development-polymicrobial-colony-biofilm-model-to-test-antimicrobials

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

University of Nottingham

Current understanding of the immune system comes primarily from laboratory-based studies. There has been substantial interest in examining how it functions in the wild, but studies have been limited by a lack of appropriate assays and study species. The three-spined stickleback (Gasterosteus aculeatus L.) provides an ideal system in which to advance the study of wild immunology, but requires the development of suitable immune assays. We demonstrate that meaningful variation in the immune response of stickleback can be measured using real-time PCR to quantify the expression of eight genes, representing the innate response and Th1-, Th2- and Treg-type adaptive responses. Assays are validated by comparing the immune expression profiles of wild and laboratory-raised stickleback, and by examining variation across populations on North Uist, Scotland. We also compare the immune response potential of laboratory-raised individuals from two Icelandic populations by stimulating cells in culture. Immune profiles of wild fish differed from laboratory-raised fish from the same parental population, with immune expression patterns in the wild converging relative to those in the laboratory. Innate measures differed between wild populations, whilst the adaptive response was associated with variation in age, relative size of fish, reproductive status and S. solidus infection levels. Laboratory-raised individuals from different populations showed markedly different innate immune response potential. The ability to combine studies in the laboratory and in the wild underlines the potential of this toolkit to advance our understanding of the ecological and evolutionary relevance of immune system variation in a natural setting.

Measuring the immune system of the three-spined stickleback - investigating natural variation by quantifying immune expression in the laboratory and the wild.

Parasitism represents one of the most widespread lifestyles in the animal kingdom, with the potential to drive coevolutionary dynamics with their host population. Where hosts and parasites evolve together, we may find local adaptation. As one of the main host defences against infection, there is the potential for the immune response to be adapted to local parasites. In this study, we used the three-spined stickleback and its Gyrodactylus parasites to examine the extent of local adaptation of parasite infection dynamics and the immune response to infection. We took two geographically isolated host populations infected with two distinct Gyrodactylus species and performed a reciprocal cross-infection experiment in controlled laboratory conditions. Parasite burdens were monitored over the course of the infection, and individuals were sampled at multiple time points for immune gene expression analysis. We found large differences in virulence between parasite species, irrespective of host, and maladaptation of parasites to their sympatric host. The immune system responded to infection, with a decrease in expression of innate and Th1-type adaptive response genes in fish infected with the less virulent parasite, representing a marker of a possible resistance mechanism. There was no evidence of local adaptation in immune gene expression levels. Our results add to the growing understanding of the extent of host-parasite local adaptation, and demonstrate a systemic immune response during infection with a common ectoparasite. Further immunological studies using the stickleback-Gyrodactylus system can continue to contribute to our understanding of the function of the immune response in natural populations.

No evidence of local adaptation of immune responses to Gyrodactylus in three-spined stickleback (Gasterosteus aculeatus).

Haplotypes underlying local adaptation and speciation are predicted to have numerous phenotypic effects, but few genes involved have been identified, with much work to date concentrating on visible, morphological, phenotypes. The link between genes controlling these adaptive morphological phenotypes and the immune system has seldom been investigated, even though changes in the immune system could have profound adaptive consequences. The Eda gene in three-spined stickleback is one of the best studied major adaptation genes; it directly controls bony plate architecture and has been associated with additional aspects of adaptation to freshwater. Here, we exposed F2 hybrids, used to separate Eda genotype from genetic background, to contrasting conditions in semi-natural enclosures. We demonstrate an association between the Eda haplotype block and the expression pattern of key immune system genes. Furthermore, low plated fish grew less and experienced higher burdens of a common ectoparasite with fitness consequences. Little is currently known about the role of the immune system in facilitating adaptation to novel environments, but this study provides an indication of its potential importance.

Eda haplotypes in three-spined stickleback are associated with variation in immune gene expression.

Understanding how wild immune variation covaries with other traits can reveal how costs and trade-offs shape immune evolution in the wild. Divergent life history strategies may increase or alleviate immune costs, helping shape immune variation in a consistent, testable way. Contrasting hypotheses suggest that shorter life histories may alleviate costs by offsetting them against increased mortality, or increase the effect of costs if immune responses are traded off against development or reproduction. We investigated the evolutionary relationship between life history and immune responses within an island radiation of three-spined stickleback, with discrete populations of varying life histories and parasitism. We sampled two short-lived, two long-lived and an anadromous population using qPCR to quantify current immune profile and RAD-seq data to study the distribution of immune variants within our assay genes and across the genome. Short-lived populations exhibited significantly increased expression of all assay genes, which was accompanied by a strong association with population-level variation in local alleles and divergence in a gene that may be involved in complement pathways. In addition, divergence around the eda gene in anadromous fish is likely associated with increased inflammation. A wider analysis of 15 populations across the island revealed that immune genes across the genome show evidence of having diverged alongside life history strategies. Parasitism and reproductive investment were also important sources of variation for expression, highlighting the caution required when assaying immune responses in the wild. These results provide strong, gene-based support for current hypotheses linking life history and immune variation across multiple populations of a vertebrate model.

