CopyrightC) 1994,American Societyfor Microbiology
Comparison
of
Restriction
Endonuclease Analysis,
Ribotyping,
and Pulsed-Field
Gel
Electrophoresis
for
Molecular
Differentiation of
Clostridium difficile
Strains
MARKRISTJANSSON,1tMAYTHEWH.SAMORE,2DALE N. GERDING,3j PAOLA C.DEGIROLAMI,2
KRIS M.BETTIN,3ADOLF W. KARCHMER,2ANDROBERTD.
ARBEITV*
Section of InfectiousDiseases, Medical Service, VA Medical Center and Boston University School ofMedicine,'
and Division of Infectious Diseases and Department of Pathology, New England Deaconess Hospital and Harvard MedicalSchool,2Boston, Massachusetts, and Medical Service, VA Medical Center and
UniversityofMinnesota, Minneapolis, Minnesota3
Received9 February 1994/Returned for modification 22 March 1994/Accepted5 May1994
Acombined clinical and molecular epidemiologic
analysis
of 46 strains of Clostridiumdijfile,
including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13community-acquired isolates, was performed.
HindIll
digests of totalcellular DNA were analyzed by restriction enzymeanalysis
(REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates wereassigned to typing groups on the basis of the profiles detected; isolates withclosely
related profiles were assignedtosubgroups. The 16isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate waspersistently
partially
degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups,respectively),
with two isolates notevaluable by PFGE. The 13epidemiologically
unrelated community-acquired isolates were assignedto 11groups by REA and to 12 groups by PFGE. Overall, ribotyping identifiedonly
nine groups among the46 isolates. We conclude that REA and PFGE have comparablediscriminatory powers forepidemiologic typing of C.difile
isolates and thatribotyping
isappreciably
lessdiscriminatory. For a few isolates, partial DNAdegradation prevented analysis by PFGE but not by REA orribotyping; the cause ofthe degradation is unknown.Clostridium difficile causes a spectrum of colonic diseases
rangingfromantibiotic-associated diarrhea to pseudomembra-nous colitis and produces significant morbidity among hospi-talized patients (4). Sporadiccases ofC.
difficile
disease may represent overgrowth of endogenous organisms (7), butnoso-comial clusters of cases have been ascribed to interpatient transfer of organisms through contaminated environmental sources as well as by hospital personnel (9, 26). Multiple techniques have been used to identify distinct strains ofC.
dificile
and to resolveepidemiologically
related cases. Theearliertypingsystemsbasedon
phenotypic
traitsor onplasmid
andphage profileswereinsufficientforcomprehensive
analy-ses, since phenotypes lack discriminatory power and not all strains aretypeable byphageandplasmid studies
(10,
14, 16, 21, 33, 37, 38). Subsequently, C. difficile typing based onrestriction endonuclease analysis
(REA)
was shown to bereproducible, highly discriminatory,anduniversally
applicable
(8, 15, 17,20).
Inthistechnique,
totalcellular DNA isdigested
-withafrequentlycuttingrestriction enzyme, and the
resulting
fragments are resolvedby agarosegel
electrophoresis.
How-*Corresponding author. Mailingaddress: Research Service
(151),
VA Medical Center, 150 S. Huntington Ave., Boston, MA 02130. Phone: (617)278-4416.Fax:(617)739-6394. Electronic mailaddress: rarbeit@bu.edu.
tPresentaddress:
Department
ofMedicine,Borgarspitali,Reykja-vi'k, Iceland.
tPresent address: Medical Service,VA Lakeside Medical Center, Chicago, IL60611.
ever, the visual assessment of hundreds of restriction frag-mentsinasingle gelcanbedifficultand may be confounded by the presence ofextrachromosomal DNA(6, 15).Southernblot
analysis using a ribosomal probe (ribotyping) detects fewer fragments andcantherefore beeasiertoread,but it may have lessdiscriminatorypower(8, 34). ChromosomalDNAdigests
prepared withaninfrequently cutting restriction endonuclease can be resolved by pulsed-field gel electrophoresis (PFGE)
into 10to 20 discernible bands representing theentire
chro-mosome (1,25). PFGE has been showntobe more
discrimi-natorythanribotyping in thestudy of several bacterial
patho-gens,includingEscherichia coli
(1), Staphylococcus
aureus(29),
and
Kiebsiella
spp. (23),but hasnotpreviouslybeensystemat-icallyevaluated forC.
difficile.
