Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a CysticFibrosis Patient Treated with b-Lactams

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C β-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A “gain of function” of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC β-lactamase is the main mechanism driving resistance in this notorious pathogen to β-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the β-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.Fil: Colque, Claudia A. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Albarracín Orio, Andrea G. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Hedemann, Laura G. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Feliziani, Sofía. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Moyano, Alejandro J. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Smania, Andrea M. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Colque, Claudia A. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Albarracín Orio, Andrea G. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Hedemann, Laura G. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Feliziani, Sofía. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Moyano, Alejandro J. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Smania, Andrea M. Universidad Nacional de Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET); Argentina.Fil: Tomatis, Pablo E. Universidad Nacional de Rosario. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Dotta, Gina. Universidad Nacional de Rosario. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Tomatis, Pablo E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Moreno, Diego M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Albarracín Orio, Andrea G. Universidad Católica de Córdoba. Facultad de Ciencias Agropecuarias. (IRNASUS-CONICET); Argentina.Fil: Moreno, Diego M. Universidad Nacional de Rosario. Instituto de Química de Rosario (IQUIR-CONICET); Argentina.Fil: Hickman Rachel A. Department of Clinical Microbiology; Denmark.Fil: Sommer, Lea M. Department of Clinical Microbiology; Denmark.Fil: Johansen, Helle K. Department of Clinical Microbiology; Denmark.Fil: Hickman Rachel A. Technical University of Denmark, Lyngb. Novo Nordisk Foundation Centre for Biosustainability; Denmark.Fil: Sommer, Lea M. Technical University of Denmark, Lyngb. Novo Nordisk Foundation Centre for Biosustainability; Denmark.Fil: Johansen, Helle K. Technical University of Denmark, Lyngb. Novo Nordisk Foundation Centre for Biosustainability; Denmark.Fil: Bonomo, Robert A. Case Western Reserve University. Departments of Molecular Biology and Microbiology, Medicine, Biochemistry, Pharmacology, and Proteomics and Bioinformatics; United States.Fil: Bonomo, Robert A. Senior Clinical Scientist Investigator. Louis Stokes Cleveland Department of Veterans Affairs; United States.Fil: Johansen, Helle K. University of Copenhagen. Department of Clinical Medicine; Denmark

High Prevalence, Coinfection Rate, and Genetic Diversity of Retroviruses in Wild Red Colobus Monkeys (Piliocolobus badius badius) in Taï National Park, Côte d'Ivoire▿

Simian retroviruses are precursors of all human retroviral pathogens. However, little is known about the prevalence and coinfection rates or the genetic diversity of major retroviruses—simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus type 1 (STLV-1), and simian foamy virus (SFV)—in wild populations of nonhuman primates. Such information would contribute to the understanding of the natural history of retroviruses in various host species. Here, we estimate these parameters for wild West African red colobus monkeys (Piliocolobus badius badius) in the Taï National Park, Côte d'Ivoire. We collected samples from a total of 54 red colobus monkeys; samples consisted of blood and/or internal organs from 22 monkeys and additionally muscle and other tissue samples from another 32 monkeys. PCR analyses revealed a high prevalence of SIV, STLV-1, and SFV in this population, with rates of 82%, 50%, and 86%, respectively. Forty-five percent of the monkeys were coinfected with all three viruses while another 32% were coinfected with SIV in combination with either STLV or SFV. As expected, phylogenetic analyses showed a host-specific pattern for SIV and SFV strains. In contrast, STLV-1 strains appeared to be distributed in genetically distinct and distant clades, which are unique to the Taï forest and include strains previously described from wild chimpanzees in the same area. The high prevalence of all three retroviral infections in P. b. badius represents a source of infection to chimpanzees and possibly to humans, who hunt them

Origin of Human T-Lymphotropic Virus Type 1 in Rural Côte d’Ivoire

Simian T-lymphotropic virus type 1 (STLV-1) strains occasionally infect humans. However, the frequency of such infections is unknown. We show that direct transmission of STLV-1 from nonhuman primates to humans may be responsible for a substantial proportion of human T-lymphotropic virus type 1 infections in rural Côte d’Ivoire, where primate hunting is common

Interspecies Transmission of Simian Foamy Virus in a Natural Predator-Prey System ▿

Simian foamy viruses (SFV) are ancient retroviruses of primates and have coevolved with their host species for as many as 30 million years. Although humans are not naturally infected with foamy virus, infection is occasionally acquired through interspecies transmission from nonhuman primates. We show that interspecies transmissions occur in a natural hunter-prey system, i.e., between wild chimpanzees and colobus monkeys, both of which harbor their own species-specific strains of SFV. Chimpanzees infected with chimpanzee SFV strains were shown to be coinfected with SFV from colobus monkeys, indicating that apes are susceptible to SFV superinfection, including highly divergent strains from other primate species

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

Bacteria are endowed with a unique ability to adapt to challenging environments. The evolution of bacterial populations during chronic infections involves a large diversity of adaptive mechanisms that cannot always be reproduced upon controlled laboratory conditions. This creates a gap between the phenotypical description and the underlying biochemical processes that drive that phenotype. Herein, we address the complexity of the bacterial adaptive response to antibiotic selective pressures by studying the in-patient evolution of a broad diversity of β-lactam resistant Pseudomonas aeruginosa hypermutator clones. By using mutational and ultra-deep amplicon sequencing analysis, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis patient. We identify the accumulation of multiple alterations targeting the chromosomally encoded class C β-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, ?a gain of function? of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work pinpoints the considerable evolutionary potential of hypermutator strains and uncovers the link between the antibiotic prescription history and the in-patient evolution of resistance.Fil: Colque, Claudia Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Tomatis, Pablo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Albarracín Orio, Andrea Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Dotta, Gina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Hedemann, Laura Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Hickman, Rachel A.. Technical University of Denmark; Dinamarca. Novo Nordisk Foundation Centre Biosustainability Dtu; DinamarcaFil: Sommer, L. M. Novo Nordisk Foundation Centre Biosustainability Dtu; Dinamarca. Technical University of Denmark; DinamarcaFil: Feliziani, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Moyano, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Bonomo, Robert A.. Cleveland Va Medical Center; Estados UnidosFil: Johansen, Helle K.. Novo Nordisk Foundation Centre Biosustainability Dtu; Dinamarca. Technical University of Denmark; DinamarcaFil: Molin, Soren. Novo Nordisk Foundation Centre Biosustainability Dtu; DinamarcaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Smania, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin