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Xiaolin Zhang,1,2,* Jiankang Song,3,* Alexey Klymov,3,* Yang Zhang,3 Leonie de Boer,1 John A Jansen,3 Jeroen JJP van den Beucken,3 Fang Yang,3 Sebastian AJ Zaat,1,* Sander CG Leeuwenburgh3,* 1Department of Medical Microbiology, Amsterdam Infection and Immunity Institute, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 2Department of Biomaterials Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands; 3Department of Biomaterials, Radboud University Medical Centre, Nijmegen, the Netherlands *These authors contributed equally to this work Background: Infections such as biomaterial-associated infection and osteomyelitis are often associated with intracellular survival of bacteria (eg, Staphylococcus aureus). Treatment of these infections remains a major challenge due to the low intracellular efficacy of many antibiotics. Therefore, local delivery systems are urgently required to improve the therapeutic efficacy of antibiotics by enabling their intracellular delivery. Purpose: To assess the potential of gelatin nanospheres as carriers for local delivery of vancomycin into macrophages of zebrafish larvae in vivo and into THP-1-derived macrophages in vitro using fluorescence microscopy. Materials and methods: Fluorescently labeled gelatin nanospheres were prepared and injected into transgenic zebrafish larvae with fluorescent macrophages. Both the biodistribution of gelatin nanospheres in zebrafish larvae and the co-localization of vancomycin-loaded gelatin nanospheres with zebrafish macrophages in vivo and uptake by THP-1-derived macrophages in vitro were studied. In addition, the effect of treatment with vancomycin-loaded gelatin nanospheres on survival of S. aureus-infected zebrafish larvae was investigated. Results: Internalization of vancomycin-loaded gelatin nanospheres by macrophages was observed qualitatively both in vivo and in vitro. Systemically delivered vancomycin, on the other hand, was hardly internalized by macrophages without the use of gelatin nanospheres. Treatment with a single dose of vancomycin-loaded gelatin nanospheres delayed the mortality of S. aureus-infected zebrafish larvae, indicating the improved therapeutic efficacy of vancomycin against (intracellular) S. aureus infection in vivo. Conclusion: The present study demonstrates that gelatin nanospheres can be used to facilitate local and intracellular delivery of vancomycin. Keywords: in vivo real-time monitoring, fluorescence microscopy, biodistribution, cell-material interaction, Staphylococcus aureus, intracellular infection

Vaginal reconstruction is necessary for various congenital and acquired conditions, including vaginal aplasia, trauma, tumors, and gender incongruency. Current surgical and non-surgical treatments often result in significant complications. Decellularized vaginal matrices (DVMs) from human tissue offer a promising alternative, but require effective sterilization to ensure safety and functionality. This study aimed to develop a sterilization method for decellularized human vaginal wall scaffolds. Based on our previously implemented decellularization technique with minor modifications, we designed and examined three sterilization methods consisting of (i) chemical decellularization, (ii) decellularization with additional peracetic acid/hydrogen peroxide (PAA/H 2 O 2 ); (iii) decellularization with antibiotic and antimycotic (AAE) based treatment. Sterilization efficacy was evaluated through controlled contamination with common vaginal microbes and sterility testing subsequent to each sterilization method. The extracellular matrix (ECM) structure was assessed via histological staining. Decellularization alone reduced some added bacterial contaminants but did not achieve complete sterilization. PAA/H 2 O 2 -sterilization resulted in severe ECM damage, rendering it unsuitable. The AAE-treatment demonstrated effective sterilization without compromising the ECM structure. Combined decellularization and AAE-based treatment forms a viable sterilization method for human vaginal wall tissue, maintaining ECM integrity and achieving effective micro-organism elimination. This method holds potential for clinical application in vaginal transplantation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Treating bone infections with common antibiotics is challenging, since pathogens like Staphylococcus aureus can reside inside macrophages. To target these intracellular bacteria, we have proposed nanoparticles (NPs) as drug carriers. This study aims to investigate the efficacy of hydroxyapatite and gelatin NPs, selected in view of their bone mimicry and potential for targeted delivery, as carriers for the antibacterial agents zinc and vancomycin. Therefore, two distinct NPs are fabricated: zinc-doped hydroxyapatite (ZnHA) and vancomycin-loaded gelatin (VGel) NPs. The NPs are characterized based on morphology, size, chemical composition, cellular internalization, and intracellular bactericidal efficacy. Specifically, the intracellular bactericidal efficacy is tested using a validated coculture model of human THP-1 derived macrophages and phagocytosed S. aureus bacteria. Scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FTIR) results show that the spherical NPs are synthesized successfully. These NPs are internalized by THP-1 cells and show >75% colocalization with lysosomes without compromising the viability of the THP-1 cells. Both ZnHA and VGel NPs substantially reduce the intracellular survival of S. aureus compared to the direct addition of dissolved zinc and vancomycin. Concluding, our NPs are highly effective drug delivery vehicles to kill intracellular S. aureus , which stress the potential of these NPs for future clinical translation., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus , allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus , C. albicans , and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus , and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus , addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions., Importance: Interaction between microbes and the host are in the center of interest both in disease and in health. In order to study the interactions between microbes of different kingdoms and the host, alternative media are required. Synthetic media are useful as they allow addition of specific components. In addition, well-defined media are required if high-resolution analyses such as metabolomics and proteomics are desired. We describe the development of a synthetic medium to study the interactions between C. albicans, S. aureus, and human oral epithelial cells. Our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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., (© 2024 The Authors. Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)

