Molecular physiological characterization of the dynamics of persister formation in Staphylococcus aureus.

Bacteria possess the ability to enter a growth-arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely, vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences, depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy.

Merging Modular Molecular Design with High Throughput Screening of Cell Adhesion on Antimicrobial Supramolecular Biomaterials.

A polymer microarray based on the supramolecular ureido-pyrimidinone (UPy) moiety is fabricated to screen antimicrobial materials for their ability to support cell adhesion. UPy-functionalized additives, either cell-adhesive, antimicrobial or control peptides, are used, and investigated in different combinations at different concentrations, resulting in a library of 194 spots. These are characterized on composition and morphology to evaluate the microarray fabrication. Normal human dermal fibroblasts are cultured on the microarrays and cell adhesion to the spots is systematically analyzed. Results demonstrate enhanced cell adhesion on spots with combinations including the antimicrobial peptides. This study clearly proves the power of the high throughput approach in combination with supramolecular molecules, to screen additive libraries for desired biological response.

Transvaginal Mesh-related Complications and the Potential Role of Bacterial Colonization: An Exploratory Observational Study.

STUDY OBJECTIVE: This study aimed to investigate the potential role of transvaginal mesh bacterial colonization in the development of mesh-related complications (MRCs). DESIGN: An observational and exploratory study. SETTING: Tertiary referral center (Amsterdam UMC, location AMC, Amsterdam, The Netherlands). PATIëNTS: 49 patients indicated for mesh removal and 20 women of whom vaginal tissue was retrieved during prolapse surgery as a reference cohort. INTERVENTIONS: collection of mesh-tissue complex (patient cohort) or vaginal tissue (reference cohort) MEASUREMENTS AND MAIN RESULTS: Homogenized samples were used for quantitative microbiological culture. Inflammation and fibrosis were semiquantitatively histologically scored; Gram staining and fluorescence in situ hybridization were used to detect bacteria and bacterial biofilms. Of the 49 patients, 44 samples (90%) were culture positive, with a higher diversity of species and more Gram-negative bacteria and polymicrobial cultures in the MRC cohort than the reference cohort, with mostly staphylococci, streptococci, Actinomyces spp., Cutibacterium acnes, and Escherichia coli. Patients with clinical signs of infection or exposure had the highest bacterial counts. Histology demonstrated moderate to severe inflammation in most samples. Gram staining showed bacteria in 57% of culture-positive samples, and in selected samples, fluorescence in situ hybridization illustrated a polymicrobial biofilm. CONCLUSION: In this study, we observed distinct differences in bacterial numbers and species between patients with MRCs and a reference cohort. Bacteria were observed at the mesh-tissue interface in a biofilm. These results strongly support the potential role of bacterial mesh colonization in the development of MRCs.

Current Concepts of Osteomyelitis: From Pathologic Mechanisms to Advanced Research Methods.

Osteomyelitis is an inflammation of the bone and bone marrow that is most commonly caused by a Staphylococcus aureus infection. Much of our understanding of the underlying pathophysiology of osteomyelitis, from the perspective of both host and pathogen, has been revised in recent years, with notable discoveries including the role played by osteocytes in the recruitment of immune cells, the invasion and persistence of S. aureus in submicron channels of cortical bone, and the diagnostic role of polymorphonuclear cells in implant-associated osteomyelitis. Advanced in vitro cell culture models, such as ex vivo culture models or organoids, have also been developed over the past decade, and have become widespread in many fields, including infectious diseases. These models better mimic the in vivo environment, allow the use of human cells, and can reduce our reliance on animals in osteomyelitis research. In this review, we provide an overview of the main pathologic concepts in osteomyelitis, with a focus on the new discoveries in recent years. Furthermore, we outline the value of modern in vitro cell culture techniques, with a focus on their current application to infectious diseases and osteomyelitis in particular.

C-Reactive Protein Promotes Inflammation through FcγR-Induced Glycolytic Reprogramming of Human Macrophages.

C-reactive protein (CRP) is an acute-phase protein produced in high quantities by the liver in response to infection and during chronic inflammatory disorders. Although CRP is known to facilitate the clearance of cell debris and bacteria by phagocytic cells, the role of CRP in additional immunological functions is less clear. This study shows that complexed CRP (phosphocholine [PC]:CRP) (formed by binding of CRP to PC moieties), but not soluble CRP, synergized with specific TLRs to posttranscriptionally amplify TNF, IL-1ß, and IL-23 production by human inflammatory macrophages. We identified FcγRI and IIa as the main receptors responsible for initiating PC:CRP-induced inflammation. In addition, we identified the underlying mechanism, which depended on signaling through kinases Syk, PI3K, and AKT2, as well as glycolytic reprogramming. These data indicate that in humans, CRP is not only a marker but also a driver of inflammation by human macrophages. Therefore, although providing host defense against bacteria, PC:CRP-induced inflammation may also exacerbate pathology in the context of disorders such as atherosclerosis.

