The emergence of antibiotic resistance in clinically significant pathogens, coupled with their ability to form persistent biofilms, presents a severe challenge in the management of burns and wound infections. To help mitigate the overuse of antibiotics and reduce the prevalence of antimicrobial-resistant bacteria, we have developed a modular theranostic system by blending two types of biocompatible copolymer building blocks, a chromogenic and an antimicrobial component, respectively. For this purpose, a parent active ester copolymer, poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(pentafluorophenyl acrylate)-co- (ECOSURF EH-3 acrylate)] was synthesized via free radical polymerization. Two different chromogenic building blocks for bacterial enzyme detection were obtained from the parent polymer by functionalization of the pentafluorophenyl acrylate units with enzyme-labile chromogenic compounds, either 5-bromo-4-chloro-3-indolyl β-d-glucuronide (X-GLUC) or 4-nitrophenyl-β-d-glucuronide (PNPG). The antimicrobial building block for infection prevention and treatment was prepared from the parent active ester polymer by modification with antimicrobial peptide SAAP-148. The chromogenic-antimicrobial polymer blend responded to the bacterial enzyme β-glucuronidase with the release of chromophores that produce a visible colour change, enabling rapid, equipment-free proof-of-concept chromogenic detection of Escherichia coli, a major cause of healthcare-associated infections. This polymer blend also demonstrated potent antimicrobial activity against E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, including multidrug-resistant strains.

ABSTRACT Interkingdom interactions between Candida albicans and Staphylococcus aureus promote lethal dissemination of the bacterium. During this process, C. albicans hypha invasion aids S. aureus dissemination through Als1p/Als3p-facilitated co-invasion. The effects of S. aureus on C. albicans hypha formation and invasion are, however, unknown. In this study, we used both liquid mDMEM-DMP as well as a previously constructed semi-solid adaptation of the medium (mDMEM-DMPA) to study the effects of C. albicans/S. aureus co-culturing on hypha formation and invasion. Semi-solid-based co-culturing significantly increased colony size and generally increased hypha invasion. Liquid growth-based time-lapse microscopy showed that S. aureus significantly promoted both C. albicans hypha length and elongation rate. Further semi-solid-based growth results revealed that >3 kDa-secreted S. aureus factors were accountable for the increase in C. albicans hypha growth. A newly constructed in vitro assay confirmed the co-invasion of S. aureus during co-culturing and showed that deletion of C. albicans Als1p/Als3p abolished the co-invasion of S. aureus during co-culturing. In conclusion, our study shows that S. aureus affects C. albicans virulence by actively stimulating C. albicans hypha extension through the production of, presently unknown, secreted factors and sequentially using hypha proteins Als1p and Als3p to co-invade. Therefore, S. aureus can stimulate C. albicans epithelial invasion even prior to attaching to its hyphae, providing the foundation for subsequent co-invasion.IMPORTANCEEpithelial barriers normally protect against invasion and systemic infection by S. aureus, but frequently, such infections occur without a port of entry. One route of S. aureus epithelial traversal is through co-invasion with the highly invasive Candida albicans. Understanding this interaction in detail is of high importance in view of the prevention of these infections. Our study shows how the S. aureus and C. albicans interaction results in mutual benefit. S. aureus appeared to affect C. albicans virulence by actively stimulating C. albicans hypha extension through the production of, presently unknown, secreted factors and sequentially using hypha proteins Als1p and Als3p to bind to the hyphae and co-invade. These insights are important from a microbial ecological perspective and offer important potential targets for interfering with the interaction and reducing the virulence of these opportunistic pathogens.

Candidozyma auris is an emerging opportunistic fungal pathogen that can cause serious catheter-related blood stream infections associated with high morbidity and mortality. The traditional antifungal treatment with polyenes, azoles or echinocandins is becoming less effective due to both intrinsic and developed resistance, complicating treatment. This study demonstrates the potent fungicidal activity of carboxyl-functionalized graphene quantum dots (cGQDs) against a panel of C. auris strains, spanning clades I to V, and a Candida albicans reference strain. Photoactivation of cGQDs in suspension with 435 nm blue light killed 99.9% of the fungi within 30 min even though the majority of test strains were resistant to at least one conventional antifungal. Moreover, cGQDs coated on flexible polydimethylsiloxane surfaces and commercial catheters via electrostatic layer-by-layer deposition with alternating positively charged polydiallyldimethylammonium polymer showed strong fungicidal activity against C. auris and C. albicans. These findings show that the cGQDs, both in suspension and in a thin film coating, have potential for future clinical development. In particular, their application to catheters may help prevent Candidozyma and Candida catheter-related infections.

