微生物学

Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).

A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an in vitro microtiter-based assay to quantify the minimal bactericidal concentration for biofilms (MBC-B) for short exposure times (2 h). This exposure period is significantly shorter than standard over-night and 24-hour treatments described in traditional protocols. This assay was developed to approximate the time an antibiotic is available during a one-time treatment before it is metabolized, sequestered by host proteins, or digested.

In many environments, bacteria favor a sessile, surface-attached community lifestyle. These communities, termed biofilms, are ubiquitous among many species of bacteria. In some cases, biofilms form under flow conditions. Flow chambers, and in particular microfluidic channels, can be used to observe biofilm development and physiological effects while varying nutrient conditions, flow velocities, or introducing antimicrobials to the biofilm in real time. Here, we describe a microfluidic-based kill-kinetics assay for the observation of antimicrobial effects on biofilms under flowing conditions.

Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized by marked tolerance to many biocides, including oxidants such as hydrogen peroxide. Hydrogen peroxide is both produced by host phagocytic cells, and used as an antimicrobial compound. Understanding biofilm tolerance to hydrogen peroxide is therefore relevant to the persistence of Pseudomonas aeruginosa in human infections (such as chronic Pseudomonas aeruginosa infections in cystic fibrosis airways) as well as in environmental settings (such as water pipes).

This protocol was developed to determine the tolerance of Pseudomonas aeruginosa biofilms to hydrogen peroxide (H₂O₂) killing. The bacteria are grown as colony biofilms on polycarbonate membranes, as previously described in Walters et al. 2003. The protocol may be adapted for other bacterial, with appropriate changes in H₂O₂ concentrations, since different bacterial species may be more or less susceptible to H₂O₂ than Pseudomonas aeruginosa.