Review
Development, standardization, and validation of a biofilm efficacy test: The single tube method

https://doi.org/10.1016/j.mimet.2019.105694Get rights and content

Abstract

Methods validated by a standard setting organization enable public, industry and regulatory stakeholders to make decisions on the acceptability of products, devices and processes. This is because standard methods are demonstrably reproducible when performed in different laboratories by different researchers, responsive to different products, and rugged when small (usually inadvertent) variations from the standard procedure occur. The Single Tube Method (ASTM E2871) is a standard method that measures the efficacy of antimicrobials against biofilm bacteria that has been shown to be reproducible, responsive and rugged. In support of the reproducibility assessment, a six-laboratory study was performed using three antimicrobials: a sodium hypochlorite, a phenolic and a quaternary/alcohol blend, each tested at low and high efficacy levels. The mean log reduction in viable bacteria in this study ranged from 2.32 to 4.58 and the associated reproducibility standard deviations ranged from 0.89 to 1.67. Independent follow-up testing showed that the method was rugged with respect to deviations in sonication duration and sonication power but slightly sensitive to sonicator reservoir degassing and tube location within the sonicator bath. It was also demonstrated that when a coupon was dropped into a test tube, bacteria can splash out of reach of the applied antimicrobials, resulting in substantial bias when estimating log reductions for the products tested. Bias can also result when testing products that hinder the harvesting of microbes from test surfaces. The culmination of this work provided recommended changes to the early version of the standard method E2871-13 (ASTM, 2013b) including use of splashguards and microscopy checks. These changes have been incorporated into a revised ASTM method E2871-19 (ASTM 2019) that is the basis for the first regulatory method (ATMP-MB-20) to substantiate “kills biofilm” claims for antimicrobials registered and sold in the US.

Introduction

Antimicrobial products must pass performance criteria specific for different use sites before entering the marketplace. In the US, antimicrobial products for use on hard, non-porous surfaces have historically been tested using microorganisms dried onto a surface using semi-quantitative methods like the Use Dilution Method (Tomasino et al., 2012). In the last decade, a considerable effort has been made to update these historical tests with more quantitative test methods (Tomasino, 2013). Validated quantitative methods are needed that test antimicrobials against planktonic bacteria in suspension, planktonic bacteria dried onto a surface, loosely surface-associated bacteria and biofilm bacteria. (Tomasino, 2013).

Fundamental research has demonstrated that biofilm bacteria are phenotypically different than planktonic bacteria of the same genotype (Sauer et al., 2002; Mah and O'Toole, 2001). Biofilm not only consists of the bacteria, but also contains extracellular matrix (EPS) that glues the cells together and provides a boundary layer of protection. Depending upon where the biofilm is living, the EPS may also contain a variety of inorganic and organic components. Because the bacteria are in close proximity to each other, they can share genetic material and communicate via quorum sensing (Lewandowski and Beyenal, 2013). A consequence of these differences from the planktonic state, biofilm bacteria are more tolerant to antimicrobials and antibiotics (Stewart, 2015; Davies, 2003; Buckingham-Meyer et al., 2007; Behnke et al., 2011) and more variable in their responses (Parker et al., 2018). A practical outcome of this understanding of biofilms is that researchers, antimicrobial manufacturers, pharma industries, and regulators must use newer biofilm methods, not conventional planktonic methods, to measure the efficacy of antimicrobials against biofilm bacteria (EPA, 2016b). Since the manner in which a biofilm is grown impacts its tolerance (Buckingham-Meyer et al., 2007; Stewart, 2015; Manner et al., 2017; Stewart and Parker, 2019), then standard biofilm methods using different reactor systems that represent a variety of environmental conditions are necessary to predict how an antimicrobial will perform.

Standard(ized) methods, i.e., those taken through the standardization process with a consensus-setting organization such as ASTM International, are the tools researchers, industry and regulators use to evaluate antimicrobial product performance. Standard methods development is the creation of laboratory protocols for the purpose of comparison, both within a single laboratory and among laboratories. The impetus for the development of many microbial standard methods is efficacy testing for product registration with a regulatory agency such as the US Environmental Protection Agency (EPA) or the US Food and Drug Administration (Tomasino, 2013). To protect public health, regulatory agencies require efficacy data when a product is registered. Table 1 gives the definition of the “statistical R's” that encapsulate the desirable attributes of an antimicrobial test method. For product registration, standard methods that are repeatable, reproducible, rugged and responsive are absolutely required (Hamilton et al., 2013). A standard method should also be reasonable, meaning it should utilize equipment that is “typical” for a laboratory and it should not require an excessive amount of time, supplies or highly specialized training. Most importantly, standard methods, unlike research methods, undergo rigorous multi-laboratory statistical evaluations of these attributes.

An effective strategy in biofilm methods development is to partition the methods into a sequence of 4 discrete steps: Grow, Treat, Sample and then Analyze. The first step is to grow a reproducible and relevant biofilm. The second step is to expose the mature biofilm to an antimicrobial product. The treated biofilm is then sampled (harvested) and then the effects of the antimicrobial are analyzed by quantifying the biofilm bacteria that survived the treatment.

