Isolation of Listeria monocytogenes from Food and Water: Official and Experimental Protocols

Reha O. Azizoglu1, Lisa Gorski2, Sophia Kathariou2

1 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, 2 Produce Safety and Microbiology Research Unit, USDA‐ARS, Albany, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9B.5
DOI:  10.1002/9780471729259.mc09b05s33
Online Posting Date:  May, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Listeria monocytogenes is frequently encountered in foods but often at low concentrations and typically in the presence of other microbiota, including nonpathogenic Listeria spp. The potential of L. monocytogenes to cause severe human disease mandates sensitive, accurate, and rapid detection in foods. Isolation of L. monocytogenes from foods is critical, not only for routine surveillance, but also for epidemiologic investigations. Isolation of the pathogen from water (especially surface water used for irrigation) is similarly important, as produce has been implicated in listeriosis outbreaks and contaminated water can be involved in contamination of produce. This unit provides basic protocols for the isolation of L. monocytogenes from foods and water. Curr. Protoc. Microbiol. 33:9B.5.1‐9B.5.19. © 2014 by John Wiley & Sons, Inc.

Keywords: Listeria; Listeria monocytogenes; food; water; selective enrichment; differential selective medium

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Listeria monocytogenes Isolation from Food: Official FDA BAM Protocol
  • Alternate Protocol 1: Listeria monocytogenes Isolation from Food: Official ISO Protocol
  • Alternate Protocol 2: Listeria monocytogenes Isolation from Food: Official USDA‐FSIS Protocol
  • Alternate Protocol 3: Listeria monocytogenes Isolation from Food: Experimental L. monocytogenes Blood Agar (LMBA) Protocol
  • Alternate Protocol 4: Listeria monocytogenes Isolation from Food: Experimental Immunomagnetic Separation (IMS)
  • Basic Protocol 2: Listeria monocytogenes Isolation from Water: Official EPA/FDA Protocol
  • Alternate Protocol 5: Listeria monocytogenes Isolation from Water: Experimental Moore Swabs and IMS Protocol
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Listeria monocytogenes Isolation from Food: Official FDA BAM Protocol

  Materials
  • Food sample of concern
  • Basal buffered Listeria enrichment broth (BLEB; BD, cat. no. 290720)
  • Selective agents:
    • Acriflavine (Sigma‐Aldrich, cat. no. A8126)
    • Nalidixic acid (sodium salt; Sigma‐Aldrich, cat. no. N4382)
    • Cycloheximide (Sigma‐Aldrich, cat. no. C7698)
  • Esculin‐containing selective agar plates, chosen on the basis of availability and cost from the following:
    • OXA (Listeria selective agar base; Oxoid, cat. no. CM0856; Oxford selective supplement; Oxoid, cat. no. SR0140)
    • PALCAM (PALCAM agar base; Oxoid, cat. no. CM0877; PALCAM selective supplement; Oxoid, cat. no. SR0150)
    • Modified Oxford agar (MOX; Listeria selective agar base; Oxoid, cat. no. CM0856; Oxford selective supplement; Oxoid, cat. no. SR0206)
    • LPM agar (Sigma‐Aldrich, cat. no. 62653), fortified with 1 g/liter esculin and 0.5 g/liter ferric ammonium citrate
  • Differential selective agar medium chosen on the basis of availability and cost from the following:
    • ALOA (Biomerieux, cat. no. AEB120082)
    • RAPID'L. mono (BioRad, cat. no. 355‐5294)
    • BBL CHROMagar Listeria (BD, cat. no. 215085)
    • BCM (BCM Listeria monocytogenes plating medium I; Biosynth, cat. no. C‐0608; supplement for plating medium I; Biosynth, cat. no. C‐0610)
  • Sheep blood agar (Remel, cat. no. R01198, or equivalent)
  • Trypticase soy agar (TSA; BD, cat. no. 236950) with 0.6% (w/v) yeast extract (BD, cat. no. 212750)
  • Blender or stomacher and 400‐ml stomacher bags (Seward)
  • 30°, 35°, and 37°C incubators
  • 10‐µl bacteriological loop (Nunc, cat. no. I7898‐1PAK)
  • Additional reagents and equipment for carrying out serotyping (FDA BAM; see Internet Resources), pulse‐field gel electrophoresis (http://www.cdc.gov/pulsenet/PDF/listeria‐pfge‐protocol‐508c.pdf), and ribotyping (e.g., http://www2.dupont.com/Qualicon/en_US/products/BAX_System/bax4listeria.html)

