Southeast Florida and Florida Keys Antibiotic Resistance Study

Sediment, coral mucus and water samples acquired from Southeast Florida between 2018 and 2019 were analyzed for 15 different antibiotic resistant gene targets via PCR presence/absence assays. The SCoRR standard operating procedure (SOP) (Fisher and others, 2015) details a related study using similar collection methods to those utilized for this project with the exception of the coral mucus samples, which were collected by aspiration, using a sterile 60 cubic centimeter plastic syringe. The SOP provides step-by-step instructions for site preparation, sample processing, quality assurance, and shipping of soil and surficial bed sediment.

DNA extraction was performed by first calibrating the Mettler scale with a 50-gram (g) certified weight and then documenting that value on the Mettler QA/QC sheet. Using sterile-techniques, approximately 0.25 g of sediment or 200 microliters (µl) of water from each sample was weighted out or pipetted and transferred to a MoBio PowerSoil bead beating tube. Sample weight or volume was logged in the project electronic laboratory notebook (ELN). The MoBio PowerSoil kit protocol was used to elute DNA to a volume of 100 µl using Qiagens AE buffer recipe (10 millimolar (mM) Tris-HCL, 0.5 mM EDTA, pH 9.0, filter sterilized and autoclaved). Eluted DNA was stored at -70 degrees Celsius (¡C) until use.

The following recipes and plate(s) layouts were used to set up quantitative polymerase chain reaction (qPCR) reactions. All reactions were run in duplicate and positives were those reactions where amplification was noted in both reactions that reached a normalized reporter signal (Rn) value of 0.1 (with an exponential trend). All duplicate positives were screened with digital PCR. Master Mix recipes (volumes per reaction) are as follows: Single probe reactions Ð 20 µl reactions - row 1 Internal Positive Control (IPC): 2x TaqMan Fast Universal Master Mix (10 µl); Template (2 µl); Primer/probe (10 micrometers (µm)/5 µm working stock) (1 µl each); 10x Exo Internal Positive Control (2 µl); 50x EXo IPC DNA (0.4 µl); PCR grade water (4.6 µl). IPC control reactions - row 8: 2x TaqMan Fast Universal Master Mix (10 µl); Template (2 µl); 10x Exo Internal Positive Control (2 µl); 50x EXo IPC DNA (0.4 µl); PCR grade water (5.6 µl). Mµltiplex (two primer and probe sets) Ð rows 2 through 7: 2x TaqMan Fast Universal Master Mix (10 µl); Template (2 µl); primer/probe (10 µm/5 µm working stock) (1 µl each); PCR grade water (6.0 µl). PCR amplification profile Master details are as follows: Pre PCR read Ð 60 ¡C for 0.5 minutes (min); Holding stage Ð 50 ¡C for 2 min then 95 ¡C for 5 min; Cycling stage Ð 45 cycles of 95 ¡C for 0.25 min then 60 ¡C for 1 min; Post PCR read Ð 60 ¡C for 0.5 min. Ninety-six well PCR plate 1 layout - row 1 = tetB/IPC, row 2 = tetL/M, row 3 = tetO/W, row 4 = ampC/vanA, row 5 = ermB/mecA, row 6 = blaSHV/blaPSE, row 7 = floR/aadA2, row 8 = controls (PCR-grade water for template Ð columns 9 and 10) and IPC negative control (2 µl of IPC block Ð columns 11 and 12). Ninety-six well PCR plate 2 layout - row 1-7 = tetG/tetQ for all the samples and the same layout of controls in row 8 as described for plate 1. Two dyes, FAM and VIC, were used to monitor amplification in the real-time PCR reaction assays. FAM-labeled probes are tetB, tetL, tetO, ampC, ermB, blaSHV, floR, and tetG. VIC-labeled probes are the IPC, TetM, TetW, vanA, mecA, blaPSE, aadA2, and tetQ.

Added keywords section with USGS persistent identifier as theme keyword.