Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Intraclonal competitive fitness of longitudinal cystic fibrosis Pseudomonas aeruginosa airway isolates in liquid cultures.

The metabolically versatile Pseudomonas aeruginosa inhabits biotic and abiotic environments including the niche of cystic fibrosis (CF) airways. This study investigated how the adaptation to CF lungs affects the within-clone fitness of P. aeruginosa to grow and persist in liquid cultures in the presence of the clonal ancestors. Longitudinal clonal P. aeruginosa isolates that had been collected from 12 CF donors since the onset of colonization for up to 30 years was subjected to within-clone competition experiments. The relative quantities of individual strains were determined by marker-free amplicon sequencing of multiplex PCR products of strain-specific nucleotide sequence variants, a novel method that is generally applicable to studies in evolutionary genetics and microbial ecology with real-world strain collections. For 10 of the 12 examined patient courses, P. aeruginosa isolates of the first years of colonization grew faster in the presence of their clonal progeny than alone. Single growth of individual strains showed no temporal trend with colonization time, but in co-culture, the early isolates out-competed their clonal progeny. Irrespective of the genetic make-up of the clone and its genomic microevolution in CF lungs, the early isolates expressed fitness traits to win the within-clone competition that were absent in their progeny.

Long-Term Microevolution of Pseudomonas aeruginosa Differs between Mildly and Severely Affected Cystic Fibrosis Lungs.

Chronic airway infections with Pseudomonas aeruginosa determine morbidity in most individuals with cystic fibrosis (CF). P. aeruginosa may persist for decades in CF lungs, which provides a rare opportunity to study the long-term within-host evolution of a bacterial airway pathogen. In this work, we sought to resolve the genetic adaptation of P. aeruginosa in CF lungs from the onset of colonization until the patient's death or permanent replacement by another P. aeruginosa clone. We followed the microevolution of the first persisting P. aeruginosa clone by whole-genome sequencing of serial isolates from highly divergent clinical courses of airway infection, i.e., a fatal outcome because of respiratory insufficiency within less than 15 years, or a rather normal daily life 25-35 years after acquisition of P. aeruginosa. Nonneutral mutations predominantly emerged in P. aeruginosa genes relevant for protection against and communication with signals from the lung environment, i.e., antibiotic resistance, cell wall components, and two-component systems. Drastic and loss-of-function mutations preferentially happened during the severe courses of infection, and the bacterial lineages of the mild courses more proficiently incorporated extra metabolic genes into their accessory genome. P. aeruginosa followed different evolutionary paths depending on whether the bacterium had taken up residence in a patient with CF and normal or already compromised lung function.

Vendor effects on murine gut microbiota influence experimental abdominal sepsis.

BACKGROUND: Experimental animal models are indispensable components of preclinical sepsis research. Reproducible results highly rely on defined and invariant baseline conditions. Our hypothesis was that the murine gut microbiota varies among different distributors of laboratory animals and that these variations influence the phenotype of abdominal sepsis derived from a bacterial inoculum model (intraperitoneal stool injection). MATERIALS AND METHODS: Male C57BL/6 mice (8-wk old) purchased from Charles River (CR), Janvier (J), and Harlan (H) were sacrificed, and the bacterial composition of feces was analyzed using CHROMagar orientation medium. Stool was injected intraperitoneally into CR mice, followed by clinical observation and gene expression analysis. Experiments were repeated 16 mo later under the same conditions. RESULTS: Stool analysis revealed profound intervendor differences in bacterial composition, mainly regarding Staphylococcus aureus and Bacillus licheniformis. Mice challenged with CR as well as H feces developed significantly higher severity of disease and died within the observation period, whereas stool from J mice did not induce any of these symptoms. Real-time polymerase chain reaction revealed corresponding results with significant upregulation of proinflammatory cytokines and vascular leakage-related mediators in CR and H injected animals. Sixteen months later, the bacterial fecal composition had significantly shifted. The differences in clinical phenotype of sepsis after intraperitoneal stool injection had vanished. CONCLUSIONS: We are the first to demonstrate vendor and time effects on the murine fecal microbiota influencing sepsis models of intraabdominal stool contamination. The intestinal microbiota must be defined and standardized when designing and interpreting past and future studies using murine abdominal sepsis models.