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., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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., Competing Interests: The authors declare no conflict of interest.

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 -/- Sirpa NOD Flk2 -/- (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., Competing Interests: The authors have no conflict of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Background: The origin of surgical site and biomaterial-associated infection is still elusive. Micro-organisms contaminating the wound may come from the air in the operating theatre, the surgical team or the skin of the patient. The skin of patients is disinfected prior to surgery, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands) may not be reached., Methods: A preliminary cohort study was performed to study the origin of surgical site and biomaterial-associated infection between May 2020 and February 2021. In order to investigate whether cutaneous microbiota colonize the wound when released from the skin upon cutting, aerobic and anaerobic bacteria were isolated, quantified and identified from the skin of 99 patients undergoing trauma surgery, before and after skin disinfection, from knife blades and from the wound directly after the first cut., Results: Ninety-nine percent of the patients were culture-positive before disinfection with chlorhexidine. Of these, 40% were still culture-positive after disinfection. Of these, 54% had a positive culture of the wound after cutting the skin. Twenty percent of the patients with a negative culture after disinfection had a positive wound culture after cutting the skin. Staphylococcus epidermidis and Cutibacterium acnes were the most commonly cultured bacterial species. In 9% of cases, more than 100 bacterial colonies were cultured from the wound; this may cause biomaterial-associated infection., Conclusion: Bacteria residing in the skin and not eradicated by disinfection may enter the surgical wound upon cutting, resulting in contamination which may cause biomaterial-associated infection., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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.

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., (Copyright © 2022 AAGL. Published by Elsevier Inc. All rights reserved.)

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 (Rif R ) or Van (Van R ). The GelMA hydrogel co-delivering Rif and Van eradicated S. aureus RN4220 Rif R and RN4220 Van R 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., Competing Interests: The authors declare no conflicts of interest., (Copyright: © 2023 Martínez-Pérez D, Guarch-Pérez C, Purbayanto MAK, et al.)

Biomaterial-associated infections are a major healthcare challenge as they are responsible for high disease burden in critically ill patients. In this study, we have developed drug-eluting antibacterial catheters to prevent catheter-related infections. Niclosamide (NIC), originally an antiparasitic drug, was incorporated into the polymeric matrix of thermoplastic polyurethane (TPU) via solvent casting, and catheters were fabricated using hot-melt extrusion technology. The mechanical and physicochemical properties of TPU polymers loaded with NIC were studied. NIC was released in a sustained manner from the catheters and exhibited in vitro antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis. Moreover, the antibacterial efficacy of NIC-loaded catheters was validated in an in vivo biomaterial-associated infection model using a methicillin-susceptible and methicillin-resistant strain of S. aureus. The released NIC from the produced catheters reduced bacterial colonization of the catheter as well as of the surrounding tissue. In summary, the NIC-releasing hot-melt extruded catheters prevented implant colonization and reduced the bacterial colonization of peri-catheter tissue by methicillin sensitive as well as resistant S. aureus in a biomaterial-associated infection mouse model and has good prospects for preclinical development., (© 2022. The Author(s).)

