Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings.

The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.

Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm.

Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well-established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0·6 logs during a 3-h contact time. Efficacy testing showed biofilms exposed to 400 µmol l-1 penicillin G decreased by only 0·3 logs. Interestingly, time-lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96-well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well-characterized methods will be crucial for the discovery of new anti-biofilm strategies. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms are tolerant to antimicrobial treatments and can lead to persistent infections. Finding new anti-biofilm strategies and understanding their mode-of-action is therefore of high importance. Historically, antimicrobial testing has focused on measuring the decrease in viability. While kill data are undeniably important, measuring biofilm disruption provides equally useful information. Starting with biofilm grown in the same reactor, we paired assessment of biofilm removal using a new treatment-flow-cell and real-time microscopy with kill data collected using the single tube method (ASTM E2871). Pairing these two methods revealed efficient biofilm removal properties of Penicillin G which were not detected during efficacy testing.

Evaluation of Petrifilm™ aerobic count plates as an equivalent alternative to drop plating on R2A agar plates in a biofilm disinfectant efficacy test.

This paper compares Petrifilm™ aerobic count (AC) plates to drop plating on R2A agar plates as an alternative method for biofilm bacteria enumeration after application of a disinfectant. A Pseudomonas aeruginosa biofilm was grown in a Centers for Disease Control and Prevention biofilm reactor (ASTM E2562) and treated with 123 ppm sodium hypochlorite (as free chlorine) according to the Single Tube Method (ASTM E2871). Aliquots from the same dilution tubes were plated on Petrifilm™ AC plates and drop plated on R2A agar plates. The Petrifilm™ AC and R2A plates were incubated for 48 and 24 h, respectively, at 36 ± 1 °C. After nine experimental runs performed by two technicians, the mean difference in biofilm log densities [log biofilm density (LD) = log10(CFU/cm(2))] between the two methods for control coupons, treated coupons, and log reduction (LR) was 0.052 (p = 0.451), -0.102 (p = 0.303), and 0.152 (p = 0.313). Equivalence testing was used to assess equivalence of the two plating methods. The 90 % confidence intervals for the difference in control and treated mean LDs between methods were (-0.065, 0.170) and (-0.270, 0.064), both of which fall within a (-0.5, +0.5) equivalence criterion. The 90 % confidence interval for the mean LR difference (-0.113, 0.420) also falls within this equivalence criterion. Thus, Petrifilm™ AC plates were shown to be statistically equivalent to drop plating on R2A agar for the determination of control LDs, treated LDs, and LR values in an anti-biofilm efficacy test. These are the first published results that establish equivalency to a traditional plate counting technique for biofilms and for a disinfectant assay.

Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test.

The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16 to 18h), treatment temperature (20-24°C), sonication duration (25-35min), and sonication power (130-480W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm(2)), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.

Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model.

Laboratory reactor systems designed to model specific environments enable researchers to explore environmental dynamics in a more controlled manner. This paper describes the design and operation of a reactor system built to model a swimming pool in the laboratory. The model included relevant engineering parameters such as filter loading and turn-overs per day. The water chemistry in the system's bulk water was balanced according to standard recommendations and the system was challenged with a bacterial load and synthetic bather insult, formulated to represent urine and perspiration. The laboratory model was then used to evaluate the efficacy of six chemical treatments against biofilm and planktonic bacteria. Results showed that the biofilm was able to accumulate on coupons and in the filter systems of reactors treated with either 1-3 mg/L free chlorine or 10 mg/L polyhexamethylene biguanide (PHMB). All the treatments tested resulted in at least a 4 log reduction in biofilm density when compared to the control, but shock treatments were the most effective at controlling biofilm accumulation. A once weekly shock dose of 10 mg/L free chlorine resulted in the greatest log reduction in biofilm density. The research demonstrated the importance of studying a biofilm in addition to the planktonic bacteria to assess the microbial dynamics that exist in a swimming pool model.