A Whole-Cell Biosensor for Detection of 2,4-Diacetylphloroglucinol (DAPG)-Producing Bacteria from Grassland Soil.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) are ecologically important in the rhizosphere, as they can control phytopathogens and contribute to disease suppression. DAPG can also trigger a systemic resistance response in plants and stimulate root exudation and branching as well as induce plant-beneficial activities in other rhizobacteria. While studies of DAPG-producing Pseudomonas have predominantly focused on rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk soil and grassland, where the level of DAPG producers are predicted to be low. In this study, we constructed a whole-cell biosensor for detection of DAPG and DAPG-producing bacteria from environmental samples. The constructed biosensor contains a phlF response module and either lacZ or lux genes as output modules assembled on a pSEVA plasmid backbone for easy transfer to different host species and to enable easy future genetic modifications. We show that the sensor is highly specific toward DAPG, with a sensitivity in the low nanomolar range (>20 nM). This sensitivity is comparable to the DAPG levels identified in rhizosphere samples by chemical analysis. The biosensor enables guided isolation of DAPG-producing Pseudomonas Using the biosensor, we probed the same grassland soil sampling site to isolate genetically related DAPG-producing Pseudomonas kilonensis strains over a period of 12 months. Next, we used the biosensor to determine the frequency of DAPG-producing pseudomonads within three different grassland soil sites and showed that DAPG producers can constitute part of the Pseudomonas population in the range of 0.35 to 17% at these sites. Finally, we showed that the biosensor enables detection of DAPG produced by non-Pseudomonas species. Our study shows that a whole-cell biosensor for DAPG detection can facilitate isolation of bacteria that produce this important secondary metabolite and provide insight into the population dynamics of DAPG producers in natural grassland soil.IMPORTANCE The interest in bacterial biocontrol agents as biosustainable alternatives to pesticides to increase crop yields has grown. To date, we have a broad knowledge of antimicrobial compounds, such as DAPG, produced by bacteria growing in the rhizosphere surrounding plant roots. However, compared to the rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk and grassland soil. Currently, we are restricted to chemical methods with detection limits and time-consuming PCR-based and probe hybridization approaches to detect DAPG and its respective producer. In this study, we developed a whole-cell biosensor, which can circumvent the labor-intensive screening process as well as increase the sensitivity at which DAPG can be detected. This enables quantification of relative amounts of DAPG producers, which, in turn, increases our understanding of the dynamics and ecology of these producers in natural soil environments.

Intergenic evolution during host adaptation increases expression of the metallophore pseudopaline in Pseudomonas aeruginosa.

Regulating intracellular levels of biological metal ions is essential for all bacterial species, as they are needed for virulence and a range of metabolic processes. Zinc is the second most abundant metal ion in Pseudomonas aeruginosa, but little is known about its regulation. Recent studies have identified a novel operon, zrmABCD (also called cntOLMI), encoding a metallophore system (pseudopaline) involved in zinc acquisition. Expression of this operon has been implicated in human infections and is regulated by the transcriptional regulator Zur (Zn2+ uptake regulator). In this study, we show that the intergenic promoter region in front of zrmABCD is a target for recurrent adaptive mutations during chronic infection of cystic fibrosis (CF) patients. We characterize the inter- and intraclonal sequence polymorphisms found in the promoter region of the metallophore system and find that most alterations increase promoter activity. One of the evolved promoters displays a more than 10-fold increase compared to the ancestral strain due to the combined effect of an altered binding site of Zur and changes to the RpoD-binding motif. This specific evolved promoter responds differently to changes in metal ion concentrations in chelated medium. We have previously shown that P. aeruginosa evolves toward iron acquisition from haemoglobin during long-term CF infections. We hereby provide the second example of adaptive mutations targeting intergenic regions that affect metal ion uptake systems during CF infections, and the first involving zinc uptake. Our results suggest that the scarcity of metal ions (including iron and zinc) is an important evolutionary driver in CF host adaptation.

Genomic Epidemiology of a Major Mycobacterium tuberculosis Outbreak: Retrospective Cohort Study in a Low-Incidence Setting Using Sparse Time-Series Sampling.

Since 1992, Denmark has documented the largest outbreak of tuberculosis in Scandinavia ascribed to a single genotype, termed C2/1112-15. As of spring 2017, the International Reference Laboratory of Mycobacteriology in Copenhagen has collected and identified isolates from more than a thousand cases belonging to this outbreak via routine mycobacterial interspersed repetitive units-variable number of tandem repeats typing. Here, we present a retrospective analysis of the C2/1112-15 dataset, based on whole-genome data from a sparse time series consisting of 5 randomly selected isolates from 23 years of sampling. Even if these data are derived from only 12% of the collected isolates, we have been able to extract important key information, such as mutation rate and conserved single-nucleotide polymorphisms to identify discrete transmission chains, as well as the possible historical origins of the outbreak.

