Intra- and Interlaboratory Performances of Two Commercial Antimicrobial Susceptibility Testing Methods for Bifidobacteria and Nonenterococcal Lactic Acid Bacteria

In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the type strains of 25 species of NELAB and bifidobacteria and MICs of vancomycin for a selection of relevant taxa were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media. The overall agreement of median MIC ranges ± 1 log₂ dilution determined by the VetMIC and Etest methods with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for acceptable intralaboratory reproducibility being defined as the median MIC ± 1 log₂ dilution, VetMIC results (with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlaboratory reproducibility for the former method. Apart from some agent-specific effects, the majority of VetMIC and Etest replicate data sets were situated within a 1- to 2-log₂ dilution range, suggesting that the two methods can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further validated the standard use of LSM and related mixed-medium formulations with commercially available systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility testing of NELAB and bifidobacteria.Because of their distinctive fermentative, functional, and potentially health-promoting properties, bifidobacteria and nonenterococcal lactic acid bacteria (NELAB) such as lactobacilli, lactococci, and Streptococcus thermophilus are intensively used in the food industry as starter cultures, adjunct cultures, and probiotics (29). Although the majority of NELAB and Bifidobacterium species are food-grade organisms, the large-scale application and deliberate introduction of such cultures into the food chain has opened the debate over whether or not criteria that document their safety for human and animal use should be defined (35). Despite the overall low pathogenic potential of these organisms, several studies have indicated that especially NELAB can act as reservoirs of potentially transferable antimicrobial resistance genes (1, 10, 15, 31). In the field of probiotics, the absence of acquired resistance traits has been recommended as a safety criterion in the selection of new commercial culture probiotic strains for human use (12, 27, 28, 33).Due mainly to the limited clinical relevance of NELAB and bifidobacteria, the development and optimization of methods for antimicrobial susceptibility testing of these organisms have long been underappreciated. Moreover, the fact that many of these organisms have specific nutritional and atmospheric requirements for growth does not allow uniform use of standardized susceptibility test media such as Mueller-Hinton broth and Iso-Sensitest (IST) broth. There are indications that de Man, Rogosa, and Sharpe (MRS) medium, which is commonly used as a growth medium for most of these organisms, may exhibit antagonistic effects with supplemental antimicrobials in susceptibility testing (13). To address the apparent limitations of using single media, a mixed formulation of IST broth (90%, vol/vol) and MRS broth (10%, vol/vol) referred to as lactic acid bacterium susceptibility test medium (LSM) was developed recently (16). This new formulation has proven to support the growth of a wide taxonomic range of lactobacilli and Bifidobacterium spp. (when supplemented with 0.03% cysteine) and minimizes potential antagonism between medium components and tested antimicrobials. So far, LSM and related mixed-medium formulations, all containing IST broth as the basic component, have been successfully used to determine MICs for members of the genera Bifidobacterium, Lactobacillus, Lactococcus, Pediococcus, and Streptococcus by microdilution and Etest methods (2, 5, 7, 8, 17, 18, 20-24, 32).In the near future, it is expected that the increased use of LSM as the standard medium for susceptibility testing of NELAB and bifidobacteria will produce a large amount of MIC data, enabling the definition of epidemiological cutoffs (ECOFFS). As proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST http://www.eucast.org/]), ECOFFS provide an objective basis to differentiate wild-type organisms, which lack acquired and mutational resistance mechanisms, from non-wild-type members of the same species that contain one or more mechanisms conferring antimicrobial resistance. For this purpose, evaluation of the internal and external quality assurance procedures for reference methods and commercial systems for susceptibility testing is absolutely crucial for the correct interpretation of these ECOFFS. Several harmonization studies of susceptibility testing of clinical (14, 30), veterinary (26, 34), and aquatic (11, 25) organisms have provided important insights into the reproducibility of results from standardized methods at the intra- and/or interlaboratory level. Although such studies would also be highly valuable to all with an interest in evidence-based biosafety assessments of NELAB and bifidobacteria for human and animal use, the performances of susceptibility test methods using LSM within and across laboratories have to our knowledge not been evaluated.Within the framework of an international research project, the European Union Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain (EU-ACE-ART), a small-scale harmonization study involving nine laboratories in eight European countries was conducted. The study set out to examine the performances of two commercial susceptibility test methods widely used throughout the EU-ACE-ART project, i.e., the VetMIC system (broth microdilution) and Etest (agar diffusion), at the intra- and interlaboratory levels. For this purpose, MICs of six antimicrobials for the type strains of 25 NELAB and Bifidobacterium species and MICs of vancomycin for a selection of relevant taxa were determined using LSM and related mixed-medium formulations as test media.