Bacterial infections are a serious healthcare complication in orthopedic and trauma surgery worldwide. Compared to systemic, local antibiotic prophylaxis has been shown to provide a higher antibiotic dose and bioavailability at the bone site with minimum toxic effects. However, there are still not enough biomaterial and antibiotic combinations available for personalized implant sizes for patients. The aim of this study was to develop a bone fixation plate coating made of a composite of poly-ε-caprolactone, hydroxyapatite and halloysite nanotubes loaded with gentamicin sulphate and fabricated via fused filament fabrication 3D printing technology. The mechanical and thermal properties of the biomaterial were analyzed. The in vitro release kinetics of gentamicin sulphate were evaluated for 14 days showing a burst release during the first two days that was followed by a sustained release of bactericidal concentrations. The composite loaded with 2 and 5% gentamicin sulphate exhibited complete antimicrobial killing of Staphylococcus aureus in an ex vivo mouse femur fixation plate infection model. Moreover, a fixation plate of the composite loaded with 5% of gentamicin sulphate was able to prevent S. aureus infection in the bone and surrounding tissue in an in vivo mouse bone fixation plate infection model 3 days post-surgery. In conclusion, the newly developed composite material successfully prevented infection in vivo. Additionally, the ability to use fused filament fabrication 3D printing to produce patient-specific implants may provide a wider range of personalized solutions for patients.

Polypropylene (PP) implants for the vaginal surgical correction of pelvic organ prolapse (POP) are known for adverse events, like vaginal or visceral exposures. It is hypothesized that this is a result of a prolonged inflammatory response. One of the triggering factors of prolonged inflammation might be bacterial contamination. A possible solution might lie in an absorbable biomaterial, which provides initial mechanical support while being gradually replaced by the host tissue. With this study we aimed to compare the host response, in a subcutaneous mouse implant infection model, to delayed absorbable poly-4-hydroxybutyrate (P4HB) and a latest generation PP implant. By comparing non-infected to Staphylococcus aureus infected mice, we assessed how bacterial contamination affects the host response and its role in the development of complications. Further, we included sham surgery as a control, mimicking the wound response in native tissue repair. Despite the higher surface area of the P4HB implants, the clearance of infection was similarly delayed in the presence of a P4HB or PP implant, as compared to sham. Further, the host response towards P4HB and PP was quite comparable, yet collagen deposition was significantly increased around infected P4HB implants at early time points. Adverse event rates were similar, though implant exposures were only seen in infected mice and more often with PP (11.1%) than P4HB implants (5.6%). Infected mice overall had significantly higher levels of infiltration of inflammatory cells and lower levels of vascularization and collagen deposition compared to non-infected mice. Thus, for both P4HB and PP, bacterial contamination negatively affected mesh integration by increased inflammation and an increased adverse event rate. Altogether, our results from this subcutaneous mouse implant infection study suggest that P4HB could be a promising degradable alternative to PP, warranting further research to study its potential as a new surgical solution for women with POP., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.P.W.R Roovers reports financial support and equipment, drugs, or supplies were provided by Tepha Inc. and Coloplast., (© 2022 Published by Elsevier Ltd.)

Candida albicans and Staphylococcus aureus account for most invasive fungal and bacterial bloodstream infections (BSIs), respectively. However, the initial point of invasion responsible for S. aureus BSIs is often unclear. Recently, C. albicans has been proposed to mediate S. aureus invasion of immunocompromised hosts during co-colonization of oral mucosal surfaces. The status of the oral immune system crucially contributes to this process in two distinct ways: firstly, by allowing invasive C. albicans growth during dysfunction of extra-epithelial immunity, and secondly following invasion by some remaining function of intra-epithelial immunity. Immunocompromised individuals at risk of developing invasive oral C. albicans infections could, therefore, also be at risk of contracting concordant S. aureus BSIs. Considering the crucial contribution of both oral immune function and dysfunction, the aim of this review is to provide an overview of relevant aspects of intra and extra-epithelial oral immunity and discuss predominant immune deficiencies expected to facilitate C. albicans induced S. aureus BSIs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest., (Copyright © 2022 Pasman, Krom, Zaat and Brul.)

