Biology subjects

Edwards, A. M.

Publications and source records attributed to Edwards, A. M..

34 records · Page 2Linked to original sources

bioRxiv2022

E. faecalis acquires resistance to antimicrobials and insect immunity via common mechanisms

Enterococcus faecalis is a normal member of the gut microbiota and an opportunistic pathogen of many animals, including mammals, birds, and insects. It is a common cause of nosocomial infections, and is particularly troublesome due to extensive intrinsic and acquired antimicrobial resistance. Using experimental evolution, we generated Drosophila-adapted E. faecalis strains, which exhibited immune resistance, resulting in increased in vivo growth and virulence. Resistance was characterised by mutations in bacterial pathways responsive to cell envelope stress. Drosophila-adapted strains exhibited changes in sensitivity to relevant antimicrobials, including daptomycin and vancomycin. Evolved daptomycin-resistant strains harboured mutations in the same signalling systems, with some strains showing increased virulence similar to Drosophila-adapted strains. Our results show that common mechanisms provide a route to resistance to both antimicrobials and host immunity in E. faecalis and demonstrate that the selection and emergence of antibiotic resistance in vivo does not require antibiotic exposure. One sentence summaryHost interaction can promote antimicrobial resistance and antimicrobial treatment can promote virulence in E. faecalis.

microbiology↗

bioRxiv2022

Growth arrest of Staphylococcus aureus induces daptomycin tolerance via cell wall remodelling

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill non-dividing bacteria. However, these studies employed only brief periods of growth arrest. We found that a bacteriostatic concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with ~100,000-fold increase in survival after 16 h growth arrest relative to exponential phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, whilst accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whilst tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling.

microbiology↗

bioRxiv2022

A statistical censoring approach accounts for hook competition in abundance indices from longline surveys

Fishery-independent longline surveys provide valuable data to monitor fish populations. However, competition for bait on the finite number of hooks leads to biased estimates of relative abundance when using simple catch-per-unit-effort methods. Numerous bias-correcting instantaneous-catch-rate methods have been proposed, modelling the bait removal times as independent random variables. However, experiments have cast doubts on the many assumptions required for these to accurately infer relative abundance. We develop a new approach by treating some observations as right-censored, acknowledging that observed catch counts are lower bounds of what they would have been in the absence of hook competition. Through simulation experiments we confirm that our approach consistently outperforms previous methods. We demonstrate performance of all methods on longline survey data of eleven species. Accounting for hook competition leads to large differences in relative indices (often -50% to +100%), with effects of hook competition varying between species (unlike other methods). Our method can be applied using existing statistical packages and can include environmental influences, making it a general and reliable method for analyzing longline survey data.

ecology↗

bioRxiv2022

An open source plant kinase chemogenomics set

129 protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in E. coli. 40 of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. 37 active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length and two slightly increased root elongation compared with control plants. Two inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. Inhibition could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Of the compounds acting as root growth inhibitors in Arabidopsis, six conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases.

plant biology↗

bioRxiv2022

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin Against Cystic Fibrosis Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate anti-pseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers; potentiating the activities of antibiotics when given in combination restoring and/or enhancing antibiotic efficacy. Growth, viability, minimum inhibitory concentration (MIC)-determination and synergy analysis showed that GA improved the efficacy of TOB against reference strains of P. aeruginosa, reducing TOB MICs and synergising with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the concentration of TOB required to inhibit 50% (MIC50) of viable cells (from 1.69 [95%CI 0.26-8.97] to 0.62 [95%CI 0.15-3.94] mg/L, p=0.002) and inhibit 90% (MIC90) (from 7.00 [95%CI 1.18-26.50] to 2.20 [95%CI 0.99-15.03] mg/L, p=0.001) compared with TOB-only. Investigating mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarisation of cytoplasmic membranes and permeabilisation of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three.

microbiology↗

bioRxiv2021

Human serum triggers antibiotic tolerance in Staphylococcus aureus

Staphylococcus aureus is a frequent cause of bloodstream infections. Treatment can be challenging, even when isolates appear to be drug susceptible, with high rates of persistent and relapsing infection. This is particularly the case with infections caused by methicillin resistant S. aureus (MRSA) strains, which are resistant to frontline antibiotics. To understand how the host environment influences treatment outcomes in MRSA infections, we studied the impact of human serum on staphylococcal susceptibility to daptomycin, an antibiotic of last resort. This revealed that serum triggered a very high degree of tolerance to daptomycin, as well as several other classes of antibiotics and antimicrobial peptides, including gentamicin, nitrofurantoin, vancomycin, nisin and gramicidin. Serum-induced daptomycin tolerance was due to two independent mechanisms. Firstly, the host defence peptide LL-37 present in serum induced tolerance by triggering the staphylococcal GraRS two component system. This led to increased cell wall accumulation that reduced access of daptomycin to its membrane target. Secondly, GraRS-independent changes to the membrane resulted in increased cardiolipin abundance that also contributed to daptomycin tolerance. When both mechanisms were blocked, serum exposed S. aureus cells were as susceptible to daptomycin as bacteria growing in laboratory media. These data demonstrate that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, which may in turn contribute to treatment failure. The inhibition of serum-induced cell wall accumulation by fosfomycin reduced tolerance, suggesting that this antibiotic may form a useful combination therapy with daptomycin.