The purposeofthisstudywas to compareREA,ribotyping, andPFGEassystemsforanalyzing
epidemiologicallyrelated and unrelated isolatesof C.difficile.
MATERIALSAND METHODS
Study population.A
prospective
epidemiologic study
of C.difficile
colonizationanddiarrheawasinitiatedatNewEngland
Deaconess Hospital
(NEDH)
toinvestigate
thepersistently
high incidence of C.difficile
diarrhea(-15
cases per1,000
admissions).
Patients admitted or transferred to two wards(one
predominantly
medical andonepredominantly
vascular-surgical)
andthree intensive careunits(two surgical
andonemedical) constituted the
study
population.
FromJanuary
through May
1991,
forpatients
enrolled in thestudy,
speci-mensfor culturewerecollected
by
rectalswab within 72 h of admissionto thestudy
sites and thenweekly
untildischarge.
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During
thestudy period,
all NEDHpatients
who had diarrhea and whose stoolswerepositive
forcytotoxin
also hadsamples taken for culture for C.dijficile.
Details of the epidemiologicanalyses
for the entirestudy
population
have been presentedelsewhere
(30, 31).
An isolatewas considered
community acquired
ifC.difficile
was recovered from stool cultures obtained within 72 h of admission and thepatient
hadnotbeenhospitalized
within theprevious
60days.
Anisolatewasdesignated NEDH-acquired
if(i)
theinitial culturespecimen
wasnegative
and asubsequentculture
specimen
obtained >72 h afteradmissionwaspositive;(ii)
theinitial culturespecimen
waspositive
but thepatient
had beenhospitalized
at NEDH within theprevious
60days;
(iii)the initial culture
specimen
waspositive
and a culturespeci-menobtained >72 h after admission
yielded
a differentstrain,in which case the
subsequent
isolate was considered NEDHacquired;
or(iv)
fornonstudy patients
for whom no initialculture
specimen
wasobtained,
diarrheabegan
>72 h after admission and apositive
culturespecimen
was obtained. An isolate wasdesignated
asacquired
at anotherhospital
if aculture
specimen
obtainedwithin72 hof admissiontoNEDH waspositive
and thepatient
had beenhospitalized
elsewhere within theprevious
60days.
The present
study
examined 46 isolates selected to includepatients
with differentepidemiologic
exposures.The incidenceof C.
difficile
diarrhea washighest
on the ward housingpredominantly patients
on thevascular-surgical
service(out-break
ward);
16 of the isolatesanalyzed
in thisstudy
werecultured from
patients
located on thisward,
including
fivesymptomatic patients hospitalized
inJanuary 1991,
threesymptomatic patients hospitalized
over a 6-weekperiod
be-tween 21 March and 3May,
1991,
andeight
asymptomatic
patients
admitted overthecourse of theepidemiologic
study.The
remaining
30isolates included 17additionalnosocomially
acquired
isolates(5
cultured frompatients
on other NEDH wards and 12 cultured frompatients
atthetime of transferto NEDH from otherhospitals)
and 13community-acquired
strains.
Microbiologic
characterization. Allprimary
cultures wereperformed
at NEDH with rectal swabs or stoolspecimens.