Alternate Protocol 1: Listeria monocytogenes Isolation from Food: Official ISO Protocol

  Materials
  • Food sample of concern
  • Half‐Fraser broth (Fraser broth base; Oxoid, cat. no. CM0895; Half Fraser supplement: Oxoid, cat. no. SR0166)
  • Fraser broth (Fraser broth base; Oxoid, cat. no. CM0895; Fraser supplement; Oxoid, cat. no. SR0156)
  • PALCAM Listeria spp. selective agar plates (PALCAM agar base; Oxoid, cat. no. CM0877; PALCAM selective supplement; Oxoid, cat. no. SR0150)
  • ALOA differential selective agar medium (ALOA; Biomerieux, cat. no. AEB120082), RAPID'L. mono (BioRad, cat. no. 355‐5294)
  • 400‐ml stomacher bag (Seward)
  • Stomacher (Seward)
  • 30° and 37°C incubator
  • 15‐ml culture tubes
  • 10‐µl bacteriological loop (Nunc, cat. no. I7898‐1PAK)
  • Additional reagents and equipment for carrying out confirmation tests for L. monocytogenes e.g., hemolysis assays (Scotter et al., ) and the CAMP test (FDA BAM; see Internet Resources)

Alternate Protocol 2: Listeria monocytogenes Isolation from Food: Official USDA‐FSIS Protocol

  Materials
  • Food sample of concern
  • Disinfectant, e.g., 3% (v/v) hydrogen peroxide, 70% (v/v) ethanol, or 70% (v/v) isopropanol
  • Modified University of Vermont broth (UVM; Sigma‐Aldrich, cat. no. 90554)
  • Morpholinepropanesulfonic acid–buffered Listeria enrichment broth (MOPS‐BLEB; BBL Listeria enrichment broth; Difco, cat. no. 222220; MOPS free acid; Sigma‐Aldrich, cat. no. M1254; MOPS sodium salt, Sigma‐Aldrich, cat. no. M9381)
  • Fraser broth (FB; Fraser broth base; Oxoid, cat. no. CM0895; Fraser supplement; Oxoid, cat. no. SR0156)
  • Modified Oxford agar (MOX; Listeria selective agar base; Oxoid, cat. no. CM0856; Oxford selective supplement; Oxoid, cat. no. SR0206)
  • 50‐ or 250‐ml stomacher bag, sterile (Seward)
  • Blender or stomacher (Seward)
  • 30° and 35°C incubators
  • Additional reagents and equipment for carrying out bacteriological, biochemical, or molecular tests for identifying L. monocytogenes (see Internet Resources)

Alternate Protocol 3: Listeria monocytogenes Isolation from Food: Experimental L. monocytogenes Blood Agar (LMBA) Protocol

  Additional Materials (also see protocol 1)
  • Buffered peptone water (LabM, cat. no. LAB46)
  • Trypticase soy agar (TSA; BD, cat. no. 236950)
  • Lithium chloride (Sigma‐Aldrich, cat. no. L0505)
  • 10 mg/liter polymyxin B sulfate (Sigma‐Aldrich, cat. no. P1004), dissolve in water and sterilize by passing through a 0.2‐µm filter
  • Ceftazimide (Sigma‐Aldrich, cat. no. C3809): dissolve in water and sterilize by passing through a 0.2‐µm filter; store up to 6 months at −20°C
  • Defibrinated sheep blood (BD, cat. no. 211945)
  • 3% (v/v) hydrogen peroxide

Alternate Protocol 4: Listeria monocytogenes Isolation from Food: Experimental Immunomagnetic Separation (IMS)

  Materials
  • Food samples of concern
  • Listeria enrichment broth (LEB; LEB base, Oxoid, cat. no. CM0862; LEB supplement, Oxoid, cat. no. SR0141)
  • Dynabeads anti‐Listeria immunomagnetic beads (Life Technologies, cat. no. 71006)
  • Phosphate‐buffered saline ( appendix 2A)/0.5% (v/v) Tween‐20 (PBS/Tween): autoclave and store at room temperature
  • Differential selective agar medium (ALOA; Biomerieux, cat. no. AEB120082)
  • 400‐ml stomach bags and stomacher (Seward)
  • 30°C and 35°C incubator
  • Dynabeads BeadRetriever tube strip (Life Technologies, cat. no. 159‐50)
  • Additional reagents and equipment for performing IMS using anti‐Listeria Dynabeads anti‐Listeria immunomagnetic beads according to the supplier's instructions, and confirmation testing for L. monocytogenes (e.g., protocol 1, step 10)

