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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 with 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.

Background and Objectives 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. Methodology 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 hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results 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. Conclusions and implications The results show how 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.

Recent research on antimicrobial resistance in the environment has emphasized targeted methods. Increased use of non-targeted methods is needed to determine how frequently novel resistance genes arise.

A new Dethiosulfovibrio strain, designated F2BT, was isolated from an anaerobic digester for treating solid waste from a marine recirculating aquaculture system. The motile, Gram-negative, non-spore-forming curved rods were 2–7 µm long and 1 µm in diameter. Growth occurred at temperatures ranging from 20 to 40 °C with a maximum rate of growth at 30 °C. The pH range for growth was pH 6.0–8.0, with a maximum rate of growth at pH 7.5. This isolate was halotolerant growing in NaCl concentrations ranging from 0 to 1.6 M with a maximum rate of growth at 0.4 M. Similarly to the five described Dethiosulfovibrio species, this obligate anaerobe isolate was fermentative, capable of utilizing peptides, amino acids and some organic acids for growth, but unlike described strains in the genus did not reduce thiosulphate or elemental sulphur to hydrogen sulphide during fermentation of organic substrates. The G+C content of 55 mol% is similar to the described Dethiosulfovibrio species. The average nucleotide identity analysis between whole genome sequences showed less than 93.15% sequence similarity between strain F2BT and the five other described Dethiosulfovibrio species. Differences in the physiological and phylogenetic characteristics between the new strain and other Dethiosulfovibrio specied indicate that F2BT represents a novel species of this genus and the epithet Dethiosulfovibrio faecalis sp. nov. is proposed. The type strain is F2BT (=DSM 112078T=KCTC25378T).

The price for renewable electricity is rapidly decreasing, and the availability of such energy is expected to increase in the coming years. This is a welcomed outcome considering that mitigation of climate disruption due to the use of fossil carbon is reaching a critical stage. However, the economy will remain dependent on carbon-based chemicals and the problem of electricity storage persists. Therefore, the development of electrosynthetic processes that convert electricity and CO2 into chemicals and energy dense fuels, perhaps even food, would be desirable. Electrochemistry has been applied to the manufacture of many valuable products and at a large industrial scale, but it is difficult to produce multicarbon chemicals from CO2 by chemistry alone. Being that the biological world possesses expertise at the construction of C-C bonds, it is being examined in conjunction with electrochemistry to discover new ways of synthesizing chemicals from electricity and CO2. One approach is microbial electrosynthesis. This Account describes the development of a microbial electrosynthesis system by the authors. A biocathode consisting of a carbon-based electrode and a microbial community produced short chain fatty acids, primarily acetate. The device works by electrolysis of water, but microbes facilitate electron transfer from the cathode while reducing CO2 by the Wood-Ljungdahl pathway possessed by an Acetobacterium sp. While this acetogenic microorganism dominates the microbiome growing on the cathode surface, 13 total species of microbes overall were ecologically selected on the cathode and genomes for each have been assembled. The combined species may contribute to the stability of the microbiome, a common feature of naturally selected microbial communities. The microbial electrosynthesis system was demonstrated to operate continuously at a cathode for more than 2 years and could also be used with intermittent power, thus demonstrating the stability of the microbiome living at the cathode. In addition to the description of reactor design and startup procedures, the possible mechanisms of electron transfer are described in this Account. While mysteries remain to be solved, much evidence indicates that the microbiome may facilitate electron transfer by supplying catalyst(s) external to the bacterial cells and onto the cathode surface. This may be in the form of a hydrogen-producing catalyst that enhances hydrogen generation by an inert carbon-based electrode. Through the enrichment of the electrosynthetic microbiome along with several modifications in reactor design and operation, the productivity and efficiency were improved. In addition to the intrinsic value of the current products, coupling the process with a secondary stage might be used to produce more valuable products from the acetic acid stream such as lipids, biocrude oil, or higher value food supplements. Alternatively, additional work on the mechanism of electron transfer, reactor design/operation, and modification of the microbes through synthetic biology, particularly to enhance carbon efficiency into higher value chemicals, are the needed next steps to advance microbial electrosynthesis so that it may be used to transform renewable electrons and CO2 directly into products and help solve the problem of climate disruption.

