In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models.

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host-pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air-liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models.

Expanding the squaramide library as mycobacterial ATP synthase inhibitors: Innovative synthetic pathway and biological evaluation.

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.

In-depth biological characterization of two black soldier fly anti-Pseudomonas peptides reveals LPS-binding and immunomodulating effects.

As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.

In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens.

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

Isolation and Identification of Dominant Bacteria From Black Soldier Fly Larvae (Hermetia illucens) Envisaging Practical Applications.

This study aimed to establish a representative strain collection of dominant aerobic bacteria from black soldier fly larvae (Hermetia illucens, BSFL). The larvae were fed either chicken feed or fiber-rich substrates to obtain a collection of BSFL-associated microorganisms. Via an approach based on only considering the highest serial dilutions of BSFL extract (to select for the most abundant strains), a total of 172 bacteria were isolated. Identification of these isolates revealed that all bacteria belonged to either the Proteobacteria (66.3%), the Firmicutes (30.2%), the Bacteroidetes (2.9%) or the Actinobacteria (0.6%). Twelve genera were collected, with the most abundantly present ones (i.e., minimally present in at least three rearing cycles) being Enterococcus (29.1%), Escherichia (22.1%), Klebsiella (19.8%), Providencia (11.6%), Enterobacter (7.6%), and Morganella (4.1%). Our collection of dominant bacteria reflects largely the bacterial profiles of BSFL already described in literature with respect to the most important phyla and genera in the gut, but some differences can be noticed depending on substrate, biotic and abiotic factors. Furthermore, this bacterial collection will be the starting point to improve in vitro digestion models for BSFL, to develop mock communities and to find symbionts that can be added during rearing cycles to enhance the larval performances, after functional characterization of the isolates, for instance with respect to enzymatic potential.

Microbial symbionts of insects as a source of new antimicrobials: a review.

To stop the antimicrobial resistance crisis, there is an urgent need for increased investment in antimicrobial research and development. Currently, many researchers are focussing on insects and their microbiota in the search for new antimicrobials. This review summarizes recent literature dedicated to the antimicrobial screening of insect symbionts and/or their metabolites to uncover their value in early drug discovery. We summarize the main steps in the methodology used to isolate and identify active insect symbionts and have noted substantial variation among these studies. There is a clear trend in isolating insect Streptomyces bacteria, but a broad range of other symbionts has been found to be active as well. The microbiota of many insect genera and orders remains untargeted so far, which leaves much room for future research. The antimicrobial screening of insect symbionts has led to the discovery of a diverse array of new active biomolecules, mainly peptides, and polyketides. Here, we discuss 15 of these symbiont-produced compounds and their antimicrobial profile. Cyphomycin, isolated from a Streptomyces symbiont of a Cyphomyrmex fungus-growing ant, seems to be the most promising insect symbiont-derived antimicrobial so far. Overall, insect microbiota appears to be a promising search area to discover new antimicrobial drug candidates.