APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

May 2000, p. 1769–1776 Vol. 66, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Immobilization with Metal Hydroxides as a Means ToConcentrate Food-Borne Bacteria for Detection

by Cultural and Molecular Methods†LISA A. LUCORE,‡ MARK A. CULLISON, AND LEE-ANN JAYKUS*

Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695-7624

Received 11 October 1999/Accepted 17 January 2000

The application of nucleic acid amplification methods to the detection of food-borne pathogens could befacilitated by concentrating the organisms from the food matrix before detection. This study evaluated theutility of metal hydroxide immobilization for the concentration of bacterial cells from dairy foods prior todetection by cultural and molecular methods. Using reconstituted nonfat dry milk (NFDM) as a model, twofood-borne pathogens (Listeria monocytogenes and Salmonella enterica serovar Enteritidis) were concentratedfrom 25-ml samples by the sequential steps of clarification and high-speed centrifugation (designated primaryconcentration) and immobilization with zirconium hydroxide and low-speed centrifugation (designated sec-ondary concentration). Sample volume reduction after immobilization with zirconium hydroxide was 50-fold,with total bacterial recoveries ranging from 78 to 96% of input for serovar Enteritidis and 65 to 96% of inputfor L. monocytogenes. Immobilized bacteria remained viable and could be enumerated by standard culturalprocedures. When followed by RNA extraction and subsequent detection by reverse transcription (RT)-PCR,detection limits of 101 to 102 CFU/25 ml of reconstituted NFDM were achieved for both organisms. Thebacterial-immobilization step was relatively nonspecific, resulting in recovery of >50% of the input cells whenevaluated on a panel of representative bacterial strains of significance to foods. The method could be adaptedto more complex dairy products, such as whole milk and ice cream, for which bacterial recoveries afterimmobilization ranged from 64 to >100%, with subsequent RT-PCR detection limits of >102 CFU/ml for wholemilk and >101 CFU for ice cream for both serovar Enteritidis and L. monocytogenes. The bacterial-immobi-lization method is easy, rapid, and inexpensive and may have applications for the concentration of a widevariety of food-borne bacteria prior to detection by both conventional and alternative methods.

Food-borne pathogens are a major cause of disease in theUnited States, resulting in substantial costs to individuals, foodprocessors, and the national economy. Since contaminationlevels are generally low and there is a zero tolerance for manybacterial pathogens in foods, detection methods requirelengthy culture enrichment steps to increase target bacterialnumbers before isolation and identification using standard cul-tural procedures. These conventional food microbiologicaltechniques often require several days or weeks to complete andmay fail to detect important bacterial pathogens present infoods at low levels. Although the alternative immunological(enzyme-linked immunosorbent assay) and nucleic acid-based(DNA or RNA hybridization) detection protocols are faster,they remain limited by relatively high detection limits, andhence, culture enrichment has not been eliminated.

The introduction of nucleic acid amplification techniques,such as PCR, has stimulated a flurry of activity by food micro-biologists seeking to adapt this methodology to the detectionof food-borne pathogens, with hopes of increased detectionsensitivity and significantly reduced testing time (1, 27, 33).Disappointingly, detection limits have remained higher than

desired and most PCR applications for the detection of food-borne pathogens still require enrichment steps. Specifically,the widespread application of PCR in food microbiology hasbeen limited by (i) high sample volumes compared to amplifi-cation volumes, (ii) residual food components which inhibitPCR enzymatic reactions, (iii) low levels of contaminatingpathogens, and (iv) the inability to discriminate between liveand dead pathogens (1).

It has been suggested that the application of many rapidmolecular methods could be improved if the bacteria wereseparated, concentrated, and purified from the food matrixbefore detection (20, 27, 33). Although methods such as cen-trifugation (2), filtration (4), cationic- and anionic-exchangeresins (29), aqueous two-phase partitioning (11, 21), immobi-lized lectins (19), and immunomagnetic separation (16, 25)have been reported for bacterial concentration in food systems,none of these methods is ideal, and in many cases, a techniqueoptimized for one food system or microorganism is not readilyadaptable to others.

Immobilization of bacterial cells with metal hydroxides wasfirst reported by Kennedy et al. (10) and was later applied inthe concentration of cells from culture media, clinical samples,and foods prior to detection by solid-phase immunoassay (8,9). The method was rapid, efficient, and inexpensive, resultingin the removal of compounds that caused nonspecific bindingin immunoassays and increasing assay detection limits greaterthan 100-fold. More recently, Berry and Siragusa (3) havereported the use of hydroxyapatite to concentrate indigenousbacteria from meat slurries and environmental samples. Thepurpose of this study was to evaluate the utility of bacterialimmobilization using metal hydroxides for the concentration

* Corresponding author. Mailing address: Department of Food Sci-ence, Box 7624, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 515-2971. Fax: (919) 515-7124. E-mail: leeann_jaykus@ncsu.edu.

† This paper is number FSR-99-38 in the journal series of the De-partment of Food Science, North Carolina State University, Raleigh,NC 27695-7624.

‡ Present address: Department of Food Science and Technology,The Ohio State University, Columbus, OH 43210.

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and purification of bacterial cells from dairy foods in prepara-tion for detection by nucleic acid amplification. Thus, we re-port data supporting the feasibility of metal hydroxide immo-bilization for the concentration of representative food-bornemicroorganisms, the use of the method in conjunction withdetection by reverse transcription (RT)-PCR, and the poten-tial adaptability of the method to multiple dairy commodities.