Asian rice is one of the most important crops because it is a staple food for almost half of the world’s 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 plant’s 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.

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.

A strain of Staphylococcus succinus was sampled from the floor of the basement of a house and isolated in an undergraduate classroom in Milwaukee, WI. Here, we report the draft genome sequence of this strain.

A combined approach involving microbial bioaugmentation and enhanced sorption was demonstrated to be effective for in situ treatment of polychlorinated biphenyls (PCBs). A pilot study was conducted for 409 days on PCB impacted sediments in four 400 m2 plots located in a watershed drainage pond in Quantico, VA. Treatments with activated carbon (AC) agglomerate bioamended with PCB dechlorinating and oxidizing bacteria decreased the PCB concentration in the top 7.5 cm by up to 52% and the aqueous concentrations of tri- to nonachlorobiphenyl PCB congeners by as much as 95%. Coplanar congeners decreased by up to 80% in sediment and were undetectable in the porewater. There was no significant decrease in PCB concentrations in non-bioamended plots with or without AC. All homologue groups decreased in bioamended sediment and porewater, indicating that both anaerobic dechlorination and aerobic degradation occurred concurrently. The titer of the bioamendments based on quantitative PCR of functional marker genes dec...

Patient: Male, 21 Final Diagnosis: Bacterial pneumonia Symptoms: Cough • fever Medication: — Clinical Procedure: — Specialty: Critical Care Medicine Objective: Unusual clinical course Background: Severe pneumonia requiring admission to an intensive care unit carries high morbidity and mortality. Evidence-based management includes early administration of empiric antibiotics against plausible bacterial pathogens while awaiting results of microbiologic cultures. However, in over 60% of pneumonia cases, no causative pathogen is identified with conventional diagnostic techniques. In this case report, we demonstrate how direct-from-sample sequencing of bacterial DNA could have identified the multiple culprit pathogens early in the disease course to guide appropriate antibiotic management. Case Report: A previously healthy, 21-year-old man presented with neck pain and fever and rapidly developed acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. He was started on broad-spectrum antibiotics and was found to have septic thrombophlebitis of the left internal jugular vein (Lemierre syndrome), with blood cultures growing Fusobacterium necrophorum. While his antibiotics were narrowed to piperacillin-tazobactam monotherapy, his clinical condition worsened, but repeated efforts to define an additional/alternative respiratory pathogen resulted in negative cultures. He eventually developed bilateral empyemas growing Mycoplasma hominis. Once azithromycin was added to the patient’s regimen, he improved dramatically. Retrospective sequencing of consecutive endotracheal aspirates showed Fusobacterium as the dominant pathogen early in the course, but with significant and increasing Mycoplasma abundance several days prior to clinical detection. Conclusions: Had sequencing information been available to the treating clinicians, the causative pathogens could have been detected earlier, guiding appropriate antibiotic therapy and perhaps preventing his clinical complications. Real-time bacterial DNA sequencing has the potential to shift the diagnostic paradigm in severe 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.

Bacteriophages are rapidly shared among hosts and act as key genetic regulators for establishing chronic infections., 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.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host-restriction. Using an advanced imaging technique (MiPACT-HCR), we found population structure tracked with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa(the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection site biogeography to pathogen evolution.

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.

