Exploiting cooperative pathogen behaviour for enhanced antibiotic potency: A Trojan horse approach.

Antimicrobial resistance poses an escalating global threat, rendering traditional drug development approaches increasingly ineffective. Thus, novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats, effectively reducing virulence in vitro and in vivo. The idea of harnessing cooperative behaviours for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study, we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats, followed by increased antibiotic effectiveness against cheat-invaded populations, thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

The role of hypermutation and collateral sensitivity in antimicrobial resistance diversity of Pseudomonas aeruginosa populations in cystic fibrosis lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient) and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity, (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure, (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.IMPORTANCEUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe.

Exploiting Cooperative Pathogen Behavior for Enhanced Antibiotic Potency: A Trojan Horse Approach.

Antimicrobial resistance poses an escalating global threat, rendering traditional drug development approaches increasingly ineffective. Thus, novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats, effectively reducing virulence in vitro and in vivo. The idea of harnessing cooperative behaviors for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study, we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats, followed by increased antibiotic effectiveness against cheat-invaded populations, thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.

High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections.

IMPORTANCE: Cystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistant Pseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity of P. aeruginosa populations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections.

Microbial Primer: LuxR-LuxI Quorum Sensing.

Quorum sensing is a term describing bacterial cell-to-cell communication systems for monitoring and responding to changes in population density. This primer serves as an introduction to the canonical LuxR-LuxI-type quorum sensing circuits common to many species of Gram-negative bacteria. Quorum sensing can synchronize behaviours across a community. Different species employ quorum sensing strategies to control specific behaviours such as bioluminescence, virulence factor production, secondary metabolite production, and biofilm formation.

The Role of Hypermutation and Collateral Sensitivity in Antimicrobial Resistance Diversity of Pseudomonas aeruginosa Populations in Cystic Fibrosis Lung Infection.

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient), and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity; (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure; (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.

High prevalence of lipopolysaccharide mutants and R2-Pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections.

Chronic, highly antibiotic-resistant infections in cystic fibrosis (CF) lungs contribute to increasing morbidity and mortality. Pseudomonas aeruginosa, a common CF pathogen, exhibits resistance to multiple antibiotics, contributing to antimicrobial resistance (AMR). These bacterial populations display genetic and phenotypic diversity, but it is unclear how this diversity affects susceptibility to bacteriocins. R-pyocins, i.e. bacteriocins produced by P. aeruginosa, are phage tail-like antimicrobials. R-pyocins have potential as antimicrobials, however recent research suggests the diversity of P. aeruginosa variants within CF lung infections leads to varying susceptibility to R-pyocins. This variation may be linked to changes in lipopolysaccharide (LPS), acting as the R-pyocin receptor. Currently, it is unknown how frequently R-pyocin-susceptible strains are in chronic CF lung infection, particularly when considering the heterogeneity within these strains. In this study, we tested R2-pyocin susceptibility of 139 P. aeruginosa variants from 17 sputum samples of seven CF patients and analyzed LPS phenotypes. We found that 83% of sputum samples did not have R2-pyocin-resistant variants, while nearly all samples contained susceptible variants. there was no correlation between LPS phenotype and R2-pyocin susceptibility, though we estimate that about 76% of sputum-derived variants lack an O-specific antigen, 40% lack a common antigen, and 24% have altered LPS cores. The absence of a correlation between LPS phenotype and R-pyocin susceptibility suggests LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Our research supports the potential of R-pyocins as therapeutic agents, as many infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations.

Frequency of quorum-sensing mutations in Pseudomonas aeruginosa strains isolated from different environments.

Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. It has previously been shown that lasR QS mutations frequently arise in cystic fibrosis (CF) lung infections, however, there has been far less emphasis on determining whether other QS system mutations arise during infection or in other environments. To test this, we utilized 852 publicly available sequenced P. aeruginosa genomes from the Pseudomonas International Consortium Database (IPCD) to study P. aeruginosa QS mutational signatures. To study isolates by source, we focused on a subset of 654 isolates collected from CF, wounds, and non-infection environmental isolates, where we could clearly identify their source. We also worked with a small collection of isolates in vitro to determine the impact of lasR and pqs mutations on isolate phenotypes. We found that lasR mutations are common across all environments and are not specific to infection nor a particular infection type. We also found that the pqs system proteins PqsA, PqsH, PqsL and MexT, a protein of increasing importance to the QS field, are highly variable. Conversely, RsaL, a negative transcriptional regulator of the las system, was found to be highly conserved, suggesting selective pressure to repress las system activity. Overall, our findings suggest that QS mutations in P. aeruginosa are common and not limited to the las system; however, LasR is unique in the frequency of putative loss-of-function mutations.

