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Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats.

ABSTRACT: During treatment of infections with antibiotics in critically ill patients in the intensive care resistance often develops. This study aims to establish whether under those conditions this resistance can develop de novo or that genetic exchange between bacteria is by necessity involved. Chemostat cultures of Pseudomonas aeruginosa were exposed to treatment regimes with ceftazidime and meropenem that simulated conditions expected in patient plasma. Development of antibiotic resistance was monitored and mutations in resistance genes were searched for by sequencing PCR products. Even at the highest concentrations that can be expected in patients, sufficient bacteria survived in clumps of filamentous cells to recover and grow out after 3 to 5 days. At the end of a 7 days simulated treatment, the minimal inhibitory concentration (MIC) had increased by a factor between 10 and 10,000 depending on the antibiotic and the treatment protocol. The fitness costs of resistance were minimal. In the resistant strains, only three mutations were observed in genes associated with beta-lactam resistance. The development of resistance often observed during patient treatment can be explained by de novo acquisition of resistance and genetic exchange of resistance genes is not by necessity involved. As far as conclusions based on an in vitro study using P. aeruginosa and only two antibiotics can be generalized, it seems that development of resistance can be minimized by treating with antibiotics in the highest concentration the patient can endure for the shortest time needed to eliminate the infection.

SUBMITTER: Feng Y

PROVIDER: S-EPMC4752458 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats.

Feng Yanfang Y Hodiamont Caspar J CJ van Hest Reinier M RM Brul Stanley S Schultsz Constance C Ter Kuile Benno H BH

PloS one 20160212 2

During treatment of infections with antibiotics in critically ill patients in the intensive care resistance often develops. This study aims to establish whether under those conditions this resistance can develop de novo or that genetic exchange between bacteria is by necessity involved. Chemostat cultures of Pseudomonas aeruginosa were exposed to treatment regimes with ceftazidime and meropenem that simulated conditions expected in patient plasma. Development of antibiotic resistance was monitor ...[more]

PMID: 26872140

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Project description:The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed the inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro, and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multidrug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.IMPORTANCE Over the past decades, pan-resistant strains of major bacterial pathogens have emerged and have rendered clinically available antibiotics ineffective, putting at risk many of the major achievements of modern medicine, including surgery, cancer therapy, and organ transplantation. A thorough understanding of processes leading to the development of antibiotic resistance in human patients is thus urgently needed. We show that drug tolerance, the ability of bacteria to survive prolonged exposure to bactericidal antibiotics, rapidly evolves in the opportunistic human pathogen Pseudomonas aeruginosa upon recurrent exposures to antibiotics. Our studies show that tolerance protects P. aeruginosa against different classes of antibiotics and that it generally precedes and promotes resistance development. The rapid evolution of tolerance during treatment regimens may thus act as a strong driving force to accelerate antibiotic resistance development. To successfully counter resistance, diagnostic measures and novel treatment strategies will need to incorporate the important role of antibiotic tolerance.

Project description:UnlabelledElongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational ?-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which ?-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ?earP, or from a ?rmlC::acc1 strain deficient in dTDP-L-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-L-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in ?efp, ?earP, and ?rmlC::acc1 strains. ?rmlC::acc1, ?earP, and ?efp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival.ImportanceInfections with pathogenic Salmonella, E. coli, and Pseudomonas isolates can all lead to infectious disease with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility, and other traits that play key roles in establishing virulence. In Salmonella spp. and E. coli, the attachment of ?-Lys is required for EF-P activity, but the proteins required for this posttranslational modification pathway are absent from many organisms. Instead, bacteria such as P. aeruginosa activate EF-P by posttranslational modification with rhamnose, revealing a new role for protein glycosylation that may also prove useful as a target for the development of novel antibiotics.

Dataset Information

Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats.

Publications

Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats.

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