The phenotypic evolution of Pseudomonas aeruginosa populations changes in the presence of subinhibitory concentrations of ciprofloxacin.

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of Pseudomonas aeruginosa infections. The immediate response of P. aeruginosa to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of P. aeruginosa populations compared to unexposed populations. Three replicate populations of P. aeruginosa PAO1 and its hypermutable mutant ΔmutS were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at - 80 °C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates.

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.

Substantial molecular evolution and mutation rates in prolonged latent Mycobacterium tuberculosis infection in humans.

The genome of Mycobacterium tuberculosis (Mtb) of latently infected individuals may hold the key to understanding the processes that lead to reactivation and progression to clinical disease. We report here analysis of pairs of Mtb isolates from putative prolonged latent TB cases. We identified two confirmed cases, and used whole genome sequencing to investigate the mutational processes that occur over decades in latent Mtb. We found an estimated mutation rate between 0.2 and 0.3 over 33 years, suggesting that latent Mtb accumulates mutations at rates similar to observations from cases of active disease.

Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

BACKGROUND: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. RESULTS: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids. CONCLUSION: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts.

The genetic basis of bacterial adaptation to a natural environment has been investigated in a highly successful Pseudomonas aeruginosa lineage (DK2) that evolved within the airways of patients with cystic fibrosis (CF) for more than 35 y. During evolution in the CF airways, the DK2 lineage underwent substantial phenotypic changes, which correlated with temporal fixation of specific mutations in the genes mucA (frame-shift), algT (substitution), rpoN (substitution), lasR (deletion), and rpoD (in-frame deletion), all encoding regulators of large gene networks. To clarify the consequences of these genetic changes, we moved the specific mutations, alone and in combination, to the genome of the reference strain PAO1. The phenotypes of the engineered PAO1 derivatives showed striking similarities with phenotypes observed among the DK2 isolates. The phenotypes observed in the DK2 isolates and PAO1 mutants were the results of individual, additive and epistatic effects of the regulatory mutations. The mutations fixed in the σ factor encoding genes algT, rpoN, and rpoD caused minor changes in σ factor activity, resulting in remodeling of the regulatory networks to facilitate generation of unexpected phenotypes. Our results suggest that adaptation to a highly selective environment, such as the CF airways, is a highly dynamic and complex process, which involves continuous optimization of existing regulatory networks to match the fluctuations in the environment.

Genome analysis of a transmissible lineage of pseudomonas aeruginosa reveals pathoadaptive mutations and distinct evolutionary paths of hypermutators.

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections.

Bacteriocin-mediated competition in cystic fibrosis lung infections.

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations,especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that(i) isolates from later infection stages inhibited earlier infecting strains less,but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage overtime; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.

Staphylococcus aureus alters growth activity, autolysis, and antibiotic tolerance in a human host-adapted Pseudomonas aeruginosa lineage.

Interactions among members of polymicrobial infections or between pathogens and the commensal flora may determine disease outcomes. Pseudomonas aeruginosa and Staphylococcus aureus are important opportunistic human pathogens and are both part of the polymicrobial infection communities in human hosts. In this study, we analyzed the in vitro interaction between S. aureus and a collection of P. aeruginosa isolates representing different evolutionary steps of a dominant lineage, DK2, that have evolved through decades of growth in chronically infected patients. While the early adapted P. aeruginosa DK2 strains outcompeted S. aureus during coculture on agar plates, we found that later P. aeruginosa DK2 strains showed a commensal-like interaction, where S. aureus was not inhibited by P. aeruginosa and the growth activity of P. aeruginosa was enhanced in the presence of S. aureus. This effect is mediated by one or more extracellular S. aureus proteins greater than 10 kDa, which also suppressed P. aeruginosa autolysis and prevented killing by clinically relevant antibiotics through promoting small-colony variant (SCV) formation. The commensal interaction was abolished with S. aureus strains mutated in the agr quorum sensing system or in the SarA transcriptional virulence regulator, as well as with strains lacking the proteolytic subunit, ClpP, of the Clp protease. Our results show that during evolution of a dominant cystic fibrosis lineage of P. aeruginosa, a commensal interaction potential with S. aureus has developed.

