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.

Emerging Multiscale Biofabrication Approaches for Bacteriotherapy.

Bacteriotherapy is emerging as a strategic and effective approach to treat infections by providing putatively harmless bacteria (i.e., probiotics) as antagonists to pathogens. Proper delivery of probiotics or their metabolites (i.e., post-biotics) can facilitate their availing of biomaterial encapsulation via innovative manufacturing technologies. This review paper aims to provide the most recent biomaterial-assisted strategies proposed to treat infections or dysbiosis using bacteriotherapy. We revised the encapsulation processes across multiscale biomaterial approaches, which could be ideal for targeting different tissues and suit diverse therapeutic opportunities. Hydrogels, and specifically polysaccharides, are the focus of this review, as they have been reported to better sustain the vitality of the live cells incorporated. Specifically, the approaches used for fabricating hydrogel-based devices with increasing dimensionality (D)-namely, 0D (i.e., particles), 1D (i.e., fibers), 2D (i.e., fiber meshes), and 3D (i.e., scaffolds)-endowed with probiotics, were detailed by describing their advantages and challenges, along with a future overlook in the field. Electrospinning, electrospray, and 3D bioprinting were investigated as new biofabrication methods for probiotic encapsulation within multidimensional matrices. Finally, examples of biomaterial-based systems for cell and possibly post-biotic release were reported.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

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.

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.

Biopharmaceutical Assessment of Mesh Aerosolised Plasminogen, a Step towards ARDS Treatment.

Acute respiratory distress syndrome (ARDS) is a severe complication of lung injuries, commonly associated with bacterial, fungal and viral infections, including SARS-CoV-2 viral infections. ARDS is strongly correlated with patient mortality and its clinical management is very complex, with no effective treatment presently available. ARDS involves severe respiratory failure, fibrin deposition in both airways and lung parenchyma, with the development of an obstructing hyaline membrane drastically limiting gas exchange. Moreover, hypercoagulation is related to deep lung inflammation, and a pharmacological action toward both aspects is expected to be beneficial. Plasminogen (PLG) is a main component of the fibrinolytic system playing key roles in various inflammation regulatory processes. The inhalation of PLG has been proposed in the form of the off-label administration of an eyedrop solution, namely, a plasminogen-based orphan medicinal product (PLG-OMP), by means of jet nebulisation. Being a protein, PLG is susceptible to partial inactivation under jet nebulisation. The aim of the present work is to demonstrate the efficacy of the mesh nebulisation of PLG-OMP in an in vitro simulation of clinical off-label administration, considering both the enzymatic and immunomodulating activities of PLG. Biopharmaceutical aspects are also investigated to corroborate the feasibility of PLG-OMP administration by inhalation. The nebulisation of the solution was performed using an Aerogen® SoloTM vibrating-mesh nebuliser. Aerosolised PLG showed an optimal in vitro deposition profile, with 90% of the active ingredient impacting the lower portions of a glass impinger. The nebulised PLG remained in its monomeric form, with no alteration of glycoform composition and 94% of enzymatic activity maintenance. Activity loss was observed only when PLG-OMP nebulisation was performed under simulated clinical oxygen administration. In vitro investigations evidenced good penetration of aerosolised PLG through artificial airway mucus, as well as poor permeation across an Air-Liquid Interface model of pulmonary epithelium. The results suggest a good safety profile of inhalable PLG, excluding high systemic absorption but with good mucus diffusion. Most importantly, the aerosolised PLG was capable of reversing the effects of an LPS-activated macrophage RAW 264.7 cell line, demonstrating the immunomodulating activity of PLG in an already induced inflammatory state. All physical, biochemical and biopharmaceutical assessments of mesh aerosolised PLG-OMP provided evidence for its potential off-label administration as a treatment for ARDS patients.

Betaine- and L-Carnitine-Based Ionic Liquids as Solubilising and Stabilising Agents for the Formulation of Antimicrobial Eye Drops Containing Diacerein.

