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Open AccessTechnical Note

Improved Positive Predictive Performance of Listeria Indicator Broth: A Sensitive Environmental Screening Test to Identify Presumptively Positive Swab Samples

1
Paradigm Diagnostics, Inc., 800 Transfer Road Suite 12, St. Paul, MN 55114, USA
2
Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2019, 7(5), 151; https://doi.org/10.3390/microorganisms7050151
Received: 18 April 2019 / Revised: 20 May 2019 / Accepted: 25 May 2019 / Published: 27 May 2019

Abstract

PDX-LIB, Listeria Indicator Broth, was developed as a proprietary sensitive screening test to identify presumptively positive environmental swab samples for Listeria sp. The original formulation, while sensitive, initially proved to exhibit acceptable levels of false positive test results. Paradigm Diagnostics has been undertaken to modify the medium formulation to render it more selective while not sacrificing its sensitivity. After identification of a candidate formulation through laboratory studies, a field trial was conducted to validate the test performance parameters, including the true positive frequency and false positive frequency in several different food-processing facilities. Identical swab samples were enriched in both the original medium formulation and the new formulation. Presumptive positive samples were confirmed by plating on selective differential agar and qPCR analysis. The field trial data demonstrate that the new formulation significantly reduces the frequency of false positive samples compared to the original Listeria Indicator Broth formulation, without compromising the sensitivity of the original formulation. The new medium formulation resulted in no false positive samples compared to the 54% increased presumptive positive samples obtained with the original medium formulation.
Keywords: food safety; environmental Listeria; Listeria detection food safety; environmental Listeria; Listeria detection

1. Introduction

In a risk assessment study, the U.S. Department of Agriculture Food Safety Inspection Service provided the rationale for mandating a national surveillance program for Listeria occurrence in USDA-regulated facilities [1]. These new regulations mandated environmental surveillance for the presence of Listeria sp. in food processing facilities to minimize the risk of foodborne illness associated with contaminated food. This development impelled many firms, including Paradigm Diagnostics, to develop simple Listeria screening tests to enable the growing demand for this test volume [2].
A comprehensive study by the Center for Disease Control in 2012 provided evidence that the implementation of environmental controls in food processing facilities coupled with robust public health monitoring (Pulse Net) helped to reduce the burden of foodborne Listeriosis [3]. Despite these encouraging results, foodborne illnesses due to pathogens, including Salmonella, STEC, and Listeria, continue to be a challenge in the national food production system [4,5,6]. Figure 1 demonstrates that the frequency of Listeriosis outbreaks in the US has experienced a marked increase in the past few years. Consequently, accurate simple screening methods for foodborne illness pathogens must be available to address the on-going need for facility environmental surveillance.
In this study, we intend to demonstrate that an improved Listeria enrichment formulation can help to eliminate uncertainty when screening environmental samples for the presumptive presence of Listeria sp. Field trial data collected from eight different food-processing facilities supports the laboratory data, showing that the new formulation, LIB v.2.0, is more accurate than the antecedent test, LIB. Specifically, the false positives observed using LIB were completely eliminated using LIB v.2.0 without a loss of sensitivity for the detection of true Listeria positive samples. Appendix A was included to provide detailed location information of where the samples were obtained.

2. Materials and Methods

PDX-LIB and Listeria Indicator Broth v.2.0 and Securswabs were supplied by Paradigm Diagnostics, Inc. St. Paul, MN. Swabs were collected as duplicates from the same locations in food processing facilities and enriched in 20 mL of either LIB, the original formulation, or LIB v.2.0, the new medium formulation, for 48 h at 37 °C. Blackened samples were streaked onto modified MOX (modified Oxford) medium and incubated for an additional 18 h at 37 °C. The modified MOX medium was prepared by substituting the esculin in the standard MOX formulation with 5 g/L D-arabitol and 0.02 g/L bromcresol purple as the indicator system for Listeria sp. [7].
MOX-positive plates were confirmed as Listeria sp. by qPCR using primers and probes as detailed in the Food and Drug Administration Bacteriological Assay Manual [8]. Statistical analysis was conducted and pairwise comparisons between pathogen isolation rates using LIB v2.0 and LIB (original formulation) were made using the Mantel-Haenszel chi-square formula for unmatched test portions [9]. A Chi-Square value of less than 3.84 was considered to indicate no significant numerical difference between the two methods being compared. The formula for χ2 is
χ2= (|a-b|-1)2/(a+b)
  • a = The number of presumptively positive samples using LIB v.2.0.
  • b = The number of presumptively positive samples using LIB.

