Evaluating the recovery of pan-susceptible and antibiotic-resistant Escherichia coli in synthetic test agricultural water using membrane filtration and Most Probable Number-based methods

Authors: XIONG, ZIRUI - University Of Delaware; BROOKS, AJANI - Alabama A & M University; GABRIEL, ELLEN - Orise Fellow; GUTIERREZ, ALAN - Orise Fellow; JOHNSON, SHALYA - Orise Fellow; East, Cheryl; MOSS, ELICA - Alabama A & M University; Durso, Lisa; Sharma, Manan

Submitted to: BMC Research Notes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Microbial indicators for foodborne pathogens in agricultural water are commonly used by farmers growing fruits and vegetables to determine their irrigation water requirements. Testing for microbial indicators of fecal contamination can be more efficient that testing for foodborne pathogens in irrigation water. In addition, testing for antimicrobial resistance (AMR) in agricultural environments is becoming prevalent to understand how AMR spreads. Escherichia coli is a bacterial species associated with fecal contamination and AMR, and therefore, the methods used to determine its levels in water are critical. In the research presented here, methods used to determine E. coli levels in water with different levels of turbidity (cloudiness), which varies according to multiple environmental factors, were evaluated using two different, widely used methods:1)membrane filtration (MF) and a 2) Colilert/Quantitray assay system. We added pan-susceptible (not antibiotic-resistant) and antibiotic -resistant E. coli to separate water samples. Our results showed that turbidity levels affected the recovery of E. coli from water by both MF and Colilert/Quantitray methods. Higher turbidity levels affected the recovery of E. coli on some selective microbiological media but not others. . For the Colilert/Quantitray system, higher turbidity levels in water decreased levels of E. coli recovered. Antibiotic-resistant E. coli were consistently recovered at lower levels than pan-susceptible E. coli. Work presented here provides scientific background for fruit and vegetable farmers to understand that turbidity can inhibit the recovery of E. coli when using MF methods or decrease the levels of observed recovery when using Colilert/Quantitray methods.

Technical Abstract: Membrane filtration and Colilert/Quantitray assays are commonly used to quantify Escherichia coli levels in agricultural water. These methods have not been evaluated in test agricultural water (TAW), a formulation used for assessing effectiveness of chemical sanitizer treatments on pathogens in water. The recovery efficiency of these methods for pan-susceptible and antibiotic-resistant E. coli in TAW may be affected by turbidity levels. TAW was formulated using EPA/FDA protocol with a pH of 6.5 at two turbidity levels: no added turbidity (< 5 NTU) and 50 NTU, achieved by adding PTI Arizona test dust. Pan-susceptible E. coli ARS C101 and cefotaxime-resistant E. coli ARS C301 were added separately to TAW (200 CFU/100 mL). Inoculated TAW was either 1) filtered using a 0.45 µm cellulose ester membrane filter and placed onto Tryptone Bile X-Glucuronide (TBX) agar, CHROMagar-ECC (ECC), or 2) tested by IDEXX Quanti-Tray/2000 Colilert. For recovery of E. coli C301, media were supplemented with 4 µg/mL cefotaxime. Recovery percentages of E. coli C101 and C301 at both turbidity levels were similar on TBX and ECC, between 88.8% and 121.7%, but E. coli C101 in TAW at 50 NTU on TBX was not quantifiable. For Colilert assays, the recovery percentages of E. coli C101 from 0 NTU and 50 NTU TAW were 118.3±6.7% and 94.6±18.6%, respectively, while recovery percentages of E. coli C301 in 0 NTU and 50 NTU TAW were 127.0±10.5% and 93.4±4.4%, respectively. A significantly (p = 0.0233) higher recovery percentage for pan-susceptible E. coli C101 was observed on CHROMagar ECC compared to cefotaxime-resistant E. coli C301. A significantly (p = 0.0016) higher recovery percentage of both E. coli 101 and 301 was observed in TAW with 0 NTU compared to 50 NTU using Colilert assay. Overall, CHROMagar ECC and Quanti-Tray/2000 Colilert provided quantifiable results for recovery of pan-susceptible and cefotaxime-resistant E. coli from artificial agricultural water at different turbidity levels. Higher levels of turbidity affected the ability to quantify E. coli by MF and affected the level of recovery by Quanti-Tray/2000 Colilert.