MATERIALS AND METHODS

Bacterial cultures. Stock cultures of Salmonella enterica serovar Enteritidis,Listeria monocytogenes Scott A, Escherichia coli O157:H7 (HC 122), and E. coliATCC 25922 were obtained courtesy of Brian Sheldon, Department of FoodScience, North Carolina State University. Cultures were grown overnight at 35°Cin brain heart infusion (BHI) broth (Difco, Detroit, Mich.) before their use inrecovery experiments. Lactobacillus sp. strain ATCC 4356, Lactococcus lactissubsp. lactis NCK 203, and Bacillus cereus NCK 143 were provided by ToddKlaenhammer, Department of Food Science, North Carolina State University.These overnight cultures were grown in MRS broth (Difco) at 37°C, Elliker broth(Difco) at 30°C, and BHI broth at 30°C, respectively. Pseudomonas aeruginosa(ATCC 10145) was obtained from the American Type Culture Collection (Ma-nassas, Va.) and grown overnight in BHI broth at 37°C. In recovery experiments,serial 10-fold dilutions were done in 0.9% NaCl (sterile saline), and plating forrecovery was performed by the spread plate technique on the agar-solidifiedbroth medium designated for each organism.

Preparation of metal hydroxides. Metal hydroxide solutions were prepared aspreviously reported with minor modifications (8, 9). For zirconium hydroxideand hafnium hydroxide, a 40-ml volume of distilled water was added to 2.0 g ofzirconium(IV) chloride or hafnium chloride 98% (Aldrich Chemical Co., Mil-waukee, Wis.). For titanous hydroxide, a 1.3 mM solution was prepared by theaddition of 200 ml of distilled water to 356 ml of titanium(III) chloride (AldrichChemical Co.). The solutions were adjusted to pH 7.0 6 0.2 by the dropwiseaddition of ammonium hydroxide (5 M) and continuous agitation. Each metalhydroxide solution was then washed three times with 200 ml of sterile salinesolution to remove excess ammonium ions (10). In the washing procedure, thehydroxide was mixed gently with the sterile saline solution and allowed to settleover a 10-min period, and then approximately 40% of the top phase (consistingof saline solution and debris) was decanted. The final volume of each hydroxidewas between 200 and 300 ml, and the hydroxide solutions were stored in the darkat room temperature for up to 6 months.

Immobilization Studies. (i) Feasibility studies with serovar Enteritidis and L.monocytogenes. In the initial immobilization studies, 200 ml of each metal hydrox-ide was mixed with 100 ml of an overnight culture of serovar Enteritidis or L.monocytogenes serially diluted in sterile saline solution to approximately 107, 105,and 103 CFU/100 ml. This represented a 1:2 volume ratio of sample to metalhydroxide. The suspensions were gently agitated at room temperature for 10 minto keep the metal hydroxides in suspension, followed by a brief vortex andcentrifugation at 500 3 g for 5 min at 7°C using an Eppendort microfuge(Brinkmann Instrument Co., Westbury, N.Y.). The supernatants were poured offand retained, and the bacterium-containing pellets were reconstituted in 100 mlof sterile saline solution. Bacterial loss to the supernatant was determined afterthe serial dilution of supernatants and subsequent plating. Percent recovery wascalculated as previously reported (8): [percent immobilization 5 (total popula-tion in sample before immobilization 2 total population in supernatant afterimmobilization) 3 100/(total population in sample before immobilization)]. Plat-ing was also performed on dilutions that were treated identically except withoutthe addition of the metal hydroxide (control). All experiments were done intriplicate.

(ii) Bacterial immobilization applied to dairy products. The efficacy of bac-terial concentration with metal hydroxides was initially investigated using anonfat dry milk (NFDM) model. Twenty-five-milliliter samples of NFDM recon-stituted in sterile water (11% [wt/vol]) were seeded with a 1-ml volume of dilutedovernight cultures of serovar Enteritidis or L. monocytogenes to achieve finalinoculum concentrations of 104, 103, 102, or 101 CFU/25 ml of NFDM. Serialdilutions of the NFDM samples were plated on BHI agar both before and afterinoculation to evaluate the level of the indigenous microflora and to confirm thepathogen levels, respectively. Sample clarification was achieved by the additionof 1.5 ml of 25% (wt/vol) sodium citrate (Fisher Chemical Co., Fair Lawn, N.J.)(22) with 5 min of shaking by hand at room temperature. An initial separationstep (designated primary concentration) was performed by centrifugation at10,000 3 g for 10 min at 7°C in a Sorvall RC-2 refrigerated centrifuge (DuPont,Wilmington, Del.). The resulting pellet was resuspended in 3.0 ml of sterile salineby gentle mixing with a pipette. Zirconium hydroxide solution was added to thereconstituted sample in a 1:2 sample-to-hydroxide volume ratio. The suspensionwas agitated by horizontal shaking on a vortexer (speed setting, 2.5) for 10 minto promote bacterial immobilization. A second separation step, to pellet thehydroxide (designated secondary concentration), was performed by centrifuga-tion (on a Sorvall RC-2 unit) at 500 3 g at 7°C for 10 min. The supernatant wascarefully removed by pipette, and the bacterium-containing pellet was reconsti-tuted to 1 ml in sterile saline and vortexed gently to thoroughly mix it. The two

supernatants (from the primary and secondary concentration steps) and the finalreconstituted pellet were plated in duplicate for recovery on BHI agar after serialdilution. Total bacterial recoveries after immobilization were calculated based onloss to the supernatants (see the formula above). In instances where recovery wasbased on direct plating of the pellet obtained from bacterial immobilization, itwas calculated using the following formula: [percent immobilization 5 (totalpopulation in pellet after immobilization) 3 100/(total population in samplebefore immobilization)]. Control samples consisted of inoculated NFDM sam-ples treated for bacterial concentration using the above scheme but without theaddition of metal hydroxide. Duplicate and parallel samples were extracted forRNA isolation and subsequent nucleic acid amplification by RT-PCR. All ex-periments were done in triplicate. The entire concentration scheme is outlined inFig. 1.

To demonstrate the applicability of the method to other dairy commodities,900-ml samples of pasteurized whole milk or melted vanilla ice cream were

FIG. 1. Flow chart of bacterial-immobilization method. For NFDM, wholemilk, and ice cream, the immobilization efficiencies of L. monocytogenes andserovar Enteritidis were evaluated with zirconium hydroxide. For specificitystudies, zirconium, titanous, and hafnium hydroxides were evaluated for theconcentrations of E. coli, E. coli O157:H7, serovar Enteritidis, P. aeruginosa, L.monocytogenes, Lactobacillus spp., B. cereus, and L. lactis.

