosus against Mono-Species and Dual-Species Biofilms of Wound Pathogens in In Vivo-like Conditions.

It is widely agreed that microbial biofilms play a major role in promoting infection and delaying healing of chronic wounds. In the era of microbial resistance, probiotic strains or their metabolic products are emerging as an innovative approach for the treatment of hard-to-heal (chronic) wounds due to their antimicrobial, healing, and host immune-modulatory effects. In this study, we aimed to investigate the potential of cell-free supernatants (CFS) from Lacticaseibacillus rhamnosus GG against mono- and dual-species biofilms of wound pathogens in a 3D in vitro infection model. Mature biofilms of Pseudomonas aeruginosa and Staphylococcus aureus were obtained on collagen scaffolds in the presence of a simulant wound fluid (SWF) and treated with CFS at different doses and time intervals. At 1:4 dilution in SWF, CFS caused a marked reduction in the colony forming-unit (CFU) numbers of bacteria embedded in mono-species biofilms as well as bacteria released by the biofilms in the supernatant. CFU count and electron microscopy imaging also demonstrated a marked antibiofilm effect against dual-species biofilms starting from 8 h of incubation. Furthermore, CFS exhibited acceptable levels of cytotoxicity at 24 h of incubation against HaCaT cells and, differently from ciprofloxacin, failed to induce resistance after 15 passages at sub-inhibitory concentrations. Overall, the results obtained point to L. rhamnosus GG postbiotics as a promising strategy for the treatment of wound biofilms.

Cell-free supernatants from Lactobacillus strains exert antibacterial, antibiofilm, and antivirulence activity against Pseudomonas aeruginosa from cystic fibrosis patients.

Chronic lung infections caused by Pseudomonas aeruginosa play a significant role in the mortality and morbidity of cystic fibrosis (CF) patients. The widespread bacterial resistance to conventional antimicrobials demands identifying new strategies to complement or replace current antibiotic therapies. In this study, we evaluated the antibacterial, antibiofilm, and antivirulence properties of cell-free supernatants (CFS) from several Lactobacillus probiotic strains against P. aeruginosa isolated from the sputum of CF patients. A strong and fast antibacterial activity of CFS from different strains of lactobacilli was observed at acidic pH towards P. aeruginosa, both in planktonic and biofilm mode of growth, in conditions mimicking CF lung. Interestingly, although when adjusted at pH 6.0, CFS lost most of their antibacterial potential, they retained some antivirulence activity towards P. aeruginosa, largely dependent on the dose, exposure time, and the Lactobacillus-P. aeruginosa strain combination. In vivo testing in the invertebrate Galleria mellonella model disclosed the lack of toxicity of acidic CFS and their ability to prevent P. aeruginosa infection. For the first time, the results revealed lactobacilli postbiotic activities in the context of the pulmonary environment, pointing to innovative postbiotics' uses in anti-infective therapy.

Antibacterial and Antibiofilm Effects of Lactobacilli Strains against Clinical Isolates of Pseudomonas aeruginosa under Conditions Relevant to Cystic Fibrosis.

Therapy of lung infections sustained by Pseudomonas aeruginosa in cystic fibrosis (CF) patients is challenging due to the presence of a sticky mucus in the airways and the ability of the bacterium to form biofilm, which exhibits increased antibiotic tolerance. A lung-directed bacteriotherapy through the airway administration of probiotics could represent an alternative approach to probiotic diet supplementation to improve the benefits and clinical outcomes of this kind of intervention in CF patients. This study aims to evaluate the ability of probiotic strains to grow in artificial sputum medium (ASM), mimicking the CF lung microenvironment, and to affect the planktonic and biofilm growth of CF clinical strains of P. aeruginosa in the same conditions. The results demonstrate that Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum (LP) can grow in ASM. LP inhibited the planktonic growth of P. aeruginosa, while both lactobacilli reduced the pre-formed biofilm of P. aeruginosa. Interestingly, LP was demonstrated to reduce the amount of polysaccharides in the extracellular matrix of P. aeruginosa biofilms and to potentiate the antibiofilm effects of tobramycin. Overall, the results indicated that LP is a promising candidate as an adjuvant in the antimicrobial therapy of P. aeruginosa infections in CF patients.