The escalating incidence of fungal infections, coupled with the emergence of antifungal resistance, represents a formidable global public health challenge. Candida albicans is a common opportunistic fungus that causes life-threatening, widespread candidiasis, especially in immunocompromised patients. Conventional antifungals, including azoles, echinocandins, and polyenes, have some drawbacks, such as host toxicity and the rapid appearance of resistant fungal strains. To overcome these limitations, we engineered a stimuli-responsive gated antimicrobial nanoparticle using mesoporous silica nanoparticles, functionalized with the antimicrobial peptide histatin 5 (Hst5) and loaded with the surfactant (3-(methyl)-1-tetradecylimidazolium) [C14MIM]+. The nanoparticles exhibit potent antimicrobial activity against C. albicans, with minimum inhibitory concentration values comparable to current clinical antifungals. Crucially, the nanodevice exhibits a dual mechanism of action, causing membrane disruption and inducing reactive oxygen species generation, which effectively hinders the development of resistance. Furthermore, the nanoparticles display strong synergistic interactions with conventional antifungals, significantly enhancing their efficacy. In addition to its planktonic activity, the system effectively inhibits biofilm formation and demonstrates high biocompatibility with human dermal cell lines. Based on these results, the nanodevice is successfully incorporated into a wound dressing matrix, demonstrating its promising translational potential for the topical treatment of cutaneous candidiasis.

IntroductionOrthopedic implant-associated infections, predominantly caused by S. aureus, pose significant challenges due to biofilm formation and antibiotic resistance. Bioactive Glass (BAG) S53P4 is a unique material with antimicrobial and bone regenerative properties. We aimed to characterize a novel BAG S53P4 cream, consisting of BAG powder and a binder, for its capacity to kill Staphylococcus aureus in suspension and biofilms in the absence or presence of titanium implant material. Since the BAG antimicrobial activity depends on ions eluted, we also analyzed the eluates of the cream and of powder and binder.MethodsBAG cream, BAG powder, and binder were evaluated for antimicrobial activity against planktonic S. aureus in the presence or absence of titanium implant material, and against S. aureus biofilms. Eluates collected at different time points were tested against a panel of bacterial and fungal pathogens. Elemental ion release and pH changes were measured over time. Time-kill and biofilm assays were performed. Additionally, the applicability and antimicrobial efficacy of BAG cream were evaluated in a cadaver mouse bone defect model.ResultsThe BAG cream and BAG powder applied to titanium implant material, as well as their respective eluates eradicated planktonic S. aureus. Elemental release from BAG cream and powder showed time-dependent shifts in levels of silicon, sodium, calcium and phosphorous together with stable alkaline pH levels, reflecting continuous ion release from the glass network and concurrent precipitation of calcium phosphate and silica phases. BAG cream and powder eluates collected as early as at 2 h were highly effective against S. aureus, the ESKAPE panel of multidrug resistant pathogens, colistin-resistant Escherichia coli and Cutibacterium acnes, and against the fungi Candidozyma auris and Candida albicans. The eluates displayed time-dependent bactericidal activity with significant bacterial killing starting already at 30 min and increasing with longer exposure times. Moreover, significant reduction in S. aureus biofilm was observed with the cream and powder eluates. BAG cream was easy to apply to the bone defect of a cadaver mouse using a syringe and it effectively prevented S. aureus growth.ConclusionThese findings show the potential of BAG cream as an innovative application form of BAG S53P4 offering a promising approach against orthopedic implant-associated infections.

