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Prosthetic joint infections (PJIs) can have disastrous consequences for patient health, including removal of the device, and placement of cemented implants is often required during surgery to eradicate PJIs. In translational research, in vivo models are widely used to assess the biocompatibility and antimicrobial efficacy of antimicrobial coatings and compounds. Here, we aim to utilize Galleria mellonella implant infection models to assess the antimicrobial activity of antibiotic-loaded bone cement (ALBC) implants. Therefore, we used commercially available bone cement loaded with either gentamicin alone (PALACOS R+G) or with a combination of gentamicin and vancomycin (COPAL G+V), compared to bone cement without antibiotics (PALACOS R). Firstly, the in vitro antimicrobial activity of ALBC was determined against Staphylococcus aureus. Next, the efficacy of ALBC implants was analyzed in both the G. mellonella hematogenous and early-stage biofilm implant infection model, by monitoring the survival of larvae over time. After 24 h, the number of bacteria on the implant surface and in the tissue was determined. Larvae receiving dual-loaded COPAL G+V implants showed higher survival rates compared to implants loaded with only gentamicin (PALACOS R+G) and the control implants without antibiotics (PALACOS R). In conclusion, G. mellonella larvae infection models with antibiotic-loaded bone cements are an excellent option to study (novel) antimicrobial approaches.
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Aims: Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods: For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results: In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion: Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required.
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The biofilm lifestyle of bacterial pathogens is a hallmark of chronic lung infections such as in cystic fibrosis (CF) patients. Bacterial adaptation to the complex conditions in CF-affected lungs and repeated antibiotherapies lead to increasingly tolerant and hard-to-treat biofilms. In the context of growing antimicrobial resistance and restricted therapeutic options, antimicrobial photodynamic therapy (aPDT) shows great promise as an alternative to conventional antimicrobial modalities. Typically, aPDT consists in irradiating a non-toxic photosensitizer (PS) to generate reactive oxygen species (ROS), which kill pathogens in the surrounding environment. In a previous study, we reported that some ruthenium (II) complexes ([Ru(II)]) can mediate potent photodynamic inactivation (PDI) against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates. In the present work, [Ru(II)] were further assayed to evaluate their ability to photo-inactivate such bacteria under more complex experimental conditions better recapitulating the microenvironment in lung infected airways. Bacterial PDI was tentatively correlated with the properties of [Ru(II)] in biofilms, in mucus, and following diffusion across the latter. Altogether, the results obtained demonstrate the negative impacting role of mucus and biofilm components on [Ru(II)]-mediated PDT, following different possible mechanisms of action. Technical limitations were also identified that may be overcome, making this report a pilot for other similar studies. In conclusion, [Ru(II)] may be subjected to specific chemical engineering and/or drug formulation to adapt their properties to the harsh micro-environmental conditions of the infected respiratory tract.
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For cystic fibrosis gene therapy, the aerosolization of genetic materials is the most relevant delivery strategy to reach the airway epithelium. However, aerosolized formulations have to resist shear forces while maintaining the integrity of plasmid DNA (pDNA) during its journey from the nebulization to the epithelial cells. Herein, we compared the efficiency of gene delivery by aerosolization of two types of formulations: (i) BSV163, a branched cationic amphiphilic compound, co-formulated with different DOPE ratios (mol/mol) and DMPE-PEG5000 and (ii) 25 KDa branched polyethylenimine (b-PEI)-based formulation used as control. This study also aims to determine whether BSV163-based formulations possess the ability to resist the nebulization mechanisms and protect the nucleic acids (pDNA) cargo. Therefore, two CpG free plasmids (pGM144 or pGM169) encoding either the luciferase reporter gene or hCFTR respectively were used. Air-Liquid Interface (ALI) cell-culture was selected as an in-vitro model for aerosol experiments due to its closer analogy with in vivo morphology. Results highlighted that DOPE ratio influences the capacity of the BSV163 based-formulations to mediate high transfection efficacies. Furthermore, we proved that addition of DMPE-PEG5000 upon the formation of the BSV163/DOPE (1/1) lipid film instead of post-insertion led to a higher transgene expression. The aerosolization of this formulation on ALI cell-culture was more efficient than the use of b-PEI-based formulation.
Assuntos
Fibrose Cística , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Aerossóis e Gotículas Respiratórios , Plasmídeos , Transfecção , DNA , Técnicas de Transferência de Genes , PolietilenoiminaRESUMO
Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.
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From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3â³)-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9 genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.
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Antimicrobial photodynamic therapy (aPDT) has become a fundamental tool in modern therapeutics, notably due to the expanding versatility of photosensitizers (PSs) and the numerous possibilities to combine aPDT with other antimicrobial treatments to combat localized infections. After revisiting the basic principles of aPDT, this review first highlights the current state of the art of curative or preventive aPDT applications with relevant clinical trials. In addition, the most recent developments in photochemistry and photophysics as well as advanced carrier systems in the context of aPDT are provided, with a focus on the latest generations of efficient and versatile PSs and the progress towards hybrid-multicomponent systems. In particular, deeper insight into combinatory aPDT approaches is afforded, involving non-radiative or other light-based modalities. Selected aPDT perspectives are outlined, pointing out new strategies to target and treat microorganisms. Finally, the review works out the evolution of the conceptually simple PDT methodology towards a much more sophisticated, integrated, and innovative technology as an important element of potent antimicrobial strategies.