Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
VETERINARY CLINICAL SCIENCES
Non Technical Summary
The development of antimicrobial resistant pathogens is considered a serious health epidemic that affects humans and animals.Antimicrobial resistance can occur due to innate or inherent properties about the bacterium or they can be due to acquired and transferrable genetic elements such as antimicrobial resistance (AMR) genes. The presence of these transferrable AMR genes is of particular concern as bacteria carrying these genes can transfer them to other non-pathogenic, potentially pathogenic and pathogenic bacteria.. The European Food Safety Authority has recognized the potential threat of introducing AMR genes to the gastrointestinal tract microbiota via probiotics and requires testing for and assurance of the absence of AMR genes in probiotics marketed for humans and animals. Currently, the same is not true in the United States. Thus, independent studies are necessary to evaluate probiotics for their safety and potential risks associated with their use.The objective of this study is to identify bacteria in probiotics marketed for use in major food animal species with phenotypic antimicrobial resistance and determine if these isolates contain transferrable AMR genes. We hypothesize that commercially available probiotics marketed for use in major food animal species will contain phenotypically resistant bacteria and have corresponding transferrable AMR genes. Twenty commercially available probiotics marketed for use in major food animal species will be chosen. Bacteria will be isolated, quantified, and in vitro susceptibility testing performed. DNA will be extracted from isolates with resistance profiles consistent with the presence of AMR genes and evaluated for the presence of known AMR genes via gene specific polymerase chain reaction (PCR).
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The development of antimicrobial resistant pathogens is considered a serious health epidemic that affects humans and animals. As emphasized by the American Veterinary Medical Association, as veterinarians we play a crucial role in antimicrobial stewardship. It is our personal and professional obligation to safeguard animal, public and environmental health from antimicrobial resistance. Unfortunately, human literature suggests that probiotics may be a source of transferrable antimicrobial resistance (AMR) genes. The European Food Safety Authority has recognized the potential threat of introducing AMR genes to the gastrointestinal tract microbiota via probiotics and requires testing for and assurance of the absence of AMR genes in probiotics marketed for humans and animals. Currently, the same is not true in the United States. Thus, independent studies are necessary to evaluate probiotics for their safety and potential risks associated with their use.The objective of this study is to identify bacteria in probiotics marketed for use in major food animal species with phenotypic antimicrobial resistance and determine if these isolates contain transferrable AMR genes. We hypothesize that commercially available probiotics marketed for use in major food animal species will contain phenotypically resistant bacteria and have corresponding transferrable AMR genes. Twenty commercially available probiotics marketed for use in major food animal species will be chosen. Bacteria will be isolated, quantified, and in vitro susceptibility testing performed. DNA will be extracted from isolates with resistance profiles consistent with the presence of AMR genes and evaluated for the presence of known AMR genes via gene specific polymerase chain reaction (PCR). Based on our preliminary data, we expect that these probiotics marketed for use in major food animal species will contain bacteria with phenotypic resistance determined via in vitro testing which will correspond with the presence of transferable AMR genes.
Project Methods
Probiotics: In addition to the 8 probiotics listed above an additional 12 will be chosen via random number generator from our master list of 40 products commercially available products marketed for use in major food animal species in the US for a total of 20 products for evaluation. We have retained the initial 8 products because, it provides us reasonable assurances that we will be successful in identifying AMR bacteria and genes in this project.Specific Aim 1: Identification of Isolates with in vitro Phenotypic Resistance Specific aim 1 will take place at the Washington Animal Disease and Diagnostic Laboratory (WADDL) under the direction and supervision of Dr. Claire Burbick, a board-certified veterinary microbiologist.Probiotic Culture techniques: A 1:10 dilution of probiotic (1 gram to 9 ml sterile saline) will be made and homogenized using a stomacher system (Seward, Behemia NY). Five, 10-fold dilutions are then made from the stomached suspension. Additional dilutions may be needed to obtain plates with 30-300 colonies. One-hundred microliters of each dilution will be inoculated onto Columbia Blood agar and Columbia Naladixic agar (Hardy Diagnostic, Santa Maria, CA) and spread evenly over the plates with a plastic spreader. Plates will be incubated at 35°C and examined daily for 3 days. Plates with between 30-300 colonies will be counted and a cfu/g calculated.Bacterial identification will be performed using a Biotyper Matrix-assisted Laser Desorption Time-of-Flight Mass Spectrometer per the manufacturer's instructions (MALDI-TOF, Bruker Daltonics, Billerica, MA). Briefly, one well-isolated colony will be sampled with a toothpick and spotted onto a 96-spot stainless steel target plate (Bruker Daltonics). Once the bacteria dries, it will be overlaid with 1µL of70% FormicAcid and allowed to dry again at room temperature (RT). The spot is then overlaid with 1 µL of Matrix HCCA solution within1 hour and allowed to dry at RT a final time. The target plate is then read by the MALDI-TOF and spectra interpreted using Biotyper RTC and Flex Control programs (Bruker Daltonics). The following MALDI-TOF scores correspond to level of confidence in genus- or species-level identification. Some isolates may require additional testing.