Biology subjects

Johansen, H. K.

Publications and source records attributed to Johansen, H. K..

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bioRxiv2022

Core and accessory effectors of type VI secretion systems contribute differently to the intraspecific diversity of Pseudomonas aeruginosa

Bacteria use type VI secretion systems (T6SSs) to deliver effector proteins into other cells or the extracellular space. Those effectors kill microbes1, manipulate eukaryotic cells2, and sequester nutrients3. Which T6SS-mediated functions are generalisable across bacteria of a species or are specific to particular strains is little known. Here, we use genomics to test for the intraspecific diversity of T6SS effectors in the opportunistic pathogen Pseudomonas aeruginosa. We found effectors that are omnipresent and conserved across strains acting as core effectors, while additional accessory effectors vary. In vitro and in vivo experiments demonstrate different roles of the two types of effectors in bacterial killing and virulence. Further, effectors compose various effector combinations. Within one local population of clinical isolates, we observed 36 combinations among 52 bacterial lineages. These findings show the distinct contribution of T6SS effectors to strain-level variation of a bacterial pathogen and might reveal conserved targets for novel antibiotics.

microbiology↗

bioRxiv2022

Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance

BackgroundCystic fibrosis (CF) patients have reduced mucociliary clearance resulting in recurring and chronic bacterial lung infections. Pseudomonas aeruginosa is one of the most common pathogens to colonize the airways of CF patients and can persist in the lungs for decades. CF patients infected with P. aeruginosa are treated with macrolides to inhibit quorum sensing, mucoidity and has additional immunomodulatory effects. However, according to the EUCAST committee, P. aeruginosa is not susceptible to macrolides leaving resistance mechanisms largely overlooked. MethodsUsing a modified susceptibility testing protocol, P. aeruginosa isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics. Quorum sensing related properties, alterations in proteome composition and ribosome subunits distribution were further analysed to characterize the effect of the uL4 mutations on the physiology of the bacteria. FindingsSeveral uL4 mutations were identified in isolates from P. aeruginosa collections from various sources and geographical locations. Most of them mapped to the conserved loop region of uL4 and resulted in increased survival upon macrolide exposure. uL4 mutations did not negatively impact the physiology of the bacteria and greater concentrations of antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity. Proteome analysis revealed that pathways involved in ribosome adaptation displayed altered expression levels possibly to compensate for the uL4 mutations, which changed the subunit distribution of the ribosome. InterpretationMacrolides used against P. aeruginosa cause selection of macrolide resistant mutants which is a widespread - but uncharacterized - phenomenon. Using a modified susceptibility test, revealed that macrolides are indeed effective bacteriostatic antibiotics against P. aeruginosa and that this effect - along with macrolide-induced quorum sensing modulation - are drastically reduced in uL4 mutants. Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated especially when patients are treated with prolonged courses of macrolides. FundingCystic Fibrosis Foundation, Independent Research Fund Denmark, Novo Nordisk Foundation Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMacrolide antibiotics are readily prescribed as anti-inflammatory therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients. This treatment strategy has largely overlooked the direct antimicrobial effect of this drug class on this pathogen, which is considered non-susceptible according to the European Committee on Antimicrobial Susceptibility Testing. This is because standardized antimicrobial susceptibility testing is sub-optimal for the quantification of macrolide minimum inhibitory concentrations due to the interference of the growth medium. Mutations in ribosomal RNA and increased efflux has been shown to reduce the effect of macrolides on P. aeruginosa, however, the involvement of ribosomal proteins has not been investigated in P. aeruginosa. Work done in other bacterial species such as Escherichia coli, Streptococcus pneumoniae, Legionella pneumophila and Neisseria gonorrhoeae has identified the ribosomal proteins uL4 and uL22 as targets for evolved macrolide resistance in vitro and in vivo. Especially the extended loop region of uL4 has been identified as important for macrolide susceptibility. We found no other studies published addressing the emergence of macrolide resistance through uL4 or uL22 mutations in P. aeruginosa. Added value of this studyWe present evidence that macrolide antibiotics used as anti-inflammatory agents and bacterial modulators against P. aeruginosa infection in cystic fibrosis (CF) patients cause the emergence of resistant strains through mutations in the ribosomal protein uL4. The importance of this finding is underlined by the identification of uL4 mutations in multiple strains not only within our strain collection comprised of CF associated isolates but throughout all available sequences of clinical P. aeruginosa isolates spanning several continents and different infection types. uL4 mutations resulted in significantly reduced susceptibility towards macrolide antibiotics with respect to bacteriostatic and quorum sensing modulation effects. The generation time in presence of macrolide antibiotics was unaffected in strains harbouring uL4 mutations, while it was reduced in wild type strains indicating a fitness advantage of the mutation. Implications of all the available evidenceThe uL4 mutants identified in this study were significantly more resistant towards macrolide antibiotics than strains harbouring a wild type uL4 hereby revealing that not only are macrolides effective as antimicrobial agents against P. aeruginosa but also that the imposed selective pressure causes resistant mutants to arise through mutations in the ribosomal uL4 protein. These mutations are also present but uncharacterized in other collections of clinical P. aeruginosa isolates from patient groups who typically receive long courses of macrolide treatment. The effect of the uL4 and possibly other hitherto unknown mutations on macrolide susceptibility can be determined via modification of the standard susceptibility testing protocol. Along with evidence describing other types of macrolide resistance mechanisms in P. aeruginosa such as rRNA modification and increased efflux activity, our results demonstrate that macrolide resistance development is widespread in P. aeruginosa and macrolide antibiotics should be considered as antimicrobial agents against P. aeruginosa with the same precautions being taken to avoid resistance development as for any other antimicrobial agent.