A genetics-based approach confirms immune associations with life history across multiple populations of an aquatic vertebrate (Gasterosteus aculeatus).

The switch from egg-laying to retaining and giving birth to live young is a major transition in the history of life. Despite its repeated evolution across the fishes, records of intermediate phenotypes are vanishingly rare, with only two known cases in existence of normally egg-laying fish species retaining embryos within the ovaries. We report the discovery of a third occurrence, in which well-developed embryos were found in the ovaries of a three-spined stickleback (Gasterosteus aculeatus), a non-copulatory, normally oviparous species. Extracted from the parent fish, these embryos hatched and grew to adulthood. Genetic and physiological examination of the parent fish and offspring ruled out development by parthenogenesis and hermaphroditism, therefore implicating internal fertilisation by a male stickleback. This extremely rare phenomenon may have been facilitated in this population by an unusual tendency for females to become egg-bound, and suggests that some major transitions may arise almost spontaneously.

Internal embryonic development in a non-copulatory, egg-laying teleost, the three-spined stickleback, Gasterosteus aculeatus.

Carbohydrate-based materials are increasingly investigated for a range of applications spanning from healthcare to advanced functional materials. Synthetic glycopolymers are particularly attractive as they possess low toxicity and immunogenicity and can be used as multivalent ligands to target sugar-binding proteins (lectins). Here, we utilised RAFT polymerisation to synthesize two families of novel diblock copolymers consisting of a glycopolymers block containing either mannopyranose or galactopyranose pendant units, which was elongated with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS) to generate a polyanionic block. The latter enabled complexation of cationic aminoglycoside antibiotic tobramycin through electrostatic interactions (loading efficiency in the 0.5-6.3 wt% range, depending on the copolymer). The resulting drug vectors were characterized by dynamic light scattering, zeta-potential, and transmission electron microscopy. Tobramycin-loaded complexes were tested for their ability to prevent clustering or disrupt biofilm of the Pseudomonas aeruginosa Gram-negative bacterium responsible for a large proportion of nosocomial infection, especially in immunocompromised patients. P. aeruginosa possesses two specific tetrameric carbohydrate-binding adhesins, LecA (PA-IL, galactose/N-acetyl-D-galactosamine-binding) and LecB (PA-IIL, fucose/mannose-binding), and the cell-associated and extracellular adhesin CdrA (Psl/mannose-binding) thus ideally suited for targeted drug delivery using sugar-decorated tobramycin-loaded complexes here developed. Both aliphatic and aromatic linkers were utilised to link the sugar pendant units to the polyacrylamide polymer backbone to assess the effect of the nature of such linkers on bactericidal/bacteriostatic properties of the complexes. Results showed that tobramycin-loaded complexes efficiently suppressed (40 to 60% of inhibition) in vitro biofilm formation in PAO1-L P. aeruginosa and that preferential targeting of PAO1-L biofilm can be achieved using mannosylated glycopolymer-b-AMPS.

Tobramycin-loaded complexes to prevent and disrupt Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa.

Previously, metabolites diffused or secreted from microbial samples have been analyzed via liquid chromatography-mass spectrometry (LC-MS) approaches following lengthy extraction protocols. Here, we present a model system for growing biofilms on discs before utilizing rapid and direct surface sampling MS, namely, liquid extraction surface analysis, to study the microbial exometabolome. One of the benefits of this approach is its surface-specific nature, enabling mimicking biofilm formation in a way that the study of planktonic liquid cultures cannot imitate. Even though  (),  and  () have been studied previously in isolation, very few studies consider the complexity of the interplay between these pathogens, which are commonly combined causative agents of infection. Our model system provides a route to investigate changes in the exometabolome, such as metabolites that become circulatory in the presence of multiple pathogens. Our results agree with previous reports showing that 2-alkyl-4(1)-quinolone signal molecules produced by  are important markers of infection and suggest that methods for monitoring levels of 2-heptyl-4-hydroxyquinoline and 2,4-dihydroxyquinoline, as well as pyocyanin, could be beneficial in the determination of causative agents in interkingdom infection including . Furthermore, studying changes in exometabolome metabolites between  quorum sensing antagonists in treated and nontreated samples suggests suppression of phenazine production by . Hence, our model provides a rapid analytical approach to gaining a mechanistic understanding of bacterial signaling.

Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis-Mass Spectrometry.

 is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the  system, one of three QS systems in , results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor  (IC 3.2 μM in  PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1-benzo[]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile () as a potent PqsR antagonist. Compound  inhibited the PqsR-controlled P- transcriptional reporter fusion in  at low submicromolar concentrations. Moreover,  showed improved efficacy against  CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1)-quinolones production.

Design, Synthesis, and Evaluation of New 1-Benzo[]imidazole Based PqsR Inhibitors as Adjuvant Therapy for  Infections.

To tackle the emerging antibiotic resistance crisis, novel antimicrobial approaches are urgently needed. Bacterial biofilms are a particular concern in this context as they are responsible for over 80% of bacterial infections and are inherently more recalcitrant toward antimicrobial treatments. The high tolerance of biofilms to conventional antibiotics has been attributed to several factors, including reduced drug diffusion through the dense exopolymeric matrix and the upregulation of antimicrobial resistance machinery with successful biofilm eradication requiring prolonged high doses of multidrug treatments. A promising approach to tackle bacterial infections involves the use of polymer drug conjugates, shown to improve upon free drug toxicity and bioavailability, enhance drug penetration through the thick biofilm matrix, and evade common resistance mechanisms. In the following study, we conjugated the antibiotic ciprofloxacin (CIP) to a small library of biodegradable and biocompatible poly(β-amino ester) (PBAE) polymers with varying central amine functionality. The suitability of the polymers as antibiotic conjugates was then verified in a series of assays including testing of efficacy and resistance response in planktonic Gram-positive and Gram-negative bacteria and the reduction of viability in mono- and multispecies biofilm models. The most active polymer within the prepared PBAE-CIP library was shown to achieve an over 2-fold increase in the reduction of biofilm viability in a  monospecies biofilm and superior elimination of all the species present within the multispecies biofilm model. Hence, we demonstrate that CIP conjugation to PBAEs can be employed to achieve improved antibiotic efficacy against clinically relevant biofilm models.

Ciprofloxacin Poly(β-amino ester) Conjugates Enhance Antibiofilm Activity and Slow the Development of Resistance.

Chronic wound infections are generally of polymicrobial nature with aerobic and anaerobic bacteria, as well as fungi frequently observed in them. Wound treatment involves a series of steps, including debridement of the wound, flushing, and often the use of multiple wound dressings many of which are antimicrobial. Yet, many wound dressings are tested versus single species of planktonic microbes, which fails to mirror the real-life presence of biofilms.

Article	Year
Measuring the immune system of the three-spined stickleback - investigating natural variation by quantifying immune expression in the laboratory and the wild. Molecular ecology resources\| PubMed ID: 26646722	2016
No evidence of local adaptation of immune responses to Gyrodactylus in three-spined stickleback (Gasterosteus aculeatus). Fish & shellfish immunology\| PubMed ID: 27913248	2017
Eda haplotypes in three-spined stickleback are associated with variation in immune gene expression. Scientific reports\| PubMed ID: 28195171	2017
A genetics-based approach confirms immune associations with life history across multiple populations of an aquatic vertebrate (Gasterosteus aculeatus). Molecular ecology\| PubMed ID: 29924437	2018
Internal embryonic development in a non-copulatory, egg-laying teleost, the three-spined stickleback, Gasterosteus aculeatus. Scientific reports\| PubMed ID: 30787313	2019
Tobramycin-loaded complexes to prevent and disrupt Pseudomonas aeruginosa biofilms. Drug delivery and translational research\| PubMed ID: 34841492	2022
ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa. NPJ biofilms and microbiomes\| PubMed ID: 35982053	2022
Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis-Mass Spectrometry. Analytical chemistry\| PubMed ID: 36881460	2023
Design, Synthesis, and Evaluation of New 1-Benzo[]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Infections. Journal of medicinal chemistry\| PubMed ID: 38170170	2024
Ciprofloxacin Poly(β-amino ester) Conjugates Enhance Antibiofilm Activity and Slow the Development of Resistance. ACS applied materials & interfaces\| PubMed ID: 38289032	2024
Development, characterization, and evaluation of a simple polymicrobial colony biofilm model for testing of antimicrobial wound dressings. Journal of applied microbiology\| PubMed ID: 38366933	2024

Shaun Robertson

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