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When examining bacterial genomes for evidence of past selection, the results depend heavily on the mutational distance between chosen genomes. Even within a bacterial species, genomes separated by larger mutational distances exhibit stronger evidence of purifying selection as assessed by d N /d S , the normalized ratio of nonsynonymous to synonymous mutations. Here, we show that the classical interpretation of this scale dependence, weak purifying selection, leads to problematic mutation accumulation when applied to available gut microbiome data. We propose an alternative, adaptive reversion model with opposite implications for dynamical intuition and applications of d N /d S . Reversions that occur and sweep within-host populations are nearly guaranteed in microbiomes due to large population sizes, short generation times, and variable environments. Using analytical and simulation approaches, we show that adaptive reversion can explain the d N /d S decay given only dozens of locally fluctuating selective pressures, which is realistic in the context of Bacteroides genomes. The success of the adaptive reversion model argues for interpreting low values of d N /d S obtained from long timescales with caution as they may emerge even when adaptive sweeps are frequent. Our work thus inverts the interpretation of an old observation in bacterial evolution, illustrates the potential of mutational reversions to shape genomic landscapes over time, and highlights the importance of studying bacterial genomic evolution on short timescales., Competing Interests: PT, TL No competing interests declared, (© 2024, Torrillo and Lieberman.)

When examining bacterial genomes for evidence of past selection, the results obtained depend heavily on the mutational distance between chosen genomes. Even within a bacterial species, genomes separated by larger mutational distances exhibit stronger evidence of purifying selection as assessed by d N / d S , the normalized ratio of nonsynonymous to synonymous mutations. Here, we show that the classical interpretation of this scale-dependence, weak purifying selection, leads to problematic mutation accumulation when applied to available gut microbiome data. We propose an alternative, adaptive reversion model with exactly opposite implications for dynamical intuition and applications of d N / d S . Reversions that occur and sweep within-host populations are nearly guaranteed in microbiomes due to large population sizes, short generation times, and variable environments. Using analytical and simulation approaches, we show that adaptive reversion can explain the d N / d S decay given only dozens of locally-fluctuating selective pressures, which is realistic in the context of Bacteroides genomes. The success of the adaptive reversion model argues for interpreting low values of d N / d S obtained from long-time scales with caution, as they may emerge even when adaptive sweeps are frequent. Our work thus inverts the interpretation of an old observation in bacterial evolution, illustrates the potential of mutational reversions to shape genomic landscapes over time, and highlights the importance of studying bacterial genomic evolution on short time scales.

