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Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance.

ABSTRACT: Streptococcus pneumoniaeis one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence ofS. pneumoniaein chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesized that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and that higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO did not result inS. pneumoniaebiofilm dispersal. Instead, treatment of bothin vitrobiofilms andex vivoadenoid tissue samples (a reservoir forS. pneumoniaebiofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO, therefore, appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms.

SUBMITTER: Allan RN

PROVIDER: S-EPMC4808185 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance.

Allan Raymond N RN Morgan Samantha S Brito-Mutunayagam Sanjita S Skipp Paul P Feelisch Martin M Hayes Stephen M SM Hellier William W Clarke Stuart C SC Stoodley Paul P Burgess Andrea A Ismail-Koch Hasnaa H Salib Rami J RJ Webb Jeremy S JS Faust Saul N SN Hall-Stoodley Luanne L

Antimicrobial agents and chemotherapy 20160325 4

Streptococcus pneumoniaeis one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing ...[more]

PMID: 26856845

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Project description:Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Project description:Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip flow reactors on a medium composed to mimic the exudate from a chronic wound. After 4 days, the biofilm was 114 μm thick with 9.45 log10 CFU cm-2 These biofilms exhibited tolerance, relative to exponential-phase planktonic cells, to subsequent treatment with ciprofloxacin. The specific growth rate of the biofilm was estimated via elemental balances to be approximately 0.37 h-1 and with a reaction-diffusion model to be 0.32 h-1, or one-third of the maximum specific growth rate for planktonic cells. Global analysis of gene expression indicated lower transcription of ribosomal genes and genes for other anabolic functions in biofilms than in exponential-phase planktonic cells and revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing. Metabolic pathways for phenazine biosynthesis and denitrification were transcriptionally activated in biofilms. A customized reaction-diffusion model predicted that steep oxygen concentration gradients will form when these biofilms are thicker than about 40 μm. Mutant strains that were deficient in Psl polysaccharide synthesis, the stringent response, the stationary-phase response, and the membrane stress response exhibited increased ciprofloxacin susceptibility when cultured in biofilms. These results support a sequence of phenomena leading to biofilm antibiotic tolerance, involving oxygen limitation, electron acceptor starvation and growth arrest, induction of associated stress responses, and differentiation into protected cell states.IMPORTANCE Bacteria in biofilms are protected from killing by antibiotics, and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.

Dataset Information

Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance.

Publications

Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance.

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