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 bacteriumPseudomonas aeruginosa. During chronic CF lung infections, a single clone ofP. aeruginosacan 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 whichP. aeruginosaevolves 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 highlightsPseudomonas aeruginosaundergoes adaptive evolution in the sinuses of people with CFOver time, pathoadapted strains display early signatures of genome degradation consistent with recent host restrictionMutations previously thought to occur in CF lungs may have first evolved in sinuses

Acetogens are anaerobic bacteria capable of fixing CO2 or CO to produce acetyl coenzyme A (acetyl-CoA) and ultimately acetate using the Wood-Ljungdahl pathway (WLP). Acetobacterium woodii is the type strain of the Acetobacterium genus and has been critical for understanding the biochemistry and energy conservation in acetogens. 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 on the unique and various metabolisms of the genus. To gain a better appreciation for the metabolic breadth of the genus, we sequenced the genomes of 4 isolates (A. fimetarium, A. malicum, A. paludosum, and A. tundrae) and conducted a comparative genome analysis (pan-genome) of 11 different Acetobacterium genomes. A unifying feature of the Acetobacterium genus is the carbon-fixing WLP. The methyl (cluster II) and carbonyl (cluster III) branches of the Wood-Ljungdahl pathway are highly conserved across all sequenced Acetobacterium genomes, but cluster I encoding the formate dehydrogenase is not. In contrast to A. woodii, all but four strains encode two distinct Rnf clusters, Rnf being the primary respiratory enzyme complex. Metabolism of fructose, lactate, and H2:CO2 was conserved across the genus, but metabolism of ethanol, methanol, caffeate, and 2,3-butanediol varied. Additionally, clade-specific metabolic potential was observed, such as amino acid transport and metabolism in the psychrophilic species, 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. IMPORTANCE 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 and, if harnessed, can help reduce greenhouse gas emissions. Overall, the data presented here provide a framework for examining the ecology and evolution of the Acetobacterium genus and highlight the potential of these species as a source for production of fuels and chemicals from CO2 feedstocks.

We present here the draft genome sequence of a pyridine-degrading bacterium

We present here the draft genome sequence of a pyridine-degrading bacterium, Micrococcus luteus ATCC 49442, which was reclassified as Pseudarthrobacter sp. strain ATCC 49442 based on its draft genome sequence. Its genome length is 4.98 Mbp, with 64.81% GC content.

Here, we report the draft genome sequence of Arthrobacter sp. strain ATCC 49987, consisting of three contigs with a total length of 4.4 Mbp. Based on the genome sequence, we suggest reclassification of Arthrobacter sp. strain ATCC 49987 as Pseudarthrobacter sp. strain ATCC 49987.

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. Previously, we showed that selection of the pathogen Acinetobacter baumannii in both structured and unstructured environments containing the antibiotic ciprofloxacin produced distinct genotypes and phenotypes, with lower resistance in biofilms as well as collateral sensitivity to β-lactam drugs (Santos-Lopez et al., 2019). Here we study how this prior history influences subsequent evolution in new β-lactam antibiotics. Selection was imposed by increasing concentrations of ceftazidime and imipenem 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 revealing important contingencies. A history of selection in structured environments constrained resistance to new drugs and led to frequent loss of resistance to the initial drug by genetic reversions and not compensatory mutations. This research demonstrates that despite strong selective pressures of antibiotics leading to genetic parallelism, history can etch potential vulnerabilities to orthogonal drugs.

TheBurkholderia cepaciacomplex (BCC) is composed of multiple species, includingB. multivoransandB. dolosa,that are significant pathogens for people with cystic fibrosis (CF) and are extensively resistant to many antibiotics. ThefixLgene of thefixLJ2-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 theB. dolosa fixLJsystem 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 infixLJpathway-mediated phenotypes inB. dolosaandB. multivorans.BCC carrying the ancestralfixLsequence are less pathogenic than constructs carrying evolved variants in both a macrophage infection model and a murine pneumonia model.In vitrophospho-transfer experiments demonstrate that the evolvedB. dolosaFixL variants are able to reducefixLJpathway activity by either having lower levels of kinase activity or increased phosphatase activity. Notably, the ancestralfixLgenotype has increased ability to survive within the soil compared to isogenic constructs with evolvedfixLgenotypes, 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.