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seeded with 100 ml of serial dilutions of an overnight culture of serovar Enter-itidis or L. monocytogenes to achieve final concentrations of 104, 103, 102, or 101

CFU/ml of milk or ice cream. These samples were subjected to the bacterial-immobilization scheme outlined in Fig. 1 except that the volumes were adjustedto account for the reduced sample size. As with the NFDM model, the twosupernatants (from the primary and secondary concentration steps) and the finalreconstituted pellet were plated for recovery and parallel samples were extractedfor RNA isolation and subsequent nucleic acid amplification by RT-PCR.

(iii) Immobilization of other food-borne bacteria. In order to evaluate thespecificity of metal hydroxide adsorption for the recovery of various gram-positive and gram-negative bacteria of significance to foods, overnight cultures ofrepresentative food-borne bacteria (serovar Enteritidis, L. monocytogenes ScottA, E. coli O157:H7, E. coli ATCC 25922, Lactobacillus sp. strain ATCC 4356, L.lactis subsp. lactis NCK 203, B. cereus NCK 143 and P. aeruginosa ATCC 25922)were serially diluted in sterile growth media to approximately 106 CFU/ml.One-milliliter volumes were mixed gently by vortexing and subjected to bacterialimmobilization according to the scheme outlined in Fig. 1 except that volumeswere adjusted to account for the reduced sample sizes. Zirconium hydroxide,titanous hydroxide, and the heavier hafnium hydroxide were evaluated in thesestudies. Due to the large size of the hafnium hydroxide complex, centrifugationspeeds were reduced to 300 3 g for the bacterial-immobilization experimentsusing that agent. Bacterial loss to the supernatant and recovery in precipitateswere calculated as described above. Tubes containing bacterial suspensions butno metal hydroxide (control) were treated in the same manner to evaluate theefficacy of centrifugation alone.

(iv) Viability of immobilized cells. To confirm that bacterial cells remainedviable after exposure to metal hydroxide solutions, 1-ml volumes of overnightcultures of serovar Enteritidis and L. monocytogenes, serially diluted 100-fold insterile saline solution, were seeded with 2 ml of zirconium hydroxide and platedon BHI agar at 0, 2, 6, 12, 24, and 48 h. Control tubes consisted of the dilutedovernight cultures without the addition of zirconium hydroxide. Plate counts fortreated and untreated samples were compared at each time point to evaluate cellsurvival in the presence of zirconium hydroxide.

(v) Statistical analyses. When statistical comparisons were necessary, analysisof variance and the Tukey-Kramer multiple comparisons test, or Student’s t test,were done on the log-transformed bacterial population or percent recovery datausing the InStat 2 statistical analysis package (GraphPad Software, San Diego,Calif.).

Molecular biological methods. (i) RNA extraction. Total RNA in the recon-stituted metal hydroxide pellets was extracted directly with TRI reagent BD(Molecular Research Center, Inc., Cincinnati, Ohio) using the method describedby the manufacturer for extraction of RNA from whole blood. Briefly, cells in themetal hydroxide pellet were lysed by the addition of 0.75 ml of TRI reagent BDsupplemented with 20 ml of 5 N acetic acid. The lysed samples were extractedwith 0.2 ml of chloroform with vigorous vortexing for 15 s and spun at 10,000 3g for 15 min at 4°C. The aqueous phase was supplemented with 5 ml of a10-mg/ml solution of yeast tRNA (Sigma Chemical Co., St. Louis, Mo.) to act asa carrier, and the RNA was precipitated in 0.5 ml of isopropanol for 5 to 10 minat room temperature followed by centrifugation at 10,000 3 g for 8 min at 4°C.The RNA pellet was washed in 75% ethanol and centrifuged again at 7,500 3 gfor 5 min at 4°C. The precipitated RNA was air dried for 5 min to removeresidual alcohol, resuspended in 30 ml of sterile diethyl pyrocarbonate-treatedwater, and used directly in RT-PCR amplifications.

(ii) RT-PCR primers and oligoprobes. The oligonucleotide primer and probesequences for L. monocytogenes and serovar Enteritidis have been previouslydescribed (12, 30, 31) and corresponded to unique 16S rRNA sequences for eachorganism. All primers and probes for PCR were synthesized by GenoSys (TheWoodlands, Tex.). Primers for Salmonella were based upon two regions ofsequence variability between the V3 and V5 regions of the 16S rRNA specific toSalmonella (12) (59 primer, 59-TGTTGTGGTTAATAACCGCA-39; 39 primer,59-CACAAATCCATCTCTGGA-39) and generated a 575-bp amplicon. L.monocytogenes primers were based on region 1228 to 1297 of the 16S rRNA (30,31) (primer L1, 59-CACGTGCTACAATGGATAG-39; primer L2, 59-AGAATAGTTTTATGGGATTAG-39) and selectively amplified a 70-bp region specificfor L. monocytogenes to the exclusion of other Listeria species. The internaloligoprobe (RLM3; 59-GTCGCGAAGCCGCGAGGT-39) for the confirmationof L. monocytogenes amplicons by Southern hybridization was also previouslyreported (30, 31).

