RESUMEN
Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.
Asunto(s)
Fibrosis Quística , Estudio de Asociación del Genoma Completo , Macrófagos , Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Macrófagos/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Fibrosis Quística/microbiología , Mutación , Virulencia/genética , Factores de Virulencia/genética , Adaptación FisiológicaRESUMEN
Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed in vitro selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing sigB. Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (icaR, spdC, and codY), while the remaining three (manA, narH, and fruB) were newly implicated by this study. Plasmid-mediated genetic complementation of manA, narH, and fruB transposon mutants corrected biofilm deficiencies, with high-level expression of manA and fruB further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/metabolismo , Plásmidos , Mutación , BiopelículasRESUMEN
Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Vías Biosintéticas/genética , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Flagelos/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Polisacáridos Bacterianos/biosíntesis , Pseudomonas aeruginosa/patogenicidad , Selección GenéticaRESUMEN
Rationale:Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation have been described for Pseudomonas aeruginosa, resulting in improved diagnosis and treatment, these features remain more poorly defined for S. aureus.Objectives: To characterize the molecular epidemiology and genetic adaptation of S. aureus during chronic CF airway infection and in response to antibiotic therapy.Methods: We performed whole-genome sequencing of 1,382 S. aureus isolates collected longitudinally over a mean 2.2 years from 246 children with CF at five U.S. centers between 2008 and 2017. Results were integrated with clinical and demographic data to characterize bacterial population dynamics and identify common genetic targets of in vivo adaptation.Measurements and Main Results: Results showed that 45.5% of patients carried multiple, coexisting S. aureus lineages, often having different antibiotic susceptibility profiles. Adaptation during the course of infection commonly occurred in a set of genes related to persistence and antimicrobial resistance. Individual sequence types demonstrated wide geographic distribution, and we identified limited strain-sharing among children linked by common household or clinical exposures. Unlike P. aeruginosa, S. aureus genetic diversity was unconstrained, with an ongoing flow of new genetic elements into the population of isolates from children with CF.Conclusions: CF airways are frequently coinfected by multiple, genetically distinct S. aureus lineages, indicating that current clinical procedures for sampling isolates and selecting antibiotics are likely inadequate. Strains can be shared by patients in close domestic or clinical contact and can undergo convergent evolution in key persistence and antimicrobial-resistance genes, suggesting novel diagnostic and therapeutic approaches for future study.
Asunto(s)
Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Adolescente , Antibacterianos/uso terapéutico , Niño , Estudios de Cohortes , Femenino , Humanos , Masculino , Epidemiología Molecular , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/genética , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
BACKGROUND: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. OBJECTIVES: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. METHODS: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. RESULTS: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. CONCLUSIONS: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.
Asunto(s)
Fibrosis Quística , Infecciones por Pseudomonas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fibrosis Quística/complicaciones , Fibrosis Quística/tratamiento farmacológico , Humanos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/genética , TemperaturaRESUMEN
RATIONALE: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments. OBJECTIVES: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics. METHODS AND MEASUREMENTS: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories. MAIN RESULTS: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. CONCLUSIONS: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.
Asunto(s)
Antibacterianos/farmacología , Bacterias , Fibrosis Quística/microbiología , Microbiota/efectos de los fármacos , Esputo/microbiología , Tobramicina/farmacología , Administración por Inhalación , Adolescente , Adulto , Anciano , Antibacterianos/uso terapéutico , Bacterias/genética , Bacterias/aislamiento & purificación , Infecciones Bacterianas/prevención & control , Niño , Fibrosis Quística/fisiopatología , Volumen Espiratorio Forzado , Humanos , Quimioterapia de Mantención , Metagenoma/efectos de los fármacos , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Factores de Tiempo , Tobramicina/uso terapéutico , Adulto JovenRESUMEN
RATIONALE: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. OBJECTIVES: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. METHODS: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. MEASUREMENTS AND MAIN RESULTS: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. CONCLUSIONS: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.