BACKGROUND: Acinetobacter baumannii is a healthcare-associated pathogen with high rates of carbapenem resistance. Colistin is now routinely used for treatment of infections by this pathogen. However, colistin use has been associated with development of resistance to this agent. OBJECTIVES: To elucidate the phylogenomics of colistin-susceptible and -resistant A. baumannii strain pairs from a cohort of hospitalized patients at a tertiary medical centre in the USA. METHODS: WGS data from 21 pairs of colistin-susceptible and -resistant, XDR clinical strains were obtained and compared using phylogeny of aligned genome sequences, assessment of pairwise SNP differences and gene content. RESULTS: Fourteen patients had colistin-resistant strains that were highly genetically related to their own original susceptible strain with a median pairwise SNP distance of 5.5 (range 1–40 SNPs), while seven other strain pairs were divergent with ≥84 SNP differences. In addition, several strains from different patients formed distinct clusters on the phylogeny in keeping with closely linked transmission chains. The majority of colistin-resistant strains contained non-synonymous mutations within the pmrAB locus suggesting a central role for pmrAB mutations in colistin resistance. Excellent genotype–phenotype correlation was also observed for carbapenems, aminoglycosides and tetracyclines. CONCLUSIONS: The findings suggest that colistin resistance in the clinical setting arises through both in vivo evolution from colistin-susceptible strains and reinfection by unrelated colistin-resistant strains, the latter of which may involve patient-to-patient transmission.

This report describes results of a bench-scale treatability study to evaluate the efficacy of bioaugmentation with bioamended activated carbon (AC) for in situ treatment of polychlorinated biphenyl (PCB) impacted sediments. To this end, the ability of PCB transforming microorganisms to degrade and reduce the overall concentration of PCBs in sediment was determined in 2 L recirculating mesocosms designed to simulate conditions in Abraham’s Creek in Quantico, Virginia. Ten sediment mesocosms were tested for the effects of AC alone, AC with slow release electron donor (cellulose) and different concentrations and combinations of PCB dehalogenating and degrading microorganisms added as bioamendments. A 78% reduction of total PCBs was observed using a cell titer of 5 × 105 Dehalobium chlorocoercia and Paraburkholderia xenovorans cells g–1 sediment with 1.5% AC as a delivery system. Levels of both higher and lower chlorinated congeners were reduced throughout the sediment column indicating that both anaerobic re...

The rise of antimicrobial resistance is a leading medical threat, motivating efforts to forecast both its evolutionary dynamics and its genetic causes. Aminoglycosides are a major class of antibiotics that disrupt translation, but resistance may occur by a number of mechanisms. Here, we show the repeated evolution of resistance to the aminoglycoside tobramycin in both P. aeruginosa and A. baumannii via mutations in fusA1, encoding elongation factor G, and ptsP, encoding the nitrogen-specific phosphotransferase system. Laboratory evolution and whole-population genome sequencing were used to identify these targets, but mutations at identical amino acid positions were also found in published genomes of diverse bacterial species and clinical isolates. We also identified other resistance mechanisms associated with growth in biofilms that likely interfere with drug binding or uptake. Characterizing the evolution of multiple species in the presence of antibiotics can identify new, repeatable causes of resistance that may be predicted and counteracted by alternative treatment., Different species exposed to a common stress may adapt by mutations in shared pathways or in unique systems, depending on how past environments have molded their genomes. Understanding how diverse bacterial pathogens evolve in response to an 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 identify many contending routes 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. Independently of the pathogen, the 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, mutations to either are unexpected and underreported causes of resistance. Additionally, both species acquired antibiotic resistance-associated mutations that were more prevalent in the biofilm lifestyle than in the planktonic lifestyle; these mutations were in electron transport chain components in A. baumannii and lipopolysaccharide biosynthesis enzymes in P. aeruginosa populations. Using existing databases, we discovered site-specific parallelism of fusA1 mutations that extends across bacterial phyla and clinical isolates. 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 backgrounds and environments.