Lactobacillus Probiotic Strains Differ in Their Ability to Adhere to Human Lung Epithelial Cells and to Prevent Adhesion of Clinical Isolates of Pseudomonas aeruginosa from Cystic Fibrosis Lung.

The field of probiotic applications is rapidly expanding, including their use for the control of respiratory tract infections. Nevertheless, probiotics ability to colonize the lung environment and to compete with pulmonary pathogens is still a poorly investigated research area. In this study, we aimed to evaluate the adhesion ability of a number of commercial probiotic strains to the human lung epithelial cell line A549. Furthermore, we assessed probiotic ability to prevent host cell adhesion of one of the major lung pathogens in cystic fibrosis, Pseudomonas aeruginosa, and to reduce the pathogen-induced inflammatory response of human peripheral blood mononuclear cells (PBMCs) in terms of cytokine release. Lactobacillus acidophilus displayed the highest adhesion ability to A549 cells evaluated as percent of adhered bacteria compared to the inoculum. In agreement with such an observation, L. acidophilus was the most efficient in preventing adhesion to A549 cells of a P. aeruginosa isolate from CF sputum. Three-color fluorescence labeling of A549 cells, P. aeruginosa, and L. acidophilus, and confocal microcopy image analyses revealed a likely exclusion effect played by both live and UV-killed L. acidophilus towards P. aeruginosa. Such results were confirmed by CFU count. When co-cultured with PBMCs, both live and UV-killed L. acidophilus reduced the amount of IL-1ß and IL-6 in culture supernatants in a statistically significant manner. Overall, the results obtained point to L. acidophilus as an interesting candidate for further studies for a potential aerogenous administration to control P. aeruginosa infections.

The antibacterial and antibiofilm activity of Granudacyn in vitro in a 3D collagen wound infection model.

OBJECTIVE: It is widely agreed that infection and the formation of biofilms play a major role in increasing inflammation and delaying wound healing. The aim of this study was to evaluate, in vitro, the antimicrobial activity of the wound irrigation solution, Granudacyn (Mölnlycke Health Care AB, Sweden) against planktonic bacteria and mature biofilms of clinically relevant bacterial species. METHOD: Quantitative evaluation of bacterial numbers and confocal and/or scanning electron microscopy were used to evaluate the wound irrigation solution's antimicrobial/antibiofilm activity in standard laboratory conditions as well as in a three-dimensional (3D) collagen wound infection model. RESULTS: The wound irrigation solution exhibited a rapid and strong antibacterial activity against both Gram-positive and Gram-negative strains isolated from infected wounds in planktonic form, with a reduction in bacterial number of >4 Logs after as little as one minute of treatment. The wound irrigation solution also exerted an evident activity against preformed biofilms of Pseudomonas aeruginosa and Staphylococcus aureus (>3 Log and >1 Log reduction in colony forming unit number, respectively, after 15 minutes of incubation). Although the wound irrigation solution was partially inhibited in the presence of simulated wound fluid, it maintained a marked antibiofilm activity in in vivo-like conditions (ie. in a 3D collagen wound infection model) with a strong killing and a mild debridement effect, which was superior to standard saline. CONCLUSION: The results obtained in this study suggest that although the wound irrigation solution used might be partially inhibited by wound exudate, it has the potential to effectively kill wound infecting planktonic as well as biofilm bacteria.

Microbial Biofilms and Antibiofilm Agents 2.0.

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Lung-Directed Bacteriotherapy in Cystic Fibrosis: Could It Be an Option?