Agar plates containing
cycloserine (500 ,ug/ml),
cefoxitin (16,ug/ml),
and fructosesupplemented
with sodium taurocholatewere incubated at
37°C
for 48 h in an anaerobicjar
(31).Presumptive
isolates of C.difficile
were identified by charac-teristic odor andcolony
morphology. Toxigenicity
was deter-mined with an in-housecytotoxin
B tissueculture assay (13)andan enzyme
immunoassay
for toxin A(Premier
MeridianDiagnostics, Cincinnati,
Ohio);
nontoxigenicstrains were con-firmed to be C.difficile
with a latex agglutination test(Cul-turette
CDT;
BectonDickinson,
Cockeysville, Md.) and bybiochemical
testing
(RapIDS
AnaIl
System;Innovative Diag-nosticSystems,
Inc., Atlanta, Ga.).
AfterisolationofC.
difficile, three coloniesweresampled
and stored at roomtemperature inchopped
meat media. REA typing was performed at theMinneapolis
VA MedicalCenter;
samples from original iso-lates weretransported
overnight in chopped meat media. PFGE andribotyping
were done at the Boston VA Medical Center.Single
coloniesofstrains tobe typed were isolated at NEDHon anaerobic blood agar plates and inoculated into 5 ml ofbrain heartinfusionbroth underCO2
with a cannula and grownovernight.
Thefollowingmorning the turbid broth wassubcultured
(1:33
dilution)
into a fresh brain heart infusion broth under anaerobicconditionsandimmediately transported tothe BostonVAMedical Center. PFGE and ribotyping wereperformed
withoutknowledge
of source, prior REA results, orepidemiologic
relationship
of the isolates.Restriction endonuclease digestion. Whole-cell DNA was
prepared aspreviouslydescribed
(8, 9,
28).
Briefly,
brain heart infusion broth was inoculated with asingle
colony
from ananaerobic blood agarplateandthen incubated
overnight.
Cellswerewashed in TE
(10
mMTris-HCl,
1 mM EDTA[pH
8.0]),
resuspended in0.1 mlof TE withlysozyme(50 mg/ml;
Sigma),
incubated for 30 min at35°C,
mixed with 0.5 ml of GES solution (guanidinethiocyanate,
0.6g/ml; EDTA,
100mM;
sarcosyl,
0.5%,
vol/vol),
incubated for 10 minat roomtemper-ature, mixed with 0.75 ml of ammonium acetate
(7.5
M),
and held on ice for 10 min. DNA was extracted withphenol-chloroform-isoamyl alcohol
(25:24:1)
andprecipitated
with cold2-propanol. For restrictiondigestion,
DNA(10
to20RIl)
wasincubatedwithHindIlI(Bethesda
ResearchLaboratories,
Gaithersburg, Md.)
according
to the manufacturer'srecom-mendations, except that20 U of enzymewasused and 3
RI
ofspermidine (100 ,ug/ml;
Sigma)
was added. Theresulting
restriction fragmentswere resolved in a 0.7% agarose gel aspreviously described; the gel was stained with ethidium bro-mide and photographedunder UV
light.
PFGEanalysis.Theapplicationof PFGEtothestudyof the molecularepidemiology of bacterialisolateshasrecently been
described indetail (25).The procedurewas appliedwith the followingspecific modifications. Isolates were inoculated into
prereducedbrainheartinfusionbroths and incubated at 37°C
in astationaryposition, and theoptical densitywasmonitored hourly in a Spectronic 21 spectrophotometer (Milton Roy
Company, Rochester,N.Y.).Whengrowthreached
mid-expo-nential phase(opticaldensityat540nm20.500), typically7h afterinoculation, theorganismswerepelleted
at4°Cand then processed as previously described (25). C.difficile
DNA in agarosewasdigestedwith SmaI(New England Biolabs, Cam-bridge, Mass.), and the resulting macrorestrictionfragments
wereresolvedby PFGE. In ourexperience, overnight
broths,
although suitable forotherbacterial species (25), yield insuf-ficient DNA in agarose for PFGE analysis of C.
difficile
isolates.
