JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/97/$04.0010

Nov. 1997, p. 2977–2980 Vol. 35, No. 11

Copyright © 1997, American Society for Microbiology

Rapid Pulsed-Field Gel Electrophoresis Protocol for Typingof Escherichia coli O157:H7 and Other Gram-Negative

Organisms in 1 DayROMESH K. GAUTOM*

Washington State Department of Health, Public Health Laboratories, Seattle, Washington 98155

Received 23 April 1997/Returned for modification 30 June 1997/Accepted 28 July 1997

Genomic DNA patterns generated by pulsed-field gel electrophoresis are highly specific for different strainsof an organism and have significant value in epidemiologic investigations of infectious-disease outbreaks.Unfortunately, time-consuming and tedious specimen processing is an inherent problem which limits the useof this powerful technology as a real-time epidemic investigational tool. Here, I describe a rapid method toimprove the response time and provide specific bacterial strain identification for the typing of Escherichia coliO157:H7 and other gram-negative organisms in a single day.

The ability to characterize and determine relatedness amongbacterial isolates involved in an infectious-disease outbreak isa prerequisite for epidemiologic investigations. Several mark-ers have been used to distinguish isolates of a given bacterialgenus or species involved in such outbreaks (1, 21). Histori-cally, clinical and public health laboratories have used conven-tional epidemiologic typing systems, usually ones based onspecific phenotypic characterizations (1, 21). Unfortunately, asa result of inconsistently expressed phenotypic traits, theseclassical typing approaches are often not able to discriminatebetween related outbreak strains. The rapidity with which peo-ple and goods move within and between countries has in-creased the threat of epidemic spread of infectious diseases. Assuch, there is a need for rapid and accurate typing techniquesto assist public health authorities in the detection and trackingof these diseases.

Since the advent of molecular fingerprinting, there has beena great deal of effort directed toward developing molecularmethods suitable for use in clinical and public health labora-tories. Pulsed-field gel electrophoresis (PFGE) is one suchapproach that identifies organisms by their molecular geno-types (4, 18, 25, 26). It involves the use of rare-cutter restrictionenzymes to generate a limited number (10 to 20) of high-molecular-weight restriction fragments. These fragments arethen separated by agarose gel electrophoresis with pro-grammed variations in both the direction and the duration ofthe electric field (the pulsed field). The resulting electro-phoretic patterns are highly specific for strains from a varietyof organisms and also provide an opportunity to examine mul-tiple variations throughout the genome of the organism so asto identify specific strains and accurately link them with diseaseoutbreaks.

PFGE has great value in epidemiological analysis, in thedifferentiation of pathogenic strains, and in the monitoring oftheir spread among communities. The technique has been suc-cessfully employed in tracking diseases caused by a numberof different bacterial pathogens, including Escherichia coliO157:H7 (3, 5), Neisseria meningitidis (27, 28), Pseudomonasaeruginosa (16), Listeria monocytogenes (6), Bordetella pertussis(11), Legionella pneumophila (15), Vibrio cholerae (7), Staphy-lococcus aureus (23), and enterococcal isolates (20). PFGE has

also permitted the characterization of isolates indistinguish-able by restriction enzyme analysis, phage typing, ribotyping,plasmid analysis, and randomly amplified polymorphic DNAanalysis (2, 3, 16, 22, 24, 25).

PFGE has become a standard technique among publichealth agencies due to its accuracy and reproducibility betweendifferent laboratories. However, current PFGE protocols in-volve time-consuming, tedious procedures for the purificationof intact genomic DNA trapped in agarose, lengthy restrictionenzyme digests, and extended electrophoresis times (12, 18).These time-consuming steps preclude the use of current PFGEprotocols in monitoring the often rapid evolution of eventsduring ongoing outbreaks (12, 18). However, recent eventssuch as disease epidemics associated with the ingestion of foodcontaminated with either E. coli O157:H7 or Salmonella spp. inseveral western states, along with outbreaks of meningococcaldisease (8–10, 14), underscore the pressing need to develop analternative protocol for PFGE that would improve the re-sponse time and allow this powerful molecular typing system tobecome a more versatile epidemiologic tool. In this paper, Idescribe such a rapid method for PFGE analysis and demon-strate that it provides specific identification of bacterial strainsin 1 day with an accuracy and a reproducibility equivalent tothose of conventional multiday procedures.

Bacterial strains and culture conditions. Isolates of E. coliO157:H7 were submitted to the Washington State Departmentof Health Public Health Laboratories for confirmation ofstrain identification or for subtyping by standard PFGE. Otherbacterial isolates were obtained from clinical specimens sub-mitted to the Public Health Laboratories for other purposes.The isolates included in this study are listed in Table 1. Theisolates were subcultured on appropriate growth media andincubated at 37°C overnight or longer (in the cases of Borde-tella and Legionella).

