The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Plasmids affect microindel mutations in Acinetobacter baylyi ADP1.

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can affect the level of microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid vector pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, results with the IncP plasmid vector pRK415 (theta replication mechanism) show that the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study (belonging to IncA/C2, IncW, pBBR incompatibility groups) confer ambiguous or no quantifiable mutagenic effects.

Piggybacking on Niche Adaptation Improves the Maintenance of Multidrug-Resistance Plasmids.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made toward resolving this "plasmid paradox" but mainly in a nonclinical context. Here, we propose an additional explanation for the maintenance of multidrug-resistance plasmids in clinical Escherichia coli strains. After coevolving two multidrug-resistance plasmids encoding resistance to last resort carbapenems with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (carbon catabolite repression) and ArcAB (aerobic respiration control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional rewiring during bacterial niche adaptation may facilitate plasmid maintenance.

Tn1 transposition in the course of natural transformation enables horizontal antibiotic resistance spread in Acinetobacter baylyi.

Transposons are genetic elements that change their intracellular genomic position by transposition and are spread horizontally between bacteria when located on plasmids. It was recently discovered that transposition from fully heterologous DNA also occurs in the course of natural transformation. Here, we characterize the molecular details and constraints of this process using the replicative transposon Tn1 and the naturally competent bacterium Acinetobacter baylyi. We find that chromosomal insertion of Tn1 by transposition occurs at low but detectable frequencies and preferably around the A. baylyi terminus of replication. We show that Tn1 transposition is facilitated by transient expression of the transposase and resolvase encoded by the donor DNA. RecA protein is essential for the formation of a circular, double-stranded cytoplasmic intermediate from incoming donor DNA, and RecO is beneficial but not essential in this process. Absence of the recipient RecBCD nuclease stabilizes the double-stranded intermediate. Based on these results, we suggest a mechanistic model for transposition during natural transformation.

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates.

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal.

Low biological cost of carbapenemase-encoding plasmids following transfer from Klebsiella pneumoniae to Escherichia coli.

OBJECTIVES: The objective of this study was to determine the biological cost, stability and sequence of two carbapenemase-encoding plasmids containing blaKPC-2 (pG12-KPC-2) and blaVIM-1 (pG06-VIM-1) isolated from Klebsiella pneumoniae when newly acquired by uropathogenic Escherichia coli clinical isolates of different genetic backgrounds. METHODS: The two plasmids were transferred into plasmid-free E. coli clinical isolates by transformation. The fitness effect of newly acquired plasmids on the host cell was assessed in head-to-head competitions with the corresponding isogenic strain. Plasmid stability was estimated by propagating monocultures for ∼312 generations. Plasmid nucleotide sequences were determined using next-generation sequencing technology. Assembly, gap closure, annotation and comparative analyses were performed. RESULTS: Both plasmids were stably maintained in three of four E. coli backgrounds and resulted in low to moderate reductions in host fitness ranging from 1.1% to 3.6%. A difference in fitness cost was observed for pG12-KPC-2 between two different genetic backgrounds, while no difference was detected for pG06-VIM-1 between three different genetic backgrounds. In addition, a difference was observed between pG12-KPC-2 and pG06-VIM-1 in the same genetic background. In general, the magnitude of biological cost of plasmid carriage was both host and plasmid dependent. The sequences of the two plasmids showed high backbone similarity to previously circulating plasmids in K. pneumoniae. CONCLUSIONS: The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Costs and benefits of natural transformation in Acinetobacter baylyi.

BACKGROUND: Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. RESULTS: We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. CONCLUSIONS: Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.

CO chemisorption at vacancies of supported graphene films: a candidate for a sensor?

We investigate CO adsorption at single vacancies of graphene supported on Ni(111) and polycrystalline Cu. The borders of the vacancies are chemically inert but, on the reactive Ni(111) substrate, CO intercalation occurs. Adsorbed CO dissociates at 380 K, leading to carbide formation and mending of the vacancies, thus preventing their effectiveness in sensor applications.

The Long-Term Multicenter Observational Study of Dabigatran Treatment in Patients With Atrial Fibrillation (RELY-ABLE) Study.

BACKGROUND: During follow-up of between 1 and 3 years in the Randomized Evaluation of Long-term Anticoagulation Therapy (RE-LY) trial, 2 doses of dabigatran etexilate were shown to be effective and safe for the prevention of stroke or systemic embolism in patients with atrial fibrillation. There is a need for longer-term follow-up of patients on dabigatran and for further data comparing the 2 dabigatran doses. METHODS AND RESULTS: Patients randomly assigned to dabigatran in RE-LY were eligible for the Long-term Multicenter Extension of Dabigatran Treatment in Patients with Atrial Fibrillation (RELY-ABLE) trial if they had not permanently discontinued study medication at the time of their final RE-LY study visit. Enrolled patients continued to receive the double-blind dabigatran dose received in RE-LY, for up to 28 months of follow up after RE-LY (median follow-up, 2.3 years). There were 5851 patients enrolled, representing 48% of patients originally randomly assigned to receive dabigatran in RE-LY and 86% of RELY-ABLE-eligible patients. Rates of stroke or systemic embolism were 1.46% and 1.60%/y on dabigatran 150 and 110 mg twice daily, respectively (hazard ratio, 0.91; 95% confidence interval, 0.69-1.20). Rates of major hemorrhage were 3.74% and 2.99%/y on dabigatran 150 and 110 mg (hazard ratio, 1.26; 95% confidence interval, 1.04-1.53). Rates of death were 3.02% and 3.10%/y (hazard ratio, 0.97; 95% confidence interval, 0.80-1.19). Rates of hemorrhagic stroke were 0.13% and 0.14%/y. CONCLUSIONS: During 2.3 years of continued treatment with dabigatran after RE-LY, there was a higher rate of major bleeding with dabigatran 150 mg twice daily in comparison with 110 mg, and similar rates of stroke and death.