(iii) RT-PCR. RT-PCRs were done with the Gene-Amp kit (Roche MolecularSystems, Branchburg, N.J.) according to the manufacturer’s instructions exceptthat the volumes for RT were increased from 20 to 30 ml to accommodate a 10-mlsample size and Ampli-Taq Gold (Applied Biosystems, Foster City, Calif.) wasused as the source of thermostable DNA polymerase. RT was done at 42°C for1 h with random hexamer primers, and then the tubes were heated to 99°C for5 min to inactivate the enzyme. After being chilled on ice, the tubes weresupplemented with 2.5 U of AmpliTaq Gold and 100 ng of each primer asappropriate. PCR amplification was done in a DNA thermal cycler (Perkin-Elmer, Norwalk, Conn.). For L. monocytogenes, amplification was performed aspreviously reported (30) and consisted of one cycle at 90°C for 3 min followed by40 cycles of 95°C for 20 s, 48°C for 20 s, 73°C for 40 s, and a final extension at73°C for 3 min. PCR amplification for serovar Enteritidis was also performed aspreviously reported (12) and consisted of one cycle of 90°C for 10 min followed

by 40 cycles of 95°C for 1.5 min, 54°C for 1.5 min, and 72°C for 1.5 min and a finalextension cycle of 73°C for 3 min. A 10- to 15-ml portion of RT-PCR product,which represented 10 to 15% of the total reaction volume, was analyzed by gelelectrophoresis on 2% (serovar Enteritidis) or 4% (L. monocytogenes) agarose,stained with ethidium bromide, and visualized by UV light. In some experiments,the inhibitory effect of food components was evaluated by serial dilution ofsample concentrates (i.e., pellets after primary and secondary concentrationsteps) and the addition of a constant amount of bacterial RNA immediatelyfollowed by nucleic acid amplification by RT-PCR.

(iv) Oligoprobe hybridization. RT-PCR products from L. monocytogenes am-plification were transferred to nylon membranes using the method of Southern(26). The DNA was bound by cross-linking with shortwave UV light (UltravioletProducts, Inc., San Gabriel, Calif.) for 3 to 5 min at a distance of 15 cm. Theoligoprobes for target amplicons were 39-end labeled with digoxigenin-dUTPusing terminal transferase, purified by ethanol precipitation, and used in hybrid-ization reactions according to the instructions in the Genius nonradioactiveend-labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.).Briefly, membranes were prehybridized for 4 h and hybridized overnight at 55°Cwith the recommended hybridization solutions (53 SSC [13 SSC is 0.15 M NaClplus 0.015 M sodium citrate], 0.1% [wt/vol] N-laurylsarcosine, 0.02% [wt/vol]sodium dodecyl sulfate, and 1% [wt/vol] Boehringer Mannheim Biochemical’sproprietary blocking reagent) containing labeled probe at a concentration of 5 to10 ng per ml of hybridization solution. The blots were washed five times in 63SSC–0.05% pyrophosphate at 55°C. Immunological detection of RT-PCR pro-duct–oligoprobe hybrids was performed using an anti-digoxigenin–alkaline phos-phatase antibody conjugate and enzyme-catalyzed colorimetric reaction with5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium salt as sub-strates.

(v) LCR. The identities of Salmonella RT-PCR amplicons were confirmed bythe ligase chain reaction (LCR) (23). A set of four primers (SEA1, SEA2, SEA3,and SEA4) were designed to specifically detect a single-base-pair differencewithin the Salmonella PCR amplicon. Primers with the following sequences weresynthesized by GenoSys: SEA1, 59-TAGTCCACGCCGTAAACGATGTCT-39;SEA2, 59-ACTTGGAGGTTGTGCCCTTGAG-39; SEA3, 59-GACATCGTTTACGGCGTGGACTA-39; SEA4, 59-CTCAAGGGCACAACCTCCAAGTA-39.

SEA2 and SEA3 were 59 phosphorylated during manufacture; SEA1 wassynthesized with a universal primer sequence (59-TGGCACTGGCCGTCGTTTTAC-39), designated UP, at the 59 end, and SEA4 was biotinylated duringmanufacture. LCR was performed with the LCR kit (Stratagene, La Jolla, Calif.)according to the manufacturer’s instructions. Briefly, 1 ml of RT-PCR productwas added to 20 ml of LCR mixture (2 ml of 103 LCR buffer, 20 ng of LCRprimers, 4 U of Pfu ligase, and 15 ml of sterile, diethyl pyrocarbonate-treatedwater) and incubated at 94°C for 5 min (denaturation) and 60°C for 4 min(annealing). The reaction mixtures were then cycled 25 times at 95°C for 1 minand 60°C for 2 min. LCR products were detected with the AmpliTek DetectionModule (Bio-Rad, Hercules, Calif.) according to the manufacturer’s instructions.In this method, LCR products diluted 1:10 or 1:50 in 13 SSC were incubated for1 h at 37°C in streptavidin-coated microtiter plate wells. The wells were washedfive times with 13 proprietary wash buffer and then incubated for 1 h at 37°Cwith alkaline phosphatase conjugated to an oligonucleotide sequence compli-mentary to the UP sequence of the SEA1 primer. The wells were again washedfive times to remove unbound enzyme and then incubated for 1 h at 37°C with theNADPH substrate. Addition of a second substrate activated a secondary enzymesystem, creating a redox cycle which produced a molecule of colored formazan.The reaction products were detected colorimetrically by endpoint determinationat 490 nm.

RESULTS

Initial immobilization studies. Initial bacterial-immobiliza-tion studies were done using diluted overnight cultures of L.monocytogenes and serovar Enteritidis. Small sample volumesof 200 ml were concentrated fourfold to 50 ml. While immobi-lization efficiency was partially dependent upon the initial con-centration of bacterial cells and the concentration of metalhydroxide (data not shown), once these parameters were op-timized, consistently high recoveries could be achieved. Per-cent recovery data for L. monocytogenes and serovar Enteriti-dis immobilized by titanous or zirconium hydroxide were basedon loss to the supernatant (Table 1) (8). Immobilization effi-ciencies ranged from 95 to 99%, and there were no statisticallysignificant differences between the metal hydroxides, the or-ganisms, or the initial inoculum levels when evaluated by theTukey-Kramer pairwise statistical comparison (P . 0.05). Theimmobilized cells could be enumerated by direct plating of thereconstituted pellet on BHI agar (data not shown).

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Bacterial recovery and detection in the NFDM model. Re-constituted NFDM was used as a model to investigate theefficiency of bacterial immobilization in foods. The entire sam-ple concentration scheme was applied to 25-ml samples andincluded the sequential steps of clarification and high-speedcentrifugation (designated primary concentration) followed byimmobilization with metal hydroxides and low-speed centrifu-gation (designated secondary concentration) (Fig. 1). Thecombined steps of primary and secondary concentration re-sulted in an overall 50-fold sample volume reduction from 25ml to 500 ml. By following the bacterial concentration with anRNA extraction step, a final sample concentrate of 20 to 30 mlcould be obtained, representing an additional 20-fold concen-tration factor.