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients.

The chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and ß-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host.

Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment.

The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

Intraclonal diversity of the Pseudomonas aeruginosa cystic fibrosis airway isolates TBCF10839 and TBCF121838: distinct signatures of transcriptome, proteome, metabolome, adherence and pathogenicity despite an almost identical genome sequence.

Microevolution of closely related Pseudomonas aeruginosa was compared in the clone TB strains TBCF10839 and TBCF121838 which had been isolated from two unrelated individuals with cystic fibrosis who had acquired clone TB during a local outbreak. Compared with the strain PAO1 reference sequence the two clone TB genomes shared 23 155 nucleotide exchanges, 32 out-of-frame indels in the coding region and another repertoire of replacement and genomic islands such as PAGI-1, PAGI-2, PAGI-5, LESGI-1 and LES-prophage 4. Only TBCF121838 carried a genomic island known from Ralstonia pickettii. Six of the seven strain-specific sequence variations in the core genome were detected in genes affecting motility, biofilm formation or virulence, i.e. non-synonymous nucleotide substitutions in mexS, PA3729, PA5017, mifR, a frameshift mutation in pilF (TBCF121838) and an intragenic deletion in pilQ (TBCF10839). Despite their almost identical genome sequence the two strains differed strongly from each other in transcriptome and metabolome profiles, mucin adherence and phagocytosis assays. TBCF121838 was susceptible to killing by neutrophils, but TBCF10839 could grow in leucocytes. Microevolution in P. aeruginosa apparently can generate novel complex traits by few or even single mutations provided that predisposing mutational events had occurred before in the clonal lineage.

Microevolution of Pseudomonas aeruginosa in the airways of people with cystic fibrosis.

The chronic infections of cystic fibrosis (CF) airways with Pseudomonas aeruginosa are a paradigm of how environmental bacteria can conquer, adapt, and persist in an atypical habitat and successfully evade defense mechanisms and chemotherapy in a susceptible host. The within-host evolution of intraclonal diversity has been examined by whole-genome sequencing, phenotyping, and competitive fitness experiments of serial P. aeruginosa isolates collected from CF airways since onset of colonization for a period of up to 40 years. The spectrum of de novo mutations and the adaptation of phenotype and fitness of the bacterial progeny were more influenced by the living conditions in the CF lung than by the clone type of their ancestor and its genetic repertoire.

Pseudomonas aeruginosa population genomics among adults with bronchiectasis across Germany.

Genome sequencing of 130 Pseudomonas aeruginosa isolates from 110 bronchiectasis patients identified a few dominant clones common in the global bacterial population and numerous rare clones infrequently seen in the environment or other human infections https://bit.ly/3lIfD2X.

Competitive survival of clonal serial Pseudomonas aeruginosa isolates from cystic fibrosis airways in human neutrophils.

Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Effective prevention of Pseudomonas aeruginosa cross-infection at a cystic fibrosis centre - results of a 10-year prospective study.