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., eLife digest A bacterium known as Acinetobacter baumannii causes serious lung infections in people with weakened immune systems. These illnesses are becoming more common largely because A. baumannii is increasingly developing resistance to antibiotics. Inside the airways, individual A. baumannii cells can stick together and coat themselves in a slimy substance to form a structure called biofilm, which physically protects bacteria from antibiotics. This may be one of the reasons why it is often harder to treat bacterial infections associated with biofilms. Another possibility is that bacteria may evolve differently in biofilms compared with cells living independently. For example, A. baumannii may colonize several regions of the lungs during an infection, leading to distinct groups of bacteria that experience different conditions and evolve separately. Each population may therefore respond differently to an antibiotic. In contrast, bacteria living independently in a well-mixed population – such as in the bloodstream of their host – would be more likely to all evolve in the same way. Santos-Lopez, Marshall et al. tested this theory by exposing populations of A. baumannii that lived either independently or in biofilms to increasing levels of an antibiotic called ciprofloxacin. The genetic information of these cells was examined as the populations were evolving, and the bacteria were also put in contact with other types of antibiotics. The analyses revealed that bacteria in well-mixed populations shared the same limited number of mutations: these gave the bacteria high levels of resistance to the antibiotic’s primary target, an enzyme involved in DNA processes. The bacteria had also become resistant to other classes of antibiotics. In contrast, the bacteria in biofilm populations evolved to be more genetically diverse, exhibiting different types of mutations that helped the cells to pump out the drug. These bacteria were less resistant to ciprofloxacin and more sensitive to other types of antibiotics. Further experiments looked into the fitness of the bacteria – their ability to survive, reproduce and compete with each other. High levels of antibiotic resistance came with lower fitness: biofilm bacteria had evolved to become being fitter than those from well-mixed population. Even in the absence of drugs, these populations were in fact fitter than the original cells. Overall, understanding how the lifestyles of bacteria affect the way they respond to drugs may help researchers to develop new approaches that limit the spread of antibiotic resistance and improve treatment.

Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes., Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.

We report here the 4.9-Mb genome sequence of a quinoline-degrading bacterium, Rhodococcus sp. strain ATCC 49988. The draft genome data will enable the identification of genes and future genetic modification to enhance traits relevant to heteroaromatic compound degradation.

We report here the 4.9-Mb genome sequence of a quinoline-degrading bacterium, Rhodococcus sp. strain ATCC 49988. The draft genome data will enable the identification of genes and future genetic modification to enhance traits relevant to heteroaromatic compound degradation.

Objective To develop a method to perform multiple tests on single nasopharyngeal (NP) swab. Study design We collected a NP swab on children aged 2–12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA sequencing, which we accomplished by cutting the tip of the swab. Results Of the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121(69.5%) tested positive for a virus. Cytokine measurement, as judged by the adequate levels of a housekeeping enzyme (GAPDH), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2,646 operational taxonomic units across all samples sequenced. Samples used for RNA sequencing had a mean RNA Integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis. Conclusion We describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

Anaerobic ammonium oxidation coupled to iron(III) reduction, termed Feammox, is a newly discovered nitrogen cycling process. However, little is known about the roles of electron shuttles in the Feammox reactions. In this study, two forms of Fe(III) (oxyhydr)oxide ferrihydrite (ex situ ferrihydrite and in situ ferrihydrite) were used in dissimilatory Fe(III) reduction (DIR) enrichments from paddy soil. Evidence for Feammox in DIR enrichments was demonstrated using the (15)N-isotope tracing technique. The extent and rate of both the (30)N2-(29)N2 and Fe(II) formation were enhanced when amended with electron shuttles (either 9,10-anthraquinone-2,6-disulfonate (AQDS) or biochar) and further simulated when these two shuttling compounds were combined. Although the Feammox-associated Fe(III) reduction accounted for only a minor proportion of total Fe(II) formation compared to DIR, it was estimated that the potentially Feammox-mediated N loss (0.13-0.48 mg N L(-1) day(-1)) was increased by 17-340% in the enrichments by the addition of electron shuttles. The addition of electron shuttles led to an increase in the abundance of unclassified Pelobacteraceae, Desulfovibrio, and denitrifiers but a decrease in Geobacter. Overall, we demonstrated a stimulatory effect of electron shuttles on Feammox that led to higher N loss, suggesting that electron shuttles might play a crucial role in Feammox-mediated N loss from soils.