To evaluate the effect of food-related RT-PCR inhibition,reconstituted NFDM samples at various points in the concen-tration process were serially diluted and seeded with RNAcorresponding to approximately 107 CFU of L. monocytogenesjust prior to RT reactions. The ability to obtain detectableamplicons at various dilutions by RT-PCR was indicative of thedegree of sample inhibition. These RT-PCR compatibilitystudies indicated minimal PCR inhibition (10-fold dilution ofthe sample was required for positive amplification) from un-treated NFDM and NFDM treated by primary concentrationalone. There was little to no inhibition after bacterial immo-

bilization with titanous or zirconium hydroxide, as evidencedby the ability to amplify target rRNA sequences without priorsample dilution (data not shown).

To evaluate both bacterial recovery and RT-PCR detectionin this model system, 25-ml samples of reconstituted NFDMwere seeded with a 1-ml aliquot of 104, 103, 102, or 101 CFU ofan overnight culture of L. monocytogenes or serovar Enteritidisand processed by the combined clarification, centrifugation,and bacterial-immobilization procedure. Recoveries were as-sessed in parallel by both bacteriological plating (supernatantsafter primary and secondary concentration and the final im-mobilized pellet) and RT-PCR amplification. Plating of theuninoculated NFDM samples revealed background microfloralevels of ,101 CFU/ml. Total bacterial recoveries after theprimary concentration steps were 85 to 98 and 78 to 98% forserovar Enteritidis and L. monocytogenes, respectively (Table2). The secondary concentration step, which employed bacte-rial immobilization, resulted in efficient recovery of 51 to 96%of inoculated serovar Enteritidis and 95 to 98% of inoculatedL. monocytogenes. The immobilized cells were apparently via-ble and could be enumerated by direct plating of the reconsti-tuted pellet. These recoveries ranged from 65 to 95% forserovar Enteritidis and 78 to 96% for L. monocytogenes (Table2). Low-speed centrifugation without prior immobilization wasextremely inefficient and variable, resulting in recoveries of,36% (data not shown). Total RNA from the immobilizationsample concentrates (500 ml) was extracted to a final volume of25 ml, representing an overall 1,000-fold sample volume reduc-tion. When subjected to nucleic acid amplification by RT-PCR,direct detection of both serovar Enteritidis and L. monocyto-genes was consistently possible at initial bacterial input levels of102 CFU/25 ml and higher cell concentrations in reconstitutedNFDM (Fig. 2). At low input levels of 101 CFU/25 ml ofNFDM, RT-PCR detection was possible for serovar Enteritidisin one out of three samples and in two out of three samples forL. monocytogenes. The identities of RT-PCR amplicons wereconfirmed by internal oligoprobe hybridization (L. monocyto-genes) or LCR (serovar Enteritidis).

Specificity of bacterial concentration. The specificity ofmetal hydroxide adsorption for the concentration of variousgram-positive and gram-negative bacteria of significance tofoods was evaluated using eight representative bacterial strainsin pure culture. Percent recovery was based on both loss to thesupernatant and recovery from direct plating of the precipitate.

TABLE 1. Immobilization of L. monocytogenes and serovarEnteritidis in pure culture using titanous hydroxide

and zirconium hydroxide

Organism Inoculum(CFU/ml)

Avg % immobilizationa

Titanoushydroxide

Zirconiumhydroxide

L. monocytogenes 107 98 6 2 98 6 2105 98 6 3 97 6 2103 95 6 3 98 6 2

Serovar Enteritidis 107 98 6 1 97 6 2105 98 6 1 98 6 1103 98 6 1 99 6 1

a Average percent immobilization (mean 6 standard deviation) of three rep-licate samples; calculated as previously reported (8): [percent immobilization 5(total population in sample before immobilization 2 total population in super-natant after immobilization) 3 100/(total population in sample before immobi-lization)].

TABLE 2. Recovery and detection of serovar Enteritidis and L. monocytogenes in 25-ml reconstituted NFDM samples after bacterialimmobilization with zirconium hydroxide and subsequent RT-PCR

Inoculuma

(CFU/25 ml)

Serovar Enteritidis L. monocytogenes

Avg % recoverybDetection

(PCR/LCR)c

Avg % recoverybDetection

(PCR/Hyb)c1o concn 2o concn Ppt 1o concn 2o concn Ppt

104 97 6 2x 96 6 3xy 69 6 4z 3/3 98 6 4x 98 6 1x 96 6 1x 3/3103 85 6 3y 95 6 3yz 85 6 3y 3/3 96 6 1x 96 6 1x 92 6 1y 3/3102 98 6 2x 96 6 1xy 95 6 1x 3/3 89 6 2y 97 6 2x 89 6 9xy 3/3101 94 6 6xy 51 6 41z 65 6 16yz 1/3 78 6 10y 95 6 5x 78 6 12y 2/3

a Total CFU at input is taken as 100%; input level confirmed by plating samples before bacterial concentration.b Average percent recovery (mean 6 standard deviation) of three replicate samples; for primary concentration (1o concn) and secondary concentration (2o concn),

percent recovery was calculated as previously reported (8): [percent recovery 5 (total population in sample before concentration 2 total population in supernatant after1o concentration or after 2o concentration {i.e., immobilization}) 3 100/(total population in sample before concentration)]. For the immobilized pellet (Ppt), percentrecovery was calculated as follows: [percent recovery 5 (total population in pellet after immobilization {i.e., after 1o and 2o concentrations} 3 100/(total populationin sample before concentration)]. Different superscript letters (x, y, and z) identify statistically significant differences (P # 0.05) in percent recovery at different inputlevels of each organism. Boldface data identify statistically significant differences (P # 0.05) between percent recovery values when calculations based on loss to thesupernatant versus direct plating of the immobilized pellet were compared.

c Detection was done by agarose gel electrophoresis of PCR products and subsequent confirmation by DNA hybridization (PCR/Hyb for L. monocytogenes) or LCR(PCR/LCR for serovar Enteritidis).