The skin microbiome can both trigger beneficial immune stimulation and pose a potential infection threat. Previous studies have shown that colonization of mouse skin with the model human skin commensal Staphylococcus epidermidis is protective against subsequent excisional wound or pathogen challenge. However, less is known about concurrent skin damage and exposure to commensal microbes, despite growing interest in interventional probiotic therapy. In this study, we address this open question by applying commensal skin bacteria at a high dose to abraded skin. Although depletion of the skin microbiome through antibiotics delayed repair from damage, probiotic-like application of commensals-including the mouse commensal Staphylococcus xylosus, 3 distinct isolates of S. epidermidis, and all other tested human skin commensals-also significantly delayed barrier repair. Increased inflammation was observed within 4 hours of S. epidermidis exposure and persisted through day 4, at which point the skin displayed a chronic wound-like inflammatory state with increased neutrophil infiltration, increased fibroblast activity, and decreased monocyte differentiation. Transcriptomic analysis suggested that the prolonged upregulation of early canonical proliferative pathways inhibited the progression of barrier repair. These results highlight the nuanced role of members of the skin microbiome in modulating barrier integrity and indicate the need for caution in their development as probiotics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The opportunistic pathogen Staphylococcus aureus frequently colonizes the inflamed skin of people with atopic dermatitis (AD) and worsens disease severity by promoting skin damage. Here, we show, by longitudinally tracking 23 children treated for AD, that S. aureus adapts via de novo mutations during colonization. Each patient's S. aureus population is dominated by a single lineage, with infrequent invasion by distant lineages. Mutations emerge within each lineage at rates similar to those of S. aureus in other contexts. Some variants spread across the body within months, with signatures of adaptive evolution. Most strikingly, mutations in capsule synthesis gene capD underwent parallel evolution in one patient and across-body sweeps in two patients. We confirm that capD negativity is more common in AD than in other contexts, via reanalysis of S. aureus genomes from 276 people. Together, these findings highlight the importance of the mutation level when dissecting the role of microbes in complex disease., Competing Interests: Declaration of interests The lab of T.D.L. has received funding from Colgate PalmOlive for studies following this work. The lab of I.M.C. has received sponsored research work from Abbvie pharmaceuticals., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Infections are relatively rare following cutaneous surgical procedures, despite the potential for wound exposure to pathogens both during surgery and throughout the healing process. Although gut commensals are believed to reduce the risk of intestinal infections, an analogous role for skin commensals has not been described. In fact, the microbiome of normally healing surgical skin wounds has not yet been profiled using culture-independent techniques. We characterized the wound microbiome in 53 patients who underwent skin cancer surgery and healed without signs or symptoms of infection. A week after surgery, several bacterial species displayed significant differences in relative abundance when compared to control, nonoperated skin from the same patient. The relative abundance of the most common bacterium found on intact skin, Cutibacterium acnes, was reduced in wounds 5-fold. Staphylococcus aureus, a frequent cause of postoperative skin infections, was enriched 6.4-fold in clinically noninfected wounds, suggesting active suppression of pathogenicity. Finally, members of the Corynebacterium genus were the dominant organism in postoperative wounds, making up 37% of the average wound microbiome. The enrichment of these bacteria in normally healing wounds suggests that they might be capable of providing colonization resistance. Future studies focused on the biological and clinical significance of the wound microbiome may shed light on normal wound healing and potential therapeutic opportunities to mitigate infection risk. IMPORTANCE Commensal bacteria on skin may limit the ability of pathogenic bacteria to cause clinically significant infections. The bacteria on healing acute wounds, which might provide such a protective effect, have not been described using culture-independent approaches in the absence of antibiotics. We compare the microbiome of wounds a week after skin cancer removal surgery with intact skin from the same patient. We find that the potentially pathogenic species S. aureus is common on these healing wounds despite the absence of symptoms or signs of infection. We report that bacteria often considered as potential skin probiotics, including Staphylococcus epidermidis, do not reach high relative abundance in wound microbiomes. In contrast, specific members of the Corynebacterium genus, rarely associated with infections, were significantly enriched in healing wounds compared to intact skin. Future work is needed to see if Corynebacterium species or derivatives thereof could be employed to lower the risk of wound infection.

The human microbiome harbours a large capacity for within-person adaptive mutations. Commensal bacterial strains can stably colonize a person for decades, and billions of mutations are generated daily within each person's microbiome. Adaptive mutations emerging during health might be driven by selective forces that vary across individuals, vary within an individual, or are completely novel to the human population. Mutations emerging within individual microbiomes might impact the immune system, the metabolism of nutrients or drugs, and the stability of the community to perturbations. Despite this potential, relatively little attention has been paid to the possibility of adaptive evolution within complex human-associated microbiomes. This review discusses the promise of studying within-microbiome adaptation, the conceptual and technical limitations that may have contributed to an underappreciation of adaptive de novo mutations occurring within microbiomes to date, and methods for detecting recent adaptive evolution. This article is part of a discussion meeting issue 'Genomic population structures of microbial pathogens'.