Due to the alarming spread of bacterial resistance to conventional drugs, the sole use of antibiotics to fight lung infections in cystic fibrosis (CF) is not resolutive, and novel strategies to replace or complement the use of antibiotics are highly desirable. Among these strategies, the use of probiotics is emerging as a particularly attractive approach. Probiotic administration via the oral route has demonstrated an ability to improve lung function and to reduce infection and exacerbation rates in CF patients through mechanisms mainly attributable to the gut-lung axis. Nevertheless, some studies reported no beneficial effect of probiotic intake suggesting that there is margin for improvement of such innovative intervention in CF. The present review aims to address the rationale behind probiotic use in CF and discuss the hypothesis that nasal/aerosol administration of appropriate probiotic strains may help to exert a direct beneficial effect on the respiratory tract, increasing the effectiveness of probiotic interventions in CF patients.

Combination of Two Kinds of Medicated Microparticles Based on Hyaluronic Acid or Chitosan for a Wound Healing Spray Patch.

Olive leaves extract (OLE) has been extensively studied as antioxidant and antibiotic and these characteristics make it particularly interesting for use on wounds. For this reason, the aim of this study was to introduce OLE in microparticles (MP) of hyaluronic acid (MPHA-OLE) or chitosan (MPCs-OLE) to obtain a spray patch for the treatment of wounds in anatomical areas that are difficult to protect with traditional patches. The MP were characterized for particle size and ability to protect OLE from degradation, to absorb water from wound exudate, to control OLE release from MP. The MPHA and MPCs medicated or not and mixtures of the two types in different proportions were studied in vitro on fibroblasts by the scratch wound healing assay. The MP size was always less than 5 µm, and therefore, suitable for a spray patch. The MPCs-OLE could slow down the release of OLE therefore only about 60% of the polyphenols contained in it were released after 4 h. Both MPHA and MPCs could accelerate wound healing. A 50% MPHA-OLE-50% MPCs-OLE blend was the most suitable for accelerating wound healing. The MPHA-OLE-MPCs-OLE blends studied in this work were shown to have the characteristics suitable for a spray patch, thus giving a second life to the waste products of olive growers.

Planktonic and Biofilm-Associated Pseudomonas aeruginosa and Staphylococcus epidermidis Elicit Differential Human Peripheral Blood Cell Responses.

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1ß, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host-cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections.

Correction: Piras, A.M., et al. Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives. Int. J. Mol. Sci. 2019, 20, 6297.

The authors wish to make the following corrections to this paper [...].

Antivirulence Properties of a Low-Molecular-Weight Quaternized Chitosan Derivative against Pseudomonas aeruginosa.

The co-occurrence of increasing rates of resistance to current antibiotics and the paucity of novel antibiotics pose major challenges for the treatment of bacterial infections. In this scenario, treatments targeting bacterial virulence have gained considerable interest as they are expected to exert a weaker selection for resistance than conventional antibiotics. In a previous study, we demonstrated that a low-molecular-weight quaternized chitosan derivative, named QAL, displays antibiofilm activity against the major pathogen Pseudomonas aeruginosa at subinhibitory concentrations. The aim of this study was to investigate whether QAL was able to inhibit the production of relevant virulence factors of P. aeruginosa. When tested in vitro at subinhibiting concentrations (0.31-0.62 mg/mL), QAL markedly reduced the production of pyocyanin, pyoverdin, proteases, and LasA, as well as inhibited the swarming motility of three out of four P. aeruginosa strains tested. Furthermore, quantitative reverse transcription PCR (qRT-PCR) analyses demonstrated that expression of lasI and rhlI, two QS-related genes, was highly downregulated in a representative P. aeruginosa strain. Confocal scanning laser microscopy analysis suggested that FITC-labelled QAL accumulates intracellularly following incubation with P. aeruginosa. In contrast, the reduced production of virulence factors was not evidenced when QAL was used as the main polymeric component of polyelectrolyte-based nanoparticles. Additionally, combination of sub-MIC concentrations of QAL and tobramycin significantly reduced biofilm formation of P. aeruginosa, likely due to a synergistic activity towards planktonic bacteria. Overall, the results obtained demonstrated an antivirulence activity of QAL, possibly due to polymer intracellular localization and QS-inhibition, and its ability to inhibit P. aeruginosa growth synergizing with tobramycin.