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Bacterial recoveries based on loss to the supernatant were 88to 100% for titanous hydroxide, 89 to 98% for zirconium hy-droxide, and 70 to 97% for hafnium hydroxide (data notshown). Standard deviations for the loss-to-supernatant recov-eries were generally small, and statistically significant differ-ences (P # 0.05) in recoveries were seen with only one organ-

ism (E. coli) for the hafnium hydroxide treatment. In all cases,the efficacy of centrifugation alone was highly erratic.

When calculations were based on direct plating of the pre-cipitate, recoveries ranged from 25 to 143% for titanous hy-droxide, 58 to 135% for zirconium hydroxide, and 25 to 102%for hafnium hydroxide (Fig. 3). Standard deviations for theserecoveries were much higher than when recovery was calcu-lated by loss to the supernatant. Statistically significant differ-ences (P # 0.05) between organisms were apparent for tita-nous and hafnium hydroxides, whereas zirconium hydroxiderecoveries were reasonably consistent (P . 0.05) regardless ofthe organism tested. As with the loss-to-supernatant data, thebacterial recoveries from pellets obtained after centrifugationalone were inconsistent and highly organism dependent. Sta-tistical comparison among metal hydroxides revealed that,while all metal hydroxides were capable of removing all of theorganisms to varying degrees, in almost all cases, zirconiumhydroxide performed better than titanous and hafnium hydrox-ides in providing equal or better recoveries by direct plating ofthe precipitate. Furthermore, subsequent experiments indi-cated that hafnium hydroxide was inhibitory to RT-PCRs (datanot shown). Because of its consistent performance in bacterialimmobilization, as well as its compatibility with enzymatic nu-cleic acid amplification, zirconium hydroxide was the only im-mobilization agent chosen for subsequent studies.

Viability of immobilized cells. To confirm that bacterial cellsremained viable after exposure to metal hydroxide solutions,serially diluted overnight cultures of serovar Enteritidis andL. monocytogenes were seeded with zirconium hydroxide and

FIG. 2. Detection of L. monocytogenes and serovar Enteritidis in artificiallycontaminated reconstituted NFDM after bacterial immobilization with zirco-nium hydroxide, RNA extraction, and RT-PCR. Reconstituted NFDM (25-ml)samples were inoculated with 104, 103, 102, or 101 CFU of L. monocytogenes (A)or serovar Enteritidis (B) and processed for bacterial concentration followed byRNA isolation and RT-PCR amplification. The corresponding initial inoculumlevel (CFU/25 ml of reconstituted NFDM) is given above each gel lane. Southernhybridization results (L. monocytogenes) and LCR absorbance readings (serovarEnteritidis) for confirmation of RT-PCR amplicons are displayed below eachamplicon. Lanes: M, marker; C, uninoculated 25-ml reconstituted-NFDM sam-ple processed for bacterial concentration; 2, complete RT-PCR cocktail withoutsample (i.e., water); 1, positive control reaction for amplification (i.e., RNAextracted from approximately 106 CFU of L. monocytogenes or serovar Enterit-idis in pure culture).

FIG. 3. Percent recovery of selected food-borne bacteria by metal hydroxide immobilization as calculated by direct plating of the immobilized pellet. The error barsrepresent standard deviations. Control 1 corresponds to centrifugation alone at 500 3 g, while control 2 corresponds to centrifugation alone at 300 3 g. Specificfood-borne microorganisms tested include the following: E. coli, E. coli O157:H7, serovar Enteritidis (S.E.), P. (Ps.) aeruginosa, L. monocytogenes (L. mono),Lactobacillus, B. cereus, and L. lactis.

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plated for recovery at 0, 2, 6, 12, 24, and 48 h. Control tubesconsisted of the diluted overnight cultures without the additionof zirconium hydroxide. Subsequent analysis indicated no sta-tistically significant increase or reduction (P . 0.05) in totalbacterial population over time for either the control or treat-ment samples for both serovar Enteritidis and L. monocyto-genes. Furthermore, there was no significant difference (P .0.05) between log-transformed counts for the control and hy-droxide treatments at any one time point for either organism.

Bacterial recovery and detection in whole milk and icecream. To evaluate the bacterial-concentration scheme inmore complex dairy products, 900-ml samples of milk and icecream were seeded with serial dilutions of L. monocytogenes orserovar Enteritidis to achieve a final concentration of 104, 103,102, or 101 CFU/ml of milk or ice cream. These samples werethen processed by the combined clarification, centrifugation,and bacterial immobilization procedure (Fig. 1). Recoverieswere evaluated based on loss to the supernatant (after second-ary concentration) and direct plating of the immobilized cellsusing standard cultural procedures. Plating of the uninoculatedmilk and ice cream samples revealed background microfloralevels of ,101 CFU/ml (ice cream) and ,102 CFU/ml (wholemilk). Total bacterial recoveries after immobilization in milk,as calculated by loss to the supernatant, were 96 to 97 and 92to 96% for serovar Enteritidis and L. monocytogenes, respec-tively (Table 3). Recoveries from ice cream were 71 to 95 and61 to 91% for serovar Enteritidis and L. monocytogenes, re-spectively (Table 3). Recoveries based on loss to the superna-tant were extremely consistent both among organisms andamong different inoculum levels for whole milk. In the case ofice cream, these recoveries were less consistent, yet there wasno notable reduction in recovery efficiency at lower inoculumlevels. Recovery efficiencies were essentially equal for L.monocytogenes and serovar Enteritidis. The immobilized cellscould be enumerated by direct plating of the reconstitutedpellet, and recoveries in milk ranged from 77 to 107% forserovar Enteritidis and 93 to 122% for L. monocytogenes (Ta-ble 3). Recoveries for ice cream were 64 to 79 and 71 to 161%for serovar Enteritidis and L. monocytogenes, respectively (Ta-ble 3). Low-speed centrifugation without prior immobilization

was extremely inefficient, resulting in .50% recovery of theinput bacteria (data not shown). Total RNA from the immo-bilization sample precipitates was extracted to a final volume of10 ml, representing an overall 100-fold sample volume reduc-tion. When subjected to nucleic acid amplification using RT-PCR, direct detection (without sample dilution) of both sero-var Enteritidis and L. monocytogenes was possible at initialbacterial input levels of $102 CFU/ml for whole milk and atinput levels of $101 CFU/ml for ice cream (Fig. 4).