What enables strains of the same species to coexist in a microbiome? Here, we investigate whether host anatomy can explain strain co-residence of Cutibacterium acnes, the most abundant species on human skin. We reconstruct on-person evolution and migration using whole-genome sequencing of C. acnes colonies acquired from healthy subjects, including from individual skin pores, and find considerable spatial structure at the level of pores. Although lineages (sets of colonies separated by <100 mutations) with in vitro fitness differences coexist within centimeter-scale regions, each pore is dominated by a single lineage. Moreover, colonies from a pore typically have identical genomes. An absence of adaptive signatures suggests a genotype-independent source of low within-pore diversity. We therefore propose that pore anatomy imposes random single-cell bottlenecks; the resulting population fragmentation reduces competition and promotes coexistence. Our findings suggest that therapeutic interventions involving pore-dwelling species might focus on removing resident populations over optimizing probiotic fitness., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Atopic dermatitis (AD) is characterized by an altered skin microbiome dominantly colonized by S. aureus . Standard treatment includes emollients, anti-inflammatory medications and antiseptics., Objectives: To characterize changes in the skin microbiome during treatment for AD., Methods: The skin microbiomes of children with moderate-to-severe AD and healthy children were investigated in a longitudinal prospective study. Patients with AD were randomized to receive either standard treatment with emollients and topical corticosteroids or standard treatment with the addition of dilute bleach baths (DBB) and sampled at four visits over a three-month period. At each visit, severity of AD was measured, swabs were taken from four body sites and the composition of the microbiome at those sites was assessed using 16S rRNA amplification., Results: We included 14 healthy controls and 28 patients. We found high relative abundances of S. aureus in patients, which correlated with AD severity and reduced apparent alpha diversity. As disease severity improved with treatment, the abundance of S. aureus decreased, gradually becoming more similar to the microbiomes of healthy controls. After treatment, patients who received DBB had a significantly lower abundance of S. aureus than those who received only standard treatment., Conclusions: There are clear differences in the skin microbiome of healthy controls and AD patients that diminish with treatment. After three months, the addition of DBB to standard treatment had significantly decreased the S. aureus burden, supporting its use as a therapeutic option. Further study in double-blinded trials is needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Khadka, Key, Romo-González, Martínez-Gayosso, Campos-Cabrera, Gerónimo-Gallegos, Lynn, Durán-McKinster, Coria-Jiménez, Lieberman and García-Romero.)

Analysis of 772 complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from early in the Boston-area epidemic revealed numerous introductions of the virus, a small number of which led to most cases. The data revealed two superspreading events. One, in a skilled nursing facility, led to rapid transmission and significant mortality in this vulnerable population but little broader spread, whereas other introductions into the facility had little effect. The second, at an international business conference, produced sustained community transmission and was exported, resulting in extensive regional, national, and international spread. The two events also differed substantially in the genetic variation they generated, suggesting varying transmission dynamics in superspreading events. Our results show how genomic epidemiology can help to understand the link between individual clusters and wider community spread., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Our understanding of how the gut microbiome interacts with its human host has been restrained by limited access to longitudinal datasets to examine stability and dynamics, and by having only a few isolates to test mechanistic hypotheses. Here, we present the Broad Institute-OpenBiome Microbiome Library (BIO-ML), a comprehensive collection of 7,758 gut bacterial isolates paired with 3,632 genome sequences and longitudinal multi-omics data. We show that microbial species maintain stable population sizes within and across humans and that commonly used 'omics' survey methods are more reliable when using averages over multiple days of sampling. Variation of gut metabolites within people over time is associated with amino acid levels, and differences across people are associated with differences in bile acids. Finally, we show that genomic diversification can be used to infer eco-evolutionary dynamics and in vivo selection pressures for strains within individuals. The BIO-ML is a unique resource designed to enable hypothesis-driven microbiome research.

Natural selection shapes bacterial evolution in all environments. However, the extent to which commensal bacteria diversify and adapt within the human gut remains unclear. Here, we combine culture-based population genomics and metagenomics to investigate the within-microbiome evolution of Bacteroides fragilis. We find that intra-individual B. fragilis populations contain substantial de novo nucleotide and mobile element diversity, preserving years of within-person history. This history reveals multiple signatures of within-person adaptation, including parallel evolution in sixteen genes. Many of these genes are implicated in cell-envelope biosynthesis and polysaccharide utilization. Tracking evolutionary trajectories using near-daily metagenomic sampling, we find evidence for years-long coexistence in one subject despite adaptive dynamics. We used public metagenomes to investigate one adaptive mutation common in our cohort and found that it emerges frequently in Western, but not Chinese, microbiomes. Collectively, these results demonstrate that B. fragilis adapts within individual microbiomes, pointing to factors that promote long-term gut colonization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rational microbiome-based therapies may one day treat a wide range of diseases and promote wellness. Yet, we are still limited in our abilities to employ such therapies and to predict which bacterial strains have the potential to stably colonize a person. The Lieberman laboratory is working to close this knowledge gap and to develop an understanding of how individual species and strains behave in the human microbiome, including with regard to their niche ranges, survival strategies, and the degree to which they adapt to individual people. We employ system-level approaches, with a particular emphasis on using de novo mutations and evolutionary inference to reconstruct the history of bacterial lineages within individuals., Competing Interests: Conflict of Interest Disclosures: T.D.L. has nothing to disclose. Conflict of Interest Disclosures: T.D.L. has nothing to disclose.