This paper describes the development and validation of the Single Tube Method (STM, ASTM E2871), a standard laboratory method that tests the efficacy of antimicrobial products against biofilms. The STM was developed with the interconnected but discrete steps of Grow, Treat, Sample and Analyze in mind. This means, for example, that although the STM was validated using a robust P. aeruginosa biofilm grown in the high shear environment of the CDC reactor (Goeres et al., 2009; ASTM, 2007), the STM treatment efficacy step was intentionally designed to accommodate biofilm grown with any microorganisms of interest (e.g., the CDC reactor has been used to grow biofilms of Staphylococcus aureus (Lineback et al., 2018), Candida tropicalis (Fernandez-Rivero et al., 2017), or a consortia (Yoon and Lee, 2017)) in any reactor system. The STM provides a quantitative measurement of the log reduction in viable biofilm bacteria that results from exposing a mature biofilm to an antimicrobial for a specified contact time. The STM was designed to treat and sample biofilm in a single tube and volume; hence, the STM does not differentiate between cells killed on the surface and cells that are removed from the surface, for example by a surfactant in the antimicrobial product formulation, then killed by active agents in the bulk liquid. A method that exposes a biofilm-coated surface (coupon) to an antimicrobial treatment in one vessel and then transfers that coupon into a second vessel for harvesting can provide data separately on removal of the biofilm from the surface (from viable cell counts from vessel 1) and kill (remaining viable cells attached to the coupon from vessel 2). The STM measures kill only.

Here we describe the early life cycle of the STM including the results from a six-laboratory collaborative study that led to its initial standardization by ASTM as E2871-13 (ASTM, 2013b), subsequent ruggedness testing on the harvesting and disaggregation steps, and investigations of potential bias that can result from inadequate harvesting and coupon splashing. This work culminated in a revised version of the STM as E2871-19 (ASTM 2019) and the first regulatory test, ATMP-MB-20, for substantiating efficacy claims against biofilms in the US (EPA, 2016a). This evolution of the STM demonstrates that standard methods are living documents that are reviewed and updated to incorporate an improved understanding of the system under investigation and changes in technology.

Section snippets

The single tube method

ASTM E2871-19, the most current version of the Single Tube Method (ASTM 2019), is summarized in Fig. 1 and demonstrated in a video created by the Standardized Biofilm Methods Laboratory in the Center for Biofilm Engineering at Montana State University (http://www.biofilm.montana.edu/standardized-biofilm-methods-training-videos.html).

Here, we describe E2871-13 and then the subsequent steps that led to E2871-19. First, a Pseudomonas aeruginosa biofilm (see Fig. S1 in Supplementary Material for a

Multi-laboratory study

The untreated control LDs (log10(CFU/cm2)) from the inter-laboratory study of the STM were analyzed to assess the resemblance of the controls across labs, and across experiments within each lab. (Fig. S4 in the Supplementary Material provides a graph of the LDs of the control data). The reproducibility SD = 0.2442 of the LDs indicates excellent reproducibility of the control biofilms grown in the high shear environment of the CDC reactor (ASTM E3161). Based on a review, Parker and Hamilton, 2011

Discussion

It is desirable that efficacy testing in a laboratory be performed on a biofilm that adequately emulates “real use” conditions so as to predict how the antimicrobial will perform in situ. Although the intent is to develop a standard antimicrobial test method that represents the environment where an antimicrobial will be applied as closely as possible, operational factors are often either included or removed that simplify the method, because it is important that a standard method be conducted in

Conclusion

Although standardized biofilm growth protocols have been around for decades (ASTM, 2002a; Goeres et al., 2007; ASTM, 2007, ASTM, 2008), standardized biofilm efficacy methods are relatively recent. The MBEC was standardized in 2011. Here we outline the early life cycle of the STM that led to its initial standardization in 2013 (ASTM, 2013a). The subsequent ruggedness and bias testing reported here led to two substantial modifications of the STM: the development of the splashguard and the use of

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to the Industrial Associates of the Center for Biofilm Engineering for partial funding of the work presented here as well as the writing of the manuscript.

References (40)

  • ASTM

    Interlaboratory Study to Establish Precision Statements for ASTM E2871: Standard Test Method for Evaluating Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using the Single Tube Method

    (2013)
  • ASTM

    Standard Test Method for Evaluating Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method

    (2013)
  • ASTM

    Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method

    (2016)
  • ASTM

    Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm Using the MBEC Assay

    (2017)
  • ASTM

    Standard Practice for Conducting Ruggedness Tests

    (2018)
  • ASTM

    Standard Practice for Preparing a Pseudomonas aeruginosa or Staphylococcus aureus Biofilm Using the CDC Biofilm Reactor

    (2018)
  • ASTM

    Standard Test Method for Evaluating Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method

    (2019)
  • S. Behnke et al.

    Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures

    Appl. Environ. Microbiol.

    (2011)
  • H. Ceri et al.

    The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms

    J. Clin. Microbiol.

    (1999)
  • D. Davies

    Understanding biofilm resistance to antibacterial agents

    Nat. Rev. Drug Discov.

    (2003)
  • Cited by (22)

    • Calculating the limit of detection for a dilution series

      2023, Journal of Microbiological Methods
    • Influence of milk proteins on the adhesion and formation of Bacillus sporothermodurans biofilms: Implications for dairy industrial processing

      2022, Food Control
      Citation Excerpt :

      The data obtained for both control and treated surfaces (CFUs) were transformed into log densities (LD; log CFU/cm2). For all experiments, the logarithmic reduction (LR) was calculated by subtracting the mean for treated biofilm (LDT) from the mean of untreated controls (LDC) (ASTM, 2019; Goeres et al., 2019). This process was analyzed using SEM.

    View all citing articles on Scopus
    1

    ASTM study director, director of all follow-up investigations.

    2

    Study design.

    3

    Implementation of all studies.

    4

    Splashguard bias study.

    5

    Imaging.

    6

    Ruggedness study.

    7

    Harvesting bias study.

    8

    Statistical analysis.

    View full text