Isolation of Persister Cells of Bacillus subtilis and Determination of Their Susceptibility to Antimicrobial Peptides.

Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides.

Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance.

Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

Bacterial Persister-Cells and Spores in the Food Chain: Their Potential Inactivation by Antimicrobial Peptides (AMPs).

The occurrence of bacterial pathogens in the food chain has caused a severe impact on public health and welfare in both developing and developed countries. Moreover, the existence of antimicrobial-tolerant persisting morphotypes of these pathogens including both persister-cells as well as bacterial spores contributes to difficulty in elimination and in recurrent infection. Therefore, comprehensive understanding of the behavior of these persisting bacterial forms in their environmental niche and upon infection of humans is necessary. Since traditional antimicrobials fail to kill persisters and spores due to their (extremely) low metabolic activities, antimicrobial peptides (AMPs) have been intensively investigated as one of the most promising strategies against these persisting bacterial forms, showing high efficacy of inactivation. In addition, AMP-based foodborne pathogen detection and prevention of infection has made significant progress. This review focuses on recent research on common bacterial pathogens in the food chain, their persisting morphotypes, and on AMP-based solutions. Challenges in research and application of AMPs are described.

The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan.

Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

Silver-coated endotracheal tubes for prevention of ventilator-associated pneumonia in critically ill patients.

BACKGROUND: Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections in intubated and mechanically ventilated patients. Endotracheal tubes (ETTs) appear to be an independent risk factor for VAP. Silver-coated ETTs slowly release silver cations. It is these silver ions that appear to have a strong antimicrobial effect. Because of this antimicrobial effect of silver, silver-coated ETTs could be an effective intervention to prevent VAP in people who require mechanical ventilation for 24 hours or longer. OBJECTIVES: Our primary objective was to investigate whether silver-coated ETTs are effective in reducing the risk of VAP and hospital mortality in comparison with standard non-coated ETTs in people who require mechanical ventilation for 24 hours or longer. Our secondary objective was to ascertain whether silver-coated ETTs are effective in reducing the following clinical outcomes: device-related adverse events, duration of intubation, length of hospital and intensive care unit (ICU) stay, costs, and time to VAP onset. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2014 Issue 10, MEDLINE, EMBASE, EBSCO CINAHL, and reference lists of trials. We contacted corresponding authors for additional information and unpublished studies. We did not impose any restrictions on the basis of date of publication or language. The date of the last search was October 2014. SELECTION CRITERIA: We included all randomized controlled trials (RCTs) and quasi-randomized trials that evaluated the effects of silver-coated ETTs or a combination of silver with any antimicrobial-coated ETTs with standard non-coated ETTs or with other antimicrobial-coated ETTs in critically ill people who required mechanical ventilation for 24 hours or longer. We also included studies that evaluated the cost-effectiveness of silver-coated ETTs or a combination of silver with any antimicrobial-coated ETTs. DATA COLLECTION AND ANALYSIS: Two review authors (GT, HV) independently extracted the data and summarized study details from all included studies using the specially designed data extraction form. We used standard methodological procedures expected by The Cochrane Collaboration. We performed meta-analysis for outcomes when possible. MAIN RESULTS: We found three eligible randomized controlled trials, with a total of 2081 participants. One of the three included studies did not mention the amount of participants and presented no outcome data. The 'Risk of bias' assessment indicated that there was a high risk of detection bias owing to lack of blinding of outcomes assessors, but we assessed all other domains to be at low risk of bias. Trial design and conduct were generally adequate, with the most common areas of weakness in blinding. The majority of participants were included in centres across North America. The mean age of participants ranged from 61 to 64 years, and the mean duration of intubation was between 3.2 and 7.7 days. One trial comparing silver-coated ETTs versus non-coated ETTs showed a statistically significant decrease in VAP in favour of the silver-coated ETT (1 RCT, 1509 participants; 4.8% versus 7.5%, risk ratio (RR) 0.64, 95% confidence interval (CI) 0.43 to 0.96; number needed to treat for an additional beneficial outcome (NNTB) = 37; low-quality evidence). The risk of VAP within 10 days of intubation was significantly lower with the silver-coated ETTs compared with non-coated ETTs (1 RCT, 1509 participants; 3.5% versus 6.7%, RR 0.51, 95% CI 0.31 to 0.82; NNTB = 32; low-quality evidence). Silver-coated ETT was associated with delayed time to VAP occurrence compared with non-coated ETT (1 RCT, 1509 participants; hazard ratio 0.55, 95% CI 0.37 to 0.84). The confidence intervals for the results of the following outcomes did not exclude potentially important differences with either treatment. There were no statistically significant differences between groups in hospital mortality (1 RCT, 1509 participants; 30.4% versus 26.6%, RR 1.09, 95% CI 0.93 to 1.29; low-quality evidence); device-related adverse events (2 RCTs, 2081 participants; RR 0.65, 95% CI 0.37 to 1.16; low-quality evidence); duration of intubation; and length of hospital and ICU stay. We found no clinical studies evaluating the cost-effectiveness of silver-coated ETTs. AUTHORS' CONCLUSIONS: This review provides limited evidence that silver-coated ETT reduces the risk of VAP, especially during the first 10 days of mechanical ventilation.