3. Results

A total of 161 samples were obtained from eight different food-processing facilities. Presumptive positive samples were identified and confirmed. Table 1 summarizes the results of field trial samples. Of the 161 environmental samples, LIB v-2.0 yielded 35 presumptive positives, while the original formulation resulted in 55 blackened samples. The 35 LIB v-2.0 samples were confirmed as true positives by plating and PCR analysis.
The LIB (original formulation) results yielded 54 presumptive positives, of which 35 were confirmed. Twenty of the presumptive positive LIB samples were deemed false positives. One hundred and seven of the LIB samples were negative, of which 106 were true negatives. One of the negative LIB samples was deemed a false negative since the duplicate LIB v-2.0 sample yielded a true positive result. Chi square analysis (Χ2 = 30.06) of the positives and false positives generated by both sample populations indicated a significant difference at the 95% confidence level.

4. Discussion

Listeria environmental screening continues to represent a significant proportion of global Listeria testing carried out in the food microbiology laboratory [10]. Accordingly, facile methods to identify presumptively positive environmental samples reduce the cost and time required. Paradigm Diagnostics developed an environmental screening test to identify presumptive positive Listeria samples. The method has been shown to be more sensitive than the USDA method [11] and potentially avoids the risk of false negative samples due to the presence of acriflavin in the enrichment medium used by most commercial enrichment media [12].
The data set in Table 1 represent environmental samples from diverse sources of food-processing facilities, Appendix A. The data translate to a sensitivity and specificity for LIB (original formulation) of 97.2% and 86.2%, respectively. In contrast, the sensitivity and specificity data for LIB v-2.0 are 100% and 100%, respectively. The positive predictive values of the respective media are 63% for LIB and 100% for LIB v-2.0.
The field data underscore the substantially better diagnostic performance characteristics of LIB v-2.0 compared with the original LIB formulation. Furthermore, the sensitivity of the new medium appears to be comparable to or better than the original formulation. We had anticipated that the new formulation would exhibit more false negatives since LIB v-2.0 contains higher levels of lithium chloride than LIB. However, we found that the LIB v-2.0 medium exhibited a greater sensitivity, with a value of 100% versus 97.2% for LIB.
This may make sense when one considers that the growth of competitive microflora, particularly Enterococcus sp., may inhibit the growth of Listeria sp. in the sample. In a recent publication, Hanachi et al. detail the potential to use Enterococcus sp., especially E. faecalis and E. faecium, to control the growth of Listeria monocytogenes in food products [13]. In addition to Enterococcus sp., many species within the lactic acid bacteria family are capable of producing anti-listerial compounds. The ability of these organisms to compete with Listeria sp. resides in their capability to both grow more robustly and produce anti-listerial bacteriocins [14].
Appendix A provides detailed site information from which the samples were obtained at their respective facilities.
In conclusion, we have demonstrated that the new formulation of the environmental Listeria screening test, LIB v-2.0, exceeds the performance characteristics of the original formulation, LIB, in comparison field trials. LIB v-2.0 provides a greater accuracy and a higher positive predictive value without sacrificing the test sensitivity.

Author Contributions

J.M.F. developed sampling and field trial citing. A.D.O. developed formulation modifications and implementation.

Funding

This research received no external funding.

Acknowledgments

The authors wish to thank Paradigm Diagnostics, Inc. for donation of materials used to conduct this study.

Conflicts of Interest

A.O. is a Chief Scientific Officer of Paradigm Diagnostics, Inc. Paradigm Diagnostics has provided support for this project through in-kind resources.