Therapeutic Potential of Antimicrobial Peptides in Polymicrobial Biofilm-Associated Infections.

It is widely recognized that many chronic infections of the human body have a polymicrobial etiology. These include diabetic foot ulcer infections, lung infections in cystic fibrosis patients, periodontitis, otitis, urinary tract infections and even a proportion of systemic infections. The treatment of mixed infections poses serious challenges in the clinic. First, polymicrobial communities of microorganisms often organize themselves as biofilms that are notoriously recalcitrant to antimicrobial therapy and clearance by the host immune system. Secondly, a plethora of interactions among community members may affect the expression of virulence factors and the susceptibility to antimicrobials of individual species in the community. Therefore, new strategies able to target multiple pathogens in mixed populations need to be urgently developed and evaluated. In this regard, antimicrobial or host defense peptides (AMPs) deserve particular attention as they are endowed with many favorable features that may serve to this end. The aim of the present review is to offer a comprehensive and updated overview of studies addressing the therapeutic potential of AMPs in mixed infections, highlighting the opportunities offered by this class of antimicrobials in the fight against polymicrobial infections, but also the limits that may arise in their use for this type of application.

Comparison of methods for the microbiological diagnosis of totally implantable venous access port-related infections.

Introduction. Totally implanted venous access ports (TIVAPs) are widely used in patients receiving long-term chemotherapy but may lead to serious complications such as catheter-related bloodstream infections (CRBSIs). Diagnosis of CRBSI requires catheter culture, but there is no consensus on microbiological culture methods to be adopted.Aim. To compare three different procedures to recover bacterial cells from colonized catheters and to determine which section of the TIVAP (i.e. tip, septum, reservoir) is the probable source of infection. To investigate the correlation between blood culture results and TIVAP culture in order to get further evidence about the utility of differential time to positivity (DTP) as a diagnostic tool before TIVAP removal.Hypothesis/Gap statement. Comparisons of different diagnostic procedures for catheter culture have been rarely reported for TIVAPs. We hypothesized that the optimization of methods to recover micro-organisms from different parts of TIVAPs may help to decrease the number of false-negative results in the diagnosis of TIVAP-related bloodstream infections.Methodology. A total of 53 TIVAPs removed because of suspected infection (n=36) or end of use (n=17) were evaluated. The reservoir, the septum and the catheter tip were separated and subjected to different treatments for the recovery of adherent micro-organisms: (a) flushing of the catheter lumen, (b) sonication and flushing, (c) treatment with dithiothreitol and flushing. The three methods were also evaluated in an in vitro catheter infection model with Staphylococcus epidermidis. Culture results were compared to those obtained from paired blood cultures drawn from TIVAP and peripheral vein and to the relative DTP.Results. The results obtained demonstrated that vigorous flushing/vortexing of the catheter lumen/septum, allows the recovery of a number of micro-organisms comparable to that of more complex procedures such as sonication or chemical treatment. Among 24 positive TIVAP-cultures, nine were tip-culture negative, whereas the corresponding reservoirs and septa were culture positive. A good correlation was observed between DTP and TIVAP cultures (P<0.001).Conclusions. The results support the evidence that sending the port reservoir in addition to the catheter tip to the microbiology laboratory may increase the sensitivity and the accuracy of CRBSI diagnosis. Moreover, when a TIVAP-related infection is suspected, DTP is a useful diagnostic tool to decide between device removal or a more conservative approach.

The Anti-Microbial Peptide (Lin-SB056-1)2-K Reduces Pro-Inflammatory Cytokine Release through Interaction with Pseudomonas aeruginosa Lipopolysaccharide.