DISCUSSION

Despite their apparent promise as effective bacterial con-centration agents in clinical, environmental, and food samples(8, 9), little is known about the exact chemical mechanism(s)whereby metal hydroxides are able to effectively immobilizebacterial cells. Kennedy et al. (10) reported that the dissolutionof zirconium(IV) chloride in water results in a cation thatpolymerizes into a tetrameric complex, [Zr4(OH)8(H2O)16]81.With increasing pH, the complex is able to polymerize further,resulting in multiple tetrameric complexes in which the zirco-nium ions are connected by hydroxide bridges. The resultinggelatinous precipitate (zirconium hydroxide) has been used toimmobilize enzymes, most likely due to the formation of par-tial covalent bonds between the hydroxyl groups of the metalhydroxide and suitable amino acid side chains. Since thesesame ligands and many others are plentiful on the surface ofthe bacterial cell, it is likely that metal hydroxide complexesimmobilize bacterial cells by the formation of partial covalentbonds with ligands on the surface of the bacterial cell wall (10).

Previous investigators have demonstrated that bacterial cellsimmobilized on metal hydroxides remain viable. For instance,Kennedy et al. (10) demonstrated that both prokaryotic (E.coli) and eukaryotic (Saccharomyces cerevisiae) cells continuedto respire when immobilized by titanous or zirconium hydrox-ide. Using a related compound, Berry and Siragusa (3) re-ported that E. coli cells immobilized by hydroxyapatite re-mained viable as evaluated by fluorescent-staining techniques.Our results are consistent with these reports, demonstratingthat representative gram-negative and gram-positive bacterial

TABLE 3. Recovery and detection of serovar Enteritidis and L. monocytogenes in 1-ml samples of whole milk or ice cream after bacterialimmobilization with zirconium hydroxide and subsequent RT-PCR

Organism

Whole milk Ice cream

Inoculuma

(CFU/ml)

Avg % recoveryb

Detection(PCR/confirm)c

Inoculuma

(CFU/ml)

Avg % recoveryb

Detection(PCR/confirm)c2o

concn Ppt 2o concn Ppt

L. monocytogenes 104 96 6 1x 114 6 5x 3/3 104 91 6 1x 104 6 23y 3/3103 92 6 1y 122 6 12x 3/3 103 67 6 1y 78 6 7y 3/3102 92 6 1y 100 6 8x 3/3 102 61 6 3y 71 6 7y 3/3101 95 6 4x 93 6 20x 0/3 101 84 6 13x 161 6 11x 2/3

Serovar Enteritidis 104 97 6 2x 83 6 7x 3/3 104 95 6 1x 69 6 8x 3/3103 96 6 1x 90 6 19xy 3/3 103 89 6 1y 65 6 2x 3/3102 97 6 2x 77 6 11x 3/3 102 87 6 1z 64 6 4x 1/3101 96 6 5x 107 6 1y 0/3 101 71 6 14w 79 6 14x 2/3

a Total CFU at input is taken as 100%; input level confirmed by plating samples before bacterial concentration.b Average percent recovery (mean 6 standard deviation) of three replicate samples; for secondary concentration (2o concn), percent recovery was calculated as

previously reported (8): [percent recovery 5 (total population in sample before concentration 2 total population in supernatant after immobilization {i.e., after 1o and2o concentration} 3 100/(total population in sample before concentration)]. For the immobilized pellet (Ppt), percent recovery was calculated as follows: [percentrecovery 5 (total population in pellet after immobilization {i.e., after 1o and 2o concentrations} 3 100/(total population in sample before concentration)]. Differentsuperscript letters (w, x, y, and z) identify statistically significant differences (P # 0.05) in percent recovery at different input levels of each organism. Boldface dataidentify statistically significant differences (P # 0.05) between percent recovery values when calculations based on loss to supernatant versus direct plating of theimmobilized pellet were compared.

c Detection was done by agarose gel electrophoresis of PCR products and subsequent confirmation (PCR/confirm) by DNA hybridization (L. monocytogenes) or LCR(serovar Enteritidis).

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species remained viable after exposure to zirconium hydroxidefor up to 48 h.

The observation that bacterial immobilization with metalhydroxides is nonspecific is also consistent with the reports ofother investigators (3, 8, 9, 10). As with our data, these inves-tigators failed to observe a significant effect on immobilizationefficiency associated with the Gram reaction or cell morphol-ogy, frequently reporting bacterial recoveries exceeding 80 to90% for a variety of gram-positive and gram-negative bacteria(3, 8, 9). All of these previous studies have assessed recoveriesbased on loss to the supernatant, and from this perspective, therecoveries reported in our study are equal to or better thanthose reported in previous papers. However, our study alsodemonstrates that viable cells can be recovered and enumer-ated directly from precipitates collected after metal hydroxideimmobilization and low-speed centrifugation. In cases whererecoveries based on direct plating of the immobilized pelletwere on the low side, particularly when compared to calcula-tions based on loss to the supernatant, we can hypothesize thatbacterial adherence to tubes or bacterial clumping during cen-trifugation may be at least partially responsible. In instanceswhere the bacterial recovery obtained by direct plating of theprecipitated pellet was in excess of 100%, we suspect that thesimultaneous immobilization of the indigenous microflora maybe responsible. Efforts are under way to ascertain the cause ofthese variations in recovery and to further optimize metalhydroxide adsorption to effectively reduce sample volumes yetstill maintain cellular viability, and hence detectability by cul-tural, immunological, and nucleic acid-based methodologies.

