European Food Safety Authority's (EFSA) Guidance on Antimicrobial Susceptibility Testing

Hello everyone. My name is Janet Liu and I am the Director of Microbiology at Emory Pharma. Today I will be giving a brief overview of the European Food Safety Authority's guidance on antimicrobial susceptibility testing of microbes used in animal feed. Within the European Union, the EFSA is the agency responsible for providing advice, risk assessment and guidance on matters related to the food supply chain. EFSA has created a scientific panel that offers advice and guidance on additives and products or substances used in animal feed. The name of the scientific panel is the Panel on Additives and Products or Substances Used in Animal Feed, abbreviated FeedAP. The guidance document that we will be discussing in this presentation is the guidance on the characterization of microorganisms used as feed additives or as production organisms. Additives include familiar products such as Lactobacillus species commonly found in fermented foods such as yogurt, to less familiar products such as microbial produced enzymes like amylase. This document serves to support applicants in the submission of microbes or microbial product additives for use in animal nutrition. Although the EFSA feedback guidance is generated for and by members of the European Union, each individual country within the Union may have additional guidance and requirements. There are many different requirements in the EFSA guidance listed in Table 1. For bacteria, this includes identification, antimicrobial susceptibility, antimicrobial production, and toxicity, and pathogenicity. In this presentation, we will only be focusing on section 2.2, antimicrobial susceptibility, specifically subsection 2.2.1. Phenotypic testing this section is applicable for live bacteria and bacteria fermentation derived products used in feed additives. Antimicrobial susceptibility testing is the determination of the susceptibility of a microbe to an antimicrobial agent such as an antibiotic. This information is important for limiting the spread of antibiotic resistance from feed additive microbes to microbes that have colonized humans and animals, which can render important antibiotic drugs and used to treat infections unusable. Antimicrobial susceptibility is assessed by determining the minimum inhibitory concentration MIC of an antimicrobial agent against the microbe. With regards to microbes used in feed additives, the applicant is required to determine and submit the MIC of their organism against a panel of antimicrobials. To fulfill the requirements of the EFSA PDAP guideline, MIC testing should be performed according to internationally recognized standards such as clinical and laboratory standard. Institute MIC determination should utilize a broth or auger based serial dilution method with MIC values expressed as milligram per liter or micrograms per milliliter. Qualitative or semi qualitative assessments such as zone of diffusion methods are not accepted of the acceptable methods. Emery Pharma utilizes the broth microdilution method which is well suited for testing multiple antimicrobials against the test organism at the same time. The procedure for the microdilution method is depicted in the schematic shown in the slide. In Step one, the antimicrobial in this case a set of antibiotics to be tested are two fold serially diluted in a 96 well microdilution plate. In step two, the bacteria being assessed is collected from an agar plate with a sterile swab and a suspension is prepared. The serially diluted antibiotic and the bacteria suspension are combined in step 3. After incubation, the MIC is determined in step 4 by checking the 96 well plate for bacterial growth either by eye or by using a spectrophotometer. The MIC is defined as the lowest concentration of an antimicrobial that inhibits bacterial growth. At Emory Pharma we often have clients that request antimicrobial testing against their bacterial feed additives. Shown in the current slide is a table listing the required antimicrobials to be tested for various microorganisms as required by the EFSA FeedAP guideline. The table also lists the MIC cutoff values for various combinations of antimicrobials and bacteria. The MIC cutoff values serve as a guide to determine whether the bacteria being assessed is susceptible or resistant to an antimicrobial agent and are defined based on published data. Although a total of 13 different antimicrobials are listed in Table 2, not all are required for MIC testing and determination against all species and are shown in the table as NR for not required. However, if a specific antimicrobial MIC cutoff value is listed for a microorganism, then that antimicrobial must be tested. For example, Bacillus species are not required to be tested against ampicillin, tylosine, ciprofloxacine, colistin, and fosfomycin. While colistin is the only antimicrobial required for testing for any bacteria in the family Enterobacteriaceae. For species not listed in Table 2, the EFSA feedback Panel recommends testing gram positive organisms against the antimicrobials link listed for Corinibacterium and other gram positive and in the case of gram negative organisms against the antimicrobials listed for Enterobacteriaceae. The resulting MIC values should then be compared to what has been published in the literature for that specific or related species. At Emory Pharma, our scientists have extensive experience in antimicrobial susceptibility testing and follow CLSI guidelines. To find out about how we can help you conduct EFSA feed DAB required MIC testing for your microorganism, please contact us through our website at www.emripharma.com or call us at 1-888-9.