Biology subjects

Marshall, C. W.

Publications and source records attributed to Marshall, C. W..

17 recordsLinked to original sources

bioRxiv2026

Long-term exposure to polyamines leads to bacteriophage resistance in Pseudomonas aeruginosa

Bacteria often evolve resistance to phage infection by altering the cell-surface structures required for viral adsorption. However, the role extracellular metabolites play in influencing phage susceptibility and the evolution of phage resistance remains unclear. Here, we evaluated whether sustained exposure to putrescine, a polyamine released during phage-mediated cell lysis, alters susceptibility of the pathogen Pseudomonas aeruginosa to the type IV pili-dependent phage DMS3vir. Using adaptive laboratory evolution over [~]66 generations, we evolved P. aeruginosa with or without putrescine and with or without DMS3vir. As expected, direct phage exposure rapidly led to complete phage resistance. Interestingly, populations exposed to putrescine also developed phage resistance by the end of the experiment, despite having never encountered the phage. Whole-population genome sequencing revealed parallel mutations in genes associated with type IV pili and the global transcriptional regulator mexT. Using transposon insertion mutants in the type IV ATPases pilT and pilB, we confirmed that disruption of these genes leads to DMS3vir phage resistance. We also used a type IV pilus biogenesis factor fimV transposon mutant, which showed a putrescine-dependent reduction in phage susceptibility. These findings show that sustained exposure to a host-derived metabolite can drive the evolution of phage resistance through modification of key phage-adsorption sites and regulatory genes. Our work identifies elevated polyamine exposure as a selective pressure that promotes type IV pili-mediated phage resistance, even in the absence of phage exposure. IMPORTANCEPseudomonas aeruginosa is a major cause of hospital-acquired infections and a key priority for phage-based therapies. Previous work has shown that the polyamine putrescine is released into the extracellular environment during cell lysis. These signals can then transiently reduce susceptibility to bacteriophage infection and alert neighboring cells to danger. Our research demonstrates that long-term exposure to putrescine can drive heritable phage resistance without prior exposure to phage. We show that resistance is linked to mutations in genes involved in type IV pili assembly. This work further demonstrates the critical role that polyamines can play in promoting phage resistance in bacterial communities.

microbiology↗

bioRxiv2026

Anoxia selects for high fitness biofilms and increased antibiotic resistance in Pseudomonas aeruginosa

Antibiotic resistance is a growing global health crisis, yet resistance is almost exclusively quantified under aerobic laboratory conditions that fail to reflect the complex microenvironments bacteria encounter during infection. Many clinically important infection sites, such as airways of individuals with cystic fibrosis or chronic wounds, are microaerobic to anoxic. To address this, we investigated how anoxia alters antibiotic resistance determinants, hypothesizing that anaerobic metabolism might change the fitness effects and selection of resistance mutations. We used experimental evolution to propagate Pseudomonas aeruginosa populations for approximately 200 generations under conditions differing in oxygen availability (oxic vs. anoxic), growth mode (biofilm vs. planktonic), and tobramycin (TOB) exposure (subinhibitory increasing to inhibitory concentrations). Subinhibitory exposure was sufficient to achieve resistance 2-4x greater than ancestral levels, with anoxic populations consistently having higher minimum inhibitory concentrations than oxic populations. While resistance mutations in fusA1 and ptsP arose across all conditions, mutations in amgS were only selected in oxic populations - indicating condition-specific resistance mutations. Notably, mexT mutations were observed in anoxic or tobramycin-exposed populations. The presumed inactivation of mexT may also enhance virulence through altered quorum sensing and increased rhamnolipid production. Anoxic populations additionally exhibited significantly increased biofilm formation, reduced twitching motility driven by type IV pilus gene mutations, and greater competitive fitness. Together, these findings demonstrate that oxygen availability shapes resistance evolution in P. aeruginosa, with the anoxic environment selecting for a more virulent, sessile, and antibiotic-resistant phenotype. ImportancePseudomonas aeruginosa is an opportunistic pathogen capable of acute and chronic infections that develop antibiotic resistance. One aspect of P. aeruginosa that makes it so challenging to treat is its metabolic versatility. We wanted to understand how growth in different infection-relevant conditions, specifically anoxia and surface attachment, would alter the evolutionary pathways of antibiotic resistance. We found that the mutations causing resistance to the commonly used antibiotic tobramycin were condition dependent. We also observed that adaptation to anoxia resulted in P. aeruginosa populations that had high biofilm forming capacity and were highly fit compared to its ancestor. This indicates that anoxic infection environments can lead to P. aeruginosa variants with increased resistance, increased recalcitrance, and potentially more virulence.