Clostridium beijerinckii strain degeneration is driven by the loss of Spo0A activity.

Solventogenic clostridia represent a diverse group of anaerobic, spore-forming bacteria capable of producing acetone, butanol and ethanol through their unique biphasic metabolism. An intrinsic problem with these organisms however is their tendency to degenerate when repeatedly subcultured or when grown continuously. This phenomenon sees cells lose their ability to produce solvents and spores, posing a significant problem for industrial applications. To investigate the mechanistic and evolutionary basis of degeneration we combined comparative genomics, ultra-deep sequencing, and concepts of sociomicrobiology using Clostridium beijerinckii NCIMB 8052 as our model organism. These approaches revealed spo0A, the master regulator gene involved in spore and solvent formation, to be key to the degeneration process in this strain. Comparative genomics of 71 degenerate variants revealed four distinct hotspot regions that contained considerably more mutations than the rest of the genome. These included spo0A as well as genes suspected to regulate its expression and activity. Ultra-deep sequencing of populations during the subculturing process showed transient increases in mutations we believe linked to the spo0A network, however, these were ultimately dominated by mutations in the master regulator itself. Through frequency-dependent fitness assays, we found that spo0A mutants gained a fitness advantage, relative to the wild type, presumably allowing for propagation throughout the culture. Combined, our data provides new insights into the phenomenon of clostridial strain degeneration and the C. beijerinckii NCIMB 8052 solvent and spore regulation network.

O-Specific Antigen-Dependent Surface Hydrophobicity Mediates Aggregate Assembly Type in Pseudomonas aeruginosa.

Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses, and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here, we show that in an in vitro model of cystic fibrosis (CF) containing the polymers extracellular DNA (eDNA) and mucin, O-specific antigen is a major factor determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, the interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs. IMPORTANCE During chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways; however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study, we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.

Heterogenous Susceptibility to R-Pyocins in Populations of Pseudomonas aeruginosa Sourced from Cystic Fibrosis Lungs.

Bacteriocins are proteinaceous antimicrobials produced by bacteria that are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa Due to their antipseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa organisms from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection; however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1 to R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources, (ii) a large number of strains lack R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of intrastrain diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.IMPORTANCE R-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection; however, little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics and to evolve phenotypic and genotypic diversity over time; thus, it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

The evolution of virulence in Pseudomonas aeruginosa during chronic wound infection.

Opportunistic pathogens are associated with a number of chronic human infections, yet the evolution of virulence in these organisms during chronic infection remains poorly understood. Here, we tested the evolution of virulence in the human opportunistic pathogen Pseudomonas aeruginosa in a murine chronic wound model using a two-part serial passage and sepsis experiment, and found that virulence evolved in different directions in each line of evolution. We also assessed P. aeruginosa adaptation to a chronic wound after 42 days of evolution and found that morphological diversity in our evolved populations was limited compared with that previously described in cystic fibrosis (CF) infections. Using whole-genome sequencing, we found that genes previously implicated in P. aeruginosa pathogenesis (lasR, pilR, fleQ, rpoN and pvcA) contained mutations during the course of evolution in wounds, with selection occurring in parallel across all lines of evolution. Our findings highlight that: (i) P. aeruginosa heterogeneity may be less extensive in chronic wounds than in CF lungs; (ii) genes involved in P. aeruginosa pathogenesis acquire mutations during chronic wound infection; (iii) similar genetic adaptations are employed by P. aeruginosa across multiple infection environments; and (iv) current models of virulence may not adequately explain the diverging evolutionary trajectories observed in an opportunistic pathogen during chronic wound infection.

Bacterial Quorum Sensing During Infection.