microbiology↗

bioRxiv2021

Characterization of antibiotic resistance in clinical isolates of Klebsiella pneumoniae in Denmark

Klebsiella pneumoniae (KP) is a major global health problem as it leads to hospital outbreaks all over the world and is becoming more difficult to treat due to its increasing antimicrobial resistance (AMR). Optimization and development of new treatments of KP requires understanding of its population structure and AMR properties. Therefore, in this study, we collected and sequenced 491 KP strains from four major Danish microbiology departments covering 51% of the Danish population. The isolates were whole genome sequenced (WGS), phenotypically characterized and compared with 2,124 KP strains from 13 different countries (PATRIC strains). We found that while genomic content varies significantly across the Danish strains, they also differ significantly from strains from other countries, due to the lack of certain AMR sequence types (e.g. ST258 and ST307) in Denmark. Genomic and experimental analysis suggest that Danish strains contain fewer virulence mechanisms and are more susceptible to antimicrobials compared to strains from other countries, likely due to the relatively low antibiotic usage in Denmark where 70% of hospital antibiotic usage is penicillins. We also identified potential novel AMR determinants to tigecycline through statistical analysis of genomic and phenotypic data. To conclude, we obtained a more comprehensive understanding of the KP strains in Denmark and provided valuable insights for future experiments and strategies to combat AMR in KP.

genomics↗

bioRxiv2021

Long-term evolution of antibiotic persistence in P. 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 persistence. While a variety of resistance mechanisms and underlying genetics are well characterised in vitro and in vivo, the evolution of persistence, and how it interacts with resistance in situ is less well understood. We assayed for persistence and resistance with three clinically relevant antibiotics: meropenem, ciprofloxacin and tobramycin, in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to forty years of evolution. We find evidence that persistence is under positive selection in the lung and that it can particularly act as an evolutionary stepping stone to resistance. However, this pattern is not universal and depends on the bacterial clone type and antibiotic used, indicating an important role for antibiotic mode of action.

