Carbon source competition within the wound microenvironment can significantly influence infection progression.

It is becoming increasingly apparent that commensal skin bacteria have an important role in wound healing and infection progression. However, the precise mechanisms underpinning many of these probiotic interactions remain to be fully uncovered. In this work, we demonstrate that the common skin commensal Cutibacterium acnes can limit the pathogenicity of the prevalent wound pathogen Pseudomonas aeruginosa in vivo. We show that this impact on pathogenicity is independent of any effect on growth, but occurs through a significant downregulation of the Type Three Secretion System (T3SS), the primary toxin secretion system utilised by P. aeruginosa in eukaryotic infection. We also show a downregulation in glucose acquisition systems, a known regulator of the T3SS, suggesting that glucose availability in a wound can influence infection progression. C. acnes is well known as a glucose fermenting organism, and we demonstrate that topically supplementing a wound with glucose reverses the probiotic effects of C. acnes. This suggests that introducing carbon source competition within the wound microenvironment may be an effective way to prevent or limit wound infection.

Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis.

Critical Gram-negative pathogens, like Pseudomonas, Stenotrophomonas and Burkholderia, have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multi-drug resistant Pseudomonas aeruginosa to ß-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant pathogen Stenotrophomonas maltophilia. The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.

Using the Galleria mellonella burn wound and infection model to identify and characterize potential wound probiotics.

Burn wound infection is the leading cause of mortality among burn wound patients. One of the most commonly isolated bacterial burn wound pathogens is Pseudomonas aeruginosa, a notorious nosocomial multidrug-resistant pathogen. As a consequence of its recalcitrance to frontline antibiotic therapy, there is an urgent need to develop alternative treatment avenues to tackle this pathogen. One potential alternative infection prevention measure is to seed the wound bed with probiotic bacteria. Several species of Lactobacillus, a common commensal bacterium, have been previously reported to display growth inhibition activity against wound pathogens. Various species of this genus have also been shown to augment the wound healing process, which makes it a promising potential therapeutic agent. Due to the complexity of the burn wound trauma and burn wound infection, an in vivo model is required for the development of novel therapeutics. There are multiple in vivo models that are currently available, the most common among them being the murine model. However, mammalian burn wound infection models are logistically challenging, do not lend themselves to screening approaches and come with significant concerns around ethics and animal welfare. Recently, an invertebrate burn wound and infection model using G. mellonella has been established. This model addresses several of the challenges of more advanced animal models, such as affordability, maintenance and reduced ethical concerns. This study validates the capacity of this model to screen for potential wound probiotics by demonstrating that a variety of Lactobacillus spp. can limit P. aeruginosa burn wound infection and improve survival.

Artificial sweeteners inhibit multidrug-resistant pathogen growth and potentiate antibiotic activity.

Antimicrobial resistance is one of the most pressing concerns of our time. The human diet is rich with compounds that alter bacterial gut communities and virulence-associated behaviours, suggesting food additives may be a niche for the discovery of novel anti-virulence compounds. Here, we identify three artificial sweeteners, saccharin, cyclamate and acesulfame-K (ace-K), that have a major growth inhibitory effect on priority pathogens. We further characterise the impact of ace-K on multidrug-resistant Acinetobacter baumannii, demonstrating that it can disable virulence behaviours such as biofilm formation, motility and the ability to acquire exogenous antibiotic-resistant genes. Further analysis revealed the mechanism of growth inhibition is through bulge-mediated cell lysis and that cells can be rescued by cation supplementation. Antibiotic sensitivity assays demonstrated that at sub-lethal concentrations, ace-K can resensitise A. baumannii to last resort antibiotics, including carbapenems. Using a novel ex vivo porcine skin wound model, we show that ace-K antimicrobial activity is maintained in the wound microenvironment. Our findings demonstrate the influence of artificial sweeteners on pathogen behaviour and uncover their therapeutic potential.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Burns and biofilms: priority pathogens and in vivo models.

Burn wounds can create significant damage to human skin, compromising one of the key barriers to infection. The leading cause of death among burn wound patients is infection. Even in the patients that survive, infections can be notoriously difficult to treat and can cause lasting damage, with delayed healing and prolonged hospital stays. Biofilm formation in the burn wound site is a major contributing factor to the failure of burn treatment regimens and mortality as a result of burn wound infection. Bacteria forming a biofilm or a bacterial community encased in a polysaccharide matrix are more resistant to disinfection, the rigors of the host immune system, and critically, more tolerant to antibiotics. Burn wound-associated biofilms are also thought to act as a launchpad for bacteria to establish deeper, systemic infection and ultimately bacteremia and sepsis. In this review, we discuss some of the leading burn wound pathogens and outline how they regulate biofilm formation in the burn wound microenvironment. We also discuss the new and emerging models that are available to study burn wound biofilm formation in vivo.

An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models.

The primary reason for skin graft failure and the mortality of burn wound patients, particularly those in burn intensive care centers, is bacterial infection. Several animal models exist to study burn wound pathogens. The most commonly used model is the mouse, which can be used to study virulence determinants and pathogenicity of a wide range of clinically relevant burn wound pathogens. However, animal models of burn wound pathogenicity are governed by strict ethical guidelines and hindered by high levels of animal suffering and the high level of training that is required to achieve consistent reproducible results. In this study, we describe for the first time an invertebrate model of burn trauma and concomitant wound infection. We demonstrate that this model recapitulates many of the hallmarks of burn trauma and wound infection seen in mammalian models and in human patients. We outline how this model can be used to discriminate between high and low pathogenicity strains of two of the most common burn wound colonizers Pseudomonas aeruginosa and Staphylococcus aureus, and multi-drug resistant Acinetobacter baumannii. This model is less ethically challenging than traditional vertebrate burn wound models and has the capacity to enable experiments such as high throughput screening of both anti-infective compounds and genetic mutant libraries.