Biology subjects

Allen, R. C.

Publications and source records attributed to Allen, R. C..

5 recordsLinked to original sources

bioRxiv2022

Negative interactions and virulence differences drive the dynamics in multispecies bacterial infections

Bacterial infections are often polymicrobial, leading to intricate pathogen-pathogen and pathogen-host interactions. There is increasing interest in studying the molecular basis of pathogen interactions and how such mechanisms impact host morbidity. However, much less is known about the ecological dynamics between pathogens and how they affect virulence and host survival. Here we address these open issues by co-infecting larvae of the insect model host Galleria mellonella with one, two, three or four bacterial species, all of which are opportunistic human pathogens. We found that host mortality was always determined by the most virulent species regardless of the number of species and pathogen combinations injected. In certain combinations, the more virulent pathogen simply outgrew the less virulent pathogen. In other combinations, we found evidence for negative interactions between pathogens inside the host, whereby the more virulent pathogen typically won a competition. Taken together, our findings reveal positive associations between a pathogens growth inside the host, its competitiveness towards other pathogens, and its virulence. Beyond being generalizable across species combinations, our findings predict that treatments against polymicrobial infections should target the most virulent species to reduce host morbidity, a prediction we validated experimentally. ImportanceThere is increasing evidence that polymicrobial infections are common and that host morbidity is impacted by interactions between the co-infecting pathogens. In this study, we infected the larvae of the insect host Galleria mellonella with four opportunistic human pathogens as mono or multispecies bacterial infections in all possible combinations and followed host survival and pathogen interactions over time. We discovered that species followed the exact same rank order in terms of their virulence, their growth inside the host, and their competitive ability against co-infecting pathogens. As consequence of this consistent rank order, we found that the most virulent species determines host survival dynamics in mixed infections. Beyond revealing a generalizable predictive pattern for virulence outcomes, our findings also predict that treatments should target the most virulent species in polymicrobial infections, a prediction we validated experimentally.

microbiology↗

bioRxiv2020

Collateral sensitivity interactions between antibiotics depend on local abiotic conditions

Mutations conferring resistance to one antibiotic can increase (cross resistance) or decrease (collateral sensitivity) resistance to others. Drug combinations displaying collateral sensitivity could be used in treatments that slow resistance evolution. However, lab-to-clinic translation requires understanding whether collateral effects are robust across different environmental conditions. Here, we isolated and characterized resistant mutants of Escherichia coli using five antibiotics, before measuring collateral effects on resistance to other antibiotics. During both isolation and phenotyping, we varied conditions in ways relevant in nature (pH, temperature, bile). This revealed local abiotic conditions modified expression of resistance against both the antibiotic used during isolation and other antibiotics. Consequently, local conditions influenced collateral sensitivity in two ways: by favouring different sets of mutants (with different collateral sensitivities), and by modifying expression of collateral effects for individual mutants. These results place collateral sensitivity in the context of environmental variation, with important implications for translation to real-world applications.

microbiology↗

bioRxiv2018

Myeloperoxidase and Eosinophil Peroxidase Inhibit the in vitro Endotoxin Activities of Lipopolysaccharide (LPS) and Lipid A and Increase Survival in an in vivo Mouse LPS LD90 model

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic leukocyte haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds and kills specific Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. Endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic Lipid A component, is a characteristic of all GNB. The possibility that haloperoxidases bind to and inhibit the endotoxin of GBN was considered and tested by contacting MPO and EPO with LPS and Lipid A and measuring for inhibition of endotoxin activity using either the in vitro gel or chromogenic Limulus amebocyte lysate (LAL) assays. Contacting MPO and EPO with LPS purified from Escherichia coli O55:B5 and with diphosphoryl Lipid A purified from E. coli F583 inhibited their endotoxin activities in proportion to the natural log of the MPO or EPO concentration. Although MPO is less cationic than EPO, MPO consistently demonstrated inhibition of endotoxin activity that is about threefold superior to EPO. Haloperoxidase enzymatic activity was not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. MPO and EPO inhibition of LPS endotoxin activity was also measured using a 90% lethal dose (LDO_SCPCAP90C_SCPCAP) mouse model studied over a five-day period. Based on Kaplan Meier survival analysis, MPO significantly increased mouse survival in a dose-dependent manner. EPO was less effective. In conclusion, contacting MPO and EPO with LPS and Lipid A inhibits in vitro endotoxin activities, but inhibition is independent of haloperoxidase enzymatic function. MPO significantly increases mouse survival against LPS in an in vivo LDO_SCPCAP90C_SCPCAP endotoxin model.