Pathogens such as Staphylococcus aureus are able to survive in many types of host cells including phagocytes such as neutrophils and macrophages, thereby resulting in intracellular infections. Treatment of intracellular infections by conventional antimicrobials (e.g., antibiotics) is often ineffective due to low intracellular efficacy of the drugs. Thus, novel techniques which can enhance the activity of antimicrobials within cells are highly demanded. Our recent studies have shown that photochemical internalization (PCI) is a promising approach for improving the efficacy of antibiotics such as gentamicin against intracellular staphylococcal infection. In this chapter, we describe the protocols aiming to study the potential of PCI-antibiotic treatment for intracellular infections in vitro and in vivo using a RAW 264.7 cell infection model and a zebrafish embryo infection model. Proof of concept of this approach is demonstrated. The protocols are expected to prompt further development of PCI-antimicrobial based novel therapies for clinically challenging infectious diseases associated with intracellular survival of pathogens., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Staphylococcus aureus is the main causative pathogen of subcutaneous, bone, and implant-related infections, forming structures known as staphylococcal abscess communities (SACs) within tissues that also contain immunosuppressive myeloid-derived suppressor cells (MDSCs). Although both SACs and MDSCs are present in chronic S. aureus infections, it remains unknown whether SACs directly trigger MDSC expansion. To investigate this, a previously developed 3D in vitro SAC model was co-cultured with murine and human bone marrow cells. Subsequently, it was shown that SAC-exposed human CD11b low/- myeloid cells or SAC-exposed murine CD11b + Gr-1 + cells were immunosuppressive mainly by reducing absolute CD4 + and CD8α + T cell numbers, as shown in T cell proliferation assays and with flow cytometry. Monocytic MDSCs from mice with an S. aureus bone infection also strongly reduced CD4 + and CD8α + T cell numbers. Using protein biomarker analysis and an immunoassay, we detected in SAC-bone marrow co-cultures high levels of GM-CSF, IL-6, VEGF, IL-1β, TNFα, IL-10, and TGF-β. Furthermore, SAC-exposed neutrophils expressed Arg-1 and SAC-exposed monocytes expressed Arg-1 and iNOS, as shown via immunofluorescent stains. Overall, this study showed that SACs cause MDSC expansion from bone marrow cells and identified possible mediators to target as an additional strategy for treating chronic S. aureus infections.

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.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log 10 -scale, the reproducibility SD (S R ) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/S R  = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Additive manufacturing is widely used to produce highly complex structures. Moreover, this technology has proven its superiority in producing tools which can be used in different applications. We designed and produced an extrusion nozzle that allowed us to hot melt extrude drug-loaded tubes. The tubes were an essential part of a new mouse ventilator-associated pneumonia (VAP) model. Ciprofloxacin (CPX) was selected for its expected activity against the pathogen Staphylococcus aureus and ease of incorporation into thermoplastic polyurethane (TPU). TPU was selected as the carrier polymer for its biocompatibility and use in a variety of medical devices such as tubing and catheters. The effect of loading CPX within the TPU polymeric matrix and the physicochemical properties of the produced tubes were investigated. CPX showed good thermal stability and in vitro activity in preventing S. aureus biofilm formation after loading within the tube's polymeric matrix. Moreover, the produced tubes showed anti-infective efficacy in vivo. The produced tubes, which were extruded via our novel nozzle, were vital for the validation of our mouse VAP model. This model can be adopted to investigate other antibacterial and antibiofilm compounds incorporated in polymeric tubes using hot melt extrusion.

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, β-galactosidase and β-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S . aureus (MRSA) strain N315, both producing α-glucosidase, but not β-glucuronidase and β-galactosidase, Escherichia coli strain DH5α, producing β-glucuronidase and α-glucosidase, but not β-galactosidase, and the enterohemorrhagic E . coli (EHEC) strain E32511, producing β-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

IgG Abs are crucial for various immune functions, including neutralization, phagocytosis, and Ab-dependent cellular cytotoxicity. In this study, we identified another function of IgG by showing that IgG immune complexes elicit distinct cytokine profiles by human myeloid immune cells, which are dependent on FcγR activation by the different IgG subclasses. Using monoclonal IgG subclasses with identical Ag specificity, our data demonstrate that the production of Th17-inducing cytokines, such as TNF, IL-1β, and IL-23, is particularly dependent on IgG2, whereas type I IFN responses are controlled by IgG3, and IgG1 is able to regulate both. In addition, we identified that subclass-specific cytokine production is orchestrated at the posttranscriptional level through distinct glycolytic reprogramming of human myeloid immune cells. Combined, these data identify that IgG subclasses provide pathogen- and cell type-specific immunity through differential metabolic reprogramming by FcγRs. These findings may be relevant for future design of Ab-related therapies in the context of infectious diseases, chronic inflammation, and cancer., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Knitted polypropylene (PP) implants for the correction of pelvic organ prolapse have been associated with complications such as vaginal exposure, infection, and pain. Since certain complications may be linked to bacterial contamination and persistent inflammation, there is a rationale to develop a biocompatible implant that is less prone to bacterial adhesion and biofilm formation. Delayed absorbable materials could meet these requirements and poly-4-hydroxybutyrate (P4HB) might be such a new material for future pelvic floor implants. We studied in vitro bacterial adhesion and biofilm formation on P4HB in comparison to PP. We investigated the influence of both polymers using flat films and compared P4HB and PP implants with different knitting designs. P4HB flat films were demonstrated to be hydrophilic with significantly less Staphylococcus aureus and Escherichia coli cultured from P4HB films than from hydrophobic PP films after 24 h of incubation. On the implants, a higher number of E. coli were cultured after 1 h of incubation from the knitted P4HB implant with the highest density and smallest pore size, compared to other P4HB and PP implants. No differences were observed between the implants for E. coli at later time points or for S. aureus incubation. These results show that in flat films, the polymer influences biofilm formation, demonstrated by a reduced biofilm formation on P4HB compared with PP flat films. In addition, the knitting design may affect bacterial adhesion. Despite certain design and material characteristics that give the knitted P4HB implants a higher surface area, this did not result in more bacterial adhesion and biofilm formation overall. Collectively, these results warrant further (pre)clinical investigations of P4HB pelvic floor implants.

