RESUMO
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.
Assuntos
Anticorpos Antibacterianos , Anticorpos Neutralizantes , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Anticorpos Antibacterianos/farmacologia , Microscopia Crioeletrônica , Imunoglobulinas/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Fibrose Cística , Humanos , Fibrose Cística/microbiologia , Pseudomonas aeruginosa/genética , Sistema Respiratório , Adaptação Fisiológica , FenótipoRESUMO
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.
Assuntos
Agonismo Inverso de Drogas , Quinolonas , Humanos , Animais , Camundongos , Proteínas de Bactérias , Biofilmes , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/química , Pseudomonas aeruginosaRESUMO
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.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Humanos , Pulmão/microbiologia , Camundongos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genéticaRESUMO
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.
Assuntos
Adaptação Fisiológica/fisiologia , Fibrose Cística/microbiologia , Pulmão/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Adaptação Fisiológica/genética , Sequência de Bases , Evolução Molecular , Genômica , Humanos , Fenótipo , Infecções por Pseudomonas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNAAssuntos
Bronquiectasia/complicações , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/transmissão , Antibacterianos , Bronquiectasia/microbiologia , Bronquiectasia/terapia , Infecção Hospitalar/transmissão , Humanos , Conhecimento , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa , Medição de Risco , Escarro/microbiologiaRESUMO
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.
Assuntos
Adaptação Fisiológica/fisiologia , Fibrose Cística/microbiologia , Pulmão/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Evolução Biológica , Criança , Pré-Escolar , Doença Crônica , Feminino , Humanos , Lactente , Masculino , Índice de Gravidade de DoençaRESUMO
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.
Assuntos
Fezes/microbiologia , Microbioma Gastrointestinal , Sepse/microbiologia , Abdome , Animais , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Índice de Gravidade de DoençaRESUMO
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.
Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias , Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Criança , Doença Crônica , Fibrose Cística/complicações , Fibrose Cística/diagnóstico , Fibrose Cística/epidemiologia , Fibrose Cística/microbiologia , Monitoramento de Medicamentos/métodos , Feminino , Alemanha/epidemiologia , Humanos , Lactente , Masculino , Tipagem de Sequências Multilocus/métodos , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Tempo para o Tratamento , Adulto JovemRESUMO
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.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Loci Gênicos , Genoma Bacteriano , Mutação , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Clonais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/microbiologia , Sistema Respiratório/patologia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Variação Genética , Genótipo , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Sequência Conservada , Microbiologia Ambiental , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
The population structure of the cosmopolitan Pseudomonas aeruginosa was investigated by genotyping 2921 isolates from 1448 independent habitats with a custom-made 58 binary marker microarray. Of 323 identified clone types, 109 clones made up 82% of the population. The 20 most frequent clones had an absolute share of 44% indicating that the P. aeruginosa population is dominated by few epidemic clonal complexes. The frequency distribution of common clones was different between inanimate habitats and human niches. The three most abundant clones in the environment were rare among isolates from human infection. Conversely, disease-associated isolates either belonged to ubiquitous clones such as C and PA14 or to clones that were uncommon in the environment. The P. aeruginosa population consists of major clones that are just as versatile in their habitat and geographic origin as the whole species and of minor clones with preference for a peculiar niche.
Assuntos
Biodiversidade , Ecossistema , Pseudomonas aeruginosa/classificação , Animais , Microbiologia Ambiental , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Análise Espaço-TemporalRESUMO
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.
Assuntos
Genoma Bacteriano , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Microbiologia Ambiental , Feminino , Variação Genética , Humanos , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Virulência/genéticaRESUMO
Pseudomonas aeruginosa, the type species of pseudomonads, is an opportunistic pathogen that colonizes a wide range of niches. Current genome sequencing projects are producing previously inconceivable detail about the population biology and evolution of P. aeruginosa. Its pan-genome has a larger genetic repertoire than the human genome, which explains the broad metabolic capabilities of P. aeruginosa and its ubiquitous distribution in aquatic habitats. P. aeruginosa may persist in the airways of individuals with cystic fibrosis for decades. The ongoing whole-genome analyses of serial isolates from cystic fibrosis patients provide the so far singular opportunity to monitor the microevolution of a bacterial pathogen during chronic infection over thousands of generations. Although the evolution in cystic fibrosis lungs is neutral overall, some pathoadaptive mutations are selected during the within-host evolutionary process. Even a single mutation may be sufficient to generate novel complex traits provided that predisposing mutational events have previously occurred in the clonal lineage.
RESUMO
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.
Assuntos
Fibrose Cística/microbiologia , Variação Genética , Genoma Bacteriano/genética , Metaboloma , Proteoma , Pseudomonas aeruginosa , Transcriptoma , Substituição de Aminoácidos , Ilhas Genômicas , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidadeRESUMO
BACKGROUND/METHODS: The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years. RESULTS: Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient. CONCLUSION: The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.
Assuntos
Fibrose Cística/microbiologia , Epidemiologia Molecular/métodos , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Estudos Transversais , Feminino , Genótipo , Humanos , Masculino , Pseudomonas aeruginosa/classificaçãoRESUMO
Genomic analyses on versatility of the ubiquitous opportunistic pathogen Pseudomonas aeruginosa have been focusing on clinical strains from humans but much less on animal and environmental strains. Here, we aimed to compare genomic patterns of bovine, environmental, and human strains of P. aeruginosa. A collection of 71 strains, equally representing bovine (non-clinical), environmental (aquatic), and human (clinical) isolates from all main subregions of Hungary was genotyped by PCR microarray. Results were interpreted in comparison with internationally established human clinical and environmental clones, based on single nucleotide polymorphisms, on di- and multiallelic loci (fliC and fpvA) of the conserved core genome, and on genetic markers for the flexible accessory genome. As a result, a total of 33 clones were identified, with one bovine, 10 environmental, and five human clones regarded as new ones. In spite of general clonal diversity, bovine and human clones seemed to be habitat related. Bovine strains were characterized by significant overrepresentation of type III FpvA pyoverdine receptor, while the environmental and human strains showed the dominance of type I FpvA. Genotypes of non-clinical bovine strains of P. aeruginosa differed from those of human clinical strains, supporting the hypothesis about specific groups of strains colonizing specific habitats.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/veterinária , Pseudomonas aeruginosa/genética , Microbiologia da Água , Animais , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Análise por Conglomerados , Hibridização Genômica Comparativa , DNA Bacteriano/análise , Técnicas de Genotipagem , Humanos , Hungria , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Reação em Cadeia da PolimeraseRESUMO
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.