Grantee Research Project Results
Final Report: Development of Simple, Field-Deployable Kit for Detecting Antibiotic Resistant Bacteria in Wastewater
EPA Contract Number: 68HERC20C0026Title: Development of Simple, Field-Deployable Kit for Detecting Antibiotic Resistant Bacteria in Wastewater
Investigators: Hsu, Fu-Chih
Small Business: Scientific Methods, Inc.
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2020 through August 31, 2020
Project Amount: $99,998
RFA: Small Business Innovation Research (SBIR) - Phase I (2020) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Clean and Safe Water
Description:
Antibiotic resistant bacteria have become a major concern in hospital wastewater discharge, wastewater treatment plants, and agricultural runoff. A quick, inexpensive, field- deployable method of detecting antibiotic resistant bacteria in these waters is desirable. Types of antibiotic resistant bacteria of concern include Salmonella, Staphylococci, E. coli, and Enterococci, among others. A product is needed that can detect these bacteria on-site and at a low cost. SmartGel is an innovative product that is easy to use and readily adapted for various testing applications. The purpose of this research is to adjust the components of the SmartGel system and its standard application for use in the detection of many other types of antibiotic resistant bacteria in a range of water matrices. The goal is for end users to be able to test water samples for antibiotic resistant bacteria without using expensive laboratory equipment or sending samples to laboratories.
During the research period, the research team developed and optimized a powdered media to detect antibiotic resistant E. coli, Staphylococcus aureus and Salmonella by incorporating several antibiotics such as Cefotaxime, Ciprofloxacin, Methicillin, and Vancomycin into the powdered media. The stability study examined the shelf life of all four antibiotics and was carried out at 65℃ for 21 days. The stability results indicated that each type of powered media used with any of the four antibiotics were very stable at room temperature for up to 1 year at 65℃ and up to 3 years at 5℃. A limited field study was conducted to test the feasibility of the SmartGel kits to detect antibiotic resistant E. coli, Staphylococcus aureus, and Salmonella from municipal wastewater, animal waste, and hospital wastewater. Isolates of antibiotic resistant strains have been archived for further evaluation.
Summary/Accomplishments (Outputs/Outcomes):
The research team optimized two types of powdered media to detect Staphylococcus aureus and Salmonella successfully. The proposed medium, Baird-Parker, worked well with the SmartGel technology, and Staphylococcus aureus colonies were observed after a 24-48 hour incubation. Four antibiotics, Cefotaxime, Cefotaxime, Methicillin, and Vancomycin, have been incorporated to the powder media successfully with the concentrations from 1 to 4 mg/L. Although the amounts of antibiotics were very small, they can be mixed well and distribute evenly among the powder medium individual packets. A stability test was conducted to determine the shelf life of powder media with antibiotics. The results are surprising because it has shown that powdered media with Cefotaxime, Methicillin, Ciprofloxacin, and Vancomycin are very stable at room temperature. They could be stored at room temperature for more than 11 months and for 3.6 years at 5℃. In limited field study, E. coli, coliforms, Salmonella, and Staphylococcus aureus resistant to 4 antibiotics were detected from all samples. Overall, concentrations of Methicillin or Vancomycin resistant Staphylococci are much higher than antibiotic resistant coliforms, E. coli, and Salmonella.
Conclusions:
During Phase I research, the research team has reached every goal listed in the Phase I proposal, such as medium development with suitable chromogenic/fluorogenic substances, incorporation of antibiotics to the powdered media, a stability study, and a limited field study. Two basic powdered medias, Baird-Parker and modified mTEC were optimized to detect Staphylococcus aureus, E. coli, coliforms, and Salmonella with SmartGel technology. Baird-Parker powder medium containing Methicillin or Vancomycin can detect antibiotic resistant strains of Staphylococcus aureus. By combining different chromogenic and fluorogenic substances with two different antibiotics, E. coli, Salmonella, and coliform antibiotic resistant strains were detectable.
The results of the stability study were superior since powder media with antibiotics can be stored at room temperature for 1 year. Although this outcome was unexpected, it provided an excellent advantage over other similar products which require refrigerator storage and have a short self-life. We believe SMARTGel antibiotics resistant bacterial testing kits will be a useful tool for detecting antibiotics resistant bacteria in environment samples. Since it is easy-to-use, convenience (ready-to-use), and cost effect (less than $5 per test).
In the field study, a prototype of the SMARTGel kit was applied to the field samples including municipal wastewater, animal, and hospital wastewater. E. coli, coliforms, and Staphylococcus antibiotic resistant strains were detected from all samples tested. It is interesting that Salmonella levels were very low compared to E. coli and Staphylococcus even in animal wastewater. We also found out that concentrations of Staphylococcus that is resistant to Methicillin or Vancomycin was higher than other bacteria. Further validation is needed to determine the strains that are resistant to Methicillin or Vancomycin detected by SMARTGel kit are truly positive. Are they Staphylococcus aureus? Do they have special gene markers for Methicillin or Vancomycin resistance? These answers should be answered during the Phase II study.
From the Phase I study, two commercial applications can be achieved. First, detection of fecal indicators such as E. coli and coliforms in drinking water, surface water, and irrigation water. There is a potential for irrigation water because farmers can perform an E. coli test in house using the SMARTGel detection kit without sending samples to a laboratory. Another potential is the ability to detect Enterococci in beach water, especially in seawater. The current available methods by membrane filtration is the MPN method which will increase a false-positive rate if a total 100 mL of sea water is analyzed by using Enterolert medium. Our commercial partner, Aquagenx has conducted a third-party validation study for SMARTGel kit to detect E. coli/coliforms from drinking water in July. The results should be available in the next couple of months. Aquagenx also submitted a bid to UNICEF for an international validation study for the current available methods to detect E. coli/coliforms in drinking water. The goal is to determine the best method for use in third world countries to provide safe drinking water for the poor communities.
The second application is to incorporate certain types of antibiotics into the powdered medium to address public concern of antibiotic resistant bacteria. The results from the stability study along with the field study indicate several advantages for SMARTGel; 1) Easy-to-use, 2) Ready-to-use, 3) Storage of room temperature up to 1 year, 4) No special instruments required, 5) Cost effective (less than $5 for 100 mL samples, and $3 for 10 mL samples). SMARTGel antibiotic resistant bacterial testing kits may make it possible to conduct a nationwide survey to understand the prevalence of antibiotic resistant bacteria in the environment. This survey will provide very useful information for decisions made by policy making agencies when implementing requirements for wastewater treatment from municipal, animal farms, and hospitals.
In the field study, a prototype of the SMARTGel kit was applied to the field samples including municipal wastewater, animal, and hospital wastewater. E. coli, coliforms, and Staphylococcus antibiotic resistant strains were detected from all samples tested. It is interesting that Salmonella levels were very low compared to E. coli and Staphylococcus even in animal wastewater. We also found out that concentrations of Staphylococcus that is resistant to Methicillin or Vancomycin was higher than other bacteria. Further validation is needed to determine the strains that are resistant to Methicillin or Vancomycin detected by SMARTGel kit are truly positive. Are they Staphylococcus aureus? Do they have special gene markers for Methicillin or Vancomycin resistance? These answers should be answered during the Phase II study.
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.