Thegelswereelectrophoresedfor 22 h ina
contour-clamped
homogeneous electric field apparatus(CHEF DRII;Bio-Rad,
Richmond, Calif.) at 6.0 V/cm, with initial and final switch times of 20 and 70 s,respectively, andlinearramping. Thegels
werestainedwith ethidiumbromide and photographed under UVlight. SmaI-digested S. aureus(ATCC8325) was used asa molecular weight size standard(25).
Ribotyping. One agaroseplug prepared as describedabove
waswashed in TE buffer and then melted at65°C.The molten agar wasallowed to cool to42°C, digested with ,-agarase(New England Biolabs), and subsequently restriction digested with HindIII(New England Biolabs) according to the manufactur-er'srecommendations. The digests were electrophoresed at 1.2 V/cm for 16 h under constant field conditions in a 0.8% agarose gel (SeaKem GTG; FMC, Rockport, Maine) with Tris-acetate-EDTA running buffer. Subsequently, the DNA in the gel was transferred to a Duralon-UV membrane (Strat-agene, LaJolla, Calif.) in 10x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) using a vacuum blotter (Bio-Rad). The membranes were then baked at80°Cfor 30min and UV cross-linked by using 20 mJ/cm2 (UV Stratalinker
2400;
Stratagene). Hybridization and washes were done at65°Cin a
hybridizationchamber (Robbins Scientific, Sunnydale, Calif.) by using the manufacturer's standard protocol. Probing was performed with the 7.5-kbBamHIfragment isolated from
pC6,
which includes the entirermBE. colioperon of E. coli K-12(5).
Definitions of groups and subgroups. For each method,
isolateswereindependently assigned a group and a subgroup
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designation; "subgroups" in this article correspond to "types" in a previous paper describing REA (8). In the initial analysis
ofREApatterns,isolates were assigned to subgroups by direct visual comparison ofHindlIl restriction digests; isolates with substantially similar banding patterns among fragments of <11.5 kb were assigned to the same group, with subgroups determined by the specificpattern of fragments of
.11.5
kb (18).For thefinal analysis of REApatternsin this study, isolates were organized into groups and subgroups on the basis of a quantitative fragmentpatternsimilarity index (SI) (8). Isolates were run in a gel with up to eight strains which had been previously assigned to REA subgroups and whose patterns
werejudged to be similar to thepatternof the new isolate. The REApatternswereanalyzed by visually comparing each 1-mm segment of the top 60 mm of DNAfragmentsonthe same gel
(typically includingfragments ranging from 30 to 2 kb). An SI was calculated as the percentage of identical segments among the total of 60 segments analyzed. Isolates whosepatternswere
indistinguishable (SI = 100)fromthat of an existing subgroup wereassigned to that subgroup; isolates with an SI between 90 and 99 relative to existing subgroups were assigned to a new
subgroup within thatgroup.IsolateswithanSIof<90relative to existing subgroups were assigned to a new group and became reference strains for futurecomparisons.REAgroups were designated by one or two capital letters and REA subgroups were designated by arabic numerals.
RibotypesandPFGEpatternswereusedtoassignisolatesto groups and subgroups on the basis of differences in the
numbers and sizes of restriction fragments detected. Isolates
that differedbyone or twoband shiftsconsistent withasingle genetic event
(restriction
sitemutation,insertion, deletion,
orinversion) were designated as subgroups within the same group;isolates that differedbymultiplechangesnotconsistent
with a single genetic event were assigned to different groups
(25). All gels included molecular weight size standards to
facilitate comparisons; all subgroup assignments were
con-firmedby runningtheisolatesin thesamegel. Ribotypegroups were designated by uppercase letters and subgroups were
designated byarabicnumerals;PFGE groupsweredesignated by romannumerals andsubgroupswere designated by lower-caseletters.
RESULTS
Reproducibility.Inpreliminary studies
comparing
REAandPFGE,wenotedthattwoisolates
(698A
and730A) assigned
todifferent REA groups were
assigned
to the same groupby
PFGE, whileotherisolates(e.g.,
288A and159)
representing
thoseREA groupswereassigned
todifferent,
unrelatedPFGE groups(Fig.