< 1.7 ID not valid1.7 - 1.99 presumptive genus ID2.0 - 2.3 confident genus ID and presumptive species ID≥2.3 confident species-level IDMinimum Inhibitor Concentration (MIC) techniques: Antimicrobial susceptibility testing will be performed per manufacturer's recommendations (Thermo Fisher Scientific, Waltham, MA). Briefly, 3-5 colonies of a pure, overnight culture will be inoculated into demineralized water to a 0.5 McFarland turbidity. A 10 µl aliquot of the suspension is then inoculated into cation adjusted Mueller-Hinton broth (Hardy Diagnostics). Fifty microliters of the suspension will be inoculated into a commercial 96-well microtiter plate containing antimicrobial agents relevant for the type of bacteria tested. The plate will be incubated at 35°C for 18-24 hours depending on the isolate tested. Following overnight growth the plate will be read using an automated plate reader and software system and the minimum inhibitory concentration determined for each antimicrobial agent (Biomic, Giles Scientific, Santa Barbara, CA).Evaluation of Data: Minimum inhibitory concentrations of antimicrobial agents tested will be compared to Clinical and Laboratory Standards Institute (CLSI, Wayne, PA) documents to apply a clinical interpretation of susceptible, intermediate or resistant. Epidemiologic Cut-off Values will also be examined to determine if other phenotypic evidence of resistance is present. A bacterium will be identified as suspect for containing AMR genes if it has a resistance pattern that is not consistent with its established inherent antimicrobial resistance and further evaluated in specific aim 2.Specific Aim 2: Comparing Phenotypic Resistance Patterns with AMR gene identification Specific Aim 2 will take place at Iowa State University under the direction and supervision of Dr. Jamie Kopper. The objective of this aim will be to evaluate isolates with phenotypic antimicrobial resistance to determine if they are consistent with the presence of a corresponding transferrable AMR gene.Based on our preliminary data we expect to find isolates with phenotypic resistance patterns consistent with tetracycline, sulfonamide, trimethoprim and erythromycin resistance. Based on the phenotypic resistance patterns identified in specific aim 1 we will determine if we should test for additional AMR genes. If additional candidate genes are identified we will validate PCRs as described below.Validation of Additional AMR Gene PCRs: Positive control DNA for candidate genes of interest will be extracted from antimicrobial resistant bacteria obtained from the Center for Disease Control and FDA Antimicrobial Resistance Bank (CDC, Athens GA) containing the gene(s) of interest. Previously published PCRs will be identified or, if necessary, PCR primers designed. PCRs will be tested using the positive control DNA for the gene of interest. PCR product will be evaluated via gel electrophoresis for an appropriately sized amplification band. If appropriately sized PCR product is present the product will be submitted for sequencing and the sequence compared to known sequence for the AMR gene of interest using the NCBI BLAST sequence analysis tool. PCRs will be considered validated if they produce product of the appropriate size and sequence with 100% match to the gene of interest.DNA extraction from probiotic bacterial isolates: Frozen cultures will be propagated by plating on the appropriate medium and incubating at 37°C for 18-24 hours. Subsequently, colonies will be placed in 1.5mL Tris-EDTA buffer and DNA was extracted using QIAGEN's DNeasy Ultraclean Microbial kit (QIAGEN, Valencia CA) according to the manufacturer's protocol. DNA concentration will be quantified, and quality assessed using NanoDrop ® spectrophotometry (Thermofisher, Waltham MA).Detection of corresponding AMR genes from bacterial DNA: Bacterial DNA will be evaluated for the presence of AMR genes of interest using PCRs previously validated within our laboratory. The PCR products will be evaluated by gel electrophoresis and visualized for an appropriately sized PCR product.Pitfalls and Alternatives: Given our preliminary we expect to find bacteria with antimicrobial resistant phenotypes and corresponding genotypes. Although some studies have noted that the identification of genotypic antimicrobial resistance does not always correspond to phenotypic resistance when the bacteria are cultured, this is rare24 and would be unlikely to be the case for all of the isolates in all of the products. It is possible that we will not find viable bacteria with the AMR genes, because the genes were from dead bacteria. This is still valuable information as the genetic material can still be transferred to other organisms within the animal's GI tract and environment.Expertise: Our collaboration is uniquely poised to successfully carry out this project. Dr. Claire Burbick is a board-certified veterinary microbiologist and her laboratory has successfully cultured and quantified micro-organisms over 20 different probiotics marketed for use in animals as part of a separate collaborative project. Dr. Kopper has previously validated PCRs for the 10 AMR genes via the described methods, successfully extracted DNA from over 50 probiotics and performed the corresponding PCRs (manuscript in review).