Pseudomonas aeruginosa is the major pathogen in chronic lung infections of individuals with cystic fibrosis (CF). Unrelated CF patients may acquire P. aeruginosa from the environment or by cross-infection in the CF setting. We tested the efficacy of measures to prevent nosocomial acquisition of P. aeruginosa at a Paediatric CF centre in a prospective 10-year study. P. aeruginosa-positive and P. aeruginosa-negative patients were seen in alternating weeks at the outpatient clinic. Faucets were equipped with filters to prevent bacterial contamination of tap water. Serial isolates were collected since the first documentation of a P. aeruginosa-positive culture and genotyped with a multimarker microarray. During the 10-year study, the annual prevalence of patients with at least one P. aeruginosa-positive culture was 39±6% in a population of 149±12 patients. P. aeruginosa was detected for the first time in 54 patients of whom 11 patients became chronically colonised with P. aeruginosa. Transient colonisations were recorded 97 times. A nosocomial acquisition of P. aeruginosa at the CF centre probably happened in one case. The worldwide dominant clones in the global P. aeruginosa population were also the most abundant clones in the panel of 324 early CF isolates. No rare clone had expanded by nosocomial transmission. It can be concluded that cross-infection with P. aeruginosa was prevented with simple hygienic measures at a CF centre that had experienced local outbreaks of nosocomial spread among unrelated patients in the past.

Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF) patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF) patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT) multimarker microarray (Alere Technologies GmbH, Jena, Germany), a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were employed as reference genotyping techniques to estimate the ArrayTube resolution power. RESULTS: 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU) whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient), respectively.AT typing of this Italian collection could be easily integrated with the global P. aeruginosa AT-typed population, uncovering that most AT-genotypes identified (> 80%) belonged to two large clonal clusters, and included 12 among the most abundant clones of the global population. CONCLUSIONS: The ArrayTube (AT) multimarker array represented a robust and portable alternative to reference techniques for performing P. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an Italian clinical collection from chronic and acute infections.

Competitive fitness of Pseudomonas aeruginosa isolates in human and murine precision-cut lung slices.

Chronic respiratory infections with the gram-negative bacterium Pseudomonas aeruginosa are an important co-morbidity for the quality of life and prognosis of people with cystic fibrosis (CF). Such long-term colonization, sometimes lasting up to several decades, represents a unique opportunity to investigate pathogen adaptation processes to the host. Our studies aimed to resolve if and to what extent the bacterial adaptation to the CF airways influences the fitness of the pathogen to grow and to persist in the lungs. Marker-free competitive fitness experiments of serial P. aeruginosa isolates differentiated by strain-specific SNPs, were performed with murine and human precision cut lung slices (PCLS). Serial P. aeruginosa isolates were selected from six mild and six severe CF patient courses, respectively. MPCLS or hPCLS were inoculated with a mixture of equal numbers of the serial isolates of one course. The temporal change of the composition of the bacterial community during competitive growth was quantified by multi-marker amplicon sequencing. Both ex vivo models displayed a strong separation of fitness traits between mild and severe courses. Whereas the earlier isolates dominated the competition in the severe courses, intermediate and late isolates commonly won the competition in the mild courses. The status of the CF lung disease rather than the bacterial genotype drives the adaptation of P. aeruginosa during chronic CF lung infection. This implies that the disease status of the lung habitat governed the adaptation of P. aeruginosa more strongly than the underlying bacterial clone-type and its genetic repertoire.

Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs.

Clones C and PA14 are the worldwide most abundant clonal complexes in the Pseudomonas aeruginosa population. The microevolution of clones C and PA14 was investigated in serial cystic fibrosis (CF) airway isolates collected over 20 years since the onset of colonization. Intraclonal evolution in CF lungs was resolved by genome sequencing of first, intermediate and late isolates and subsequent multimarker SNP genotyping of the whole strain panel. Mapping of sequence reads onto the P. aeruginosa PA14 reference genome unravelled an intraclonal and interclonal sequence diversity of 0.0035% and 0.68% respectively. Clone PA14 diversified into three branches in the patient's lungs, and the PA14 population acquired 15 nucleotide substitutions and a large deletion during the observation period. The clone C genome remained invariant during the first 3 years in CF lungs; however, 15 years later 947 transitions and 12 transversions were detected in a clone C mutL mutant strain. Key mutations occurred in retS, RNA polymerase, multidrug transporter, virulence and denitrification genes. Late clone C and PA14 persistors in the CF lungs were compromised in growth and cytotoxicity, but their mutation frequency was normal even in mutL mutant clades.

