Artificial Sputum Medium

Abstract

The artificial sputum medium (ASM) was formulated to mimic the sputum of cystic fibrosis (CF) patients. The intra-and inter-patient variation in the composition of highly viscous CF sputum complicates the achievement of a reproducible culture condition to study microbial biofilms, for example, Pseudomonas aeruginosa colonization in the CF lung. However, the ASM is a homogenous non-viscous medium, which can be prepared in bulk quantities using a fixed set of ingredients. The conventional biofilm models always had cells attached to a solid biotic or abiotic surface submerged in a steady-state or continuous flow of culture medium. In contrast, P. aeruginosa grows in the CF lung under micro-aerophilic to anaerobic conditions in the form of microcolonies eventually forming a macro-aggregate/colony in which bacteria adhere to each other and to sputum components. Similarly, in ASM, P. aeruginosa forms micro-and macro-colonies and showed various phenotypes specific to CF isolates.

Bacterial biodiversity drives the evolution of CRISPR-based phage resistance in Pseudomonas aeruginosa

“…1 and Extended Data Fig. 3), or artificial sputum medium (for composition, see ref. 17) (Extended Data Fig.…”

“…1, Deviance test: Relationship between community composition and CRISPR; Residual deviance(30, n = 36) = 1.81, p < 0.001; Tukey contrasts: Monoculture v Mixed; z = −5.99, p < 0.001; Monoculture v A. baumannii ; z = −4.33, p < 0.001; Monoculture v B. cenocepacia ; z = −3.76, p < 0.01; Monoculture v S. aureus ; z = −2.38, p = 0.14; Monoculture v surface mutant; z = 2.26, p = 0.19). To explore the clinical relevance of this observation, we next co-cultured P. aeruginosa with A. baumannii in artificial sputum medium (ASM), which mimics the abiotic environment of sputum from cystic fibrosis patients 17 . This analysis revealed a similar effect of A. baumannii on the evolution of CRISPR-based resistance in both LB and ASM (Extended Data Fig.…”

A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB

“…In order to standardize a sputum matrix, either tremendous volumes of human sputum need to be pooled and extensively validated for the absence of Mycobacterium tuberculosis complex (MTBC)-DNA, or artificial sputum (AS) can be used which is produced under controlled and standardized conditions. Three major types of AS have been developed based either on polyacrylamide, on methylcellulose, or on watery dispersions [18][19][20]. Unfortunately, none of these resembles natural human sputum in consistency, chemical properties or viscosity; therefore, they do not simulate a clinical diagnostic sample.…”

Label-Free Optical Biodetection of Pathogen Virulence Factors in Complex Media Using Microtoroids with Multifunctional Surface Functionality

“…The artificial sputum medium, which mimics respiratory environment of the CF, was prepared accordingly a previously published protocol. 46 Simply, all essential Lamino acids (250 mg for each), except L-tryptophan, were added to a buffer solution having mucin, salmon sperm DNA, DTPA, NaCl, KCl, and Tris base under continuous stirring, as described in the protocol. 46 Then, the solution pH was stabilized to 7.0 using Tris base and the total volume was raised to 1000 mL by adding MB grade H 2 O.…”

“…46 Simply, all essential Lamino acids (250 mg for each), except L-tryptophan, were added to a buffer solution having mucin, salmon sperm DNA, DTPA, NaCl, KCl, and Tris base under continuous stirring, as described in the protocol. 46 Then, the solution pH was stabilized to 7.0 using Tris base and the total volume was raised to 1000 mL by adding MB grade H 2 O. Under sterile conditions, L-tryptophan and egg yolk emulsion were added respectively, following sterilization of the medium at 110°C for 15 min in an autoclave (Tuttnauer, Germany).…”