Bacteria are highly interactive and possess an extraordinary repertoire of intercellular communication and social behaviors, including quorum sensing (QS). QS has been studied in detail at the molecular level, so mechanistic details are well understood in many species and are often involved in virulence. The use of different animal host models has demonstrated QS-dependent control of virulence determinants and virulence in several human pathogenic bacteria. QS also controls virulence in several plant pathogenic species. Despite the role QS plays in virulence during animal and plant laboratory-engineered infections, QS mutants are frequently isolated from natural infections, demonstrating that the function of QS during infection and its role in pathogenesis remain poorly understood and are fruitful areas for future research. We discuss the role of QS during infection in various organisms and highlight approaches to better understand QS during human infection. This is an important consideration in an era of growing antimicrobial resistance, when we are looking for new ways to target bacterial infections.

Combinatorial quorum sensing in Pseudomonas aeruginosa allows for novel cheating strategies.

In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods, cheats can be prevented from invading populations of cooperators and this was described by Dandekar et al. (Science 2012;338:264-266) as 'a metabolic incentive to cooperate'. We hypothesized that P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate that rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual-signal input to a 3O-C12-HSL-only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signalling cheat, with higher fitness in competition with its ancestor. Our results show three important principles: first, combinatorial QS allows for diverse social strategies to emerge; second, restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations; and third, modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

Allelic polymorphism shapes community function in evolving Pseudomonas aeruginosa populations.

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of individuals with cystic fibrosis (CF) by forming antibiotic-resistant biofilms. Emergence of phenotypically diverse isolates within CF P. aeruginosa populations has previously been reported; however, the impact of heterogeneity on social behaviors and community function is poorly understood. Here we describe how this heterogeneity impacts on behavioral traits by evolving the strain PAO1 in biofilms grown in a synthetic sputum medium for 50 days. We measured social trait production and antibiotic tolerance, and used a metagenomic approach to analyze and assess genomic changes over the duration of the evolution experiment. We found that (i) evolutionary trajectories were reproducible in independently evolving populations; (ii) over 60% of genomic diversity occurred within the first 10 days of selection. We then focused on quorum sensing (QS), a well-studied P. aeruginosa trait that is commonly mutated in strains isolated from CF lungs. We found that at the population level, (i) evolution in sputum medium selected for decreased the production of QS and QS-dependent traits; (ii) there was a significant correlation between lasR mutant frequency, the loss of protease, and the 3O-C12-HSL signal, and an increase in resistance to clinically relevant ß-lactam antibiotics, despite no previous antibiotic exposure. Overall, our findings provide insights into the effect of allelic polymorphism on community functions in diverse P. aeruginosa populations. Further, we demonstrate that P. aeruginosa population and evolutionary dynamics can impact on traits important for virulence and can lead to increased tolerance to ß-lactam antibiotics.

Neoadjuvant Chemotherapy Shifts Breast Tumor Microbiota Populations to Regulate Drug Responsiveness and the Development of Metastasis.

Breast tumors have their own specific microbiota, distinct from normal mammary gland tissue. Patients with breast cancer that present with locally advanced disease often undergo neoadjuvant chemotherapy to reduce tumor size prior to surgery to allow breast conservation or limit axillary lymph node dissection. The purpose of our study was to evaluate whether neoadjuvant chemotherapy modulates the tumor microbiome and the potential impact of microbes on breast cancer signaling. Using snap-frozen aseptically collected breast tumor tissue from women who underwent neoadjuvant chemotherapy (n = 15) or women with no prior therapy at time of surgery (n = 18), we performed 16S rRNA-sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays to confirm presence of identified microbiota. Using bacteria-conditioned media, we determined the effect of bacterial metabolites on breast cancer cell proliferation and doxorubicin therapy responsiveness. We show chemotherapy administration significantly increased breast tumor Pseudomonas spp. Primary breast tumors from patients who developed distant metastases displayed increased tumoral abundance of Brevundimonas and Staphylococcus. We confirmed presence of Pseudomonas in breast tumor tissue by IHC staining. Treatment of breast cancer cells with Pseudomonas aeruginosa conditioned media differentially effected proliferation in a dose-dependent manner and modulated doxorubicin-mediated cell death. Our results indicate chemotherapy shifts the breast tumor microbiome and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and develop novel bacterial biomarkers that could predict distant metastases. IMPLICATIONS: Breast tumor microbiota are modified by therapy and affects molecular signaling.