Samples from Day 0 were used as the baseline, and samples from Day 21 were considered the maximum treatment effect time period for analysis. Microbiological assays were conducted under Institutional Biosafety Committee registration #2017-049 and #2017-021 at Texas A and M University. Samples preserved with glycerol were thawed on ice and mixed thoroughly with phosphate-buffered saline (PBS) (Gibco Life Technologies, Thermo Scientific Microbiology, Oakwood Village, OH, USA) in a 1:10 dilution, using 9 mL of PBS and 1 g of feces. An aliquot of 50 µL of this dilution was spiral-plated using an EddyJet® 2 Spiral Plater (Neutec Group Inc., Farmingdale, NY, USA) onto MacConkey agar (Difco, Becton Dickinson Sparks, MD, USA) for quantification of fecal coliforms; more specifically, we counted only lactose fermenting colonies and therefore presumptive E. coli. This dilution also was spiral plated to MacConkey agar supplemented with tetracycline (Sigma Aldrich, Merck, St. Louis, MO, USA) at 16 milligrams per liter (mg/L) as well as MacConkey agar supplemented with ceftriaxone (Sigma Aldrich, Merck, St. Louis, MO, USA) at 4 mg/L. This same dilution also was spiral-plated to m-Enterococcus agar (Difco) for quantification of enterococci, and to m-Enterococcus agar supplemented with tetracycline at 16 mg/L and to m-Enterococcus agar supplemented with erythromycin (Sigma Aldrich) at 8 mg/L. MacConkey agar plates were incubated at 37 °C for 18 h; in contrast, m-Enterococcus plates were incubated at 42 °C for 48 h. All plates were counted using the Flash & Go® System (Neutec Group Inc.).

Two random colonies from each plain (i.e., non-antibiotic) agar plate were selected and streaked to tryptic soy agar (TSA) agar with 5% sheep blood (Difco) for confirmation of species using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). Employing a single-use sterilized wooden toothpick, a single isolate of presumptive E. coli, or Enterococcus spp., was spread onto two spots of a reusable 96-spot target plate (Bruker Daltonik GmbH, Billerica, MA, USA). Once dry, one microliter (µL) of 70% formic acid was added to the first spot of each sample spot pair only for Enterococcus spp. (Gram-positive) isolates and to one empty spot to serve as a negative control. Formic acid was restricted in use to Gram-positive isolates, as it is unnecessary for Gram-negative bacteria such as E. coli. One µL of the bacterial test standard (BTS) solution (Bruker Daltonik GmbH) was applied to the first and second spots on the plate as a positive control. After drying of all spots, one µL of α-Cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonik GmbH, Billerica, MA, USA) was added to each spot, including all the sample spots, BTS spots, the formic acid negative control spot and an additional empty spot serving as a secondary negative control. The target plate was then transferred to the MALDI-TOF Microflex LT/SH (Bruker Daltonik GmbH) for reading, using MBT Compass v1.4 software (Bruker Daltonik GmbH). After confirmation of genus and species, these same isolates were used for phenotypic susceptibility testing.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.