Evolutionary dynamics of bacteria in a human host environment.

Laboratory evolution experiments have led to important findings relating organism adaptation and genomic evolution. However, continuous monitoring of long-term evolution has been lacking for natural systems, limiting our understanding of these processes in situ. Here we characterize the evolutionary dynamics of a lineage of a clinically important opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it adapts to the airways of several individual cystic fibrosis patients over 200,000 bacterial generations, and provide estimates of mutation rates of bacteria in a natural environment. In contrast to predictions based on in vitro evolution experiments, we document limited diversification of the evolving lineage despite a highly structured and complex host environment. Notably, the lineage went through an initial period of rapid adaptation caused by a small number of mutations with pleiotropic effects, followed by a period of genetic drift with limited phenotypic change and a genomic signature of negative selection, suggesting that the evolving lineage has reached a major adaptive peak in the fitness landscape. This contrasts with previous findings of continued positive selection from long-term in vitro evolution experiments. The evolved phenotype of the infecting bacteria further suggests that the opportunistic pathogen has transitioned to become a primary pathogen for cystic fibrosis patients.

In vivo evolution of antimicrobial resistance in a biofilm model of Pseudomonas aeruginosa lung infection.

The evolution of antimicrobial resistance (AMR) in biofilms has been repeatedly studied by experimental evolution in vitro, but rarely in vivo. The complex microenvironment at the infection site imposes selective pressures on the bacterial biofilms, potentially influencing the development of AMR. We report here the development of AMR in an in vivo mouse model of Pseudomonas aeruginosa biofilm lung infection. The P. aeruginosa embedded in seaweed alginate beads underwent four successive lung infection passages with or without ciprofloxacin (CIP) exposure. The development of CIP resistance was assessed at each passage by population analysis of the bacterial populations recovered from the lungs of CIP-treated and control mice, with subsequent whole-genome sequencing of selected isolates. As inflammation plays a crucial role in shaping the microenvironment at the infection site, its impact was explored through the measurement of cytokine levels in the lung homogenate. A rapid development of AMR was observed starting from the second passage in the CIP-treated mice. Genetic analysis revealed mutations in nfxB, efflux pumps (mexZ), and two-component systems (parS) contribution to CIP resistance. The control group isolates exhibited mutations in the dipA gene, likely associated with biofilm dispersion. In the initial two passages, the CIP-treated group exhibited an elevated inflammatory response compared to the control group. This increase may potentially contribute to the release of mutagenic reactive oxygen species and the development of AMR. In conclusion, this study illustrates the complex relationship between infection, antibiotic treatment, and immune response.

Resistance towards and biotransformation of a Pseudomonas-produced secondary metabolite during community invasion.

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways.

BACKGROUND: Analysis of global gene expression by DNA microarrays is widely used in experimental molecular biology. However, the complexity of such high-dimensional data sets makes it difficult to fully understand the underlying biological features present in the data.The aim of this study is to introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first "Archetypal Analysis" of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas aeruginosa isolated from the airways of cystic fibrosis patients. RESULTS: Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene expression between different groups identified adaptive changes of the bacteria residing in the cystic fibrosis lung. The analysis suggests a similar gene expression pattern between isolates with a high mutation rate (hypermutators) despite accumulation of different mutations for these isolates. This suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. CONCLUSIONS: Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering and matrix factorization made it possible to reveal minor similarities among different groups of data, which other analytical methods failed to identify. We suggest that this analysis could be used to supplement current methods used to analyze DNA microarray data.

A bittersweet fate: detection of serotype switching in Pseudomonas aeruginosa.

High-risk clone types in Pseudomonas aeruginosa are problematic global multidrug-resistant clones. However, apart from their ability to resist antimicrobial treatment, not much is known about what sets these clones apart from the multitude of other clones. In high-risk clone ST111, it has previously been shown that replacement of the native serotype biosynthetic gene cluster (O4) by a different gene cluster (O12) by horizontal gene transfer and recombination may have contributed to the global success of this clone. However, the extent to which isolates undergo this type of serotype switching has not been adequately explored in P. aeruginosa. In the present study, a bioinformatics tool has been developed and utilized to provide a first estimate of serotype switching in groups of multidrug resistant (MDR) clinical isolates. The tool detects serotype switching by analysis of core-genome phylogeny and in silico serotype. Analysis of a national survey of MDR isolates found a prevalence of 3.9 % of serotype-switched isolates in high-risk clone types ST111, ST244 and ST253. A global survey of MDR isolates was additionally analysed, and it was found that 2.3 % of isolates had undergone a serotype switch. To further understand this process, we determined the exact boundaries of the horizontally transferred serotype O12 island. We found that the size of the serotype island correlates with the clone type of the receiving isolate and additionally we found intra-clone type variations in size and boundaries. This suggests multiple serotype switch events. Moreover, we found that the housekeeping gene gyrA is co-transferred with the O12 serotype island, which prompted us to analyse this allele for all serotype O12 isolates. We found that 95 % of ST111 O12 isolates had a resistant gyrA allele and 86 % of all O12 isolates had a resistant gyrA allele. The rates of resistant gyrA alleles in isolates with other prevalent serotypes are all lower. Together, these results show that the transfer and acquisition of serotype O12 in high-risk clone ST111 has happened multiple times and may be facilitated by multiple donors, which clearly suggests a strong selection pressure for this process. However, gyrA-mediated antibiotic resistance may not be the only evolutionary driver.