This study demonstrates that metal hydroxide immobiliza-tion may be a highly feasible approach to bacterial concentra-tion in foods and one that offers significant advantages overother approaches currently under investigation. Although themost widely used bacterial-concentration method is centrifu-gation (5, 15, 17, 28), high centrifugal forces are generallynecessary to effectively sediment cells. The use of metal hy-droxides prior to centrifugation, as demonstrated in this work,enabled a reduction in centrifugal force, presumably due to anincrease in the total mass of the bacterium-hydroxide complexas opposed to the uncomplexed bacterial cells. This reduction

in centrifugation speed most likely reduces the tendency tocoprecipitate PCR inhibitors and may also aid in maintainingcellular viability, facilitating direct detection of the bacteria inthe precipitated floc by standard cultural procedures. Further-more, the adsorption of bacteria to the surfaces of the metalhydroxides allows effective removal without the need for sub-sequent elution steps, since the immobilized cells remain viableand can be enumerated by culturing. The metal hydroxidemethod also circumvents problems that may be associated withfiltration, such as clogged filters and simultaneous concentra-tion of inhibitory compounds (18) or immunomagnetic sepa-ration, which is organism specific and expensive and requiressmall sample sizes (16, 25).

We are cautiously optimistic that the metal hydroxide im-mobilization procedure will also be adaptable to more complexdairy products. Recognizing that the reconstituted NFDM sys-tem is “ideal” with respect to minimizing PCR inhibitors andcontrol of product composition, we are encouraged by thereasonably good recoveries and detection limits demonstrated.The method clearly worked for whole milk and ice cream,having achieved final RT-PCR detection levels similar to thosefor NFDM. It is important to note that the present study didnot address the volume reductions necessary for practical ap-plication of the method to larger food sample sizes ($25 ml org) of these more complex dairy products. As we continue ourefforts in this area, it is becoming increasingly clear that samplepretreatments to simultaneously remove PCR inhibitors andmaintain bacterial cell viability will remain necessary (24, 33).

The detection limits reported in this work are equal to if notbetter than those reported by other investigators. When sur-veying the literature on PCR-based detection of food-bornepathogens, we found that detection limits rarely exceed 102 to103 CFU/ml or g in samples that have not previously under-gone culture enrichment (1, 27, 33). Detection limits of #1CFU/ml or g have been reported after 8 to 48 h of cultureenrichment (1, 27, 33). Only a few investigators have examinedmore than one form of bacterial concentration, applications tolarger sample volumes, or elimination of enrichment steps (6,7, 30). Most recently, Herman et al. (6, 7) reported a chemicalextraction method that enabled the detection of as little as 1

FIG. 4. Detection of L. monocytogenes and serovar Enteritidis in artificially contaminated whole milk or ice cream after bacterial immobilization with zirconiumhydroxide, RNA extraction, and RT-PCR. One-milliliter samples of whole milk (A) or ice cream (B) were inoculated with 104, 103, 102, or 101 CFU of L. monocytogenes(LM; bottom) or serovar Enteritidis (SE; top) and processed for bacterial concentration followed by RNA isolation and subsequent RT-PCR amplification. Thecorresponding initial inoculum level (CFU/1 ml of whole milk or ice cream) is given above each gel lane. Southern hybridization results (L. monocytogenes) and LCRabsorbance readings (serovar Enteritidis) for confirmation of RT-PCR amplicons are displayed below each amplicon. Lanes: M, marker; C, 1-ml samples ofuninoculated whole milk or ice cream processed for bacterial concentration; 2, complete RT-PCR cocktail without sample (i.e., water); 1, positive control reactionfor amplification (i.e., RNA extracted from approximately 106 CFU of L. monocytogenes or serovar Enteritidis in pure culture).

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CFU of L. monocytogenes cells or Clostridium tyrobutyricumspores from 25 ml of raw milk using a nested PCR approach.While our detection limits are not quite as good as those ofHerman et al. (6, 7), we have purposely avoided nested PCRdue to the potential for cross contamination, which increasesthe probability of false-positive results. Alternatively, we usedan rRNA target for the nucleic acid amplification because ofthe high copy number of this molecule in viable cells (32). Itwas also chosen because of its relative instability, compared toDNA, when used as the target for nucleic acid amplification infood systems, thus reducing the likelihood of detecting deadcells (13, 14). Further work to refine the rRNA-based RT-PCRassay, specifically with respect to the detection of viable cells(14) should improve the overall detection limits of the assay,allowing sensitive detection of bacterial contamination withoutthe need for prior culture enrichment. Such developmentalefforts are under way in our laboratory.

Three major issues remain when considering the successfuladaptation of PCR to the detection of food-borne pathogens,i.e., reduction of sample size, concentration of viable bacteria,and removal of PCR inhibitors. Immobilization of food-bornebacteria by metal hydroxides effectively addresses all of theseissues. The method, when applied to 25-ml samples of recon-stituted NFDM, resulted in a 50-fold sample concentrationfactor and recovery of 65 to 96% of the input bacteria. Whencoupled with an RNA extraction step, the final sample volumewas reduced 1,000-fold, and the resulting RT-PCR detectionlimits for both L. monocytogenes and serovar Enteritidis wereapproximately 102 CFU/25 ml without prior culture enrich-ment. The immobilization step is rapid (,1 h), inexpensive($0.50/sample), and simple, requiring no sophisticated equip-ment or personnel training. Since it is nonspecific and appar-ently results in the recovery of viable cells, it may also beapplicable to sample preparation prior to the use of otherrapid-detection methodologies, such as enzyme-linked immu-nosorbent assay. Furthermore, the use of bacterial concentra-tion prior to conventional cultural procedures, such as enrich-ment or plating, may facilitate improved detection limits andreduced medium costs. We hope to explore some of theseissues in future studies.

ACKNOWLEDGMENTS

This work was funded by the Southeast Dairy Foods Research Cen-ter, Dairy Management, Inc.; the North Carolina Dairy Foundation;the North Carolina Agricultural Foundation; and the North CarolinaAgricultural Research Service.

We gratefully acknowledge Danielle Robins Datz for her help in thepreparation of the manuscript.

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