Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

It is important that foodborne disease agents are detected rapidly and preferably in field-based settings in order to reduce the burden of foodborne disease. Common strategies to detect foodborne bacterial pathogens include biochemical profiling, selective and differential culturing, immunological isolation and detection, and molecular detection. However, these methods are hampered by sporadic contamination, small sample sizes tested, the often low concentrations of the foodborne pathogenic bacteria, require long processing times, and/or are not applicable for field settings. Further, compounds in many food matrices are inhibitory to detection and diagnostic applications. In order to improve the likelihood of microbial detection, the United States Food and Drug Administration has suggested that testing agricultural water (such as wash water and irrigation water) which either comes in contact with a large surface area of fresh produce or serves as a vehicle for produce contamination is a viable alternative to direct testing of food¹. Even so, the often low natural pathogen-burden coupled with the dilution effect of the representative agricultural water sample makes sample preparation methods for pathogen concentration essential. Such a method would require sampling large volumes of water (≥10 L), adequate pathogen-concentration, and compatibility with downstream detection strategies.

Modified Moore swabs (MMS) are inexpensive, simple, and rugged devices used for concentrating bacteria from large volumes (≥10 L) of water^2-4. The MMS consists of a plastic cassette filled with gauze, which serves as a coarse filter for large volumes of water pumped through the cassette using a peristaltic pump. The MMS is a non-discriminatory method of bacterial concentration (≥10 fold concentration) that captures organic and inorganic particulate material including microorganisms in processed liquid samples. It is likely that the excellent efficacy of concentration of target microorganisms by the MMS can be explained by the fact that microorganisms are expected to be attached to the silt-clay fraction or organic micro-aggregates of the suspended solids³. The rugged design of the MMS allows for overcoming most shortcomings associated with other filtration methods for capture and concentration of bacteria from water, such as clogging of filters, inability to process large volumes, filter samples with high turbidity, and high costs. For these reasons, the FDA is recommending that MMS’s be incorporated into official procedures for environmental and produce-related sample collection procedures⁵.

Here, a method is described for the concentration, enrichment, and detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from agricultural waters. A MMS is used for concentration of bacteria, and also serves as a vessel for selective or non-selective bacterial enrichment. Bacterial detection is achieved biochemically using paper-based analytical devices (µPADs)⁶. µPADs can be manufactured as fluidic networks or spot tests using a variety of methods including photolithography, inkjet printing, stamping, and wax printing^7-11. Examples of fluidic designs can be dendritic channel patterns where the sample is deposited in the center and subsequently flows to distal reservoirs or single channel patterns in which the sample or substrate are pulled from the outer reservoirs of the channel by capillary action into the center¹². For this protocol, we have chosen to employ for 7-mm-diameter wax-paper spot arrays imbedded with chromogenic substrates that can be processed by enzymes indicative of the microorganisms tested here: Chlorophenol red β-D-galactopyranoside (CPRG) and 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc) for detection of β-galactosidase and β-glucuronidase produced by E. coli; 5-bromo-6-chloro-3-indolyl caprylate (magenta caprylate) for the detection of C8-esterase produced by Salmonella spp.; and 5-bromo-4-chloro-3-indolyl-myo-inositol phosphate (X-InP) for detection of phosphatidylinositol-specific phospholipase C (PI-PLC) produced by L. monocytogenes⁶. Thus, the presence of a particular bacterium can be observed visually without the need for complex equipment or data interpretation. The specificity and sensitivity of the enzyme-based colorimetric µPAD detection of these specific target bacteria has been previously explored⁶. In addition, the sensitivity of the integrated concentration-detection method for these target bacteria was evaluated by spiking of large volumes of water with pre-determined levels of microorganisms (unpublished data and Bisha et al.¹³).