Pathogens experience pressure in an infection to adapt, with selection favoring mutants that persist. Pseudomonas aeruginosa commonly adapts by evolving mutants with hyper-biofilm production that evade clearance. Despite our understanding of the adaptive phenotypes, studying their emergence and dynamics in an infection has proven challenging. Here we used a porcine full-thickness burn wound model of chronic infection to study how mixed strains of P. aeruginosa adaptively evolve. Wounds were infected with six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days post-infection. Rugose small-colony variants (RSCVs) were detected at 3-d and persisted, with the majority evolved from PA14-1. Whole genome sequencing of PA14-1 RSCVs revealed driver mutations exclusively in the wsp pathway. RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) present in the inoculum. The rapid rise of RSCVs to detectable frequencies is evidence of positive selection of the Wsp chemosensory system and suggests that RSCVs may arise earlier in an infection than originally appreciated, to facilitate infection. Given the prevalence of RSCVs in chronic infections, we predict that RSCVs may be a common, early adaptation during infections.

How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance., Microorganisms exist in a diverse ecosystem and have evolved many different mechanisms for sensing and influencing the polymicrobial environment around them, utilizing both diffusible and contact-dependent signals. Contact-dependent growth inhibition (CDI) is one such communication system employed by Gram-negative bacteria. In addition to CDI mediation of growth inhibition, recent studies have demonstrated CDI-mediated control of communal behaviors such as biofilm formation. We postulated that CDI may therefore play an active role in host-pathogen interactions, allowing invading strains to establish themselves at polymicrobial mucosal interfaces through competitive interactions while simultaneously facilitating pathogenic capabilities via CDI-mediated signaling. Here, we show that Pseudomonas aeruginosa produces two CDI systems capable of mediating competition under conditions of growth on a surface or in liquid. Furthermore, we demonstrated a novel role for these systems in contributing to virulence in acute infection models, likely via posttranscriptional regulation of beneficial behaviors. While we did not observe any role for the P. aeruginosa CDI systems in biofilm biogenesis, we did identify for the first time robust CDI-mediated competition during interaction with a mammalian host using a model of chronic respiratory tract infection, as well as evidence that CDI expression is maintained in chronic lung infections. These findings reveal a previously unappreciated role for CDI in host-pathogen interactions and emphasize their importance during infection. IMPORTANCE How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance.

Despite its importance, anammox (anaerobic ammonium oxidation) in estuarine sediment systems remains poorly understood, particularly at the continental scale. This study aimed to understand the abundance, diversity, and activity of anammox bacteria and to determine the main factors influencing the anammox process in estuarine sediments in China. Estuarine sediments were collected from 18 estuaries spanning over 4000 km. Experiments using an 15 N–tracer, quantitative PCR, and clone library construction were used to determine the activity, abundance, and diversity of anammox bacteria. The impact of environmental factors on anammox processes was also determined. The abundance of the anammox-specific hydrazine synthase (hzsB) gene ranged from 1.8 × 105 ± 3.4 × 104 to 3.6 × 108 ± 7.5 × 107 copies g−1 dw. Candidatus Scalindua, Brocadia, Kuenenia, Jettenia, and two novel unidentified clusters were detected, with Scalindua dominating the anammox population. Additionally, the abundances of Scalindua, Kuenenia, and Brocadia were found to be significantly correlated with latitude. The anammox rates ranged from 0.29 ± 0.15 to 13.68 ± 3.98 nmol N g−1 dw h−1 and contributed to 2.39–82.61% of total N2 production. Pearson correlation analysis revealed that the anammox rate was positively correlated with total nitrogen, total carbon, and temperature, and was negatively correlated with dissolved oxygen (DO). The key factors influencing the hzsB gene abundance were ammonium concentration, salinity, and DO. Ammonium concentration, pH, temperature, and latitude were main variables shaping the anammox-associated bacterial community. Our results suggested that anammox bacteria are ubiquitous in coastal estuaries in China and underline the importance of anammox resulting in N loss at a continental scale.