pharmacology and toxicology↗

bioRxiv2018

Axenic Biofilm Formation and Aggregation by Synechocystis PCC 6803 is Induced by Changes in Nutrient Concentration, and Requires Cell Surface Structure

Phototrophic biofilms are key to nutrient cycling in natural environments and bioremediation technologies, but few studies describe biofilm formation by pure (axenic) cultures of a phototrophic microbe. The cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model micro-organism for the study of oxygenic photosynthesis and biofuel production. We report here that wild-type (WT) Synechocystis caused extensive biofilm formation in a 2000 liter outdoor non-axenic photobioreactor under conditions attributed to nutrient limitation. We developed a biofilm assay and found that axenic Synechocystis forms biofilms of cells and extracellular material, but only when induced by an environmental signal, such as by reducing the concentration of growth medium BG11. Mutants lacking cell surface structures, namely type IV pili and the S-layer, do not form biofilms.\n\nTo further characterize the molecular mechanisms of cell-cell binding by Synechocystis, we also developed a rapid (8 hour) axenic aggregation assay. Mutants lacking Type IV pili were unable to aggregate, but mutants lacking a homolog to Wza, a protein required for Type 1 exopolysaccharide export in Escherichia coli, had a super-binding phenotype. In WT cultures, 1.2x BG11 induced aggregation to the same degree as 0.8x BG11. Overall, our data support that Wza-dependant exopolysaccharide is essential to maintain stable, uniform suspensions of WT Synechocystis cells in unmodified growth medium, and this mechanism is counter-acted in a pili-dependent manner under altered BG11 concentrations.\n\nImportanceMicrobes can exist as suspensions of individual cells in liquids, and also commonly form multicellular communities attached to surfaces. Surface-attached communities, called biofilms, can confer antibiotic resistance to pathogenic bacteria during infections, and establish food webs for global nutrient cycling in the environment. Phototrophic biofilm formation is one of the earliest phenotypes visible in the fossil record, dating back over 3 billion years. Despite the importance and ubiquity of phototrophic biofilms, most of what we know about the molecular mechanisms, genetic regulation, and environmental signals of biofilm formation comes from studies of heterotrophic bacteria. We aim to help bridge this knowledge gap by developing new assays for Synechocystis, a phototrophic cyanobacterium used to study oxygenic phototsynthesis and biofuel production. With the aid of these new assays, we contribute to the development of Synechocystis as a model organism for the study of axenic phototrophic biofilm formation.

microbiology↗

bioRxiv2017

Modified antibiotic adjuvant ratios can slow and steer the evolution of resistance: co-amoxiclav as a case study

As the spread of antibiotic resistance outstrips the introduction of new antibiotics, reusing existing antibiotics is increasingly important. One promising method is to combine antibiotics with synergistically acting adjuvants that inhibit resistance mechanisms, allowing drug killing. Here we use co-amoxiclav (a commonly used and clinically important drug combination of the {beta}-lactam antibiotic amoxicillin and the {beta}-lactamase inhibitor clavulanate) to ask whether treatment efficacy and resistance evolution can be decoupled via component dosing modifications.\n\nA simple mathematical model predicts that different ratios of these two drug components can produce distinct evolutionary responses despite similar initial levels of control. We test this hypothesis by selecting Escherichia coli with a plasmid encoded {beta}-lactamase (ESBL CTX-M-14), against different proportions of amoxicillin and clavulanate. Consistent with our theory, we found that while resistance evolved under all conditions, the component ratio influenced both the rate and mechanism of resistance evolution. Specifically, we found that the current clinical practice of high amoxicillin to clavulanate ratios resulted in the most rapid failure due to the evolution of gene dosing responses. Increased plasmid copy number allowed E. coli to increase {beta}-lactamase dosing and effectively titrate out the low quantities of clavulanate, restoring amoxicillin resistance. In contrast, we found high clavulanate ratios were more robust - plasmid copy number did not increase, although porin or efflux resistance mechanisms were found, as in all drug ratios. Our results indicate that by changing the ratio of adjuvant to antibiotic we can slow and steer the path of resistance evolution. We therefore suggest the use of increased clavulanate dosing regimens to slow the rate of resistance evolution.

microbiology↗