Basic Protocol 2: Listeria monocytogenes Isolation from Water: Official EPA/FDA Protocol

  Materials
  • Water sample of concern
  • Basal buffered Listeria enrichment broth (BLEB, BD cat. no. 290720)
  • Selective agents:
    • Acriflavine (Sigma‐Aldrich, cat. no. A8126)
    • Nalidixic acid (sodium salt; Sigma‐Aldrich, cat. no. N4382)
    • Cycloheximide (Sigma‐Aldrich, cat. no. C7698) or other antifungal agent
  • PALCAM Listeria spp. selective agar plates (PALCAM agar base; Oxoid, cat. no. CM0877; PALCAM selective supplement, Oxoid, cat. no. SR0150) or
  • Modified Oxford agar (MOX; Listeria selective agar base, Oxoid, cat. no. CM0856; Oxford selective supplement, Oxoid, cat. no. SR0206)
  • 1‐liter sterile jars or bottles
  • Swing sampler (Nasco, cat. no. B01310M)
  • 0.45‐µm filter
  • Scissors, flame‐sterilized
  • Whirl‐Pak bag (Nasco, cat. no. B01297WA)
  • 30°C incubator
  • Additional reagents and equipment for performing confirmation testing for L. monocytogenes ( protocol 1)

Alternate Protocol 5: Listeria monocytogenes Isolation from Water: Experimental Moore Swabs and IMS Protocol

