Combining diagnostic methods for antimicrobial susceptibility testing – A comparative approach

https://doi.org/10.1016/j.mimet.2017.11.010Get rights and content

Highlights

  • Determination of minimum inhibitory concentration after 20 h is recommended.

  • Fluorophore staining following MIC determination provides MBC in 30 min.

  • Isothermal microcalorimetry proves delayed growth after antimicrobial treatment.

Abstract

Background

The minimum inhibitory concentration (MIC) is a measure of antimicrobial susceptibility testing (AST) of a given antibiotic but provides insufficient information when bacterial killing is crucial, e.g., when treating immunocompromised patients. In these cases, the minimum bactericidal concentration (MBC) is a more reliable measure of antibiotic activity. Here, we aim to demonstrate and recommend combinations of methods for MIC and MBC measurements. We also aim to emphasize the importance of uniform protocols for these procedures including the time point for reading MIC results, which the authors suggest to be 20 h.

Methods

To address the challenges with obtaining fast and reliable readouts on MIC as well as the kinetic and end-point effects of antibiotics, the broth micro dilution method, a calorimetric method and a microscopy-based screening system (MBSS) were evaluated in this study. For MBC determination, fluorophore staining with SYTO9 and propidium iodide was compared to the broth regrowth method.

Results

Three scenarios for combining the MIC and MBC methods depending on the investigators' primary concern (time, cost or sensitivity) are presented. Further, as the MBSS and the isothermal microcalorimetry method detected delayed bacterial growth up to 18 h after initiation of experiments, the importance of reading MIC testing after a full 20 h is emphasized. A one-fold change in MIC values can be observed when comparing data obtained at 16 h and 20 h of incubation.

Conclusion

The authors suggest that combining MIC and MBC determinations will provide more detailed understanding of the bacteria susceptibility to antibiotic drugs and result in more clinically relevant data and optimized therapies. Furthermore, establishing 20 h as a time point for reading MIC results will provide more uniform data across laboratories.

Introduction

In the search for efficient antibiotic therapies, antimicrobial susceptibility testing (AST) is performed in vitro in experimental and diagnostic laboratories worldwide. Standardized experimental conditions are thus essential for obtaining reliable and reproducible results. As a measure of AST, the minimum inhibitory concentration (MIC) is used and is defined as the lowest concentration of an antibiotic which inhibits visible growth of a microorganism after 16–20 h of incubation (Andrews, 2001). However, the MIC does not reveal whether the antibiotic is bacteriostatic or bactericidal and is a weak predictor of the antibiotic efficacy in vivo (Wiegand et al., 2008).

Especially with regard to antibiotic therapy in patients with inflammatory diseases (Andrews, 2001, Brennan and Durack, 1983) or immunocompromised patients, it is crucial to determine the minimum bactericidal concentration (MBC), since an intact immune system is necessary to eliminate inhibited pathogens (Bär et al., 2009). The MBC is the lowest concentration that kills the bacteria, with a 99.9% or 103 reductions in the number of bacterial colonies on subculture (Taylor et al., 1983, Meylan et al., 1986, Lowy et al., 1983, Pankey and Sabath, 2004). Comparing MBC values helps to rationally choose the most efficacious antibiotic for compromised patients (Taylor et al., 1983).

Many studies have used the MBC and the MBC/MIC ratio to assess bactericidal activity of antibiotics, correlate in vitro data with possible outcomes of in vivo treatments (Brennan and Durack, 1983, Meylan et al., 1986, Lowy et al., 1983) and even predict outcomes of in vivo treatments (Lowy et al., 1983, Pankey and Sabath, 2004, Tuomanen et al., 1986). Determination of MBC has received some criticism, as the commonly used test has shown poor reproducibility between laboratories (Taylor et al., 1983, Meylan et al., 1986, Pelletier, 1984). The test is derived from the broth dilution procedure and involves re-growth of bacteria in antibiotic-free media (Taylor et al., 1983). The variability in reported MBC values is attributed to lack of specific guidelines and differences in technical details of the procedure, such as the size of the inoculum used (Brennan and Durack, 1983), sample mixing (Taylor et al., 1983, Pelletier, 1984), and growth phase of the bacteria (Brennan and Durack, 1983, Meylan et al., 1986). These details do not affect MIC values, yet are critical factors for the outcomes of MBC determinations (Brennan and Durack, 1983, Taylor et al., 1983). Nonetheless, reports suggest that MBC is of higher clinical relevance when predicting the response of bacteria to antibiotic therapy in vivo, and thus the determination of both MIC and MBC is advisable (Brennan and Durack, 1983, Bär et al., 2009).

