Vaughn S. Cooper

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen responsible for a wide range of infections, including ventilator-associated pneumonia, bloodstream infections, and urinary tract infections. Most studies investigating A. baumannii pulmonary pathogenesis rely on acute pneumonia models that use high bacterial inocula and do not recapitulate the prolonged infections observed in some patients. Recently, our laboratory developed a murine model of chronic pulmonary infection that enabled the identification of virulence factors specifically required for long-term lung persistence. To investigate how A. baumannii adapts during chronic pulmonary infection, we performed an experimental evolution study using this model. This approach revealed an unexpected role for the archaic chaperone-usher (Csu) pili in A. baumannii pathogenesis. At early time points, Csu expression reduced bacterial burdens in both acute and chronic pneumonia models, an effect associated with enhanced host inflammatory responses. In contrast, at later stages of chronic infection, Csu promoted bacterial persistence, potentially by facilitating adhesion to pulmonary epithelial cells. These opposing effects were conserved across two distinct A. baumannii clinical isolates. We also found that Csu contributes to bacterial colonization in a catheter-associated urinary tract infection (CAUTI) model in a strain-dependent manner. Together, these findings reveal an infection-stage-dependent role for Csu in A. baumannii pathogenesis, demonstrating that a bacterial surface structure can impair early colonization while promoting long-term persistence. Author summary: Understanding how bacterial pathogens establish and maintain infections requires identifying the factors that promote their survival within the host. Currently, most experimental models of Acinetobacter baumannii infections focus on acute infections, potentially overlooking bacterial factors that are required for prolonged infections. In this study, we investigated how A. baumannii , a major cause of hospital-acquired infections, adapts to the lung environment during chronic infection. Using experimental evolution, we identified a novel role for Csu pili, surface structures traditionally associated with biofilm formation. Rather than promoting bacterial fitness throughout infection, Csu pili exhibited opposing effects depending on disease progression. Their expression was detrimental to bacterial fitness during early infection, a phenotype associated with increased host inflammatory responses, but became advantageous during prolonged infection, potentially by enhancing bacterial association with respiratory epithelial cells. Importantly, these opposing effects were observed in distinct clinical isolates, suggesting that this phenomenon is not restricted to a particular bacterial strain. Our findings highlight how the contribution of individual virulence factors can change over the course of infection and underscore the importance of considering infection duration when studying bacterial pathogenesis.

Mirror bacteria are hypothetical synthetic organisms built from molecules of opposite chirality to those used throughout nature. This primer discusses their potential dangers: because of their chiral mismatch to the natural environment, mirror bacteria could plausibly evade predation, immunity and other ecological control mechanisms to a degree never seen before. This property could simultaneously make them unprecedented invasive species and dangerous pathogens.

The patterns and pathways of pathogen adaptation during chronic infections can reveal the gateways microbes must unlock to colonize and persist. In chronic lung infections of cystic fibrosis (CF) patients, a longtime goal has been to anticipate disease outcome and to improve treatment. Understanding of these processes for Burkholderia multivorans, the most prevalent CF pathogen of the Burkholderia cepacia complex, is limited. We investigate the evolution of seven different strains recovered from chronic airway infections of eight CF patients over 7-17 years. Despite divergent origins, each infecting population followed similar phylogenetic patterns of early diversification followed by the emergence of a dominant clade. The defining mutations of these prevalent lineages also affected the same set of global regulators, which altered clinically significant bacterial phenotypes in parallel directions. Mutated genes govern lipid metabolism, immune evasion, antibiotic resistance, biofilm production, and survival under oxygen limitation, and unite studies of chronic infections by different species of the B. cepacia complex. Most significantly, the phylodynamic signal that a dominant lineage had emerged within the infection, more so than any particular set of mutations, was associated with a more rapid decline in patient lung function. These findings reveal the importance of combining data from pathogen and host to link bacterial adaptation to disease progression, and ultimately to identify paths to reinforce host defense. Author summary: Cystic fibrosis (CF) remains a serious disease despite major advances in care, because chronic lung infections can still gradually damage the airways and reduce lung function. Among the bacteria that can cause these long-term infections, the Burkholderia cepacia complex is especially concerning because it can survive many antibiotics and is associated with poor outcomes. In this study, we focused on Burkholderia multivorans , now one of the most common Burkholderia species found in CF lungs. By following bacterial populations from eight patients over several years, we found that these infections often evolved in a similar way: they first became genetically diverse, and later one dominant lineage emerged and took over. This transition was linked to changes in genes affecting antibiotic resistance, immune evasion, biofilm formation, and survival in low-oxygen conditions. Most importantly, the appearance of these dominant bacterial lineages was associated with faster decay of patient lung function. These findings show that bacterial evolution during chronic infections can follow predictable pathways that drive disease progression, which can enable earlier detection, monitoring, and treatment of CF lung infections.