Isolation and characterization of persisters of the pathogenic microorganism Staphylococcus aureus.

The presence of antibiotic persisters is one of the leading causes of recurrent and chronic diseases. One challenge in mechanistic research on persisters is the enrichment of pure persisters. In this work, we validated a proposed method to isolate persisters with notorious Staphylococcus aureus cultures. With this, we analyzed the proteome profile of pure persisters and revealed the distinct mechanisms associated with vancomycin and enrofloxacin induced persisters. Furthermore, morphological and metabolic characterizations were performed, indicating further differences between these two persister populations. Finally, we assessed the effect of ATP repression, protein synthesis inhibition, and reactive oxygen species (ROS) level on persister formation. In conclusion, this work provides a comprehensive understanding of S. aureus vancomycin and enrofloxacin induced persisters, facilitating a better mechanistic understanding of persisters and the development of effective strategies to combat them.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Bacterial infection as a likely cause of adverse reactions to polyacrylamide hydrogel fillers in cosmetic surgery.

BACKGROUND: The etiology of long-lasting adverse reactions to gel fillers used in cosmetic surgery is not known. Bacterial infection and immunological reaction to the product have been suggested. METHODS: We performed a case-control study, with 77 biopsies and 30 cytology specimens originating from 59 patients with adverse reactions to polyacrylamide gel, and 54 biopsies and 2 cytology specimens from 28 control subjects with no adverse reactions. Samples from 5 patients and 4 controls could not be investigated for presence of bacteria owing to limited material. Samples from the remaining 54 patients and 24 controls were systematically examined for the presence of bacteria by culture, 16S rRNA gene sequencing, Gram stain, and fluorescence in situ hybridization. RESULTS: Bacteria, mostly normal skin bacteria such as Staphylococcus epidermidis and Propionibacterium acnes, were identified in bacteriologically investigated samples from 53 of 54 patients (98%), and in none of the 24 controls (0%). The bacteria were lying in small clusters, which in symptomatic lesions were detected up to 5 years postinjection. CONCLUSIONS: Commensal bacteria of low virulence are capable of producing long-term infection in the presence of polyacrylamide filler in cosmetic surgery, possibly due to a biofilm mode of growth. Adequate skin preparation and use of sterile technique in these procedures are mandatory, but antibiotic prophylaxis prior to injection of nondegradable gels like polyacrylamide should be explored as well.

A zebrafish high throughput screening system used for Staphylococcus epidermidis infection marker discovery.

BACKGROUND: Staphylococcus epidermidis bacteria are a major cause of biomaterial-associated infections in modern medicine. Yet there is little known about the host responses against this normally innocent bacterium in the context of infection of biomaterials. In order to better understand the factors involved in this process, a whole animal model with high throughput screening possibilities and markers for studying the host response to S. epidermidis infection are required. RESULTS: We have used a zebrafish yolk injection system to study bacterial proliferation and the host response in a time course experiment of S. epidermidis infection. By combining an automated microinjection system with complex object parametric analysis and sorting (COPAS) technology we have quantified bacterial proliferation. This system was used together with transcriptome analysis at several time points during the infection period. We show that bacterial colony forming unit (CFU) counting can be replaced by high throughput flow-based fluorescence analysis of embryos enabling high throughput readout. Comparison of the host transcriptome response to S. epidermidis and Mycobacterium marinum infection in the same system showed that M. marinum has a far stronger effect on host gene regulation than S. epidermidis. However, multiple genes responded differently to S. epidermidis infection than to M. marinum, including a cell adhesion gene linked to specific infection by staphylococci in mammals. CONCLUSIONS: Our zebrafish embryo infection model allowed (i) quantitative assessment of bacterial proliferation, (ii) identification of zebrafish genes serving as markers for infection with the opportunistic pathogen S. epidermidis, and (iii) comparison of the transcriptome response of infection with S. epidermidis and with the pathogen M. marinum. As a result we have identified markers that can be used to distinguish common and specific responses to S. epidermidis. These markers enable the future integration of our high throughput screening technology with functional analyses of immune response genes and immune modulating factors.