evolutionary biology↗

bioRxiv2021

β-lactamase remodeling and evolution of collateral resistance in hypermutator Pseudomonas aeruginosa upon long-term antibiotic therapy

Antibiotic resistance development has been studied using approaches that range from laboratory experimental evolution, surveillance and epidemiology, to clinical isolate sequencing. However, evolutionary trajectories depend on the environment in which selection takes place, compelling to address evolutionary analyses in antibiotic-treated patients, to embrace the whole inherent environmental complexities as well as their dynamics over time. Herein, we address the complexity of the bacterial adaptive response to changing antibiotic selective pressures by studying the long-term in-patient evolution of a broad diversity of {beta}-lactam resistant Pseudomonas aeruginosa clones. By using mutational and ultra-deep amplicon sequencing, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis (CF) patient. We identified the accumulation of multiple alterations targeting the chromosomally encoded class C {beta}-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, "a gain of function" of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the diversity of evolutionary trajectories driven by bacteria in the natural CF environmental setting, towards a multidrug resistant phenotype after years of antibiotic treatment against a formidable pathogen.

microbiology↗

bioRxiv2021

Achromobacter genetic adaptation in cystic fibrosis

Achromobacter is an emerging pathogen in patients with cystic fibrosis (CF) and Achromobacter caused infections are associated with more severe disease outcomes and high intrinsic antibiotic resistance. While conventional CF pathogens are studied extensively, little is known about the genetic determinants leading to antibiotic resistance and the genetic adaptation in Achromobacter infections. Here, we analyzed 101 Achromobacter genomes from 51 patients with CF isolated during the course of up to 20 years of infection to identify within-host adaptation, mutational signatures, and genetic variation associated with increased antibiotic resistance. We found that the same regulatory and inorganic ion transport genes were frequently mutated in persisting clone types within and between Achromobacter species indicating convergent genetic adaptation. Genome-wide association study (GWAS) of six antibiotic resistance phenotypes revealed the enrichment of associated genes involved in inorganic ion transport genes, transcription gene enrichment in {beta}-lactams, and energy production and translation gene enrichment in the trimethoprim/sulfonamide group. Overall, we provide insights into the pathogenomics of Achromobacter infections in patients with CF airways. Since emerging pathogens are increasingly recognised as an important healthcare issue, our findings on evolution of antibiotic resistance and genetic adaptation can facilitate better understanding of disease progression and how mutational changes have implications for patients with CF.

microbiology↗

bioRxiv2020

Transmission and genetic adaptation of Achromobacter in cystic fibrosis

Achromobacter species are increasingly being detected in patients with cystic fibrosis (CF), and this emerging pathogen is associated with antibiotic resistance and more severe disease outcomes. Nonetheless, little is known about the extent of transmission and antibiotic resistance development in Achromobacter infections. We sequenced the genomes of 101 clinical isolates of Achromobacter (A. xylosoxidans based on MALDI-TOF/API N20 typing) collected from 51 patients with CF--the largest longitudinal dataset to-date. We performed phylogenetic analysis on the genomes and combined this with epidemiological and antibiotic resistance data to identify patient-to-patient transmission and development of antibiotic resistance. We found that MALDI-TOF/API N20 was not sufficient for Achromobacter species-level typing, and that the population of Achromobacter isolates was composed of five different species where A. xylosoxidans accounted for 52% of infections. Most patients were infected by unique Achromobacter clone types; nonetheless, suspected patient-to-patient transmission cases identified by shared clone types were observed in 35% (N=18) of patients. In 15 of 16 cases the suspected transmissions were further supported by genome- or clinic visit-based epidemiological analysis. Finally, we found that resistance developed over time. We show that whole-genome sequencing (WGS) is essential for Achromobacter species typing and patient-to-patient transmission identification which was identified in A. ruhlandii, A. xylosoxidans and, for the first time, A. insuavis. Furthermore, we show that the development of antibiotic resistance is associated with chronic Achromobacter infections. Our findings emphasize that transmission and antibiotic resistance should be considered in future treatment strategies.