The therapeutic efficacy of topically administered drugs, however powerful, is largely affected by their bioavailability and, thus, ultimately, on their aqueous solubility and stability. The aim of this study was to evaluate the use of ionic liquids (ILs) as functional excipients to solubilise, stabilise, and prolong the ocular residence time of diacerein (DIA) in eye drop formulations. DIA is a poorly soluble and unstable anthraquinone prodrug, rapidly hydrolysed to rhein (Rhe), for the treatment of osteoarthritis. DIA has recently been evaluated as an antimicrobial agent for bacterial keratitis. Two ILs based on natural zwitterionic compounds were investigated: L-carnitine C6 alkyl ester bromide (Carn6), and betaine C6 alkyl ester bromide (Bet6). The stabilising, solubilising, and mucoadhesive properties of ILs were investigated, as well as their cytotoxicity to the murine fibroblast BALB/3T3 clone A31 cell line. Two IL-DIA-based eye drop formulations were prepared, and their efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa was determined. Finally, the eye drops were administered in vivo on New Zealand albino rabbits, testing their tolerability as well as their elimination and degradation kinetics. Both Bet6 and Carn6 have good potential as functional excipients, showing solubilising, stabilising, mucoadhesive, and antimicrobial properties; their in vitro cytotoxicity and in vivo ocular tolerability pave the way for their future use in ophthalmic applications.

Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts.

Three killer toxins that were previously investigated, one excreted by Kluyveromyces wickerhamii and two by different strains of Wickerhamomyces anomalus, were produced at the pilot scale, lyophilized and characterized, and the formulates were assessed for their zymocidial effect against Brettanomyces bruxellensis spoilage yeast. A comparative analysis allowed the evaluation of the minimum inhibitory concentration (MIC) against a sensitive strain. Fungicidal and fungistatic concentrations were used to evaluate the cytocidal effect using a cytofluorimetric approach that confirmed the lethal effect of all lyophilized formulates against B. bruxellensis spoilage yeasts. Moreover, the potential killer toxins' cytotoxicity against human intestinal cells (Caco-2) were evaluated to exclude any possible negative effect on the consumers. Finally, the effective lethal effect of all three lyophilized killer toxins toward B. bruxellensis sensitive strain were tested. The results indicated that all of them acted without dangerous effects on the human epithelial cells, opening the way for their possible commercial application. In particular, D15 showed the lowest MIC and the highest activity, was evaluated also in wine, revealing a strong reduction of Brettamonyces yeast growth and, at the same time, a control of ethyl phenols production.

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.

Novel Yeasts Producing High Levels of Conjugated Linoleic Acid and Organic Acids in Fermented Doughs.

Traditional fermented foods are obtained by a complex consortium of autochthonous microorganisms producing a wide variety of bioactive compounds, thus representing a reservoir of strains with new functional properties. Here, doughs obtained using five different wholegrain flours were singly fermented with selected yeast strains, which were evaluated for their functional traits. Lactate, volatile fatty acids and conjugated linoleic acid isomers produced by fermented doughs were detected by HPLC, while dough anti-inflammatory capacity was measured on human peripheral blood mononuclear cells by flow cytometry. Yeast potential probiotic activity was assessed by evaluating their resistance to simulated gastric and intestinal fluids. For the first time we report evidence of yeast strains producing high levels of the conjugated linoleic acid (CLA) isomer CLA 10-12tc and propionic acid, which are known for their specific health benefits. Moreover, such yeast strains showed an anti-inflammatory capacity, as revealed by a significantly decreased production of the strongly pro-inflammatory cytokine IL-1ß. All our Saccharomyces cerevisiae strains were remarkably resistant to simulated gastric and intestinal fluids, as compared to the commercial probiotic strain. The two strains S. cerevisiae IMA D18Y and L10Y showed the best survival percentage. Our novel yeast strains may be exploited as valuable functional starters for the industrial production of cereal-based innovative and health-promoting fermented foods.

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.

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.

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 [...].

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.