Bioactive glass (BAG) S53P4 is a clinically approved bone substitute with antibacterial, osteoconductive and osteostimulatory properties. Its antibacterial effect is associated with ion release, local pH elevation and osmolality, but the precise biochemical and biophysical mode-of-action is unclear. This study investigates the antibacterial mechanism of BAG S53P4 eluates. BAG eluates, collected at 2, 4, 8, and 24 h, eradicated Staphylococcus aureus. Elemental analysis revealed an early increase in concentrations of Si and Na, a later rise in Ca, depletion of P over time and rapid loss of Mg. Membrane disturbances occurred within 5 min, evident by permeability for SYTOX, aligning with time-kill kinetics for S. aureus and Bacillus subtilis. In B. subtilis, 2h-BAG-eluate induced rapid delocalization of marker proteins for cell division and DNA repair, signaling membrane potential collapse and nucleoid condensation. Transcriptomics revealed early transcription remodeling reflecting ionic and energetic imbalance, including disruption of central metabolism, redox homeostasis, and translational stability. Scanning electron microscopy revealed severe cell surface damage and particulate deposits on S. aureus. Transmission electron microscopy showed cell envelop disruptions and cytoplasmic leakage. Energy dispersive X-ray analysis identified Si on bacterial cell surface at 4 h and intracellular accumulation in punctured, empty cells at 24 h. Overall, BAG ionic dissolution products kill bacteria through a stepwise mechanism involving membrane damage, protein delocalization and metabolic impairment, accompanied by Si deposition on bacterial surfaces and loss of Mg. This finally leads to cell wall degradation, cytoplasmic content leakage and further Si deposition on the cells and inside cell ghosts.

Fracture non-union represents a complex clinical challenge resulting from an incompletely understood interplay between mechanical and biological factors. Infection frequently contributes to non-union but many cases are misdiagnosed due to a lack of classical clinical symptoms. This study characterized peripheral blood mononuclear cells (PBMCs) from aseptic non-union (NU-AS, n = 24) and fracture-related infected non-union (NU-FRI, n = 20), and compare them to healed controls (H, n = 18). High-dimensional mass cytometry (CyTOF) revealed significant elevations of regulatory T cells (Tregs; p = 0.0028) and T helper 1 (Th1) cells (p = 0.0073), and reduced expression of the activation marker CD38 in CD4+ T cells (p = 0.0016) and Tregs (p = 0.0017) in NU compared to H. In a subgroup analysis between NU-AS and NU-FRI, monocyte and CD38+ Treg cell counts provided excellent diagnostic potential, with the combination achieving a sensitivity of 100% and a specificity of 91.7%. These findings highlight an important role of the activation marker CD38 in diagnosing chronic subclinical infection, which promises earlier identification of appropriate management of these patients.

Bacterial infections are a major healthcare concern. Clinical application of photo-activated quantum dots to efficiently treat bacterial infections has been hindered by inadequate production of reactive oxygen species. In this study, photoactive antimicrobial carboxyl-functionalized graphene quantum dots (cGQDs) are synthesized with an exceptionally high singlet oxygen (1O2) quantum yield of 0.88. Compared to non-functionalized GQDs, cGQDs exhibit over a 20-fold enhancement in the 1O2 quantum yield. According to the density functional theory simulations, the dramatic increase of 1O2 quantum yield is due to significantly enhanced spin-orbit coupling between singlet and triplet excited states of GQDs with addition of & horbar;COOH groups. Under low-intensity blue light (5 mW cm- 2), Staphylococcus aureus is completely eliminated with just 0.8 mu g mL-1 of cGQDs, and a minimum bactericidal concentration (MBC) of 0.4 mu g mL-1 is determined, representing the lowest MBC reported against S. aureus using light-activated quantum dots. Layer-by-layer assembly of cGQD films also results in over a 99.9% reduction against multi-drug resistant Staphylococcus aureus and Escherichia coli under illumination. cGQDs, both in suspension and as a nano-assembled film, exhibit good cell viability in mammalian cells under both dark and light conditions. These results highlight the strong potential of cGQDs as an effective nanomaterial for antibacterial applications.