Asunto(s)
Aminofenoles/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Fibrosis Quística/tratamiento farmacológico , Inflamación/prevención & control , Quinolonas/uso terapéutico , Infecciones del Sistema Respiratorio/prevención & control , Adulto , Agonistas de los Canales de Cloruro/uso terapéutico , Fibrosis Quística/diagnóstico por imagen , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Pulmón/diagnóstico por imagen , Pulmón/metabolismo , Masculino , Infecciones del Sistema Respiratorio/metabolismo , Esputo/efectos de los fármacos , Esputo/metabolismo , Tomografía Computarizada por Rayos XAsunto(s)
Coinfección , Fibrosis Quística , Infecciones por Pseudomonas , Infecciones Estafilocócicas , Fibrosis Quística/complicaciones , Humanos , Pulmón/diagnóstico por imagen , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa , Infecciones Estafilocócicas/complicaciones , Staphylococcus aureusRESUMEN
Staphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Aminoglicósidos/farmacología , Proteínas Bacterianas/genética , Cefoxitina/farmacología , Fluoroquinolonas/farmacología , Ácido Fusídico/farmacología , Humanos , Lincosamidas/farmacología , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Rifampin/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/aislamiento & purificación , Combinación Trimetoprim y Sulfametoxazol/farmacologíaRESUMEN
RATIONALE: Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease. OBJECTIVES: To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes. METHODS: A total of 649 children with CF and newly identified P. aeruginosa were followed for a median 5.4 years during which a total of 2,594 P. aeruginosa isolates were collected. Twenty-six in vitro bacterial phenotypes were assessed among the isolates, including measures of motility, exoproduct production, colony morphology, growth, and metabolism. MEASUREMENTS AND MAIN RESULTS: P. aeruginosa phenotypes present at the time of culture were associated with both stage of infection (new onset, intermittent, or chronic) and the primary clinical outcome, occurrence of a pulmonary exacerbation (PE) in the subsequent 2 years. Two in vitro P. aeruginosa phenotypes best distinguished infection stages: pyoverdine production (31% of new-onset cultures, 48% of intermittent, 69% of chronic) and reduced protease production (31%, 39%, and 65%, respectively). The best P. aeruginosa phenotypic predictors of subsequent occurrence of a PE were mucoidy (odds ratio, 1.75; 95% confidence interval, 1.19-2.57) and reduced twitching motility (odds ratio, 1.43; 95% confidence interval, 1.11-1.84). CONCLUSIONS: In this large epidemiologic study of CF P. aeruginosa adaptation, P. aeruginosa isolates exhibited two in vitro phenotypes that best distinguished early and later infection stages. Among the many phenotypes tested, mucoidy and reduced twitching best predicted subsequent PE. These phenotypes indicate potentially useful prognostic markers of transition to chronic infection and advancing lung disease.
Asunto(s)
Fibrosis Quística/complicaciones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Adolescente , Niño , Preescolar , Fibrosis Quística/microbiología , Progresión de la Enfermedad , Femenino , Humanos , Técnicas In Vitro , Lactante , Modelos Logísticos , Masculino , Estudios Multicéntricos como Asunto , Evaluación de Resultado en la Atención de Salud , Fenotipo , Estudios Prospectivos , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
BACKGROUND: Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized. METHODS: We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation. RESULTS: Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03-3.83] and 2.14 [1.32-3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial. CONCLUSIONS: Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing.