microbiology↗

bioRxiv2025

Acinetobacter spp. with lower susceptibility to quaternary ammonium compounds enriched in microbial communities of frequently used sinks

Sanitary environments that undergo frequent cleaning and disinfection may harbor microbial communities with potential health risks. While biofilms in healthcare settings are well studied, comparatively less is known about sink microbiomes in public and educational buildings, where hundreds of people may interact with shared sink fixtures. This study characterized the spatial and temporal heterogeneity of sink drain biofilm microbiomes in academic buildings. We sampled 16 sinks from two buildings (four floors each, with sinks closest and furthest to the bathroom entrance), which are cleaned daily with quaternary ammonium compound (QAC) disinfectants, during periods of low and high student traffic (during and after academic breaks, respectively) across winter, spring, and summer. We observed significant spatial and temporal variations in microbial assemblages. Individual sinks accounted for 43% (PERMANOVA, p < 0.0001) of the variation in microbial communities. Microbiomes in each building were dominated by two genera, which together accounted for 30% of the community composition: Acinetobacter and Enhydrobacter (also classified as Moraxella) in the newer building, and Sphingomonas and Mycobacterium in the older building. Acinetobacter abundance varied seasonally and showed higher relative abundance during periods of high traffic. Metagenomic analysis of selected sinks revealed a high prevalence of qac genes and metagenome-assembled genomes (MAGs) harboring antimicrobial resistance genes (ARGs) including A. parvus. Notably, 34-53% of qac genes were co-localized on contigs associated with mobile genetic elements. These findings suggest that disinfected sink drains serve as persistent reservoirs of diverse microorganisms and potentially mobile resistance elements. IMPORTANCESink drains are recognized environmental reservoirs for multidrug-resistant bacteria and have been linked to healthcare-associated outbreaks. In public and educational buildings, these microbiomes are shaped by frequent human activity, making them potential sources of exposure and contributors to the environmental dissemination of antibiotic resistance genes. Quaternary ammonium compound (QAC) disinfectants are widely used on surfaces; however, they can select for resistant taxa and co-select for antibiotic resistance. In this study, despite routine cleaning with QACs, public restroom drains remain colonized by resilient biofilms, posing a potential risk to multiple users. Additionally, factors such as human traffic and seasonal variation may influence drain usage and microbial community composition. Elucidating how seasonal dynamics and human activity shape sink biofilms is essential for understanding their role in the environmental transmission of antimicrobial resistance and informing mitigation strategies in nonclinical settings.

microbiology↗

bioRxiv2025

Pseudomonas aeruginosa biofilm-deficient mutants undergo parallel adaptation during chronic infection

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 ({Delta}wspF{Delta}pelA{Delta}pslBCD). Porcine wounds were infected with this mutant and biopsies were sampled at days 7, 14 and 35. Small colony variants were isolated from the wound, and whole genome sequencing revealed these variants had acquired mutations in genes in lipopolysaccharide and type IV pili biosynthesis, with wzy and pilU genes being most commonly targeted. pilU mutants were associated with a hyperbiofilm phenotype that outcompeted the parental strain, and 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.

microbiology↗

bioRxiv2024

Effects of Soluble Electron Shuttles on Microbial Iron Reduction and Methanogenesis