The ability of many anti-microbial peptides (AMPs) to modulate the host immune response has highlighted their possible therapeutic use to reduce uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which were previously found to have anti-microbial activity against Pseudomonas aeruginosa in in vivo-like models mimicking the challenging environment of chronically infected lungs (i.e., artificial sputum medium and 3-D lung mucosa model). The dendrimeric derivative exerted a stronger anti-inflammatory activity than its monomeric counterpart towards lung epithelial- and macrophage-cell lines stimulated with P. aeruginosa lipopolysaccharide (LPS), based on a marked decrease (up to 80%) in the LPS-induced production of different pro-inflammatory cytokines (i.e., IL-1ß, IL-6 and IL-8). Accordingly, (lin-SB056-1)2-K exhibited a stronger LPS-binding affinity than its monomeric counterpart, thereby suggesting a role of peptide/LPS neutralizing interactions in the observed anti-inflammatory effect. Along with the anti-bacterial and anti-biofilm properties, the anti-inflammatory activity of (lin-SB056-1)2-K broadens its therapeutic potential in the context of chronic (biofilm-associated) infections.

In vitro Interaction of Pseudomonas aeruginosa Biofilms With Human Peripheral Blood Mononuclear Cells.

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was <10% upon 24 h incubation of PBMC with P. aeruginosa biofilms. PBMC incubated for 24 h with preformed P. aeruginosa biofilms were significantly more activated compared to PBMC incubated alone. Interestingly, a marked activation of CD56+CD3- natural killer (NK) cells was observed that reached 60% of NK cells as an average of different donors. In the culture supernatants of PBMC co-cultured with P. aeruginosa biofilms, not only pro-inflammatory (IL-1ß, IFN-γ, IL-6, and TNF-α) but also anti-inflammatory (IL-10) cytokines were significantly increased as compared to PBMC incubated alone. Furthermore, incubation of biofilms with PBMC, caused a statistically significant increase in the CFU number of P. aeruginosa, as compared to biofilms incubated without PBMC. In order to assess whether PBMC products could stimulate the growth of P. aeruginosa biofilms, we incubated preformed P. aeruginosa biofilms with or without supernatants obtained from the co-cultures of PBMC with biofilms. In the presence of the supernatants, the CFU count of biofilm-derived P. aeruginosa, was two to seven times higher than those of biofilms incubated without supernatants (P < 0.01). Overall, the results obtained shed light on the reciprocal interaction between human PBMC and P. aeruginosa biofilms. P. aeruginosa biofilms induced PBMC activation and cytokine secretion but, in turn, the presence of PBMC and/or PBMC-derived components enhanced the number of P. aeruginosa biofilm associated bacteria. This may indicate a successful bacterial defensive/persistence strategy against immune response.

Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential.

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives.

In the era of antimicrobial resistance, the identification of new antimicrobials is a research priority at the global level. In this regard, the attention towards functional antimicrobial polymers, with biomedical/pharmaceutical grade, and exerting anti-infective properties has recently grown. The aim of this study was to evaluate the antibacterial, antibiofilm, and antiadhesive properties of a number of quaternized chitosan derivatives that have displayed significant muco-adhesive properties and wound healing promotion features in previous studies. Low (QAL) and high (QAH) molecular weight quaternized chitosan derivatives were synthetized and further modified with thiol moieties or pendant cyclodextrin, and their antibacterial activity evaluated as minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The ability of the derivatives to prevent biofilm formation was assessed by crystal violet staining. Both QAL and QAH derivatives exerted a bactericidal and/or inhibitory activity on the growth of P. aeruginosa and S. epidermidis. The same compounds also showed marked dose-dependent anti-biofilm activity. Furthermore, the high molecular weight derivative (QAH) was used to functionalize titanium plates. The successful functionalization, demonstrated by electron microscopy, was able to partially inhibit the adhesion of S. epidermidis at 6 h of incubation. The shown ability of the chitosan derivatives tested to both inhibit bacterial growth and/or biofilm formation of clinically relevant bacterial species reveals their potential as multifunctional molecules against bacterial infections.