Critical physiological factors influencing the outcome of antimicrobial testing according to ISO 22196 / JIS Z 2801

Abstract

Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.

MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology

“…A major challenge in the management of biofilm-associated infections (BAI) is the development of adequate, standardized biofilm susceptibility testing assays that are clinically meaningful, i.e. that their results correlate with treatment outcome [3, 4]. Over the last years, a multitude of diverse laboratory methods to assess anti-biofilm treatments has been developed.…”

Antimicrobial Activity Testing Methods for Hydrophobic Patterned Surfaces

“…It has been demonstrated that the micro-patterned surface of lms reduces bacterial contamination 7 . To evaluate the antimicrobial activity of patterned lms different tests can be used and depending on the methodology used, the antimicrobial activity results could vary 7,17,18 .…”

“…However, the usefulness of the ISO 22196 assay for the evaluation of new antimicrobial surfaces is unclear as it showed discrepancies with other methods mainly due to the unrealistic way of transferring bacteria to the plastic surfaces, the high inoculum used and the amount of culture medium left in the liquid layer for 24 h for the diffusion of chemical antimicrobial products present in plastics 13,18 . In a study carried out in 8 different research facilities, differences between laboratories were found using the ISO 22196 standard method, mainly for materials with intermediate antimicrobial activity 17 . Four critical factors that in uence the outcome of antibacterial testing in a series of experiments were described: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration.…”

Permanent antimicrobial silicone rubber based on bonding guanidine polymers

“…The antibacterial performance of rubber specimens against E. coli and S. aureus were investigated with a film attachment method according to the Japanese Industrial Standard (JIS Z 2801:2000) with slight modifications . Briefly, 100 μL of cultured E. coli or S. aureus solution (10 6 CFU/mL) was dropped on the surface of a 5 × 5 cm 2 square specimen which was horizontally placed on a sterilized petri dish, and the droplet was covered with a sterilized PE film (4 × 4 cm 2 ) in case of evaporation during the following process.…”

“…transform infrared spectroscopy (FTIR) of VMQ-VPHMG and VMQ was recorded over the wavenumber range of 4000 to 400 cm −1 by Nicolet 5700 spectrometer (Perkin Elmer spectrum 100) using an attenuated total reflectance (ATR) mode. The water contact angles of vulcanized silicone rubbers were measured by the contact angle tester JC2000D3 (Shanghai Zhongchen Digital Technic Apparatus Co. Ltd, China).2.7 | Antibacterial performanceThe antibacterial performance of rubber specimens against E. coli and S. aureus were investigated with a film attachment method according to the Japanese Industrial Standard (JIS Z 2801:2000) with slight modifications 30,31. Briefly, 100 μL of cultured E. coli or S. aureus solution (10 6 CFU/mL) was dropped on the surface of a 5 × 5 cm 2 square specimen which was horizontally placed on a sterilized petri dish, and the droplet was covered with a sterilized PE film (4 × 4 cm 2 ) in case of evaporation during the following process.…”