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.

Nosocomial diseases represent a huge health and economic burden. A significant portion is associated with the use of medical devices, with 80% of these infections being caused by a bacterial biofilm. The insertion of a foreign material usually elicits inflammation, which can result in hampered antimicrobial capacity of the host immunity due to the effort of immune cells being directed to degrade the material. The ineffective clearance by immune cells is a perfect opportunity for bacteria to attach and form a biofilm. In this study, we analyzed the antibiofilm capacity of three naturally derived biofilm inhibitors when combined with immune cells in order to assess their applicability in implantable titanium devices and low-density polyethylene (LDPE) endotracheal tubes. To this end, we used a system based on the coculture of HL-60 cells differentiated into polymorphonuclear leukocytes (PMNs) and Staphylococcus aureus (laboratory and clinical strains) on titanium, as well as LDPE surfaces. Out of the three inhibitors, the one coded DHA1 showed the highest potential to be incorporated into implantable devices, as it displayed a combined activity with the immune cells, preventing bacterial attachment on the titanium and LDPE. The other two inhibitors seemed to also be good candidates for incorporation into LDPE endotracheal tubes.

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., (Copyright © 2020 American Society for Microbiology.)

Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-1-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

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., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Epidural catheters are frequently colonized by gram-positive bacteria. Although the incidence of associated epidural infections is low, their consequences can be devastating. We investigated bacterial growth on epidural catheters by quantitative bacterial culture and scanning electron microscopy (SEM) in order to explore the patterns of epidural catheter colonization., Methods: 28 patients undergoing major abdominal surgery with thoracic epidurals (treatment ≥72 hours) were studied. Before the removal of the catheter, the skin surrounding the insertion site was swabbed. The entire catheter was divided into extracorporeal, subcutaneous, and tip segments. Skin swabs and catheter segments were quantitatively cultured, bacterial species were identified, and SEM was performed on four selected catheters., Results: 27 of 28 catheters were included. The percentages of positive cultures were: skin swab 29.6%, extracorporeal segments 11.1%, subcutaneous segments 14.8%, and tip segments 33.3%. One patient was diagnosed with a catheter-associated infection. Staphylococcus epidermidis was cultured from the skin and the catheter extracorporeal, subcutaneous, and tip segments. SEM of this catheter showed bacteria-like and intraluminal host cell-like structures. SEM of two other catheters showed intraluminal fibrin networks in their tip segments., Conclusions: We present the first SEM pictures of an epidural catheter with a bacterial infection. Bacterial growth developed from the skin to the tip of this catheter, indicating the skin as a primary source of infection. By SEM, catheters with low levels of bacterial growth demonstrated an intraluminal fibrous network which possibly plays a role in catheter obstruction., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2020. Re-use permitted under CC BY. Published by BMJ.)

Staphylococcus aureus and Staphylococcus epidermidis are related species which can cause predominantly acute and subacute infections, respectively. Differences in human adaptive immune responses to these two species are not well understood. Dendritic cells (DCs) have an important role in the control and regulation of anti-staphylococcal T cell responses. Therefore, we aimed to compare the ability of S. aureus and S. epidermidis to influence the essential steps in human DC activation and subsequent antigen-specific CD4 + T cell proliferation, and to investigate the underlying mechanisms. Using multiple strains of both species, we observed that S. aureus was internalized more effectively than S. epidermidis by DCs but that both species were equally potent in activating these host cells, as evidenced by similar induction of DC maturation marker expression and antigen loading onto MHC-II molecules. The DCs stimulated by S. aureus strains not harboring superantigen (SAg) genes or by any of the S. epidermidis strains, induced low, likely physiological levels of T cell proliferation. Only DCs stimulated with S. aureus strains harboring SAg genes induced high levels of T cell proliferation. Taken together, S. aureus and S. epidermidis do not differently affect DC activation and ensuing antigen-specific T cell proliferation, unless a strain has the capacity to produce SAgs.