Standard PFGE procedure. This procedure was designed bythe Centers for Disease Control and Prevention for use in thetracing of E. coli O157:H7 outbreaks (12). Briefly, a singlecolony of the isolate to be tested was inoculated in 3 ml ofstandard Trypticase soy broth (Becton Dickinson and Co.,Cockeysville, Md.) and incubated at 37°C on a roller drum for16 to 18 h for further growth. The cells were washed in SE (75mM NaCl, pH 8.0; 25 mM EDTA, pH 8.0) by centrifugation,and the optical density of the cells at a wavelength of 610 nmwas adjusted to 1.40. Chromosomal-grade agarose (Bio-Rad* Phone: (206) 361-2885. Fax: (206) 361-2814.

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Laboratories, Hercules, Calif.) was prepared in 10 mM Trisand 0.1 mM EDTA to a final concentration of 1.2% and main-tained at 55°C. Plugs were formed by mixing 0.5 ml of cellsuspension with 0.5 ml of agarose, and this mixture was thenpipetted into plug molds (Bio-Rad Laboratories). After theplugs solidified they were transferred to lysis buffer (50 mMTris, pH 8.0; 50 mM EDTA, pH 8.0; 1% sarcosine; 1 mg ofproteinase K per ml) for 16 to 20 h of incubation in a 55°Cwater bath. The lysis buffer was removed, and the plugs werewashed with 5 ml of sterile distilled water for 5 min and thenwith 3 ml of TE (10 mM Tris, pH 8.0; 1 mM EDTA, pH 8.0)for 5 min at room temperature. A final set of four washes, 30min each, was done with 3 ml of TE per wash. Two 1-mm-thickslices of plugs were preincubated with 200 ml of 13 XbaI buffer(Boehringer Mannheim Corporation, Indianapolis, Ind.) for30 min. The buffer was then removed and replaced with a freshmixture containing 50 U of XbaI restriction enzyme (Boehr-inger Mannheim Corporation) in 13 restriction buffer andincubated at 37°C for 16 to 20 h. The plugs were then brieflysoaked in standard 0.53 Tris-borate-EDTA (TBE) prior toelectrophoresis or, if necessary, stored at 4°C in 0.5 ml of TEfor several hours. Electrophoresis of the prepared samples wasperformed on the CHEF-mapper (Bio-Rad Laboratories) sys-tem by using pulsed-field-certified agarose (Bio-Rad Labora-tories) with 2 liters of standard 0.53 TBE running buffer. Theelectrophoretic conditions used were as follows: initial switchtime, 2.16 s; final switch time, 54.17 s; run time, 22 h; angle,120°; gradient, 6.0 V/cm; temperature, 14°C; ramping factor,

linear. After electrophoresis the gels were stained for 30 min in1 liter of sterile distilled water containing 100 ml of ethidiumbromide (10 mg/ml) and destained in three washes of 30 mineach by using 1 liter of distilled water.

Rapid PFGE procedure. In working out the details of thisprotocol, a variety of specific reaction conditions and reagentswere tested. The elements that make up the final procedurewere chosen to maximize efficiency while maintaining a highlevel of accuracy of bacterial typing.

Individual bacterial colonies grown overnight on plates weresuspended directly with cotton swabs in about 2 to 3 ml of TEbuffer (100 mM Tris and 100 mM EDTA). The cell suspen-sions were adjusted with TE buffer to 20% transmittance byusing a bioMerieux Vitek (Hazelwood, Mo.) colorimeter. Ali-quots of 100 ml of the cell suspensions were transferred to1.5-ml microcentrifuge tubes. Lysozyme (10-mg/ml stock solu-tion) and proteinase K (20-mg/ml stock solution) were addedto a final concentration of 1 mg/ml each and mixed severaltimes by being pipetted up and down. The bacterial suspen-sions were incubated at 37°C for 10 to 15 min. InCert agarose(FMC BioProducts, Rockland, Maine) was prepared in waterto a final concentration of 1.2% and maintained at 55°C in awater bath. Following the lysozyme-proteinase K incubation, 7ml of 20% sodium dodecyl sulfate and 140 ml of 1.2% InCertagarose were mixed with each bacterial suspension with thehelp of a pipette. This bacterium-agarose mixture was imme-diately added to plug molds (Bio-Rad Laboratories). The plugswere allowed to solidify for 5 to 10 min at 4°C and thentransferred to 2-ml round-bottom tubes containing 1.5 ml ofESP buffer (0.5 M EDTA, pH 9.0; 1% sodium lauryl sarcosine;1 mg of proteinase K per ml). They were incubated in a waterbath at 55°C for 2 h. After the completion of proteolysis, theplugs were transferred to 50-ml tubes containing 8 to 10 ml ofsterile, preheated (50°C) distilled water and incubated for 10min at 50°C with gentle mixing in a shaker water bath. Subse-quently, four 50°C washes were done in a shaker water bath for15 min each with 8 to 10 ml of preheated (50°C) TE buffer (10mM Tris, pH 8.0; 1 mM EDTA, pH 8.0). The plugs were thencooled to room temperature in TE buffer. At this point, theycould be used immediately or stored for 3 to 4 weeks at 4°C in1 ml of TE. For restriction endonuclease digestion, two 1-mm-thick slices of each plug were incubated at 37°C for 3 h with 50or 30 U of XbaI or SpeI enzyme, respectively, in 100 ml of theappropriate (13) restriction enzyme buffer.