Comparative whole-genome analysis of China and global epidemic Pseudomonas aeruginosa high-risk clones.

OBJECTIVES: The various sequence types (STs) of Pseudomonas aeruginosa (P. aeruginosa) high-risk clones (HiRiCs) have been sporadically reported in China, but the systematic analysis of genomes for these STs remains limited. This study aimed to address the evolutionary pathways underlying the emergence of HiRiCs and their routes of dissemination from Chinese and global perspectives. METHODS: The phylogenetic analysis was performed based on 416 newly sequenced clinical P. aeruginosa strains from Guangdong (GD), published genome sequences of 282 Chinese isolates, and 868 HiRiCs isolates from other countries. The genomic comparison study of global HiRiC ST244 was conducted to detect the model of global dissemination and local separation driven by association regional-specific antibiotic resistance genes. Furthermore, the evolutionary route of the emerging, China-specific HiRiC ST1971 was explored using Most Recent Common Ancestor (MRCA) analysis. RESULTS: Based on comparative genomics analysis, we found a clear geographical separation of ST244 isolates, yet with an association between ST244 isolates from GD and America. We identified a set of 38 AMR genes that contribute to the geographical separation in ST244, and we identified genetic determinants either positively (MexB) and negatively (opmD) associated with GD ST244. For the China-unique HiRiC ST1971, its evolutionary history across different continents before emerging as ST1971 in China was also deduced. CONCLUSION: This study provides insight into the specific genetics underlying regional differences among globally disseminated P. aeruginosa HiRiCs (ST244) as well as new understanding of the dissemination and evolution of a regional HiRiC (ST1971). Understanding the genetics of these and other HiRiCs may assist in controlling their emergence and further spread.

Whole-genome sequencing reveals high-risk clones of Pseudomonas aeruginosa in Guangdong, China.

The ever-increasing prevalence of infections produced by multidrug-resistant or extensively drug-resistant Pseudomonas aeruginosa is commonly linked to a limited number of aptly-named epidemical 'high-risk clones' that are widespread among and within hospitals worldwide. The emergence of new potential high-risk clone strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. P. aeruginosa related high-risk clones have been sporadically found in China, their genome sequences have rarely been described. Therefore, the large-scale sequencing of multidrug-resistance high-risk clone strains will help us to understand the emergence and transmission of antibiotic resistances in P. aeruginosa high-risk clones. In this study, 212 P. aeruginosa strains were isolated from 2 tertiary hospitals within 3 years (2018-2020) in Guangdong Province, China. Whole-genome sequencing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing were applied to analyze the genomic epidemiology of P. aeruginosa in this region. We found that up to 130 (61.32%) of the isolates were shown to be multidrug resistant, and 196 (92.45%) isolates were Carbapenem-Resistant Pseudomonas aeruginosa. MLST analysis demonstrated high diversity of sequence types, and 18 reported international high-risk clones were identified. Furthermore, we discovered the co-presence of exoU and exoS genes in 5 collected strains. This study enhances insight into the regional research of molecular epidemiology and antimicrobial resistance of P. aeruginosa in China. The high diversity of clone types and regional genome characteristics can serve as a theoretical reference for public health policies and help guide measures for the prevention and control of P. aeruginosa resistance.

Whole-Genome Sequencing of 11 High-Risk Clone ST111 Pseudomonas aeruginosa Isolates from French Hospitals.

This study reports high-quality genomes of 11 sequence type 111 (ST111) isolates of Pseudomonas aeruginosa. This ST is known for its worldwide dissemination and high capacity to acquire antibiotic resistance mechanisms. This study used long- and short-read sequencing to provide high-quality closed genomes for most of the isolates.

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.