Asunto(s)
Fibrosis Quística/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/fisiología , Biopelículas/efectos de los fármacos , Niño , Preescolar , Fibrosis Quística/complicaciones , Femenino , Genotipo , Glicosaminoglicanos/análisis , Humanos , Lactante , Masculino , Fenotipo , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Insuficiencia del TratamientoRESUMEN
The objective of this study is to describe the epidemiology of intestinal carriage with extended-spectrum-cephalosporin-resistant Enterobacteriaceae in children with index infections with these organisms. Patients with resistant Escherichia coli or Klebsiella bacteria isolated from the urine or a normally sterile site between January 2006 and December 2010 were included in this study. Available infection and stool isolates underwent phenotypic and molecular characterization. Clinical data relevant to the infections were collected and analyzed. Overall, 105 patients were identified with 106 extended-spectrum-cephalosporin-resistant E. coli (n = 92) or Klebsiella (n = 14) strains isolated from urine or a sterile site. Among the 27 patients who also had stool screening for resistant Enterobacteriaceae, 17 (63%) had intestinal carriage lasting a median of 199 days (range, 62 to 1,576). There were no significant differences in demographic, clinical, and microbiological variables between those with and those without intestinal carriage. Eighteen (17%) patients had 37 subsequent resistant Enterobacteriaceae infections identified: 31 urine and 6 blood. In a multivariable analysis, antibiotic intake in the 91 days prior to subsequent urine culture was significantly associated with subsequent urinary tract infection with a resistant organism (hazard ratio, 14.3; 95% confidence interval [CI], 1.6 to 130.6). Intestinal carriage and reinfection were most commonly due to bacterial strains of the same sequence type and with the same resistance determinants as the index extended-spectrum-cephalosporin-resistant Enterobacteriaceae, but carriage and reinfection with different resistant Enterobacteriaceae strains also occurred.
Asunto(s)
Antibacterianos/uso terapéutico , Portador Sano/tratamiento farmacológico , Portador Sano/microbiología , Cefalosporinas/uso terapéutico , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Enterobacteriaceae/efectos de los fármacos , Adolescente , Niño , Preescolar , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Heces/microbiología , Femenino , Genotipo , Humanos , Lactante , Intestinos/microbiología , Masculino , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Factores de Riesgo , beta-Lactamasas/biosíntesis , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Cystic fibrosis (CF) lung disease is associated with diverse bacteria chronically infecting the airways. Slow-growing, antibiotic-resistant mutants of Staphylococcus aureus known as small-colony variants (SCVs) have been isolated from respiratory secretions from European adults and children with CF lung disease using specific but infrequently used culture techniques. Staphylococcus aureus SCVs can be selected either by exposure to specific antibiotics or by growth with another CF pathogen, Pseudomonas aeruginosa. We sought to determine the prevalence, clinical significance, and likely mechanisms of selection of S. aureus SCVs among a US cohort of children with CF. METHODS: We performed a 2-year study of 100 children with CF using culture techniques sensitive for S. aureus SCVs, and evaluated associations with clinical characteristics using multivariable regression models. RESULTS: Staphylococcus aureus SCV infection was detected among 24% of participants and was significantly associated with a greater drop in lung function during the study (P = .007, adjusted for age and lung function at enrollment). This association persisted after adjusting for infection with other known CF pathogens, including P. aeruginosa and methicillin-resistant S. aureus. Evidence indicated that S. aureus SCVs were likely selected in vivo by treatment with the antibiotic trimethoprim-sulfamethoxazole and possibly by coinfection with P. aeruginosa. CONCLUSIONS: Infection with SCV S. aureus was independently associated with worse CF respiratory outcomes in this pediatric cohort. As many clinical microbiology laboratories do not specifically detect S. aureus SCVs, validation and extension of these findings would require widespread changes in the usual laboratory and clinical approaches to these bacteria.
Asunto(s)
Fibrosis Quística/complicaciones , Farmacorresistencia Bacteriana , Neumonía Estafilocócica/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Adolescente , Antibacterianos/uso terapéutico , Portador Sano/microbiología , Niño , Preescolar , Fibrosis Quística/tratamiento farmacológico , Femenino , Humanos , Lactante , Masculino , Interacciones Microbianas , Neumonía Estafilocócica/tratamiento farmacológico , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/aislamiento & purificación , Resultado del Tratamiento , Estados UnidosRESUMEN
Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.