Natural transformation facilitates transfer of transposons, integrons and gene cassettes between bacterial species.

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.

A trade-off between the fitness cost of functional integrases and long-term stability of integrons.

Horizontal gene transfer (HGT) plays a major role in bacterial microevolution as evident from the rapid emergence and spread of antimicrobial drug resistance. Few studies have however addressed the population dynamics of newly imported genetic elements after HGT. Here, we show that newly acquired class-1 integrons from Salmonella enterica serovar Typhimurium and Acinetobacter baumannii, free of associated transposable elements, strongly reduce host fitness in Acinetobacter baylyi. Insertional inactivation of the integron intI1 restored fitness, demonstrating that the observed fitness costs were due to the presence of an active integrase. The biological cost of harboring class-1 integrons was rapidly reduced during serial transfers due to intI1 frameshift mutations leading to inactivated integrases. We use a mathematical model to explore the conditions where integrons with functional integrases are maintained and conclude that environmental fluctuations and episodic selection is necessary for the maintenance of functional integrases. Taken together, the presented data suggest a trade-off between the ability to capture gene cassettes and long-term stability of integrons and provide an explanation for the frequent observation of inactive integron-integrases in bacterial populations.

Gene transfer potential of outer membrane vesicles of Acinetobacter baylyi and effects of stress on vesiculation.

Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10(-6) to 10(-8), with transfer efficiencies of approximately 10(3) and 10(2) per µg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.

Stature in Holocene foragers of North India.

The Ganga Plain of North India provides an archaeological and skeletal record of semi-nomadic Holocene foragers in association with an aceramic Mesolithic culture. Prior estimates of stature for Mesolithic Lake Cultures (MLC) used inappropriate equations from an American White reference group and need revision. Attention is given to intralimb body proportions and geo-climatic provenance of MLC series in considering the most suitable reference population. Regression equations from ancient Egyptians are used in reconstructing stature for MLC skeletal series from Damdama (DDM), Mahadaha (MDH), and Sarai Nahar Rai (SNR). Mean stature is estimated at between 174 (MDH) and 178 cm (DDM and SNR) for males, and between 163 cm (MDH) and 179 cm (SNR) for females. Stature estimates based on ancient Egyptian equations are significantly shorter (from 3.5 to 7.1 cm shorter in males; from 3.2 to 7.5 cm shorter in females) than estimates using the American White reference group. Revised stature estimates from tibia length and from femur + tibia more accurately estimate MLC stature for two reasons: a) these elements are highly correlated with stature and have lower standard estimates of error, and b) uncertainty regarding methods of measuring tibia length is avoided. When compared with Holocene samples of native Americans and Mesolithic Europeans, MLC series from North India are tall. This aspect of their biological variation confirms earlier assessments and results from the synergistic influence of balanced nutrition from broad-spectrum foraging, body-proportions adapted to a seasonally hot and arid climate, and the functional demands of a mobile, semi-nomadic life-style.

The Biofilm Lifestyle Shapes the Evolution of ß-Lactamases.

The evolutionary relationship between the biofilm lifestyle and antibiotic resistance enzymes remains a subject of limited understanding. Here, we investigate how ß-lactamases affect biofilm formation in Vibrio cholerae and how selection for a biofilm lifestyle impacts the evolution of these enzymes. Genetically diverse ß-lactamases expressed in V. cholerae displayed a strong inhibitory effect on biofilm production. To understand how natural evolution affects this antagonistic pleiotropy, we randomly mutagenized a ß-lactamase and selected for elevated biofilm formation. Our results revealed that biofilm evolution selects for ß-lactamase variants able to hydrolyze ß-lactams without inhibiting biofilms. Mutational analysis of evolved variants demonstrated that restoration of biofilm development was achieved either independently of enzymatic function or by actively leveraging enzymatic activity. Taken together, the biofilm lifestyle can impose a profound selective pressure on antimicrobial resistance enzymes. Shedding light on such evolutionary interplays is of importance to understand the factors driving antimicrobial resistance.

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a ß-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

Modulation of multidrug-resistant clone success in Escherichia coli populations: a longitudinal, multi-country, genomic and antibiotic usage cohort study.