Appendix A

Table A1.
Ready to Eat Food Facility
LocationLIBLIB v.2.0MOX, PCR
Cooler 1: Aisle A: Pepper PalletNEGNEG
Squeegee in Cooler 2NEGNEG
Curtain between coolers 1 & 2; aisle ANEGNEG
Curtain between coolers 2 & 3; aisle ANEGNEG
Wood under Plate Cooler 2NEGNEG
Dampness behind Wood on floorPOSNEGNEG
Blue CHEP pallet Cooler 3 (damp)NEGNEG
Cooler 3 drainNEGNEG
Wood Pallet (damp) Cooler 3NEGNEG
Floor Under Rack (105) wet - cooler 3NEGNEG
ICE from case of Brussel Sprouts Rack 105 Cooler 3NEGNEG
Underneath Table 26; School CoolerNEGNEG
Inside of Floor Scrubber lidPOSNEGNEG
Blue Filter of Floor Scrubber reservoirNEGNEG
Inside of Floor Scrubber hosePOSNEGNEG
Floor Scrubber BrushNEGNEG
Dishwasher Floor Drain (Bin cleaning area)POSNEGNEG
Meat Processing—Fermentation/Drying
LocationLIBLIB v.2.0MOX, PCR
Drain in packaging roomNEGNEG
Vacuum MachineNEGNEG
Under Packaging Room tableNEGNEG
Dishwater room drainPOSNEGNEG
Underneath foot stoolNEGNEG
Hand sink drainNEGNEG
SqueegeeNEGNEG
RTE room drain by ECA deviceNEGNEG
Drain in cooked coolerNEGNEG
Coving in cooked coolerNEGNEG
Smoke cart wheelsPOSNEGNEG
Black cart wheelsNEGNEG
Dish sink drain rightNEGNEG
Dish sink drain middleNEGNEG
Dish sink drain leftPOSNEGNEG
RTE floor drain outside aging coolerNEGNEG
Raw Door FloorPOSNEGNEG
RTE Food Facility/Sandwiches/SaladsLIBLIB v.2.0MOX, PCR
Cooling Unit # 1NEGNEG
Cooling Unit #2NEGNEG
Cooling Unit #3NEGNEG
Cooling Unit #5NEGNEG
Cooling Unit #6NEGNEG
Drain #14NEGNEG
Drain #15POSPOSLm
Line #3 Bag holeNEGNEG
Threshold Swing Door #3NEGNEG
Threshold Swing Door #2NEGNEG
Threshold Swing Door #1NEGNEG
Above ceiling in Wash RoomNEGNEG
Threshold H&C cooler door fr. StNEGNEG
Drain #27NEGNEG
Threshold M&C cooler door fr. RecNEGNEG
Receiving ThresholdNEGNEG
Drain # 9NEGNEG
Threshold shipping cooler Door #2NEGNEG
Mat in Hallway QA officeNEGNEG
Retail Store Food Areas
Deli—Back RoomLIBLIB 2.0MOX, PCR
Drain in front of raw chicken sink, insidePOSPOSLm
Drain in front of 3-compartment sink, insideNEGNEG
Drain in back wall underneath racksNEGNEG
Inside condenser pipe in-between racks by drain #7POSNEGNEG
Drain underneath food prep sinkNEGNEG
Mop sinkNEGNEG
Drain behind ice machineNEGNEG
Top of dishwasherNEGNEG
Drain under dishwasher (no cover)NEGNEG
Drain in front of Deli coolerNEGNEG
Produce CoolerLIBLIB v.2.0MOX,PCR
Inside access port—drain plug—Produce coolerPOSNEGNEG
Wall in Produce coolerNEGNEG
Cooling unit guard inside Produce coolerNEGNEG
Frame of shelf in Produce cooler (left side)NEGNEG
PRE—by drain in produce cooler - water presentNEGNEG
PRE—water on floor of produce cooler below boxNEGNEG
Outside box of produce that was dripping bottom shelfPOSPOSLm
hole in wall right side middleNEGNEG
shelf leg by floor right sideNEGNEG
shelf leg by doorNEGNEG
bottom shelf where iced produce sitsNEGNEG
middle shelf where iced produce sitsNEGNEG
shelf where organic produce sitsNEGNEG
coving on left side by iced produceNEGNEG
hole in wall left side by iced produceNEGNEG
water on floor where cut fruit sitsNEGNEG
Deli (Front Room)LIBLIB v.2.0MOX,PCR
Food prep sink drain + underneath coverNEGNEG
Drain underneath Combi Oven (cover)NEGNEG
Drain under Food Prep SinkNEGNEG
CaféLIBLIB v.2.0MOX,PCR
Drain under soda fountainPOSNEGNEG
Drain in front of dishwasherNEGNEG
Drain under 3-compartment sinkNEGNEG
Drain under prep sinkNEGNEG
Drain by mop sinkNEGNEG
mop sinkNEGNEG
Coffee ShopLIBLIB v.2.0MOX,PCR
Drain under sinkNEGNEG
Foam drain for coffee maker machineNEGNEG