Several different kinds and concentrations of agaroses wereutilized to optimize the resolution of the fingerprint profiles.SeaKem Gold agarose (FMC BioProducts) at a concentrationof 1% provided the desired resolution of DNA fingerprints.The plug slices of the samples were loaded and electropho-resed in 1% SeaKem Gold agarose with 2 liters of standard0.53 TBE running buffer. The electrophoresis was performedwith the GenePath or CHEF-mapper system (Bio-Rad Labo-ratories) with all conditions except the run time being identicalto those used in the standard procedure described above.These conditions were designed for a run time of 22 h; how-ever, for this rapid procedure, the system was manually shutdown after 14 h of electrophoresis (which corresponded to afinal switch time of 35.07). Since the initial development of thisprocedure, several electrophoretic runs programmed to stopafter 14 h were also performed. In these runs, the initial andfinal switch times were 2.16 and 35.07 s, respectively; all otherparameters remained identical with those of the standard pro-cedure. The results obtained from these runs were always iden-tical to the ones obtained when the system was manually shutdown after 14 h. Following electrophoresis, the gels werestained for 20 min in 500 ml of sterile distilled water containing

TABLE 1. Bacterial isolates analyzed by rapid PFGE

Organism No. of isolatesanalyzed

Escherichia coli O157:H7 patient isolates ...................................40Escherichia coli O157:H7 food isolates ....................................... 2Escherichia coli non-O157:H7 isolates......................................... 1Escherichia coli animal isolates..................................................... 3

Salmonella poona............................................................................ 2Salmonella montevideo ................................................................... 2Salmonella saint-paul...................................................................... 1Salmonella enteritidis ...................................................................... 2Salmonella newport ......................................................................... 2

Shigella flexneri 1............................................................................. 1Shigella flexneri 3............................................................................. 2Shigella flexneri 2............................................................................. 2Shigella sonnei ................................................................................. 2Shigella flexneri 4............................................................................. 1

Neisseria meningitidis ...................................................................... 5

Stenotrophomonas maltophilia....................................................... 8

Enterobacter aerogenes.................................................................... 5

Pseudomonas aeruginosa ................................................................ 2

Klebsiella sp. .................................................................................... 1

Bordetella pertussis .......................................................................... 3Bordetella parapertussis................................................................... 2Bordetella bronchiseptica ................................................................ 1

Vibrio cholerae................................................................................. 2

Legionella pneumophila.................................................................. 3Legionella sp.................................................................................... 3

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50 ml of ethidium bromide (10 mg/ml) and destained in threewashes of 30 min each in 1 liter of distilled water. For com-parison, plugs from the same isolate, prepared in accordancewith both the Centers for Disease Control and Preventionstandard protocol and this rapid 1-day procedure, were elec-trophoresed in adjacent lanes on 1% SeaKem Gold gels. Thegels were photographed under UV illumination with Polaroidfilm. A well-characterized strain of E. coli O157:H7 (12) wasincluded in all runs as a control.

The focus of the present study was to develop a rapid PFGEsubtyping method for E. coli O157:H7. A total of 42 isolates ofthis pathogen, 40 from patients and 2 from a suspected foodsource, were analyzed by this new rapid procedure. Clinicalisolates (Table 1) belonging to 10 other genera were also in-cluded to assess the general applicability of this rapid PFGEprotocol to other gram-negative bacteria.

The consistency of the control DNA patterns confirmed thereproducibility of the procedure. All isolates included in thisstudy were typed by this new rapid procedure within 24 h.When used to analyze E. coli O157:H7 strains, the 24-h pro-cedure, with its maximum electrophoresis time of 14 h, wasable to achieve sufficient specificity in its banding patterns (Fig.1). Each sample of E. coli was processed by both conventionaland rapid procedures, and the resulting DNA profiles werecompared. In all cases, the results of the rapid electrophoresisapproach compared favorably with the resolution achieved bythe conventional technique (data not shown). The specific en-zyme used in each case was chosen to result in a similar sizerange of bands for each organism analyzed on the same gel.

In order to evaluate the applicability of this procedure todifferent gram-negative bacteria, samples of DNA isolated byboth techniques from 10 E. coli, 5 N. meningitidis, 5 Salmonellasp., 5 Shigella sp., 5 Stenotrophomonas maltophilia, 2 Enter-obacter aerogenes, 2 B. pertussis, and 1 Klebsiella sp. isolateswere run in adjacent wells on the same gel. The restrictionpatterns obtained by the standard and rapid methods wereindistinguishable for each strain. Examples of this are shown inFig. 2 and 3. Background smearing was less noticeable in thesamples prepared by the rapid method, suggesting that theDNA had remained intact.

The procedure was shortened at several stages. The mostnotable time savings were achieved by (i) utilizing bacterial

cells directly from the culture plates of clinical specimens, (ii)expediting cell lysis and proteinase K treatment, (iii) shorten-ing the washing times by using preheated water and TE buffer,(iv) shortening the restriction digestion time, and (v) usingSeaKem Gold agarose, which allows for more rapid electro-phoresis. It is evident that the modified procedure provides alarge amount of clean, intact chromosomal DNA that can beeasily digested with a variety of restriction enzymes in a shortperiod. From a single isolate, a comparison of the DNA fin-gerprints obtained from several-day-old and from fresh cul-tures gave indistinguishable patterns (data not shown). Thissuggests that in an outbreak situation it should be possible todirectly process isolates coming from different laboratories.

Rapid, accurate typing is of fundamental importance foreffective epidemiology and outbreak investigation. Important

FIG. 1. Representative agarose gel showing PFGE patterns of XbaI-digestedgenomic DNA of E. coli O157:H7 isolates prepared by the rapid method. Lanes1 to 6, individual patient isolates; lane 7, control E. coli O157:H7.

FIG. 2. PFGE of E. coli isolates showing XbaI-digested genomic DNA pre-pared by standard and rapid methods. Lane 1, lambda concatemers used asmolecular size markers; lanes 2 to 11, paired samples of E. coli O157:H7 DNAfrom individual patients; lanes 12 and 13, paired samples of DNA from non-O157:H7 E. coli. The first and second lanes of each pair contain patterns of DNAprepared by the standard and rapid methods, respectively.

FIG. 3. PFGE of genomic DNA from seven bacterial pathogens. GenomicDNA was prepared by standard and rapid methods and digested with XbaIenzymes (all except N. meningitidis and B. pertussis, which were digested withSpeI). DNA samples were electrophoresed in pairs. The first and second lanes ofeach pair show patterns of digested DNA prepared by the standard and rapidmethods, respectively. Lanes 1 and 2, E. aerogenes; lanes 3 and 4, Klebsiella sp.;lanes 5 and 6, Stenotrophomonas maltophilia; lanes 7 and 8, Salmonella monte-video; lanes 9 and 10, Shigella flexneri 3; lanes 11 and 12, N. meningitidis; lanes 13and 14, B. pertussis; lane 15, lambda concatemers used as molecular size markers.

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public health decisions regarding the institution of preventivemeasures or the providing of reassurance that no true outbreakexists (10) are often based primarily on molecular typing data.It is well established that different strains of one serotypecirculate in most populations. PFGE is reproducible because itlooks at a stable genotype rather than at variably expressedphenotypic characteristics. Most standard PFGE procedurestake 5 to 6 days, although more rapid methods have beendescribed (13, 19). None of these rapid approaches provideresults with the speed of the procedure described here, and allof them require trade-offs not encountered in the present ap-proach. Other rapid PFGE procedures have saved time eitherthrough the elimination of proteinase K digestion or by the useof achromopeptidase. These approaches, however, havemainly been applied to gram-positive organisms without exten-sive investigation of their applicability to gram-negative organ-isms (2, 13, 17), and the turnaround time still exceeds 1 day.The procedure described in this report allows for the successfultyping of a variety of gram-negative organisms within a 24-hperiod. This has broad implications for both epidemiologicinvestigations and therapeutic interventions in outbreak situa-tions.

I thank Jon Counts and Donna Osmond for their continuous sup-port during the development of this method. I also thank JairamLingappa for his excellent suggestions to improve the text of themanuscript and Kaye Campos and Bill Lawrence for their technicalassistance.

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