BACKGROUND: The effect of antibiotic usage on the success of multidrug-resistant (MDR) clones in a population remains unclear. With this genomics-based molecular epidemiology study, we aimed to investigate the contribution of antibiotic use to Escherichia coli clone success, relative to intra-strain competition for colonisation and infection. METHODS: We sequenced all the available E coli bloodstream infection isolates provided by the British Society for Antimicrobial Chemotherapy (BSAC) from 2012 to 2017 (n=718) and combined these with published data from the UK (2001-11; n=1090) and Norway (2002-17; n=3254). Defined daily dose (DDD) data from the European Centre for Disease Prevention and Control (retrieved on Sept 21, 2021) for major antibiotic classes (ß-lactam, tetracycline, macrolide, sulfonamide, quinolone, and non-penicillin ß-lactam) were used together with sequence typing, resistance profiling, regression analysis, and non-neutral Wright-Fisher simulation-based modelling to enable systematic comparison of resistance levels, clone success, and antibiotic usage between the UK and Norway. FINDINGS: Sequence type (ST)73, ST131, ST95, and ST69 accounted for 892 (49·3%) of 1808 isolates in the BSAC collection. In the UK, the proportion of ST69 increased between 2001-10 and 2011-17 (p=0·0004), whereas the proportions of ST73 and ST95 did not vary between periods. ST131 expanded quickly after its emergence in 2003 and its prevalence remained consistent throughout the study period (apart from a brief decrease in 2009-10). The extended-spectrum ß-lactamase (ESBL)-carrying, globally disseminated MDR clone ST131-C2 showed overall greater success in the UK (154 [56·8%] of 271 isolates in 2003-17) compared with Norway (51 [18·3%] of 278 isolates in 2002-17; p<0·0001). DDD data indicated higher total use of antimicrobials in the UK, driven mainly by the class of non-penicillin ß-lactams, which were used between 2·7-times and 5·1-times more in the UK per annum (ratio mean 3·7 [SD 0·8]). This difference was associated with the higher success of the MDR clone ST131-C2 (pseudo-R2 69·1%). A non-neutral Wright-Fisher model replicated the observed expansion of non-MDR and MDR sequence types under higher DDD regimes. INTERPRETATION: Our study indicates that resistance profiles of contemporaneously successful clones can vary substantially, warranting caution in the interpretation of correlations between aggregate measures of resistance and antibiotic usage. Our study further suggests that in countries with low-to-moderate use of antibiotics, such as the UK and Norway, the extent of non-penicillin ß-lactam use modulates rather than determines the success of widely disseminated MDR ESBL-carrying E coli clones. Detailed understanding of underlying causal drivers of success is important for improved control of resistant pathogens. FUNDING: Trond Mohn Foundation, Marie Sklodowska-Curie Actions, European Research Council, Royal Society, and Wellcome Trust.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. METHODS: Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80-200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. RESULTS: The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%-27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. CONCLUSIONS: Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid-host association can rapidly emerge.

Generativity, hierarchical action and recursion in the technology of the Acheulean to Middle Palaeolithic transition: a perspective from Patpara, the Son Valley, India.

The Acheulean to Middle Palaeolithic transition is one of the most important technological changes that occurs over the course of human evolution. Here we examine stone artefact assemblages from Patpara and two other excavated sites in the Middle Son Valley, India, which show a mosaic of attributes associated with Acheulean and Middle Palaeolithic industries. The bifaces from these sites are very refined and generally small, but also highly variable in size. A strong relationship between flake scar density and biface size indicates extensive differential resharpening. There are relatively low proportions of bifaces at these sites, with more emphasis on small flake tools struck from recurrent Levallois cores. The eventual demise of large bifaces may be attributed to the curation of small prepared cores from which sharper, or more task-specific flakes were struck. Levallois technology appears to have arisen out of adapting aspects of handaxe knapping, including shaping of surfaces, the utilization of two inter-dependent surfaces, and the striking of invasive thinning flakes. The generativity, hierarchical organization of action, and recursion evident in recurrent Levallois technology may be attributed to improvements in working memory.

Evolutionary and functional history of the Escherichia coli K1 capsule.

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.

Evolutionary Instability of Collateral Susceptibility Networks in Ciprofloxacin-Resistant Clinical Escherichia coli Strains.

Collateral sensitivity and resistance occur when resistance development toward one antimicrobial either potentiates or deteriorates the effect of others. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection, and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin-resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression are the main drivers destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks. IMPORTANCE Antimicrobial resistance occurs due to genetic alterations that affect different processes in bacteria. Thus, developing resistance toward one antimicrobial drug may also alter the response toward others (collateral effects). Understanding the mechanisms of such collateral effects may provide clinicians with a framework for informed antimicrobial treatment strategies, limiting the emergence of antimicrobial resistance. However, for clinical implementation, it is important that the collateral effects of resistance development are repeatable and temporarily stable. Here, we show that collateral effects caused by resistance development toward ciprofloxacin in clinical Escherichia coli strains are not temporarily stable because of compensatory mutations restoring the fitness burden of the initial resistance mutations. Consequently, this instability is complicating the general applicability and clinical implementation of collateral effects into treatment strategies.