Bacterial populations diversify during infection into distinct subpopulations that coexist within the human body. Yet, it is unknown to what extent subpopulations adapt to location-specific selective pressures as they migrate and evolve across space. Here we identify bacterial genes under local and global selection by testing for spatial co-occurrence of adaptive mutations. We sequence 552 genomes of the pathogen Stenotrophomonas maltophilia across 23 sites of the lungs from a patient with cystic fibrosis. We show that although genetically close isolates colocalize in space, distant lineages with distinct phenotypes separated by adaptive mutations spread throughout the lung, suggesting global selective pressures. Yet, for one gene (a distant homologue of the merC gene implicated in metal resistance), mutations arising independently in two lineages colocalize in space, providing evidence for location-specific selection. Our work presents a general framework for understanding how selection acts upon a pathogen that colonizes and evolves across the complex environment of the human body.

Mycobacterium tuberculosis remains a leading cause of death worldwide, especially among individuals infected with HIV. Whereas phylogenetic analysis has revealed M. tuberculosis spread throughout history and in local outbreaks, much less is understood about its dissemination within the body. Here we report genomic analysis of 2,693 samples collected post mortem from lung and extrapulmonary biopsies of 44 subjects in KwaZulu-Natal, South Africa, who received minimal antitubercular treatment and most of whom were HIV seropositive. We found that purifying selection occurred within individual patients, without the need for patient-to-patient transmission. Despite negative selection, mycobacteria diversified within individuals to form sublineages that co-existed for years. These sublineages, as well as distinct strains from mixed infections, were differentially distributed throughout the lung, suggesting temporary barriers to pathogen migration. As a consequence, samples taken from the upper airway often captured only a fraction of the population diversity, challenging current methods of outbreak tracing and resistance diagnostics. Phylogenetic analysis indicated that dissemination from the lungs to extrapulmonary sites was as frequent as between lung sites, supporting the idea of similar migration routes within and between organs, at least in subjects with HIV. Genomic diversity therefore provides a record of pathogen diversification and repeated dissemination across the body.

We developed a competition-based screening strategy to identify compounds that invert the selective advantage of antibiotic resistance. Using our assay, we screened over 19,000 compounds for the ability to select against the TetA tetracycline-resistance efflux pump in Escherichia coli and identified two hits, β-thujaplicin and disulfiram. Treating a tetracycline-resistant population with β-thujaplicin selects for loss of the resistance gene, enabling an effective second-phase treatment with doxycycline., Competing Interests: The authors have no competing financial interests.

A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)-plate, in which bacteria spread and evolved on a large antibiotic landscape (120 × 60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics., (Copyright © 2016, American Association for the Advancement of Science.)

Hybrid drugs are a promising strategy to address the growing problem of drug resistance, but the mechanism by which they modulate the evolution of resistance is poorly understood. Integrating high-throughput resistance measurements and genomic sequencing, we compared Escherichia coli populations evolved in a hybrid antibiotic that links ciprofloxacin and neomycin B with populations evolved in combinations of the component drugs. We find that populations evolved in the hybrid gain less resistance than those evolved in an equimolar mixture of the hybrid's components, in part because the hybrid evades resistance mediated by the multiple antibiotic resistance (mar) operon. Furthermore, we find that the ciprofloxacin moiety of the hybrid inhibits bacterial growth whereas the neomycin B moiety diminishes the effectiveness of mar activation. More generally, comparing the phenotypic and genotypic paths to resistance across different drug treatments can pinpoint unique properties of new compounds that limit the emergence of resistance., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

Alternating antibiotic therapy, in which pairs of drugs are cycled during treatment, has been suggested as a means to inhibit the evolution of de novo resistance while avoiding the toxicity associated with more traditional combination therapy. However, it remains unclear under which conditions and by what means such alternating treatments impede the evolution of resistance. Here, we tracked multistep evolution of resistance in replicate populations of Staphylococcus aureus during 22 d of continuously increasing single-, mixed-, and alternating-drug treatment. In all three tested drug pairs, the alternating treatment reduced the overall rate of resistance by slowing the acquisition of resistance to one of the two component drugs, sometimes as effectively as mixed treatment. This slower rate of evolution is reflected in the genome-wide mutational profiles; under alternating treatments, bacteria acquire mutations in different genes than under corresponding single-drug treatments. To test whether this observed constraint on adaptive paths reflects trade-offs in which resistance to one drug is accompanied by sensitivity to a second drug, we profiled many single-step mutants for cross-resistance. Indeed, the average cross-resistance of single-step mutants can help predict whether or not evolution was slower in alternating drugs. Together, these results show that despite the complex evolutionary landscape of multidrug resistance, alternating-drug therapy can slow evolution by constraining the mutational paths toward resistance.

