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1.
Nat Commun ; 13(1): 1231, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35264582

RESUMO

Acute bacterial infections are often treated empirically, with the choice of antibiotic therapy updated during treatment. The effects of such rapid antibiotic switching on the evolution of antibiotic resistance in individual patients are poorly understood. Here we find that low-frequency antibiotic resistance mutations emerge, contract, and even go to extinction within days of changes in therapy. We analyzed Pseudomonas aeruginosa populations in sputum samples collected serially from 7 mechanically ventilated patients at the onset of respiratory infection. Combining short- and long-read sequencing and resistance phenotyping of 420 isolates revealed that while new infections are near-clonal, reflecting a recent colonization bottleneck, resistance mutations could emerge at low frequencies within days of therapy. We then measured the in vivo frequencies of select resistance mutations in intact sputum samples with resistance-targeted deep amplicon sequencing (RETRA-Seq), which revealed that rare resistance mutations not detected by clinically used culture-based methods can increase by nearly 40-fold over 5-12 days in response to antibiotic changes. Conversely, mutations conferring resistance to antibiotics not administered diminish and even go to extinction. Our results underscore how therapy choice shapes the dynamics of low-frequency resistance mutations at short time scales, and the findings provide a possibility for driving resistance mutations to extinction during early stages of infection by designing patient-specific antibiotic cycling strategies informed by deep genomic surveillance.


Assuntos
Infecções Bacterianas , Fibrose Cística , Infecções por Pseudomonas , Infecções Respiratórias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana/genética , Resistência Microbiana a Medicamentos , Humanos , Mutação , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Infecções Respiratórias/tratamento farmacológico
2.
Vaccine ; 36(46): 6926-6932, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30314911

RESUMO

The Pseudomonas aeruginosa type III secretion system protein PopB and its chaperon protein PcrH, when co-administered with the adjuvant curdlan, elicit Th17 responses after intranasal immunization of mice. These PopB/PcrH-curdlan vaccines protect mice against acute lethal pneumonia in an IL-17-dependent fashion involving CD4 helper T cells secreting IL-17 (Th17 cells). In this study, we tested whether encapsulation of PopB/PcrH in poly-lactic-co-glycolic acid (PLGA) nanoparticles could elicit Th17 responses to PopB. Recombinant PopB/PcrH or PcrH alone was encapsulated into PLGA nanoparticles. Mice (FVB/N) were intranasally immunized with the PLGA-PopB/PcrH nanoparticles, PLGA-PcrH nanoparticles, PLGA alone, or PopB/PcrH alone. The protective efficacy was assessed in an acute lung infection model with a lethal dose of an ExoU-producing version of P. aeruginosa strain PAO1. Th17 responses were assayed by intracellular flow cytometry and by ELISA for IL-17 in supernatants of splenocytes co-cultured with purified PopB/PcrH. PLGA-PopB/PcrH-immunized mice showed 3-4-fold higher Th17 responses both in the lung and in the spleen compared to mice immunized with empty PLGA or PopB/PcrH alone. After challenge with P. aeruginosa, PLGA-PopB/PcrH-immunized mice showed significantly lower bacterial counts in the lungs and improved survival. In conclusion, encapsulation of PopB/PcrH in PLGA nanoparticles can elicit Th17 responses to intranasal vaccination and protect mice against acute lethal P. aeruginosa pneumonia.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Portadores de Fármacos/administração & dosagem , Pneumonia Bacteriana/prevenção & controle , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Células Th17/imunologia , Administração Intranasal , Animais , Antígenos de Bactérias/administração & dosagem , Carga Bacteriana , Proteínas de Bactérias/administração & dosagem , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-17/análise , Pulmão/microbiologia , Pulmão/patologia , Vacinas contra Pseudomonas/administração & dosagem , Análise de Sobrevida , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
3.
Infect Immun ; 83(10): 4003-14, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26216419

RESUMO

Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant Staphylococcus aureus in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe S. aureus pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22(+) TCRß(+) cells and neutrophils in BALF. Exogenous IL-22 protected mice from S. aureus pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to S. aureus pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.


Assuntos
Microbioma Gastrointestinal , Intestinos/microbiologia , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/microbiologia , Staphylococcus aureus/fisiologia , Animais , Feminino , Humanos , Interleucinas/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/imunologia , Interleucina 22
4.
Infect Immun ; 82(8): 3483-91, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24914214

RESUMO

As an immune-privileged site, the eye, and particularly the outer corneal surface, lacks resident mature immune effector cells. Physical barriers and innate mediators are the best-described effectors of immunity in the cornea. When the barriers are breached, infection can result in rapid tissue destruction, leading to loss of visual acuity and frank blindness. To determine the cellular and molecular components needed for effective adaptive immunity on the corneal surface, we investigated which immune system effectors were required for protection against Staphylococcus aureus corneal infections in mice, which are a serious cause of human eye infections. Both systemically injected and topically applied antibodies to the conserved cell surface polysaccharide poly-N-acetylglucosamine (PNAG) were effective at mediating reductions in corneal pathology and bacterial levels. Additional host factors impacting protection included intercellular adhesion molecule 1 (ICAM-1)-dependent polymorphonuclear leukocyte (PMN) recruitment, functional CD4(+) T cells, signaling via the interleukin-17 (IL-17) receptor, and IL-22 production. In germfree mice, there was no protective efficacy of antibody to PNAG due to the lack of LY6G(+) inflammatory cell coeffector recruitment to the cornea. Protection was manifest after 3 weeks of exposure to conventional mice and acquisition of a resident microbiota. We conclude that in the anterior eye, ICAM-1-mediated PMN recruitment to the infected cornea along with endogenous microbiota-matured CD4(+) T cells producing both IL-17 and IL-22 is required for antibody to PNAG to protect against S. aureus infection.


Assuntos
Imunidade Adaptativa , Anticorpos Antibacterianos/imunologia , Infecções Oculares/imunologia , Olho/imunologia , Microbiota/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Carga Bacteriana , Linfócitos T CD4-Positivos/imunologia , Córnea/microbiologia , Córnea/patologia , Modelos Animais de Doenças , Infecções Oculares/microbiologia , Humanos , Molécula 1 de Adesão Intercelular/imunologia , Interleucina-17/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Interleucina 22
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