In many aquatic and terrestrial ecosystems, iron (Fe) reduction by microorganisms is a key part of biogeochemical cycling and energy flux. The presence of redox active electron shuttles in the environment potentially enables a phylogenetically diverse group of microbes to use insoluble iron as a terminal electron acceptor. We investigated the impact that different electron shuttles had on respiration, microbial physiology, and microbial ecology. We tested eight different electron shuttles, seven quinones and riboflavin, with redox potentials between 0.217 V and -0.340 V. Fe(III) reduction coupled to acetate oxidation was observed with all shuttles. Once Fe(III) reduction began to plateau, a rapid increase in acetate consumption was observed and coincided with the onset of methane production, except in the incubations with the shuttle 9,10-anthraquinone-2-carboxylic acid (AQC). The rates of iron reduction, acetate consumption, methanogenesis, and the microbial communities varied significantly across the different shuttles independent of redox potential. In general, shuttles appeared to reduce the overall diversity of the community compared to no shuttle controls, but certain shuttles were exceptions to this trend. Geobacteraceae were the predominant taxonomic family in all enrichments except in the presence of AQC or 1,2,-dihydroxyanthraquinone (AQZ), but each shuttle enriched a unique community significantly different from the no shuttle control conditions. This suggests that the presence of different redox-active electron shuttles can have a large influence on the microbial ecology and total carbon flux in the environment. ImportanceIron is the fourth most abundant element in the Earths crust and the reduction of iron by microbes is an important component of global biogeochemical cycles. A phylogenetically diverse group of microbes are capable of conserving energy with oxidized iron as a terminal electron acceptor, but the environmental conditions favoring certain taxonomic clades in iron-reducing environments is unclear. One complicating factor often overlooked in small-scale enrichments is the influence of soluble, redox-active electron shuttles on the rate and microbial ecology of iron reduction. We tested the effects of eight different electron shuttles on the microbial physiology and ecology in iron-reducing enrichments derived from a local wetland. Each electron shuttle varied the microbial activity and enriched for a microbial community distinct from the no shuttle control condition. Therefore, in complex subsurface environments with many redox-active compounds present, we propose electron shuttles as a reason for the coexistence of multiple clades of iron-reducing bacteria.

microbiology↗

bioRxiv2023

Bacillus subtilis maintains antibiofilm activity against Staphylococcus aureus following adaptive laboratory evolution

Quorum sensing interference has been touted as an ideal mechanism for the development of new anti-virulence therapies. Recent work has established Bacillus subtilis 6D1, a Gram-positive spore forming bacterium with probiotic qualities, produces metabolites that inhibit Staphylococcus aureus virulence and biofilm formation via quorum sensing interference. However, it remains unknown how long-term exposure to these molecules drive S. aureus adaptation and evolution. S. aureus planktonic cells and biofilms were propagated in the presence of B. subtilis 6D1 cell free extracts (CFE) for [~]73 generations. Fitness, virulence, and antibiotic resistance assays of the ancestor and all evolved lineages revealed the emergence of treatment and lifestyle associated ecological traits. Compared to the ancestor and media-evolved lineages, S. aureus lineages evolved in the presence of B. subtilis 6D1 CFE were less competitive in a biofilm and exhibited increased phenotypic sensitivity to multiple antibiotics. Notably, B. subtilis 6D1 CFE maintained its ability to inhibit S. aureus biofilm growth and disassemble mature biofilm in all evolved lineages. S. aureus populations propagated in the presence of CFE acquired missense mutations in genes associated with plasmid-borne efflux systems and RNA polymerase. Furthermore, CFE-evolved lineages did not develop mutations in both competence and drug resistance pathways found in similarly evolved control lineages. Our data suggest long-term exposure to biofilm inhibitory molecules, like those produced by B. subtilis 6D1, can reduce S. aureus fitness in a biofilm and increase sensitivity to multiple antibiotics. ImportanceQuorum sensing interference (QSI) has been touted as an ideal mechanism to diminish bacterial virulence and improve antibiotic killing, however few studies investigate the genetic and phenotypic adaptations that occur after long-term exposure to QSI therapies. Recent studies revealed Bacillus subtilis reduces biofilm formation and virulence via signaling interference with the S. aureus Agr QS system; however, it remains unclear how long-term exposure to these compounds drives S. aureus adaptation and evolution. This study helps to address these gaps by investigating whether QSI strategies deployed by probiotic bacteria are viable approaches to increase antibiotic efficacy without increasing antibiotic resistance evolution.

microbiology↗

bioRxiv2021

Plant growth promoting activity of bacteria isolated from Asian rice (Oryza sativa L.) are plant subspecies dependent