microbiology↗

bioRxiv2021

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (mcr) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in E. coli is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

microbiology↗

bioRxiv2021

A Functional Menadione Biosynthesis Pathway is Required for Capsule Production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione plays a critical role in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

microbiology↗

bioRxiv2021

In pursuit of a better broiler: A comparison of the inactivity, behavior, and enrichment use of fast- and slower-growing broiler chickens

Selection for rapid growth has produced heavier, more efficient broiler chickens, but has also introduced health and welfare issues, which may cause or be caused by inactivity. Rapid growth may also limit the performance of motivated behaviors, whereas the provision of enrichment may increase these behaviors and general activity. This study aimed to evaluate the inactivity, behavior, and enrichment use of 2 fast- (CONV) and 12 slower-growing broiler strains [categorized as fastest (FAST), moderate (MOD) and slowest slow (SLOW), based on their growth rates]. To evaluate inactivity, one male and one female from 153 pens were outfitted with omni-directional accelerometers from day 21 until processing (14-24 birds/strain from 8-12 pens/strain). Additionally, to supplement inactivity data, five-minute continuous behavioral observations of four focal birds per pen (2 males, 2 females) were conducted on days 26, 42, and 56 (72-148 observations of 8-12 pens/strain) to quantify the duration and frequency of various behaviors; at the same time, 5-11 instantaneous scan samples were also performed for pen-based enrichment use. Inactivity peaked at 78-80% of the day for all strains; however, those with slower growth rates reached these levels at older ages. Compared to slower-growing strains at the same age, faster-growing strains were more inactive, spent more time sitting and feeding, spent less time standing and walking, and used enrichments less; these differences mostly occurred at younger ages. Generally, at the same age, strains with similar growth rates (within the same category) behaved similarly, with only a few exceptions. Results suggest that not all strains identified as slow-growing broilers behave differently from fast-growing broilers, nor do they all behave similarly to each other. As such, results suggest that improved broiler welfare, particularly with respect to reduced inactivity, the performance of a wider range of normal, motivated behaviors, and/or increased enrichment use, is related to the broiler strains specific growth rate.

animal behavior and cognition↗

bioRxiv2021

Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.

microbiology↗

bioRxiv2020

In Pursuit of a Better Broiler: Growth, Efficiency and Mortality of 16 Strains of Broiler Chickens

To meet the growing consumer demand for chicken meat, the poultry industry has selected broiler chickens for increasing efficiency and breast yield. While this high productivity means affordable and consistent product, it has come at a cost to broiler welfare. There has been increasing advocacy and consumer pressure on primary breeders, producers, processors and retailers to improve the welfare of the billions of chickens processed annually. Several small-scale studies have reported better welfare outcomes for slower growing strains compared to fast growing, conventional strains. However, these studies often housed birds with range access or used strains with vastly different growth rates. Additionally, there may be traits other than growth, such as body conformation, that influence welfare. As the global poultry industries consider the implications of using slower growing strains, there was a need for a comprehensive, multidisciplinary examination of broiler chickens with a wide range of genotypes differing in growth rate and other phenotypic traits. To meet this need, our team designed a study to benchmark data on conventional and slower growing strains of broiler chickens reared in standardized laboratory conditions. Over a two-year period, we studied 7,528 broilers from 16 different genetic strains. In this paper, we compare the growth, efficiency and mortality of broilers to one of two target weights (TW): 2.1 kg (TW1) and 3.2 kg (TW2). We categorized strains by their growth rate to TW2 as conventional (CONV), fastest slow strains (FAST), moderate slow strains (MOD) and slowest slow strains (SLOW). When incubated, hatched, housed, managed and fed the same, the categories of strains differed in body weights, growth rates, feed intake and feed efficiency. At 48 days of age, strains in the CONV category were 835-1264 g heavier than strains in the other categories. By TW2, differences in body weights and feed intake resulted in a 22 to 43-point difference in feed conversion ratios. Categories of strains did not differ in their overall mortality rates.

physiology↗

bioRxiv2020

Staphylococcal DNA repair confers tolerance of the bactericidal activity of both neutrophils and antibiotics

The repair of DNA damage is essential for bacterial viability and contributes to adaptation via increased rates of mutation and recombination. However, the mechanisms by which DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks via the oxidative burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

microbiology↗

bioRxiv2019

RexAB is essential for the mutagenic repair of Staphylococcus aureus DNA damage caused by co-trimoxazole

Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft-tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step towards the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. This revealed that SXT caused DNA damage in S. aureus via both thymidine limitation and the generation of reactive oxygen species. Then, using mutants defective for DNA repair, it was found that repair of this damage required the RexAB nuclease/helicase complex, indicating that SXT causes DNA double-strand breaks. Furthermore, RexAB-mediated DNA repair led to induction of the SOS response, which resulted in an increased mutation rate and may explain the frequent emergence of resistant strains during SXT therapy. In summary, this work determined that SXT causes DNA damage in S. aureus via both thymidine limitation and oxidative stress, which is repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug-resistance during treatment of infections caused by S. aureus.

microbiology↗

bioRxiv2018

Colistin requires de novo lipopolysaccharide biosynthesis for activity

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

microbiology↗

bioRxiv2017

The electron transport chain sensitises Staphylococcus aureus and Enterococcus faecalis to the oxidative burst

Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in a whole human blood model. Supplementation of the culture medium of the heme-auxotrophic SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream infection, we explored whether restoration of the electron transport chain in this organism also affected survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced growth rate but provides resistance to a key immune defence mechanism.

microbiology↗

bioRxiv2017

Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We have shown previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, Group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipid in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

microbiology↗