Advances in sequencing technologies have enabled the identification of mutations acquired by bacterial pathogens during infection. However, it remains unclear whether adaptive mutations fix in the population or lead to pathogen diversification within the patient. Here we study the genotypic diversity of Burkholderia dolosa within individuals with cystic fibrosis by resequencing individual colonies and whole populations from single sputum samples. We find extensive intrasample diversity, suggesting that mutations rarely fix in a patient's pathogen population--instead, diversifying lineages coexist for many years. Under strong selection, multiple adaptive mutations arise, but none of these sweep to fixation, generating lasting allele diversity that provides a recorded signature of past selection. Genes involved in outer-membrane components, iron scavenging and antibiotic resistance all showed this signature of within-patient selection. These results offer a general and rapid approach for identifying the selective pressures acting on a pathogen in individual patients based on single clinical samples.

Background: Flux Balance Analysis is a theoretically elegant, computationally efficient, genome-scale approach to predicting biochemical reaction fluxes. Yet FBA models exhibit persistent mathematical degeneracy that generally limits their predictive power., Results: We propose a novel objective function for cellular metabolism that accounts for and exploits degeneracy in the metabolic network to improve flux predictions. In our model, regulation drives metabolism toward a region of flux space that allows nearly optimal growth. Metabolic mutants deviate minimally from this region, a function represented mathematically as a convex cone. Near-optimal flux configurations within this region are considered equally plausible and not subject to further optimizing regulation. Consistent with relaxed regulation near optimality, we find that the size of the near-optimal region predicts flux variability under experimental perturbation., Conclusion: Accounting for suboptimal solutions can improve the predictive power of metabolic FBA models. Because fluctuations of enzyme and metabolite levels are inevitable, tolerance for suboptimality may support a functionally robust metabolic network.

Background: New therapeutic targets for antibiotic-resistant bacterial pathogens are desperately needed. The bacterial surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG) mediates biofilm formation by some bacterial species, and antibodies to PNAG can confer protective immunity. By analyzing sequenced genomes, we found that potentially multidrug-resistant bacterial species such as Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex (BCC) may be able to produce PNAG. Among patients with cystic fibrosis patients, highly antibiotic-resistant bacteria in the BCC have emerged as problematic pathogens, providing an impetus to study the potential of PNAG to be targeted for immunotherapy against pan-resistant bacterial pathogens., Methods: The presence of PNAG on BCC was assessed using a combination of bacterial genetics, microscopy, and immunochemical approaches. Antibodies to PNAG were tested using opsonophagocytic assays and for protective efficacy against lethal peritonitis in mice., Results: PNAG is expressed in vitro and in vivo by the BCC, and cystic fibrosis patients infected by the BCC species B. dolosa mounted a PNAG-specific opsonophagocytic antibody response. Antisera to PNAG mediated opsonophagocytic killing of BCC and were protective against lethal BCC peritonitis even during coinfection with methicillin-resistant Staphylococcus aureus., Conclusions: Our findings raise potential new therapeutic options against PNAG-producing bacteria, including even pan-resistant pathogens.

Bacterial pathogens evolve during the infection of their human host(1-8), but separating adaptive and neutral mutations remains challenging(9-11). Here we identify bacterial genes under adaptive evolution by tracking recurrent patterns of mutations in the same pathogenic strain during the infection of multiple individuals. We conducted a retrospective study of a Burkholderia dolosa outbreak among subjects with cystic fibrosis, sequencing the genomes of 112 isolates collected from 14 individuals over 16 years. We find that 17 bacterial genes acquired nonsynonymous mutations in multiple individuals, which indicates parallel adaptive evolution. Mutations in these genes affect important pathogenic phenotypes, including antibiotic resistance and bacterial membrane composition and implicate oxygen-dependent regulation as paramount in lung infections. Several genes have not previously been implicated in pathogenesis and may represent new therapeutic targets. The identification of parallel molecular evolution as a pathogen spreads among multiple individuals points to the key selection forces it experiences within human hosts.

Background: The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner., Results: We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages., Conclusion: Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices.