APPLIED

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ENVIRONMENTAL MICROBIOLOGY, Feb. 1992,

p.

Vol. 58, No. 2

709-712

0099-2240/92/020709-04$02.00/0 Copyright

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1992, American Society for Microbiology

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by Pulsed-Field Gel Electrophoresis PATRICIA J. HOWARD, KARTIKA D. HARSONO, AND JOHN B. LUCHANSKY* Department of Food Microbiology and Toxicology, Food Research Institute, 1925 Willow Drive, University of Wisconsin, Madison, Wisconsin 53706-1187 Received 27 September 1991/Accepted 20 November 1991 Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed speciesand serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively. TBE (10) buffer were mixed, and approximately 230 ,ul of this suspension was added to each well of a 10-sample acrylic mold (Bio-Rad) to form agarose plugs. Intact, high-molecular-weight genomic DNA was prepared from listeria cells embedded in agarose plugs essentially as described previously (14). Each plug was cut into about five inserts, and each insert was placed in a sterile microcentrifuge tube for restriction endonuclease digestion of embedded DNA as described by the enzyme manufacturer (Promega Corp., Madison, Wis.). Depending on the enzyme, 3 to 30 U (1 to 1.3 ,ul) of enzyme was added per insert. Following digestion for at least 16 h at the appropriate temperature with gentle shaking, 25 pLI of 0.5 M EDTA (pH 8) was added to stop the reaction. A CHEF-DR II (BioRad) pulsed-field system and either electrophoresis-grade (GIBCO BRL, Life Technologies, Inc., Gaithersburg, Md.) or chromosomal-grade (Bio-Rad) agarose were used to resolve high-molecular-weight restriction fragments. CHEF analysis of different Listeria species and serogroups. In preliminary experiments, the enzymes SacII, NarI, Sfil, EagI, NaeI, SacI, NcoI, BamHI, ClaI, SalI, and XbaI were evaluated without success (e.g., too many bands and/or ambiguous restriction profiles). In contrast, ApaI, AscI, NotI, and SmaI were most useful for the separation of megabase-sized restriction fragments from strains representing different Listeria species. Although several AscI fragments migrated the same relative distance for all species tested (Fig. 1), as expected, unique genomic fingerprints were discernible for L. innocua (10 AscI fragments), L. seeligeri (9 AscI fragments), L. ivanovii (8 AscI fragments), and L. monocytogenes (7 AscI fragments). Similarly, genomic DNA was prepared from L. monocytogenes strains representing serogroups 1, 3, and 4 for CHEF analysis. A few bands of the same length were common among all strains, but the overall migration patterns of AscI digests varied among strains of different serogroups and among strains within the same serogroup (data not shown). Comparison of L. monocytogenes serotype 4b strains by CHEF analysis. Genomic DNA extracted from serotype 4b strains digested with AscI generated seven fragments for three of seven strains evaluated (Fig. 2, lanes B, C, and H) and eight fragments for the remaining four of seven strains tested (Fig. 2, lanes D to G). Although several bands were common (i.e., migrated the same relative distance) in the genomic fingerprints, there were differences among restriction profiles, most notably between the patient (JBL1231)

During the past decade, Listeria monocytogenes has been incriminated in a number of food-borne disease outbreaks and several sporadic episodes of listeric illness (8). Several methods, including serotyping, plasmid typing, phage typing, monocine typing, various physiological and biochemical tests, and multilocus enzyme electrophoresis, are available for differentiating L. monocytogenes from avirulent listeriae in foods (8, 13). However, many of the aforementioned identification schemes rely on phenotypic parameters, which can present problems because phenotypically similar strains can be genotypically dissimilar. Although efforts have been made to detect and type listeriae at the DNA level by restriction endonuclease analyses (16, 20) and probe technologies (1, 2, 6, 7, 19), pulsedfield gel electrophoresis (PFGE) has not been exhausted as an alternate method for differentiating Listeria spp. This technology has been used to study the epidemiology and molecular biology of other genera, to obtain physical maps of bacterial genomes, and to determine the map locations of markers not previously mapped and/or new mutations (4, 9, 11, 12, 15). Vicente et al. (18) published a brief note on progress towards the application of PFGE to the analysis of L. monocytogenes; however, the size and number of fragments resulting from digestion with various enzymes were not established. Similarly, Brosch et al. (3) used PFGE for epidemiologic studies of L. monocytogenes serovar 4b but did not report estimations of genomic length. Carriere et al. (5) also reported on PFGE analysis of L. monocytogenes, but Listeria species other than L. monocytogenes were not evaluated and the data presented were obtained primarily with a single enzyme, Apal. We report on the use of several rarely cutting restriction enzymes for clamped homogeneous electric field (CHEF) pulsed-field analyses of Listeria spp. Preparation, digestion, and fractionation of high-molecularweight genomic DNA in agarose plugs. Listeria cultures were passed twice in brain heart infusion (Difco Laboratories, Detroit, Mich.) broth at 37°C prior to use. Cells (100 ml) from an overnight culture were harvested by centrifugation (12,400 x g, 10 min, 4°C), washed twice with NT buffer (1 M NaCl, 10 mM Tris-hydrochloride [pH 7.6]), and resuspended in 10 ml of NT buffer (ca. 8 x 108 CFU/ml). Equal volumes of cells and a 1% solution of low-melting-temperature agarose (Bio-Rad Laboratories, Richmond, Calif.) prepared in *

Corresponding author. 709

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A B

kb

C D

A

B

C

D

E

F

G H

kb

2,200

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33a5-

945-

242.5_ 194.0_ 145.5-

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460_ 245_

97.0_ 48.5_

FIG. 1. CHEF gel electrophoresis of AscI digests of genomic DNA from different Listeria species. Lanes: A, Saccharomyces cerevisiae chromosome size standard (Bio-Rad); B, L. innocua (JBL1007); C, L. monocytogenes (JBL1002); D, L. ivanovii (JBL1128); E, L. seeligeri (JBL1182). Electrophoresis was done for 16 h at 200 V with electrophoresis-grade agarose (1%) and pulse times ramped from 50 to 76.6 s. The genomic fingerprints shown are identical to the restriction profiles of each strain obtained in at least five replicate gels.

and product (cheese; JBL1232) paired isolates (compare lanes F and G). These data indicate that CHEF analysis can be used to discern obscure differences among phenotypically similar strains for epidemiologic studies. The relationship between serologically similar L. monocytogenes Scott A and 101M was more precisely established with the restriction endonucleases ApaI, NotI, and SmaI. Digestion with these enzymes produced a finite number (less than 25) of discernible bands in the size range of about 25 to 450 kb (Fig. 3). Moreover, the use of chromosomal-grade agarose (and attendant pulse times) rather than electrophore-

A B C D E F G H kb

___

2,200__ 9 41 770

460 _

!

24 5

w

436.5_

FIG. 2. CHEF gel electrophoresis of AscI digests of genomic DNA from serotype 4b strains of L. monocytogenes. Lanes: A, S. cerevisiae chromosome size standard (Bio-Rad); B, JBL1002; C, JBL1156; D, JBL1000; E, JBL1180; F, JBL1232; G, JBL1231; H, JBL1181. Electrophoresis was done for 16 h at 200 V with electrophoresis-grade agarose (1%) and pulse times ramped from 50 to 76.6 s. The genomic fingerprints shown are identical to the restriction profiles of each strain obtained in at least four replicate gels.

FIG. 3. Comparison of CHEF gel electrophoresis patterns of restriction endonuclease digests of genomic DNA of L. monocytogenes serotype 4b 101M and Scott A. Lanes: A and H, lambda ladder size standard (Bio-Rad); B, D, and F, SmaI, ApaI, and Notl digests of L. monocytogenes 101M (JBL1002); C, E, and G, SmaI, ApaI, and NotI digests of L. monocytogenes Scott A (JBL1000). Electrophoresis was done for 8 h at 200 V with chromosomal-grade agarose (0.5%) and pulse times ramped from 1.2 to 20 s. The genomic fingerprints shown are identical to the restriction profiles of each strain obtained in at least 12 replicate gels.

sis-grade agarose reduced run times by about 50% and increased the resolution of the SmaI and ApaI profiles. For 22 discrete SmaI fragments resolved by CHEF (lanes B and C), the chromosomal fingerprints of strains 101M and Scott A differed by at least six bands. The enzyme Apal produced 15 and 17 fragments for Scott A and 101M, respectively; 5 of these bands were dissimilar. Digestion of the L. monocytogenes chromosome with NotI yielded five distinct fragments, with at least three bands differing in migration between 101M and Scott A. Although the three largest Notl fragments were not resolved under the conditions described in the legend to Fig. 3, in subsequent gels these bands were resolved with switch times of 50 to 90 s over 24 h at 200 V (Table 1). Determination of genome length. The size and number of AscI and Notl fragments resulting from digestion of L. monocytogenes serotype 4b Scott A (JBL1000) and 101M (JBL1002) are listed in Table 1. Although data are reported for both AscI and Notl, the data obtained with AscI probably provide a more accurate estimate of genome size than those obtained with NotI because of the percent error involved in sizing fragments in the megabase range (1.5 to 2.2 megabases) with existing standards and technology. Summation of individually sized AscI fragments estimated the genomes of L. monocytogenes 101M (JBL1002) and Scott A (JBL1000) to be 2,925 and 3,046 kb, respectively. Other investigators (5) using ApaI estimated the genome sizes of L. monocytogenes serogroup 1 strains to be 2,660 kb (1/2a), 2,640 kb (1/2b), and 2,340 kb (1/2c); the genome size of serotype 4b strains was reported to be 2,701 kb. Sources of error in calculating genome length include the contribution of plasmid DNA and/or fragments of

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in geno...
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