genomics↗

bioRxiv2020

Gene loss and acquisition in lineages of bacteria evolving in a human host environment

While genome analyses have documented that there are differences in the gene repertoire between evolutionary distant lineages of the same bacterial species, less is known about micro-evolutionary dynamics of gene loss and acquisition within lineages of bacteria as they evolve over the timescale of years. This knowledge is valuable to understand both the basic mutational steps that on long timescales lead to evolutionary distant bacterial lineages, and the evolution of the individual lineages themselves. In the case that lineages evolve in a human host environment, gene loss and acquisition may furthermore have implication for disease. We analyzed the genomes of 45 Pseudomonas aeruginosa lineages evolving in the lungs of cystic fibrosis patients to identify genes that are lost or acquired during the first years of infection in each of the different lineages. On average, the lineage genome content changed with 88 genes (range 0-473). Genes were more often lost than acquired, and prophage genes were more variable than bacterial genes. We identified genes that were lost or acquired independently across different clonal lineages, i.e. convergent molecular evolution. Convergent evolution suggests that there is a selection for loss and acquisition of certain genes in the host environment. We find that a significant proportion of such genes are associated with virulence; a trait previously shown to be important for adaptation. Furthermore, we also compared the genomes across lineages to show that within-lineage variable genes more often belonged to genomic content not shared across all lineages. Finally, we used 4,760 genes shared by 446 P. aeruginosa genomes to develop a stable and discriminatory typing scheme for P. aeruginosa clone types (Pactyper, https://github.com/MigleSur/Pactyper). In sum, our analysis adds to the knowledge on the pace and drivers of gene loss and acquisition in bacteria evolving over multiple years in a human host environment and provides a basis to further understand how gene loss and acquisition plays a role in lineage differentiation and host adaptation. Data SummaryP. aeruginosa genome sequencing data has been made publicly available by Marvig et al. (2015) and is deposited in Sequence Read Archive (SRA) under accession ERP004853.

evolutionary biology↗

bioRxiv2019

Hypermutator Pseudomonas aeruginosa exploits multiple genetic pathways to develop multidrug resistance during long term infections in the airways of cystic fibrosis patients

Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolated from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic CF infections. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to be positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed towards the establishment of a population constituted by genotypically diversified coexisting sub-lineages, all of which converged to multi-drug resistance. These sub-lineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and fstI, encoding the {beta}-lactamase and penicillin-binding protein 3, respectively, were found among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying {beta}-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.\n\nIMPORTANCEBy increasing mutation rates, hypermutators boost antibiotic resistance evolution by enabling bacterial pathogens to fully exploit their genetic potential and achieve resistance mechanisms for almost every known antimicrobial agent. Here, we show how co-existing clones from a P. aeruginosa hypermutator lineage that evolved during 20 years of chronic infection and antibiotic chemotherapy, converged to multidrug resistance by targeting genes from alternative genetic pathways that are part of the broad P. aeruginosa resistome. Within this complex assembly of combinatorial genetic changes, in some specific cases, multiple mutations are needed in the same gene to reach a fine tuned resistance phenotype. Hypermutability enables this genetic edition towards higher resistance profiles by recurrently targeting these genes, thus promoting new epistatic relationships and the emergence of innovative resistance-conferring alleles. Our findings help to understand this link between hypermutability and antibiotic resistance, a key challenge for the design of new therapeutic strategies.

microbiology↗

bioRxiv2019

The high persister phenotype of Pseudomonas aeruginosa is associated with increased fitness and persistence in cystic fibrosis airways