Background: The escalating global crisis of antibiotic resistance necessitates the discovery of novel antimicrobial agents. Antimicrobial peptides (AMPs) represent a promising alternative to combat multidrug-resistant (MDR) pathogens. Because traditional AMP discovery is labour-intensive and costly, machine learning (ML) is applied to identify AMPs effective against MDR bacteria and skin infections. Methods: The ML-based CalcAMP model predicts the antimicrobial activity of 16,384 unique 14-amino-acid peptide sequences, resulting in a novel Guided Designed Smart antimicrobial Therapeutic (GDST) peptide catalogue. Parent sequences and retro-inverso (RI) variants of two prime GDST peptides undergo extensive testing against MDR bacteria and in skin infection models. Results: GDST-038 and GDST-045, along with their RI variants, show potent antimicrobial activity against Acinetobacter baumannii and Staphylococcus aureus, rapidly depolarizing the cytoplasmic membrane, exhibiting broad-spectrum bactericidal effects against ESKAPE pathogens, and causing minimal haemolysis. RI variants display superior A. baumannii biofilm killing compared to parent sequences, while all GDST peptides achieve >3-log reductions in S. aureus biofilm CFU within 24 h. Potent efficacy is observed in a 3D human skin epidermal infection model, with elimination of S. aureus at ≥15 μM. No resistance develops after 22 passages. Conclusions: ML-driven screening enables rapid identification of two novel candidate AMPs, highlighting the therapeutic potential of GDST peptides for MDR bacterial infections.

Background:Co-infections of Candida albicans and Staphylococcus aureus can significantly increase morbidity and mortality. However, the effect of C. albicans-S. aureus co-existence on virulence factor secretion and pro-inflammatory effects remain elusive. Methods:We systematically investigated the virulence factors released by C. albicans and S. aureus under different culturing conditions using proteomics. We characterized their pro-inflammatory effects in macrophages with transcriptomics and gene set enrichment analysis. Results and Discussion:We showed that co-culturing of C. albicans and S. aureus promoted the secretion of 7 cytolytic, 11 proteolytic, and 3 lipolytic extracellular virulence factors (ECVFs) and impacted non-ECVFs, owing to Als1/Als3-mediated interactions, the presence of C. albicans, or its pH maintenance. Co-culturing promotes C. albicans hypha formation and β-glucan masking, suggesting that co-culturing enhances both C. albicans invasion and immune evasion. Moreover, the secretome of C. albicans-S. aureus co-culture increased pro-inflammatory pathways including promoting TNF-, NFKB-, and Toll-like receptor signaling pathways, as well as cytokine-cytokine receptor interactions in macrophages. Our findings support that C. albicans and S. aureus reciprocally promote their virulence potential and pro-inflammatory effects, which may provide mechanistic insights into the increased morbidity and mortality during their co-infection in vivo.

In this work, a copolymer of poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(hexamethylene diamine acrylamide)-co-(ECOSURF EH-3 acrylate)] was synthesized via free radical polymerization, followed by multi-step modification using click chemistry. The copolymer was subsequently functionalized with either 5-bromo-4-chloro-3-indolyl beta-d-glucuronide (X-GLUC) or 4-nitrophenyl-beta-d-glucuronide (PNPG), two enzyme-labile chromogenic substrates used to visually observe bacterial beta-glucuronidase activity. This enzyme is secreted by over 98% of Escherichia coli strains, a common cause of healthcare-associated infections. The use of two substrates demonstrates the system's versatility in detecting beta-glucuronidase activity across different bacterial species, wherein enzymatic cleavage of the dye-sugar bond produces a visible chromogenic signal. Both copolymers were found to be non-cytotoxic to human lung fibroblasts and were independently crosslinked under UV light to form distinct polymer network structures. Upon water swelling, each hydrogel enabled qualitative detection of beta-glucuronidase-producing bacteria through the release of indigo or yellow dyes, corresponding to the chromogenic response of X-GLUC or PNPG, respectively. This colorimetric response was confirmed both visually and spectroscopically, underscoring the potential of these polymers for development into multiplexed enzyme-based bacterial detection platforms. The technology offers promising applications in microbiological diagnostics, particularly in food safety and medical contexts.