Asian rice is one of the most important crops because it is a staple food for almost half of the worlds population. Rice has two subspecies, JAPONICA and INDICA. To have production of rice keep pace with a growing world population, it is anticipated that the use of fertilizers will also need to increase, which may cause environmental damage through runoff impacts. An alternative strategy to increase crop yield is the use of plant growth promoting bacteria. Thousands of microbial species can exist in association with plant roots and shoots, and some are critical to the plants survival. We isolated 140 bacteria from rice and investigated whether JAPONICA and INDICA rice subspecies were positively influenced by these isolates. The bacterial isolates were screened for their ability to solubilize phosphate, a known plant growth promoting characteristic, and 25 isolates were selected for further analysis. These 25 phosphate solubilizing isolates were also able to produce other potentially growth-promoting factors. Five of the most promising bacterial isolates were chosen for whole genome sequencing. Four of these bacteria, isolates related to Pseudomonas mosselii, Microvirga sp., Paenibacillus rigui and Paenibacillus graminis, improved root and shoot growth, root to shoot ratio, and increased root dry weights of JAPONICA plants but had no effect on growth and development of INDICA plants. This indicates that while bacteria have several known plant growth promoting functions, their effects on growth parameters can be plant subspecies dependent and suggest close relationships between plants and their microbial partners.

microbiology↗

bioRxiv2021

Excess dietary sugar impairs colonic epithelial regeneration in response to damage.

The colonic epithelium requires continuous renewal by intestinal stem cells (ISCs) to restore the barrier after damage and proliferation of epithelial cells is modulated by dietary metabolites. We demonstrate that mice fed a high sugar diet failed to repair colonic barrier damage, resulting in increased intestinal pathology. Culturing ISCs in excess sugar limited murine and human colonoid development, indicating that dietary sugar can directly affect colonic epithelial proliferation. Similarly, in vivo lineage tracing experiments and transcriptomic analysis indicated that dietary sugar impeded the proliferative potential of ISCs. ISCs and their immediate daughter cells predominantly rely on mitochondrial respiration for energy; however, metabolic analysis of colonic crypts revealed that a high sugar diet primed the epithelium for glycolysis without a commensurate increase in aerobic respiration. Colonoids cultured in high-glucose conditions accumulated glycolytic metabolites but not TCA cycle intermediates, indicating that the two metabolic pathways may not be coupled in proliferating intestinal epithelium. Accordingly, biochemically inducing pyruvate flux through the TCA cycle by inhibiting pyruvate dehydrogenase kinase rescued sugar-impaired colonoid development. Our results indicate that excess dietary sugar can directly inhibit epithelial proliferation in response to damage and may inform diets that better support the treatment of acute intestinal injury.

immunology↗

bioRxiv2021

Immunosuppression broadens evolutionary pathways to treatment failure during Acinetobacter baumannii pneumonia

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

microbiology↗

bioRxiv2021

Rampant prophage movement among transient competitors drives rapid adaptation during infection

Interactions between bacteria, their close competitors, and viral parasites are common in infections but understanding of these eco-evolutionary dynamics is limited. Most examples of adaptations caused by phage lysogeny are through the acquisition of new genes. However, integrated prophages can also insert into functional genes and impart a fitness benefit by disrupting their expression, a process called active lysogeny. Here, we show that active lysogeny can fuel rapid, parallel adaptations in establishing a chronic infection. These recombination events repeatedly disrupted genes encoding global regulators, leading to increased cyclic-di-GMP levels and elevated biofilm production. The implications of prophage-mediated adaptation are broad, as even transient members of microbial communities can alter the course of evolution and generate persistent phenotypes associated with poor clinical outcomes. One Sentence SummaryBacteriophage act as genetic regulators that are key to establishing chronic infections and are rapidly shared among co-infecting strains.

microbiology↗

bioRxiv2020

Experimental evolution to identify undescribed mechanisms of resistance to a novel cationic peptide antibiotic

A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and mutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. The results show how the experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.

microbiology↗

bioRxiv2020

Evolution and adaptation of Pseudomonas aeruginosa in the paranasal sinuses of people with cystic fibrosis