Introduction: Outbreaks of healthcare-associated infections (HAI) result in substantial patient morbidity and mortality; mitigation efforts by infection prevention teams have the potential to curb outbreaks and prevent transmission to additional patients. The incorporation of whole genome sequencing (WGS) surveillance of suspected high-risk pathogens often identifies outbreaks that are not detected by traditional infection prevention methods and provides evidence for transmission. Our approach to real-time WGS surveillance, the Enhanced Detection System for Healthcare-Associated Transmission (EDS-HAT), has 1) identified serious outbreaks that were otherwise undetected and 2) shown the potential to be cost saving. Methods: We describe our cost-efficient methods to perform WGS surveillance and data analysis of pathogens for institutions that are interested in expanding infection prevention surveillance. We provide an overview of the weekly workflow of EDS-HAT during two distinct phases over three years. Results: In an average week at our tertiary healthcare system, we sequenced 60 samples at a cost of less than $100 each during Phase 1, and 80 samples for less than $70 each in Phase 2, inclusive of laboratory reagents and staff salaries. The average turnaround time, from sample collection to reporting data to infection prevention, was nine days. Conclusions: Performing EDS-HAT in real-time can be both feasible and time-efficient. Providing such timely information to aid in outbreak detection could identify transmission events sooner and thus could increase patient safety.

Background: Urinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. Results: We present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. Conclusions: The catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

Multidrug resistant (MDR) bacterial infections without antibiotic options are a public health emergency. Infections associated with medical implants serve as an example. Conventional antibiotics have limited ability to eradicate these infections as they are associated with antibiotic-tolerant biofilms. Here, we report the use of bacteriophage therapy for the treatment of a MDR, non-operable Pseudomonas aeruginosa periprosthetic joint infection that had failed multiple antibiotic and surgical interventions. Treatment with intermittent bacteriophage therapy alone without antibiotics over a 2 year time period resulted in clinical resolution of the infection, but not microbiological eradication. Bacteriophage therapy established this control, in part, by altering virulence as defined by disease severity and symptoms and disrupting biofilm. Whole genome sequencing demonstrated the continued presence of bacteriophage during treatment. This provides preliminary evidence that bacteriophage therapy can be used to treat MDR infections in salvage cases when surgical and antibiotic options do not exist.

Pseudomonas aeruginosa readily adapts to infection by acquiring stable and heritable mutations. Previously, we discovered that the first adaptations in a porcine wound model were rugose small-colony variants (RSCVs) caused by mutations in the wsp operon. These mutants overproduce Pel and Psl biofilm exopolysaccharides that improve defense against host responses. To identify other mechanisms of host adaptation that lead to hyperbiofilm phenotypes, we created a mutant with an activated wsp pathway but unable to produce these exopolysaccharides (PAO1Δ wspF Δ pelA Δ pslBCD ). Porcine wounds were infected with this mutant, and biopsies were sampled at days 7, 14, and 35. Colony variants were isolated from the wound, peaking at approximately 5% of the total P. aeruginosa population, and whole genome sequencing revealed that these variants had acquired mutations in genes in lipopolysaccharide and type IV pili biosynthesis, with wzy and pilU genes being most commonly targeted. PAO1 pilU mutants were associated with a hyperbiofilm phenotype that outcompeted the parental strain, and PAO1 wzy mutants were associated with a hyperbiofilm phenotype and increased tolerance to host antimicrobial products. We further identified that several variants had acquired large genome deletions that spanned up to 320 consecutive genes and other variants with high copy numbers of Pf6 filamentous phage. Together our results suggest that the hyperbiofilm phenotype is adaptive in chronic infections and that P. aeruginosa has redundant and diverse pathways to generate this phenotype. IMPORTANCE: We demonstrate that in a porcine full-thickness thermal injury wound model, a Pseudomonas aeruginosa mutant deficient in biofilm formation undergoes adaptive evolution by acquiring mutations that alter the outer membrane, either type IV pili (T4P) or lipopolysaccharide (LPS) mutations, that restores the deficient biofilm phenotype. We also observe a striking degree of mutational parallelism, at both the biosynthetic pathway and gene level, indicating the strong selective pressures experienced by these pathways during chronic wound infection.