1A).
Takentogether,
these resultsimplied
that isolatesrepresentingdifferentgenetic lineages
(as
resolvedby
one
technique)
could convergetothesamegenetic lineage
(as
resolvedby anothermethod).
Suchaconclusionis inconsistentwith the fundamental
premise
that DNA-basedanalyses
dif-ferentiate among isolates on the basis of
divergent
genetic
variation
(1, 24).
Thatis,
although
different methods may vary intheir ability to detectevolutionary
divergence
and,
conse-quently,
intheirdiscriminatory
power, isolatesassigned
to aparticular
genetic
lineage
by
onetechnique
should not beassigned to
substantially
different,
unrelatedlineages by
analternative
approach.
We consideredthat these
incongruent
results could repre-sent either variations inreproducibility
or technical errors,suchasthe
mislabeling
ofstrains, occurring
during
thecourse ofprocessing
in thetwo separate laboratories.Consequently,
Isolate REA PFGE
119S BM1 VIIIb Vnla
288A Ji
698A J9 XXVIa
730A Yi
159 Y16 la
Isolate REA PFGE
119S BM1
Vm%
288A BM5 Vlla
698A 730A 159
XXVia Y5
XXCVlb
Y1
Y16 Ia
FIG. 1. Increasedcorrespondencebetween PFGE and REA meth-ods for typing C. difficile isolates associated with application of SI
procedureforinterpretingREAprofiles.Inpanel A,REA groups and
subgroupswereassignedonthe basis of visualinspection;inpanelB, REA groups and subgroups were assigned on the basis of SI (as described in the text). Eachpanel represents a separate analysisin which isolateswereexaminedindependently byboth methods.Isolate 159 is representative of the strain detected among symptomatic
patientsonthe outbreak ward inJanuary1991(seethetextfordetails).
wereexaminedtheseisolates
plus
additional strainsrepresent-ing multiple
different groups andsubgroups. Duplicate
ali-quots of each isolate wereprepared simultaneously, coded,
andsentblindedtothetwo
typing
laboratories. On the repeat PFGEanalysis,
eachoftheisolateswasassigned
to thesamegroup and
subgroup
aspreviously.
Reexaminationby REA,
now
analyzed by
using
theSI,
assigned
fourisolates(including
two shown in
Fig.
1)
to groups different from those in theoriginal study;
theresults fortheremaining eight
isolateswereunchanged.
For each oftheisolates,
theoriginal
and repeatrestriction
digests
showed the same pattern of restrictionfragments,
indicating
that thechange
inassignments
wasrelatedtodifferent methods of
interpreting
thedigest
profiles.
Thenonparallel
classifications were resolvedby using
the revised REAassignments
(Fig.
1B).
Weconclude that the SIprovides
a more reliable and consistent method foranalyzing
the REAdigests
thandirect visual comparison.Typeability.
Asexpected,
all isolates could beanalyzed by
REA andby
ribotyping.
However,
threeisolatescould notbe assessedby
PFGE because the DNAprepared
in agarosewasconsistently degraded
into randomfragments ranging
from 100to200 kb insize. This mild
degradation
prevented
the resolu-tion of thelarge
restrictionfragments generated by
digestion
withinfrequently cutting
enzymes, such asSmaI,
although
thesepreparations produced distinct,
reproducible
Hindlll restrictiondigests
suitable for REA orribotyping. Multiple
attempts to avoid thisdegradation, including
eitherimmedi-ately
washing
theorganisms
in EDTAand/or
proteinase
Korimmediately
heating
them to65°C
with or withoutEDTA,
were unsuccessful. Neither of these
procedures
adversely
affected theisolationof DNA from the S.aureuscontrol strain.
This
phenomenon
has been notedby
otherinvestigators
preparing
DNA from Clostridiumperfringens
(12)
and from otherclinical isolatesofC.difficile
(36)
for PFGEanalysis.