This study reports an objective comparison of methodologies by measuring the effect of a range of antibiotics on bacterial growth inhibition and bacterial killing. The influence of the antibiotics on growth kinetics was also assessed. Thus, the implementation of isothermal microcalorimetry (IMC) and a microscopy-based screening system for real-time determination of MIC values as alternatives to traditional end-point measurements were pursued. IMC has been used previously to study growth, metabolism, and susceptibility to antibiotics used against Pseudomonas aeruginosa (Esarte López et al., 2015, Lago et al., 2011), Staphylococcus aureus (Li et al., 2012, Entenza et al., 2014) and Escherichia coli (Zaharia et al., 2013, Vazquez et al., 2014, Shi et al., 2015). The oCelloScope™, a microscopy-based screening system (MBSS) has previously been implemented and validated as a method for assessing bacterial growth (Fredborg et al., 2013). The mechanism and use of the LIVE/DEAD® Baclight™ viability staining kit has previously been described (Stocks, 2004), and has been used for assessment of bacterial viability (Berney et al., 2007), identification of MBC values (Boye et al., 1983, Gant et al., 1993), enumeration of viable bacteria in drinking water (Boulos et al., 1999) and probiotic dairy products (Auty et al., 2001).

In addition to demonstrating the advantages and disadvantages of the methods, this work proposes three courses-of-action for the investigator. The first scenario suggests methods, which will provide MIC and MBC values the fastest, the second scenario relates to using the most inexpensive methods, while the third scenario utilizes the most sensitive experimental settings. The authors envisage that combining MIC and MBC methods will result in a more detailed understanding of the antibiotic action on the tested microbes. These scenarios will also provide information regarding the effect on growth kinetics of tested bacteria during and after exposure to antibiotics.

Section snippets

Materials

Gentamicin sulfate, tobramycin and vancomycin were acquired from Sigma-Aldrich, (Brøndby, Denmark). Colistin sulfate was acquired from Serva Electrophoresis (Heidelberg, Germany). Ciprofloxacin and Mueller-Hinton broth (MHB) was from Fluka analytical (distributed by Sigma-Aldrich, Brøndby, Denmark). P. aeruginosa PA01 (reference strain), S. aureus 15981 (clinical isolate strain) and E. coli ATCC 25922 (reference strain) were kindly provided by the Institute of Immunology and Microbiology,

Results

P. aeruginosa PA01 and E. coli ATCC 25922 were tested against four antibiotics: gentamicin, tobramycin, ciprofloxacin, and colistin while S. aureus 15981 was additionally tested against vancomycin; all by using broth microdilution, the microscopy-based screening system, IMC, broth re-growth, and fluorophore staining. The methods described in this paper are recommended for testing aerobic bacteria that grow in Mueller-Hinton Broth (MHB) within 16–20 h. MHB is recommended both by the Clinical and

Discussion

In this paper, broth microdilution, IMC, and the MBSS have been described as methods for determining MIC values, while broth regrowth and fluorophore staining have been tested as methods for MBC determination. The median MIC and MBC values are presented in Table 1. In general, there is a good agreement between values obtained using the MIC methods. It is noticeable that MIC values obtained from IMC are generally twofold higher than results obtained using broth dilution or the microscopy-based

Acknowledgements

The authors acknowledge financial support from the Danish Agency for Science, Technology and Innovation (DanCARD, grant no. 06-097075), Innovation Fund Denmark (70-2013-1) (PAP) and University of Copenhagen 2016 Programme of Excellence Research Centre for Control of Antibiotic Resistance (UC-CARE) (SNK).

Glossary

AST
antimicrobial susceptibility testing
MIC
minimum inhibitory concentration
MBC
minimum bactericidal concentration
MBSS
microscopy-based screening system
EUCAST
the European Committee on Antimicrobial Susceptibility Testing
CLSI
Clinical and Laboratory Standards Institute
IMC
isothermal microcalorimetry
WT
wild type
CFU
colony forming units

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