Despite intensive antibiotic treatment of cystic fibrosis (CF) patients, Pseudomonas aeruginosa often persists in patient airways for decades, and can do so without the development of antibiotic resistance. Using a high-throughput screening assay of survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of high-persister variants (Hip) in a large patient cohort. In a screen of 467 longitudinal clinical isolates of P. aeruginosa from 40 CF patients, we classified 25.7% as Hip. Hip were identified in 26 patients, but only a few bacterial lineages were dominated by Hip. Instead, the emergence of Hip increased over time, suggesting that CF airways treated with ciprofloxacin select for Hip with an increased fitness in this environment. We generally observed diverse genetic changes in the Hip isolate population (as many co-occurring routes to increased fitness exist), but interestingly elevated mutation counts in the RpoN gene of 18 Hip isolates suggest that this sigma factor plays a role in shaping levels of antibiotic tolerance. To probe the impact of the Hip phenotype in a CF-similar environment, we tested the fitness properties of otherwise genotypically and phenotypically similar low-persister (Lop) and Hip isolates in co-culture using a specialized flow-cell biofilm system mimicking pharmacokinetic/-dynamic antibiotic dosing. Hip survived ciprofloxacin treatment far better than Lop isolates. The results of this investigation provide novel insights into persister dynamics and fitness contributions to survival in the CF lung, and show that the Hip phenotype of antibiotic susceptible bacteria plays an important role in long-term infections. SignificanceAntibiotic resistance is emphasized as a rapidly increasing health threat, but antibiotic tolerance via the occurrence of persister cells in antibiotic-treated bacterial populations is clinically and publicly neglected. In 40 CF patients representing a well-established human infection model - long-term lung infections by Pseudomonas aeruginosa - we show the emergence and accumulation of persister variants in a clinical population heavily reliant on antibiotic therapy. We observe that the high-persister (Hip) phenotype is independent of resistance and likely the consequence of numerous genetic alterations, complicating surveillance and inhibition in the clinic. Furthermore, we find Hip are selected for over time, survive better than normal bacteria, and can outcompete them in CF-similar conditions, ultimately affecting 65% of patients in an early disease cohort.

microbiology↗

bioRxiv2018

Privatisation rescues function following loss of cooperation

A single cheating mutant can lead to the invasion and eventual eradication of cooperation from a population. Consequently, cheat invasion is often considered as \"game over\" in empirical and theoretical studies of cooperator-cheat dynamics, especially when cooperation is necessary for fulfilling an essential function. But is cheat invasion necessarily \"game over\" in nature? By following a population of bacteria through loss of cooperation and beyond, we observed that individuals evolved to replace cooperation with a selfish, or \"private\" behaviour. Specifically, we show that when cheating caused the loss of cooperative iron acquisition in a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients, a private uptake system that only benefits the focal individual was upregulated. This observation highlights the importance of social dynamics of natural populations and emphasizes the potential impact of past social interaction on the evolution of private traits.

evolutionary biology↗

bioRxiv2018

Evolutionary highways to persistent infection by Pseudomonas aeruginosa

Persistent infections require bacteria to evolve from their naive colonization state by optimizing fitness in the host. This optimization involves coordinated adaptation of multiple traits, obscuring evolutionary trends and complicating infection management. Accordingly, we screen 8 infection-relevant phenotypes of 443 longitudinal Pseudomonas aeruginosa isolates from 39 young cystic fibrosis patients over 10 years. Using statistical modeling, we map evolutionary trajectories and identify trait correlations accounting for patient-specific influences. By integrating previous genetic analyses of 474 isolates, we provide a window into early adaptation to the host, finding: 1) a 2-3 year timeline of rapid adaptation after colonization, 2) variant \"naive\" and \"adapted\" states reflecting discordance between phenotypic and genetic adaptation, 3) adaptive trajectories leading to persistent infection via 3 distinct evolutionary modes, and 4) new associations between phenotypes and pathoadaptive mutations. Ultimately, we effectively deconvolute complex trait adaptation, offering a framework for evolutionary studies and precision medicine in clinical microbiology.

evolutionary biology↗