Asunto(s)
Antagonistas del Ácido Fólico , Infecciones Estafilocócicas , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , Fosfatos de Dinucleósidos , Antagonistas del Ácido Fólico/metabolismo , Inflamación , Ratones , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Timidina/metabolismoRESUMEN
Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.
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Cromosomas Bacterianos , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Humanos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genéticaRESUMEN
Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1ß by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1ß expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.
Asunto(s)
Fibrosis Quística/microbiología , Citocinas/metabolismo , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Pulmón/microbiología , Macrófagos/microbiología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Muerte Celular , Fibrosis Quística/inmunología , Fibrosis Quística/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Transducción de Señal , Células THP-1 , Factores de Tiempo , VirulenciaRESUMEN
A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla(KPC-5), was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla(KPC-5) showed significant differences from the flanking regions of other bla(KPC) variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro(103)-->Arg), while KPC-4 contained Pro(103)-->Arg plus an additional amino acid change (Val(239)-->Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.
Asunto(s)
Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , Sustitución de Aminoácidos , Antibacterianos/farmacología , Bacteriemia/microbiología , Ácido Clavulánico/farmacología , Farmacorresistencia Bacteriana/genética , Inhibidores Enzimáticos/farmacología , Variación Genética , Hidrólisis , Focalización Isoeléctrica , Pruebas de Sensibilidad Microbiana , Fenotipo , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores de beta-LactamasasRESUMEN
During a 6-month period, 37/513 (7.2%) Pseudomonas aeruginosa isolates belonging to 13 pulsed-field gel electrophoresis (PFGE) groups from Puerto Rican hospitals were carbapenem nonsusceptible. Seven of 37 isolates from four PFGE groups carried bla(IMP-18), and 25/37 isolates from seven PFGE groups carried bla(KPC). The results indicated the clonal spread of bla(KPC)-positive P. aeruginosa isolates into several Puerto Rican hospitals and the dissemination of bla(IMP-18) and bla(KPC) into genetically unrelated isolates.
Asunto(s)
Carbapenémicos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , beta-Lactamasas/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de la Membrana Bacteriana Externa/fisiología , Niño , Preescolar , Farmacorresistencia Bacteriana , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Lactante , Masculino , Proteínas de Transporte de Membrana/fisiología , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Porinas/fisiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , beta-Lactamasas/genéticaRESUMEN
OBJECTIVES: Although Pseudomonas aeruginosa from cystic fibrosis patients are well known for their antibiotic resistance, isolates that are highly susceptible to multiple drug classes have also been encountered. In this study, hypersusceptible P. aeruginosa isolates were analysed for changes in intrinsic resistance mechanisms to explain the observed phenotype. METHODS: P. aeruginosa strains PA30 and PA431 were isolated from the sputa of cystic fibrosis patients and susceptibilities were determined by agar dilution. Isolates were genetically unrelated by PFGE analysis. Expression of efflux pumps, porins, a chromosomal cephalosporinase and a gene, glmS, previously implicated in hypersusceptibility were evaluated by real-time RT-PCR, outer membrane protein analysis and beta-lactamase hydrolysis assays. RESULTS: PA30 was hypersusceptible to beta-lactams, fluoroquinolones and antimetabolites, with MICs at least 4-fold lower than those for the prototype strain PAO1, while PA431 was hypersusceptible to beta-lactams and antimetabolites. Both isolates overproduced the porin OprF but showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. PA30 had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance. PA431 showed down-regulation of oprM, the last gene in the mexAB-oprM operon, but overexpressed the mexXY pump. The ampC beta-lactamase was weakly inducible in strain PA30, corresponding to cefoxitin hypersusceptibility. CONCLUSIONS: The changes in expression of several intrinsic mechanisms in the hypersusceptible strains did not correlate with the observed phenotype. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility.
Asunto(s)
Antibacterianos/farmacología , Fibrosis Quística/complicaciones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Humanos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Fenotipo , Pseudomonas aeruginosa/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saliva/microbiología , beta-Lactamasas/metabolismoRESUMEN
Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.