Clonal epidemiology of Pseudomonas aeruginosa in cystic fibrosis.

The basic defect in cystic fibrosis (CF) predisposes to chronic bacterial airway infections, particularly with Pseudomonas aeruginosa. Airway infections with P. aeruginosa in individuals with CF are unique in that they chronically affect a host who is immunocompetent in terms of cellular and humoral responses but is immunocompromised by impaired airway clearance. The initially acquired P. aeruginosa clone typically persists for many years in the patients' airways and thereby diversifies by de novo point mutations and the composition of its accessory genome. Co-colonizations with 2 or more clones are preferentially observed during the first 3 years of colonization. Upper and lower airways are commonly colonized by the same clone suggesting that the sinuses are the reservoir and gateway for the colonization of the lower airways. Early antipseudomonal chemotherapy has an 80% chance to eradicate the P. aeruginosa clone. This regimen introduced in the late 1980s has shifted the median age of the onset of chronic airways colonization with P. aeruginosa from school age to early adulthood at the most successful CF centres. The measures to prevent and to treat the Pseudomonas infections in CF have been considerably improved during the last 20 years. Highly transmissible epidemic strains, however, that emerge within a clonal lineage remain a major, still unresolved health threat for the CF community.

Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence.

RATIONALE: During long-term lung infection in patients with cystic fibrosis (CF), Pseudomonas aeruginosa strains develop mutations leading to clonal expansion. This microevolution is believed to be correlated with a reduced virulence. OBJECTIVES: We tested this hypothesis in models of lung infection, using mice with different genetic backgrounds. METHODS: From infected airways of six patients with CF, 25 P. aeruginosa clones were isolated during a period of up to 16.3 years and genotypically and phenotypically characterized. Virulence of the 8 early, 6 intermediate, and 11 late CF isolates and 5 environmental strains was assessed by monitoring acute mortality versus survival and P. aeruginosa chronic persistence versus lung clearance in mice of different genetic backgrounds, including CF mice. MEASUREMENTS AND MAIN RESULTS: Different patients harbored clonally unrelated strains, but early, intermediate, and late P. aeruginosa isolates from single patients were clonally related, allowing comparative in vivo analysis. Although late isolates were attenuated in causing acute mortality in the mouse models, compared with early and intermediate clonal isolates and environmental strains, they did not differ from early and intermediate clonal isolates in their capacity to establish chronic infection and cause extensive inflammation in the murine respiratory tract. CONCLUSIONS: Our findings indicate that clonal expansion of P. aeruginosa strains during microevolution within CF lungs leads to populations with altered but not reduced virulence. These P. aeruginosa clones with adapted virulence play a critical role in the pathogenesis of chronic infections and may serve to define virulence determinants as targets for novel therapies.

Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy.

BACKGROUND: Early antimicrobial chemotherapy can prevent or at least delay chronic cystic fibrosis (CF) airways infections with Pseudomonas aeruginosa. METHODS: During a 10-year study period P. aeruginosa was detected for the first time in 54 CF patients regularly seen at the CF centre Hannover. Amplicon sequencing of 34 loci of the P. aeruginosa core genome was performed in baseline and post-treatment isolates of the 15 CF patients who had remained P. aeruginosa - positive after the first round of antipseudomonal chemotherapy. RESULTS: Deep sequencing uncovered coexisting alternative nucleotides at in total 33 of 55,284 examined genome positions including six non-synonymous polymorphisms in the lasR gene, a key regulator of quorum sensing. After early treatment 42 of 50 novel nucleotide substitutions had emerged in exopolysaccharide biosynthesis, efflux pump and porin genes. CONCLUSIONS: Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs.

Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14.

Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant Pseudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within-clone single nucleotide sequence diversity of 8 × 10(-6) for clone C and 2 × 10(-5) for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.