  Materials
  • Sterile water
  • 5× buffered Listeria enrichment broth (BLEB; BD, cat. no. 290720)
  • Dynabeads anti‐Listeria immunomagnetic beads (Life Technologies, cat. no. 71006)
  • Phosphate‐buffered saline ( appendix 2A) 0.5% (v/v) Tween‐20 (PBS/Tween): autoclave and store at room temperature
  • Brilliance Listeria agar (Oxoid, cat. no. CM1080)
  • Fraser broth (Fraser broth base: Oxoid, cat. no. CM0895; Fraser supplement, Oxoid, cat. no. SR0156)
  • Nonselective medium, e.g., trypticase soy agar (TSA; BD, cat. no. 236950) or brain heart infusion (BHI) agar (BD, cat. no. 241830)
  • Modified Oxford agar (Listeria selective agar base; Oxoid, cat. no. CM0856; Oxford selective supplement; Oxoid, cat. no. SR0206)
  • Moore swabs (see Fig. )
  • Whirl‐Pak bags (Nasco, cat. no. B01297WA)
  • 30° and 37°C incubators
  • Orbital shaker (e.g., VWR)
  • Automated Bead Retriever (Life Technologies), optional
  • Dynabeads BeadRetriever tube strip (Life Technologies, cat. no. 159‐50)
  • 1.5‐ml microcentrifuge tube
  • 10‐µl bacteriological loop (Nunc, cat. no. I7898‐1PAK)
  • Additional reagents and equipment for performing IMS using anti‐Listeria Dynabeads anti‐Listeria immunomagnetic beads according to the supplier's instructions and for carrying out confirmation testing for L. monocytogenes (e.g., see protocol 1, step 10)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Amagliani, G., Omiccioli, E., Campo, A., Bruce, I.J., Brandi, G., and Magnani, M. 2006. Development of a magnetic capture hybridization‐PCR assay for Listeria monocytogenes direct detection in milk samples. J. Appl. Microbiol. 100:375‐383.
  Barrett, T.J., Blake, P.A., Morris, G.K., Puhr, N.D., Bradford, H.B., and Wells, J.G. 1980. Use of Moore swabs for isolating Vibrio cholerae from sewage. J. Clin. Microbiol. 11:385‐388.
  Becker, B., Schuler, S., Lohneis, M., Sabrowski, A., Curtis, G.D.W., and Holzapfel, W.H. 2006. Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290‐1. Int. J. Food Microbiol. 109:127‐131.
  Beumer, R.R. and Hazeleger, W.C. 2003. Listeria monocytogenes: Diagnostic problems. FEMS Immunol. Med. Microbiol. 35:191‐197.
  Beumer, R.R., te Giffel, M.C., Anthony, S.V.R., and Cox, L.C. 1996. The effect of acriflavine and nalidixic acid on the growth of Listeria spp. in enrichment media. Food Microbiol. 13:137‐148.
  Bruhn, J.B., Vogel, B.F., and Gram, L. 2005. Bias in the Listeria monocytogenes enrichment procedure: Lineage 2 strains outcompete Lineage 1 strains in University of Vermont selective enrichments. Appl. Environ. Microbiol. 71:961‐967.
  Centers for Disease Control. 2013. Vital signs: Listeria illnesses, deaths, and outbreaks—United States, 2009‐2011. MMWR Weekly. 62:448‐452.
  Farber, K.R. and Peterkin, P.I. 1991. Listeria monocytogenes, a food‐borne pathogen. Microbiol. Rev. 55:476‐511.
  Gasanov, U., Hughes, D., and Hansbro, P.M. 2005. Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: A review. FEMS Microbiol. Rev. 29:851‐875.
  Gnanou‐Besse, N., Audinet, N., Kérouanton, A., Colin, P., Kalmokoff, M. 2005. Evolution of Listeria populations in food samples undergoing enrichment culturing. Int. J. Food Microbiol. 104:123‐134.
  Gnanou‐Besse, N., Barre, L., Buhariwalla, C., Vignaud, M.L., Khamissi, E., Decourseulles, E., Nirsimloo, M., Chelly, M., and Kalmokoff M. 2010. The overgrowth of Listeria monocytogenes by other Listeria spp. in food samples undergoing enrichment cultivation has a nutritional basis. Int. J. Food Microbiol. 136:345‐351.
  Gorski, L., Liang, A.S., Walker, S., Nguyen, K.M., Carychao, D., Cooley, M.B., Govoni, J., and Mandrell, R.E. 2013. Comparison of methods for Salmonella enterica and Listeria monocytogenes isolation to measure incidence in a Central California watershed. American Society for Microbiology, 113th General Meeting, Abstract 1147. ASM Press. Washington, D.C.
  Hitchins, A.D. and Jinneman, K. 2011. Detection and enumeration of Listeria monocytogenes in foods. Chapter 10. In Bacteriological Analytical Manual. U.S. Food and Drug Administration. Silver Spring, Md.
  International Organization for Standardization. 1996. Microbiology of food and animal feeding stuffs—Horizontal method for the detection and enumeration of Listeria monocytogenes—Part 1: Detection method. ISO. Geneva, Switzerland.
  International Organization for Standardization. 1998. Microbiology of food and animal feeding stuffs—Horizontal method for the detection and enumeration of Listeria monocytogenes—Part 2: Enumeration method. ISO. Geneva, Switzerland.
  International Organization for Standardization (ISO). 2004. Modification of the isolation media and the haemolysis test and inclusion of precision data. ISO. Geneva, Switzerland.
  Jadhav, S., Bhave, M., and Palombo, E.A. 2012. Methods used for the detection and subtyping of Listeria monocytogenes. J. Microbiol. Methods 88:327‐341.
  Johansson, T. 1998. Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food‐processing environments. Int. J. Food Microbiol. 40:77‐85.