IntroductionInfected chronic wounds present a dual therapeutic challenge, requiring both the eradication of pathogens and the restoration of tissue homeostasis. Often the current treatments are ineffective against multidrug-resistant (MDR) pathogens and fail to promote wound healing. Antimicrobial peptides, such as bovine lactoferricin (LfcinB), offer a promising alternative owing to their broad-spectrum activity and immunomodulatory properties. The branched tetrameric LfcinB-derived peptide (LBT; (RRWQWR)4K2Ahx2-C2) is particularly attractive, as its multivalent architecture enhances antimicrobial potency and provides a tunable branching core for structural modifications.MethodsIn this study, three novel tetrameric variants were designed by substituting the L-lysine branching residues in LBT with non-natural lysine derivatives to alter motif orientation and linker flexibility. Among the novel peptides, the diaminopropionic acid (DAP)-modified variant, (LBT-1; (RRWQWR)4DAP2Ahx2-C2) was selected as best-performing candidate based on antimicrobial and hemolytic activity assessment.ResultsCompared to LBT, the novel LBT-1 demonstrated superior activity against methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii, achieving rapid bactericidal action within 5 minutes. LBT-1 also exhibited potent activity across the ESKAPE(E) panel and against the emerging MDR fungal pathogen Candidozyma auris. Beyond direct antimicrobial effects, LBT-1 enhanced macrophage-mediated bacterial clearance, neutralized endotoxins, and accelerated wound closure in vitro. Importantly, LBT-1 showed superior pro-angiogenic activity in vitro and achieved significantly higher bactericidal activity against MRSA in an ex vivo human skin wound infection model. This study identifies LBT-1 as a multifunctional therapeutic that addresses key pathological features of chronic wounds.ConclusionTogether, these findings validate our peptide design strategy, revealing previously unknown characteristics of the LBT peptide and the enhanced multifunctionality achieved with LBT-1, supporting its continued development for chronic wound management.

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.

ABSTRACT 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.

Co-infections of Candida albicans and Staphylococcus aureus can significantly increase morbidity and mortality. This synergism is linked to the interactions between C. albicans and S. aureus that allow for staphylococcal co-invasion and dissemination. While it is known that extracellular virulence factors (ECVFs) contribute to this process, the effects of C. albicans-S. aureus co-culturing on ECVF composition remain unknown. In this study we used mass spectrometry-based proteomics to investigate the effect of co-culturing on the extracellular proteins released by the S. aureus and C. albicans. Co-culturing of C. albicans and S. aureus promoted the secretion of 7 cytolytic, 11 proteolytic, and 3 lipolytic ECVFs. Interestingly, co-culturing of C. albicans Als1p/Als3p mutant alleviated the increase for the majority of the differentially changed C. albicans ECVFs, but not for S. aureus ECVFs, highlighting the importance of Als1p/Als3p in the secretion of C. albicans ECVFs. Of 27 detected S. aureus ECVFs, 17 were significantly increased in co-culturing. Among these, maintenance of pH alone in S. aureus monoculture increased five haemolytic proteins, i.e., alpha haemolysin (Hly/Hla), beta haemolysin (Hlb), and gamma haemolysin (HlgA-C) to a similar extent as the co-culture. In contrast, maintenance of pH diminished the increase of protease-like proteins, (phospho)lipases, delta hemolysin, and leukotoxin, suggesting that both pH-dependent and pH-independent C. albicans factors affect S. aureus ECVFs. A cytotoxicity assay demonstrated that the secretome from co-culture has higher cytotoxicity towards human oral cells (Ca 9-22 and HO1N1) than monoculture. Finally, co-culturing increased the levels of non-extracellular virulence factors from both C. albicans and S. aureus. Taken together, the co-culturing of C. albicans and S. aureus reciprocally promotes their virulence potential, which may provide insights into the synergistic lethality during their co-infection in vivo. ### Competing Interest Statement The authors have declared no competing interest.

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.

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 (Delta pvl), or complemented mutant (Delta pvl+pvl). Three days post-surgery, Delta pvl-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, Delta pvl-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 Delta pvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

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.

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 mg/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.

Study ObjectiveThis study aimed to investigate the potential role of transvaginal mesh bacterial colonization in the development of mesh-related complications (MRCs).DesignAn observational and exploratory study.SettingTertiary referral center (Amsterdam UMC, location AMC, Amsterdam, The Netherlands).Patiënts49 patients indicated for mesh removal and 20 women of whom vaginal tissue was retrieved during prolapse surgery as a reference cohort.Interventionscollection of mesh-tissue complex (patient cohort) or vaginal tissue (reference cohort)Measurements and Main resultsHomogenized 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.ConclusionIn 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.