Theproblem
appearstobeparticularly
prevalent
amongclostridial isolates. In studiesinvolving
>300 isolates of thefamily
Enterobacteriaceae,
wehaveobserved similardegradation
withonly
twostrains ofKiebsiella;
wehaveneverobserved itamong >400 isolatesofstaphylococci
(2).
Discriminatory
power. Thediscriminatory
power of the differenttechniques
is indicatedby
the number of distincttyping
groupsdefined among the isolates studied and the total number of distinctpatterns
detected(i.e., subgroups).
Overall,
REA and PFGE gave
generally comparable
results,
whileribotyping
identifiedapproximately
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TABLE 1. Discriminatory power of different molecular methods for
typingC. difficileisolates
No.ofgroups/no.ofsubgroups
Isolates n identifiedby:
Ribotyping REA PFGEl Nosocomial,outbreakward 16 2/5 5/7 5/6
Nosocomial, sporadic 17 4/10 9/15 9/14
Community acquired,sporadic 13 7/12 11/13 12/13
Total 46 9/16 18/30 20/28
aOneisolate from the outbreak ward andtwoothernosocomial isolates could notbetyped by PFGE(see thetextfordetails).
terns (Table 1). Among the total series of 46 strains, PFGE
discerned slightly more groups than REA (20 versus 18, respectively). The three isolates that were nontypeable by PFGE all represented a single REA group,with each isolate
assignedto adifferent subgroup. Consequently, overall, REA distinguished a slightly greater number of distinct patterns thanPFGE(30 versus28,respectively).
Amongthe 16isolatesfrom the outbreakward, four of the five isolates from the patients with diarrhea in January 1991 were indistinguishable by each method (isolates 20s, 2s, lOs, and 159) (Fig. 2 through 4); the fifth isolate (12s) was
indistinguishable by PFGE and was assigned to a related
subgroup byREA. The three isolates obtained from patients with diarrhea on the outbreak ward during March through
Maywere also indistinguishablefrom each otherby all three methods but represented a distinctly different strain.Among
theeight isolates fromasymptomatic patientsonthe outbreak ward, REA and PFGE both detected three additional groups and ribotypingdetectedone.
REAtype Y16 Y16 Y16 Y6 M3 AD5 BN1 A2
Strainno. 20s 2s lls 160 652a494a452a633a
- 12kb
- 5kb
*~~~~~ 2k
FIG. 2. REA(Hindlll digests) ofeight
C.difficile
isolates. Isolates20s, 2s, and lOs were cultured from symptomatic patients on the outbreak ward in January 1991; isolate 160 was cultured from an
asymptomaticpatientonthe outbreakward;isolate 652arepresentsan
isolateacquiredatanotherhospital; andisolates494a,452a,and 633a representcommunity-acquired isolates. The extreme left- and
right-hand lanes represent HindIII digests of bacteriophage lambda in-cludedasmolecularweightstandards.
Ribotype A2 A2 A2 A2 RI Cl Dl El
Strainno. 20s 2s lOs 160 652a 494a 452a 633a
_,- -_ -_
wS---_ _
- 12kb
- 5kb
-2 kb
FIG. 3. Ribotypeanalysis (Southernblotof HindIIIdigestsprobed
with an E. coliribosomal operon) of the eight C.difficileisolatesused forFig.2.
Amongthe 17sporadic nosocomial isolates therewas
some-what greater diversity; REA and PFGE again distinguished
comparable numbers of distinct patterns and both were appre-ciably more discriminatory than ribotyping. As assessedby all three techniques, an isolate obtained from a symptomatic patient transferred from anotherfacility in early January was indistinguishable from the three isolates subsequently cultured fromsymptomatic patients on the outbreak ward during March through May. Thus, the patient transferred in January appears to have introduced that strain into thehospital. Three other isolates obtained from transferredpatients were also assigned
tooneof thetyping groups detected among the isolatesonthe outbreak ward; however, all of these patients were admitted after isolates of those types hadbeendetected at NEDH, and nonehad beenhospitalized at NEDH in the preceding year.