3D-printed dual drug delivery nanoparticle- loaded hydrogels to combat antibiotic-resistant bacteria.

Implant-associated infections are not easy to diagnose and very difficult to treat, due to the ability of major pathogens, such as Staphylococcus aureus, to develop biofilms and escape the immune response and antibiotic treatment. We, therefore, aimed to develop a 3D-printed dual rifampicin (Rif)- and vancomycin (Van)-loaded polylactic- co-glycolic acid (PLGA) nanoparticles (NPs) delivery system based on hydrogels made of gelatin methacrylate (GelMA). The release of Rif and Van from NPs manufactured from different PLGA molecular weights was studied in phosphate-buffered saline for 21 days. Low molecular weight PLGA NPs exhibited the fastest release of Rif and Van within the first 7 days and were selected for antimicrobial evaluation. Four different GelMA-based 3D-printed samples were successfully produced, carrying non-loaded NPs, Rif-NPs, Van-NPs, or alternating layers of Rif-NPs and Van-NP. The exposition of S. aureus against increased concentrations of Rif or Van produced new resistant strains to Rif (RifR) or Van (VanR). The GelMA hydrogel co-delivering Rif and Van eradicated S. aureus RN4220 RifR and RN4220 VanR strains. S. aureus RN4220 and S. aureus AMC 201 colonies developed resistance to Rif after contact with the GelMA hydrogel containing only Rif-NPs which appeared to be due to known mutations in the rpoB gene. In conclusion, 3D-printed GelMA hydrogel loaded with PLGA Rif-Van-NPs drug delivery system show promising in vitro results to prevent implant-associated infections caused by antimicrobial-resistant bacteria.

CalcAMP: A New Machine Learning Model for the Accurate Prediction of Antimicrobial Activity of Peptides.

To combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic organisms, including bacteria, viruses, parasites, and fungi. Here, we present the development of a novel machine learning model capable of predicting the activity of antimicrobial peptides (AMPs), CalcAMP. AMPs, in particular short ones (<35 amino acids), can become an effective solution to face the multi-drug resistance issue arising worldwide. Whereas finding potent AMPs through classical wet-lab techniques is still a long and expensive process, a machine learning model can be useful to help researchers to rapidly identify whether peptides present potential or not. Our prediction model is based on a new data set constructed from the available public data on AMPs and experimental antimicrobial activities. CalcAMP can predict activity against both Gram-positive and Gram-negative bacteria. Different features either concerning general physicochemical properties or sequence composition have been assessed to retrieve higher prediction accuracy. CalcAMP can be used as an promising prediction asset to identify short AMPs among given peptide sequences.

3D-printed wound dressings containing a fosmidomycin-derivative prevent Acinetobacter baumannii biofilm formation.

Acinetobacter baumannii causes a wide range of infections, including wound infections. Multidrug-resistant A. baumannii is a major healthcare concern and the development of novel treatments against these infections is needed. Fosmidomycin is a repurposed antimalarial drug targeting the non-mevalonate pathway, and several derivatives show activity toward A. baumannii. We evaluated the antimicrobial activity of CC366, a fosmidomycin prodrug, against a collection of A. baumannii strains, using various in vitro and in vivo models; emphasis was placed on the evaluation of its anti-biofilm activity. We also developed a 3D-printed wound dressing containing CC366, using melt electrowriting technology. Minimal inhibitory concentrations of CC366 ranged from 1 to 64 µg/mL, and CC366 showed good biofilm inhibitory and moderate biofilm eradicating activity in vitro. CC366 successfully eluted from a 3D-printed dressing, the dressings prevented the formation of A. baumannnii wound biofilms in vitro and reduced A. baumannii infection in an in vivo mouse model.

Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities.

The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.