We established a research-education partnership, EvolvingSTEM, that currently provides thousands of secondary school students the opportunity to conduct authentic research experiments centered on microbial evolution each year. Providing high school students access to research experiences improves learning, can have positive and long-lasting impacts on their attitudes toward science, and gives them the opportunity to make impactful scientific contributions. Through EvolvingSTEM, students evolve populations of Pseudomonas fluorescens in a bead model that includes daily cycles of bacterial dispersal, attachment, and biofilm growth, and observe heritable changes in colony morphology. We sequenced the genomes of 69 evolved clones with unique colony morphologies and identified parallel mutations in genes known to regulate or affect biofilm growth ( wsp , yfiBNR , morA, and fuzY ). We also uncovered novel adaptations: loss-of-function mutations in phosphodiesterase PFLU0185 that did not alter colony morphology and mutations affecting periplasmic disulfide bond formation producing small colonies. PFLU0185 mutants rapidly and consistently reached high frequencies, and phenotyping revealed roles in cyclic di-GMP regulation, biofilm formation, and motility, prompting us to name this gene bmo ( b iofilm and mo tility regulator). Competition experiments and microscopy demonstrated that bmo mutants are ecological generalists that compete with their ancestor and specialist mutants. Consequently, phenotypic diversity is maintained, with smooth (ancestral and bmo ) colonies consistently outnumbering wrinkly and fuzzy variants. This study advances our understanding of biofilm genetic architecture while demonstrating that student-led research can uncover mechanisms of microbial adaptation relevant to Pseudomonas infection biology. IMPORTANCE: Bacterial biofilms dominate microbial life; however, their evolutionary genetics remain incompletely understood. Extensive replication of biofilm selection experiments by secondary school students can provide valuable insights into mechanisms of adaptation. Notably, mutations in a conserved phosphodiesterase, PFLU0185/ bmo , dominate evolved populations without changing colony morphology. Phenotypic assays reveal that bmo plays a generalist role and competes with its ancestor and other diverse, less frequent biofilm-specialist mutants, resulting in populations that have differentiated to fill multiple niches within the biofilm life cycle. This work also demonstrates the power of distributed research networks for addressing complex biological questions while promoting scientific literacy to the public.

Long-term experimental evolution (LTEE) provides a powerful framework for dissecting how ecological interactions shape adaptive trajectories. Here, we evolved Klebsiella pneumoniae, Pseudomonas protegens and Pseudomonas aeruginosa in single- and mixed-species biofilm communities for 24 weeks and tracked changes in population dynamics, phenotypes, and genomes. In mono-species evolution, all three species exhibited similar dynamics of adaptation, with steadily increasing biofilm-associated populations. In contrast, mixed-species communities displayed striking compositional shifts, with P. protegens emerging as the dominant biofilm former and K. pneumoniae dominating the supernatant. Phenotypic assays revealed that all three species showed enhanced biofilm formation, but this increase was consistently greater in isolates from mono-species than mixed species communities, with P. protegens showing the largest gains. Beyond biofilm production, biofilm-associated isolates exhibited greater phenotypic diversification than planktonic isolates, whereas mixed-species interactions constrained diversification. Whole-genome sequencing identified species-specific putative adaptations such as csrD in K. pneumoniae, yfiBNR in P. protegens , and cheA in P. aeruginosa that arose early, persisted, and were enriched in mixed-species isolates. Functional assays confirmed that these mutations were indeed adaptive by enhancing biofilm formation, with yfiBNR mutations in P. protegens increasing cyclic-di-GMP production and producing a competitive advantage that recapitulated its dominance in LTEE biofilms. Our findings show that biofilm evolution fosters phenotypic diversification, whereas interspecific interactions shape adaptive trajectories, with specific mutations acting as keystone drivers of long-term ecological dynamics in multi-species communities.

Polymicrobial biofilms are acknowledged as evolutionary hotspots; however, the influence of interspecies interactions on adaptive processes remains ambiguous. Using custom-engineered 3D-printed flow systems, biofilms were grown over an 18-day period to explore the evolutionary dynamics over time between two competing species, Pseudomonas defluvii and Pseudomonas brenneri, within communities of differing complexity, using whole-population and whole-genome sequencing methodologies. P. defluvii demonstrated significant phenotypic and genetic diversification in simple biofilms, yet this variation was reduced in complex biofilms, implying that interspecies interactions constrained its adaptation. In contrast, P. brenneri exhibited negligible evolutionary changes irrespective of the diversity present. Genomic analysis correlated the adaptation of P. defluvii with biofilm regulation and chemotaxis. The co-culture with evolved strains indicated that the variants of P. defluvii outperformed their ancestral forms, while P. brenneri remained unchanged. These results show that while the biofilm lifestyle generally fosters adaptive evolutionary processes, interspecies diversity can restrict diversification in a manner specific to each species. This study provides novel insights into the evolution of bacteria within complex biofilm environments, with implications for understanding microbial behavior in both clinical and industrial settings.