  Johnson, J., Jinneman, K., Stelma, G., Smith, B.G., Lye, D., Messer, J., Ulaszek, J., Evsen, L., Gendel, S., Bennett, R.W., Swaminathan, B., Pruckler, J., Steigerwalt, A., Kathariou, S., Yildirim, S., Volokhov, D., Rasooly, A., Chizhikov, V., Wiedmann, M., Fortes, E., Duvall, R.E., and Hitchins, A.D. 2004. Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes. Appl. Environ. Microbiol. 70:4256‐4266.
  Kathariou, S. 2002. Listeria monocytogenes virulence and pathogenicity: A food safety perspective. J. Food Prot. 65:1811‐1829.
  Loncarevic, S., Tham, W., and Danielsson‐Tham, M.L. 1996. The clones of Listeria monocytogenes detected in food depend on the method used. Lett. Appl. Microbiol. 22:381‐384.
  Loncarevic, S., Økland, M., Sehic, E., Norli, H.S., and Johansson, T. 2008. Validation of NMKL method No. 136—Listeria monocytogenes, detection and enumeration in foods and feed. Int. J. Food Microbiol. 124:154‐163.
  MacDonald, F. and Sutherland, A.D. 1994. Important differences between the generation times of Listeria monocytogenes and Listeria innocua in two Listeria enrichment broths. J. Dairy Res. 61:433‐436.
  Mendonça, M., Conrad, N.L., Conceição, F.R., Moreira, Â.N., da Silva, W.P., Aleixo, J.A., and Bhunia, A.K. 2012. Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii. BMC Microbiology 12:275.
  Petran, R.L. and Swanson, K.M.J. 1993. Simultaneous growth of Listeria monocytogenes and Listeria innocua. J. Food Prot. 56:616‐618.
  Reissbrodt, R. 2004. New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.—an overview. Int. J. Food Microbiol. 95:1‐9.
  Rijpens, N. and Herman, L. 2004. Comparison of selective and nonselective primary enrichments for the detection of Listeria monocytogenes in cheese. Int. J. Food Microbiol. 94:15‐22.
  Ryser, E.T., Arimi, S.M., Bunduki, M.M.‐C., and Donnelly, C.W. 1996. Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. Appl. Environ. Microbiol. 62:1781‐1787.
  Schlech, W.F. 3rd. 2000. Foodborne listeriosis. Clin. Infect. Dis. 31:770‐775.
  Scotter, S.L., Langton, S., Lombard, B., Schulten, S., Nagelkerke, N., Int‘ Veld, P.H., Rollier, P., and Lahellec, C. 2001. Validation of ISO method 11290 Part 1—Detection of Listeria monocytogenes in foods. Int. J. Food Microbiol. 64:295‐306.
  Strawn, L.K., Gröhn, Y.T., Warchocki, S., Worobo, R.W., Bihn, E.A., and Wiedmann, M. 2013. Risk factors associated with Salmonella and Listeria monocytogenes contamination of produce fields. Appl. Environ. Microbiol. 79:7618‐7627.
  Swaminathan, B. and Gerner‐Smidt, P. 2007. The epidemiology of human listeriosis. Microbes Infect. 9:1236‐1243.
  Uyttendaele, M., Van Hoorde, I., and Debevere, J. 2000. The use of immuno‐magnetic separation (IMS) as a tool in a sample preparation method for direct detection of L. monocytogenes in cheese. Int. J. Food Microbiol. 54:205‐212.
  Vazquez‐Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez‐Bernal, G., Goebel, W., Gonzalez‐Zorn, B., Wehland, J., and Kreft, J. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584‐640.
  Wadud, S., Leon‐Velarde, C.G., Larson, N., and Odumeru, J.A. 2010. Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready‐to‐eat foods. J. Microbial. Methods 81:153‐159.
  Walcher, G., Stessl, B., Wagner, M., Eichenseher, F., Loessner, M.J., and Hein, I. 2010. Evaluation of paramagnetic beads coated with recombinant Listeria phage endolysin‐derived cell‐wall‐binding domain proteins for separation of Listeria monocytogenes from raw milk in combination with culture‐based and real‐time polymerase chain reaction‐based quantification. Foodborne Pathog. Dis. 7:1019‐1024.
  Yang, H., Qu, L., Wimbrow, A.N., Jiang, X., and Sun, Y. 2007. Rapid detection of Listeria monocytogenes by nanoparticle‐based immunomagnetic separation and real‐time PCR. Int. J. Food Microbiol. 118:132‐138.
  Zitz, U., Zunabovic, M, Domig, K.J., Wilrich, P.T., and Kneifel, W. 2011. Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua. J. Food Prot.74:1282‐1287.
  Zunabovic, M., Domig, K.J., and Kneifel, W. 2011. Practical relevance of methodologies for detecting and tracing of Listeria monocytogenes in ready‐to‐eat foods and manufacture environments—A review. J. Food Prot. 74:1282‐1287.
Internet Resources
  http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm071400.htm
  This Web site contains Chapter 10 of the FDA Bacteriological Analytical Manual (BAM), “Detection and Enumeration of Listeria monocytogenes in Foods,” and includes updates up to April 2013.
  http://www.fsis.usda.gov/wps/wcm/connect/1710bee8‐76b9‐4e6c‐92fc‐fdc290dbfa92/MLG‐8.pdf?MOD=AJPERES
  This Web site contains Chapter 8.09 of reference methods established by United States Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS),“Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, and Egg Products, and Environmental Samples,” with updates up to 05/01/2013. It also includes confirmatory tests mentioned in the protocols in section 8.6.
  http://www.cdc.gov/pulsenet/PDF/listeria‐pfge‐protocol‐508c.pdf
  This Web site describes the Pulse‐Net protocol for PFGE of Listeria.
  http://www2.dupont.com/Qualicon/en_US/products/BAX_System/bax4listeria.html
  This Web site describes automated ribotyping for Listeria at DuPont.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library