People with the genetic disorder cystic fibrosis (CF) harbor lifelong respiratory infections, with morbidity and mortality frequently linked to chronic lung infections dominated by the opportunistically pathogenic bacterium Pseudomonas aeruginosa. During chronic CF lung infections, a single clone of P. aeruginosa can persist for decades and dominate end-stage CF lung disease due to its propensity to adaptively evolve to the respiratory environment, a process termed "pathoadaptation". Chronic rhinosinusitis (CRS), chronic inflammation and infection of the sinonasal space, is highly prevalent in CF and the sinuses may serve as the first site in the respiratory tract to become colonized by bacteria that then proceed to seed lung infections. We identified three evolutionary genetic routes by which P. aeruginosa evolves in the sinuses of people with CF, including through the evolution of mutator lineages and proliferative insertion sequences and culminating in early genomic signatures of host-restriction. Our findings raise the question of whether a significant portion of the pathoadaptive phenotypes previously thought to have evolved in response to selective pressures in the CF lungs may have first arisen in the sinuses and underscore the link between sinonasal and lung disease in CF. Graphical abstract and highlightsO_LIPseudomonas aeruginosa undergoes adaptive evolution in the sinuses of people with CF C_LIO_LIOver time, pathoadapted strains display early signatures of genome degradation consistent with recent host restriction C_LIO_LIMutations previously thought to occur in CF lungs may have first evolved in sinuses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/359844v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@13b36aborg.highwire.dtl.DTLVardef@8230e1org.highwire.dtl.DTLVardef@1570c3eorg.highwire.dtl.DTLVardef@1e4e530_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

The roles of history, chance, and natural selection in the evolution of antibiotic resistance

History, chance, and selection are the fundamental factors that drive and constrain evolution. We designed evolution experiments to disentangle and quantify effects of these forces on the evolution of antibiotic resistance. History was established by prior antibiotic selection of the pathogen Acinetobacter baumannii in both structured and unstructured environments, selection occurred in increasing concentrations of new antibiotics, and chance differences arose as random mutations among replicate populations. The effects of history were reduced by increasingly strong selection in new drugs, but not erased, at times producing important contingencies. Selection in structured environments constrained resistance to new drugs and led to frequent loss of resistance to the initial drug. This research demonstrates that despite strong selective pressures of antibiotics leading to genetic parallelism, history can etch potential vulnerabilities to orthogonal drugs.

evolutionary biology↗

bioRxiv2020

Host adaptations in the fixLJ pathway of the Burkholderia cepacia complex increase virulence

The Burkholderia cepacia complex (BCC) is composed of multiple species, including B. multivorans and B. dolosa, that are significant pathogens for people with cystic fibrosis (CF) and are extensively resistant to many antibiotics. The fixL gene of the fixLJ 2-component system (TCS) in these BCC species shows evidence of positive selection for nonsynonymous mutations during chronic lung infection in CF. Previous work showed that the B. dolosa fixLJ system regulates 11% of the genome and modulates biofilm formation, motility, persistence within macrophages, and virulence in a murine pneumonia model. Here, we assess the impacts of clinically observed FixL evolved variants in fixLJ pathway-mediated phenotypes in B. dolosa and B. multivorans. BCC carrying the ancestral fixL sequence are less pathogenic than constructs carrying evolved variants in both a macrophage infection model and a murine pneumonia model. In vitro phospho-transfer experiments demonstrate that the evolved B. dolosa FixL variants are able to reduce fixLJ pathway activity by either having lower levels of kinase activity or increased phosphatase activity. Notably, the ancestral fixL genotype has increased ability to survive within the soil compared to isogenic constructs with evolved fixL genotypes, demonstrating that increased virulence comes at an expense. Modulation of the FixLJ system has profound effects on many BCC phenotypes including full pathogenicity, and this modulation is critical for BCC adaptation to the host.

microbiology↗

bioRxiv2020

The defining genomic and predicted metabolic features of the Acetobacterium genus

