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

Summary

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

Abstract

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Introduction

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).

Protocol

1. Concentration of Bacteria from Large Volumes of Agricultural Water Using MMS MMS Preparation Cut a rectangular section of 4-ply cheesecloth measuring 40 x 12 cm. Fold the cheesecloth along both axes to obtain a rectangle of 20 x 6 cm. Roll the cheesecloth tightly along its long axis to form a cylindrical swab of approximately 6 cm tall and 3 cm in diameter. Autoclave the cheesecloth swab in aluminum foil, do not autoclave the cassette. Decontamina…

Representative Results

As described in this protocol, concentration of bacteria using the MMS (Figure 1) can be performed within approximately 15-20 min. The MMS in constructed from acrylonitrile butadiene styrene (ABS) in two separate components; a lid and a cartridge both with an integrated spigot assembly into which a cylindrical cheesecloth swab is inserted (Figure 1A). Both components are then screwed together forming the MMS (Figure 1B). MMS-based processing is driven by a battery-powere…

Discussion

This protocol describes an integrated method for detecting E. coli, Salmonella spp., and L. monocytogenes in agricultural water. Here, MMS concentration of bacteria from large volumes (10 L) of agricultural water, is coupled with bacterial enrichment, and bacterial-indicative colorimetric detection using µPADs. The MMS procedure can cope with high particulate content in the water samples while concentrating the bacteria 10-fold, is robust and simple enough for field applications by minimal…

Materials

Agricultural water			Irrigation water, produce wash water, well water, etc.
Vinyl tubing	Wilmar	BN-CVT1005	1/4" inner diameter,  3/8" outer diameter, available at:  http://www.wilmar.com
Modified Moore Swab cartridge	Lumiere Diagnostics		11 ½ cm in length and 4 ½ cm in width, available at:  http://www.lumierediagnostics.com.  Alternativelly, a non-disposable version of the cartridge can be used (refer to the text)
Cheesecloth	Chesapeake Wiper & Supply, Inc.	CC90	Grade #90, 44 × 36 weave, available at:  www.raglady.com
Household Bleach	Various		Sodium hypochlorite concentration approx. 6%
Sodium thiosulphate 5-hydrate	Mallinckrodt Baker Inc	8100-04
Manifold	Built in-house		Optional, device can be constructed from PVC pipes and appropriate fittings
Peristaltic pump	Micron Meters	RPP1300	Available at:  http://www.micronmeters.com
Serological pipette	Various		Disposable, 10ml
Universal preenrichment broth	Difco	223510
Buffered peptone water	Difco	218105
Salmonella supplement	Biomérieux Industry	42650	http://www.biomerieux-usa.com
VIDAS UP Listeria (LPT) Broth	Biomérieux Industry	410848	http://www.biomerieux-usa.com
Vancomycin	Sigma-Aldrich	861987	http://www.sigmaaldrich.com
Pipet-Aid	Various		Drummond DP-110 used here
Shaking incubator	Various		Excella E25, New Brunswick Scientific used here
Micropipette	Various		10 μl, 1 ml
Micropipette tips	Various		Barrier, 10 μl, 1 ml
1.5 microcentrifuge tubes	Various		RNase- and DNase- free
Probe sonicator	Q Sonica LLC	XL-2000 series
µPADs	Avant		Wax printed 7 mm diameter circles, with 4 pt line thickness. Contact Dr. Charles Henry for additional information
HEPES [N-(2-Hydroxyethyl)piperazine-N′-2-ethanesulfonic acid]	Sigma-Aldrich	H3375
Bovine serum albumin	Sigma-Aldrich	A8022
Chlorophenol red-galactopyranoside (CPRG)	Sigma-Aldrich	59767
5-Bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc)	Sigma-Aldrich	B8174
5-bromo-6-chloro-3 indolylcaprylate (magenta caprylate)	Sigma-Aldrich	53451
5-Bromo-4-chloro-myo-inositol phosphate (X-InP)	Sigma-Aldrich	38896
Petri dishes, polystyrene 100mm by 15 mm	Various		Sterile
Flat bed scanner	Various		Xerox USB scanner
ImageJ software	National Institutes of Health		http://rsb.info.nih.gov/ij/