Abstract Antibiotics in development are usually tested on rapidly dividing cells in a culture medium and do not reflect the complexity of infections in vivo , while testing in vivo is limited, expensive and ethically concerning. This often results in the development and subsequent prescription of antibiotics only targeting infections in which pathogens are undergoing rapid cell division and in case of persistent infections like keratitis leads to poor clinical outcomes such as impaired vision or loss of an eye. In this study, we demonstrate antibiotic tolerance of Pseudomonas aeruginosa strains PA01 and PA14 using the ex vivo porcine keratitis model in which bacterial physiology more closely mimics infections in vivo than in a culture medium. MBEC and MIC were used as a guideline to establish the concentration of applied antibiotics on tissue. Infected ex vivo porcine corneas were treated with therapeutically relevant concentrations of gentamicin, ciprofloxacin and chloramphenicol. Ciprofloxacin was the most potent across all tests demonstrating a positive correlation with MIC but not MBEC. Nonetheless, the results demonstrated that MIC and MBEC concentrations were not sufficient to clear infection even after 18 hours of continuous exposure to the tested antibiotics reflecting the need for novel antibiotics that can target the persistent subpopulation of these pathogens and the ability of the ex vivo keratitis model to be a relevant platform to identify novel antibiotics with suitable activities. There was a clear visual distinction between corneas infected with cytotoxic strain PA14 and invasive strain PA01. In this study, both strains PA14 and PA01 showed a high level of antibiotic tolerance, which suggests that in clinical settings the treatment approach could be similar regardless of the causative strain. Data summary The authors confirm all supporting data and protocols have been provided within the article or through supplementary data files.
Introduction . Bacterial keratitis, particularly caused by Pseudomonas aeruginosa , is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo . Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis. Methodology . Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model. Results . Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l −1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l −1 ) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure. Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.
A microalgal–bacterial consortium was used for pilot scale bioremediation of landfill leachate. A techno-economic analysis was conducted using experimental results to provide a pathway for economic viability.
Abstract Purpose Our objective was to assess the efficacy of an ex vivo sheep corneal model as an alternative for live animal testing in screening drug cytotoxicity. In pursuit of this goal, we investigated the impact of two commonly used topical antibiotics, ciprofloxacin and gentamicin, on wound healing. Furthermore, we examined different antibiotic dosages and dosing regimens to understand their effects comprehensively. Methods The epithelium on ex viv o sheep corneas was removed with a scalpel, and the area was treated with ciprofloxacin (0.1, 0.3, and 1 mg mL -1 ), gentamicin (0.25, 1, and 3 mg mL -1 ), or phosphate-buffered saline (control). The corneas were exposed to treatments continuously or twice daily for ten minutes. Wound closure was observed by fluorescein retention and histological staining. Results Untreated corneas healed within 41 hours. Continuous exposure to both ciprofloxacin and gentamicin significantly reduced the corneal healing ability in a time- and concentration-dependent manner. Overall, ciprofloxacin was found to be more toxic than gentamycin. However, this model showed that the corneal epithelium could heal effectively when both antibiotics were administered intermittently. Conclusion Ciprofloxacin demonstrated greater inhibition of wound healing compared to gentamicin, aligning with in vivo studies. The administration of drops several times daily mitigated the toxic effects of antibiotics. The ex vivo sheep wound healing model holds promise as an alternative approach to in vivo toxicity testing, enabling the swift evaluation of novel antimicrobial treatments and eye drop additives.
Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m-2 s-1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species.At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present.Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.
We aimed to improve algal growth rate on leachate by optimising the algal microbiome. An algal-bacterial consortium was enriched from landfill leachate and subjected to 24 months of adaptive laboratory evolution, increasing the growth rate of the dominant algal strain, Chlorella vulgaris, almost three-fold to 0.2 d−1. A dramatic reduction in nitrate production suggested a shift in biological utilisation of ammoniacal-N, supported by molecular 16S rRNA taxonomic analyses, where Nitrosomonas numbers were not detected in the adapted consortium. A PICRUSt approach predicted metagenomic functional content and revealed a high number of sequences belonging to bioremediation pathways, including degradation of aromatic compounds, benzoate and naphthalene, as well as pathways known to be involved in algal-bacterial symbiosis. This study enhances our understanding of beneficial mechanisms in algal-bacterial associations in complex effluents, and ultimately enables the bottom-up design of optimised algal microbiomes for exploitation within industry.
Abstract As algal biotechnology develops, there is an increasing requirement to conserve cultures without the cost, time and genetic stability implications of conventional serial transfers, including issues regarding potential loss by failure to regrow, contamination on transfer, mix up and/or errors in the documentation on transfer. Furthermore, it is crucial to ensure both viability and functionality are retained by stored stock-cultures. Low temperature storage, ranging from the use of domestic freezers to storage under liquid nitrogen, is widely being used, but the implication to stability and function rarely investigated. We report for the first time, retention of functionality in the maintenance of master stock-cultures of an industrially relevant, lipid-producing alga, under a variety of cryopreservation regimes. Storage in domestic (−15 °C), or conventional −80 °C freezers was suboptimal, with a rapid reduction in viability observed for samples at −15 °C and a >50% loss of viability, within one month, for samples stored at −80 °C. No reduction in viability occurred at −196 °C. Post-thaw culture functional performance was also influenced by the cryopreservation approach employed. Only samples held at −196 °C responded to nitrogen limitation in terms of growth characteristics and biochemical profiles (lipid production and chlorophyll a ) comparable to the untreated control, cultured prior to cryopreservation. These results have important implications in microbial biotechnology, especially for those responsible for the conservation of genetic resources.
When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.
Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.
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