Acetogens are anaerobic bacteria capable of fixing CO2 or CO to produce acetyl-CoA and ultimately acetate using the Wood-Ljungdahl pathway (WLP). This autotrophic metabolism plays a major role in the global carbon cycle. Acetobacterium woodii, which is a member of the Eubacteriaceae family and type strain of the Acetobacterium genus, has been critical for understanding the biochemistry and energy conservation in acetogens. Other members of the Acetobacterium genus have been isolated from a variety of environments or have had genomes recovered from metagenome data, but no systematic investigation has been done into the unique and varying metabolisms of the genus. Using the 4 sequenced isolates and 5 metagenome-assembled genomes available, we sequenced the genomes of an additional 4 isolates (A. fimetarium, A. malicum, A. paludosum, and A. tundrae) and conducted a comparative genome analysis of 13 different Acetobacterium genomes to obtain better phylogenomic resolution and understand the metabolic diversity of the Acetobacterium genus. Our findings suggest that outside of the reductive acetyl-CoA (Wood-Ljungdahl) pathway, the Acetobacterium genus is more phylogenetically and metabolically diverse than expected, with metabolism of fructose, lactate, and H2:CO2 constant across the genus, and ethanol, methanol, caffeate, and 2,3-butanediol varying across the genus. While the gene arrangement and predicted proteins of the methyl (Cluster II) and carbonyl (Cluster III) branches of the Wood Ljungdahl pathway are highly conserved across all sequenced Acetobacterium genomes, Cluster 1, encoding the formate dehydrogenase, is not. Furthermore, the accessory WLP components, including the Rnf cluster and electron bifurcating hydrogenase, were also well conserved, though all but four strains encode for two Rnf clusters. Additionally, comparative genomics revealed clade-specific potential functional capabilities, such as amino acid transport and metabolism in the psychrophilic group, and biofilm formation in the A. wieringae clade, which may afford these groups an advantage in low-temperature growth or attachment to solid surfaces, respectively. Overall, the data presented herein provides a framework for examining the ecology and evolution of the Acetobacterium genus and highlights the potential of these species as a source of fuels and chemicals from CO2-feedstocks.

genomics↗

bioRxiv2019

Parallel evolution of tobramycin resistance across species and environments

An important problem in evolution is identifying the genetic basis of how different species adapt to similar environments. Understanding how various bacterial pathogens evolve in response to antimicrobial treatment is a pressing example of this problem, where discovery of molecular parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in environments containing antibiotics combined with periodic whole population genome sequencing can be used to characterize the evolutionary dynamics of the pathways to antimicrobial resistance. We separately propagated two clinically relevant Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing concentrations of tobramycin in two different environments each: planktonic and biofilm. Independent of the pathogen, populations adapted to tobramycin selection by parallel evolution of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate phosphotransferase. As neither gene is a direct target of this aminoglycoside, both are relatively novel and underreported causes of resistance. Additionally, both species acquired antibiotic-associated mutations that were more prevalent in the biofilm lifestyle than planktonic, in electron transport chain components in A. baumannii and LPS biosynthesis enzymes in P. aeruginosa populations. Using existing databases, we discovered both fusA1 and ptsP mutations to be prevalent in antibiotic resistant clinical isolates. Additionally, we report site-specific parallelism of fusA1 mutations that extend across several bacterial phyla. This study suggests that strong selective pressures such as antibiotic treatment may result in high levels of predictability in molecular targets of evolution despite differences between organisms genetic background and environment.

microbiology↗

bioRxiv2019

Biofilm-dependent evolutionary pathways to antibiotic resistance

Bacterial populations vary in their stress tolerance and population structure depending upon whether growth occurs in well-mixed or structured environments. We hypothesized that evolution in biofilms would generate greater genetic diversity than well-mixed environments and lead to different pathways of antibiotic resistance. We used experimental evolution and whole genome sequencing to test how the biofilm lifestyle influenced the rate, genetic mechanisms, and pleiotropic effects of resistance to ciprofloxacin in Acinetobacter baumannii populations. Both evolutionary dynamics and the identities of mutations differed between lifestyle. Planktonic populations experienced selective sweeps of mutations including the primary topoisomerase drug targets, whereas biofilm-adapted populations acquired mutations in regulators of efflux pumps. An overall trade-off between fitness and resistance level emerged, wherein biofilm-adapted clones were less resistant than planktonic but more fit in the absence of drug. However, biofilm populations developed collateral sensitivity to cephalosporins, demonstrating the clinical relevance of lifestyle on the evolution of resistance.

microbiology↗