Drain under milk/coffee barNEGNEG
Meat Plant (2)
LocationLIBLIB v.2.0MOX,PCR
Meat Rack for snack sticksNEGNEG
Drain Oven RoomPOSPOSL. mono
Door out of oven roomPOSPOSL. mono
Cooler FloorPOSPOSL. inocua
Packaging tableNEGNEG
RTE tubNEGNEG
Snack Stick CutterPOSPOSL. welshmeri
Ready to Eat Food Facility (2)
Environmental Swabs—pre-op
LocationLIBLIB v.2.0MOX,PCR
Drain G coverNEGNEG
Line 4 bearing on sprocket shaftPOSPOSL. inocua
Line 4 good bearingsPOSPOSL. mono
prep room floor grateNEGNEG
floor scrubberPOSPOSL. mono
air hose compositeNEGNEG
prep room center drainNEGNEG
squeegee in productionPOSPOSL. mono
squeegee in productionNEGNEG
squeegee in prep roomPOSPOSListeria sp
prep room meat and cheese cartsNEGNEG
Floor under rackingPOSPOSL. mono
Floor near prep room wall interfacePOSPOSListeria sp
Center Floor compositeNEGNEG
Drain composite NNEGNEG
Drain composite SPOSNEGnegative
Fork lift with scalePOSPOSL. inocua
Fork lift (stand up)NEGNEG
Cimpl Bologna PalletPOSPOSListeria sp
Cimpl Bologna CardboardNEGNEG
Cimpl Bologna PlasticPOSPOSListeria sp
ASE Ham PalletPOSNEGnegative
ASE Ham CardboardPOSNEGnegative
ASE Ham PlasticNEGNEG
Abbyland PalletPOSPOSListeria sp
Abbyland CardboardPOSPOSListeria sp
Abbyland PlasticNEGNEG
Hot Ham palletPOSNEGnegative
Toby 409/AKA T2POSPOSL. mono
line 4 bearing (all)POSPOSL. mono
Line 3 bearing (all)NEGNEG
Line 5 bearings (all)NEGNEG
Bakery
LocationLIBLIB v.2.0MOX,PCR
Dairy (“Meat“) cooler condenser pipePOSNEGnegative
Dairy (“Meat“) cooler drainPOSPOSL. mono
Bakery cooler drainPOSNEGnegative
Bakery cooler condenser pipeNEGNEG
Drain in center of bakery roomPOSPOSL. mono
Drain at end of bakery coolerPOSPOSL. mono
Long red drain in sandwich prep areaNEGNEG
Sandwich cooler condenser pipePOSPOSL. mono
Sandwich cooler drainPOSNEGNEG
Drain in middle of sandwich prep areaNEGNEG
“Fast chill“ condenser pipeNEGNEG
“Fast chill“ drainPOSPOSL. mono
Holding cooler condenser pipePOSPOSL. mono
Holding cooler drainPOSPOSL. mono
Far left “finished product“ cooler condenser pipeNEGNEG
Far left “finished product“ cooler drainPOSNEGnegative
Far right “finished product“ cooler condenser pipePOSNEGnegative
Far right “finished product“ cooler drainPOSNEGnegative
Inside tub of floor scrubberNEGNEG
Inside of hose out the top of floor scrubberNEGNEG
Scrub brush on bottom of floor scrubberPOSNEGnegative
Scrub brush on bottom of floor scrubberNEGNEG
Squeegee on back of floor scrubberNEGPOSListeria sp
List of abbreviations: MOX: Modified Oxford Medium, PCR: Polymerase Chain Reaction, PDX-LIB: Paradigm Diagnostics’ Listeria Indicator Broth. Items bold permit easier identification of positive samples in table.

References

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Figure 1. Listeria Outbreaks in the U.S. 1998–2018*. * From the NORS Dashboard Available at https://wwwn.cdc.gov/norsdashboard/. (Accessed on 9 May 2019).
Figure 1. Listeria Outbreaks in the U.S. 1998–2018*. * From the NORS Dashboard Available at https://wwwn.cdc.gov/norsdashboard/. (Accessed on 9 May 2019).
Microorganisms 07 00151 g001
Table 1. Field Trial Summary.
Table 1. Field Trial Summary.
MediumTotal SamplesPresumptive PositivesNegativesTP*TNFPFNΧ2
LIB1615410634106201
LIBv-2.016135126351260030.06
TP = true positive, TN = true negative, FP = false positive, FN = false negative. *Confirmed using MOX plating and qPCR as described in the US Food and Drug Administration Bacteriological Assay Manual [8].
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