The greatestdiversity was detected among the community-acquired isolates. By PFGE analysis, 12 of these 13 isolates wereassigned to different groups; REA detected 11 groups. By both techniques, three isolates were assigned to groups or
subgroups detected among the hospital-acquired strains. In eachinstance,the nosocomial isolate was cultured prior to the
community-acquired strainof the same type.
Correlation between toxin assays and molecular
typing.
Overall, 34 isolates were positive for both toxin A and cyto-toxin (toxin B), 11 were nontoxigenic by both assays, and 1(isolate 555) was negative for toxin A and positive for cyto-toxin. Different isolates within the same REA and PFGE group were concordant with respect to toxigenicity, with one exception: REA group A included two nontoxigenic isolates
(REA subgroup A2; PFGE group VII) and one toxigenic
isolate(REA subgroup Al; PFGE group XII). Isolate 555, a community-acquired isolate cultured from an asymptomatic
patient, represented a unique typing group according to all three methods.
DISCUSSION
Inevaluating typing systems for studying the epidemiology ofinfectious agents, several basic criteria are
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PFGCE type ATCC Ia la la lb Xllla IX IV VII AT( Strain no. 8325 20s 2s lOs 160 652a 494a 452a 633a 8325
-674 kl)
-361 kb
-208 kb
FIG. 4. PFGEanalysis(SmaIdigests) of theeight C.
dizficile
isolates used for Fig. 2 and 3. The extreme left- and right-hand lanes represent SmaI digests ofS. aureus ATCC 8325 included as molecular weight standards.ability, reproducibility, and discriminatory power (24). The
organisms causing the infection have to be typeable by the method; the characteristic being assessed by the typing system should be stable, and the method should provide consistent results. The ability to interpret the results easily facilitates
applyingthe technique. Typing systems that are more discrim-inatory for differentiating among epidemiologically unrelated
isolates are more reliable for establishing that isolates of the same type are epidemiologically related. Ideally, the typing method should be inexpensive and readily performed with
widelyavailable equipment.
Thegoal of this study was to analyze a set of
epidemiolog-icallywell-characterized isolates ofC.difficile by three different DNA-based techniques-REA, ribotyping, and PFGE. Al-though all three methods are based on restriction endonucle-asedigestionof DNA, they differ appreciably in the numbers andsizesof the restriction fragments detected. Both REA and PFGE directly analyze restriction digests ofC. difficile DNA
(-3,600 kb),but REAdigests represent hundreds of fragments
typically ranging from 0.5 to 30 kb, while PFGE digests
comprise 6 to 11 fragments ranging from 10 to 700 kb. In
ribotyping, the restriction fragment length polymorphisms
associated withthe ribosomal operons are detected by probing
Southern blots prepared from the REA digests; ribotypes comprise20to 24fragments rangingfrom 0.7 to 12.2kb.
Overall, ribotyping was appreciably less discriminatory for
analyzingC.
difficile
isolates than REA and PFGE. The latter twomethodswerecomparablefordetecting hospital outbreakstrains and for differentiating among sporadic nosocomial isolates or epidemiologically unrelated
community-acquired
isolates. The most appreciable difference between REA and
PFGEinthisstudywas theeaseof
interpreting
the differentrestriction
profiles.
The discrete number of well-resolvedbands detected by PFGEcould be
readily
analyzed by
directvisual comparison,whereas the more
complex
REA patternsrequired more intensive
segmental
analysis
of thegels
to achieve reliabletyping results.The various
phenotypic
typing
systems that have beenappliedtoC.
difficile
isolates haverecently
beenreviewed(35).
Antibiotic susceptibility, bacteriocin
production
andsuscepti-bilitypatterns, and
cytotoxicity
all have limiteddiscriminatory
power
(21,
33,38).
Theuseofpolyacrylamide
gel
electrophore-sis (PAGE) to analyze bacterial proteins is somewhat more
effective, particularly when coupledwith [35S]methionine
la-bellingorimmunoblotting(27, 35).Incomparative studies, the
discriminatorypowerof immunoblotting was superior to rou-tine PAGE andserotyping and was inferior to that of REA (8,
27). Typing systems based on the analysis of plasmids and
bacteriophage susceptibilities have also been applied to C.
difficile,
but they have limited utility as many strains are nontypeable (10, 27,33,38).REA is a relatively uncomplicated method for analyzing total cellular DNA and has been successfully applied to several bacterial species, includingStreptococcus pyogenes (11),
coag-ulase-negative staphylococci (6), andC.
difficile
(15, 17). The method isthus broadly applicable and has proven reasonablydiscriminatory. However, the numerous restriction fragments that aregenerated are often poorlyresolved, especially frag-ments of<11kb (Fig. 2). Consequently,direct interpretation of theprofilesmaybeunreliable forassigning isolates to typing groups and subgroups, as illustrated by the initial analysis in
thisstudy. Toavoid thispitfall,wenowanalyzethe restriction
profilefor eachisolate by comparing 1-mm segmentsdirectly with the restrictionprofileofareferencestrain(s)andthereby generatinganSI. Thisprocedure,althoughsomewhattedious, provides a more objective assessment than does an overall direct visual interpretation, but as the library of known sub-groupsand groups getslarger, theabilityto correctly classify
new isolates becomes more difficult. Digital scanning of gels
and subsequent analysis
using
computer softwaremight
pro-videalesslabor-intensivemeansofevaluatingtheprofiles.
Ribotypingis basedonaSouthern blot
analysis
of the subset ofrestrictionfragmentsassociatedwiththe ribosomal operons(34). C.
difficile,
like many otherbacteria,
hasmultiple
ribo-somaloperons,and the
ribotype
profiles typically
include upto 24 well-resolved bands(Fig.
3).
However, as haspreviously
beenobserved,
ribotyping
provides
only
limiteddiscriminatory
powerforidentifying
different strains withinaspecies,
presum-ablybecause therestriction sites both within and
flanking
theribosomaloperons are
relatively
well conserved(3,
29).
SmaIdigestsof C.difficile
DNAcomprised
6to11fragments
that were well resolvedby
PFGE;
these restrictionprofiles
proved to be both
relatively
uncomplicated
tointerpret
andhighly
discriminatory.
PFGE has beensuccessfully applied
inon May 15, 2020 by guest
http://jcm.asm.org/
epidemiologic analyses
ofmultiple
bacterialspecies,
including
E.coli,
staphylococci,
enterococci,
andmycobacteria
(reviewed
in reference25).
Recentstudies havesuggested
that it is alsoapplicable
toC.
difficile (7, 19),
but the results were notcompared
with those of othertechniques.
The limitations ofPFGE relate
primarily
to therelatively long processing
timerequired
to isolategenomic
DNA in agarose and to thespecialized
equipment required
toperform
the electrophore-sis. In thisstudy,
we encountered an additionalcomplication
that has not
previously
beenemphasized.
For 3 of the 46strains,
thegenomic
DNApreparations
weremildly
degraded,
sothatdistinctSmaI
restrictionfragments
forPFGEanalysis
could not be obtained. The same DNA could be
effectively
digested
with a morefrequently
cutting
restriction enzyme,such as
HindlIl,
and thus REA andribotyping
were not affected. Numerous modifications of the DNA isolationpro-cedure were unsuccessful at
eliminating
thisproblem,
which,
onthe basis ofexperience
inseverallaboratories,
appearstobeparticularly
prevalent
among clostridial isolates. The mecha-nism of thisdegradation
remains unknown butpresumably
involves aparticularly
active endonuclease. The observationthat all three
isolates,
whichwereepidemiologically
unrelated,
represented
the same, distinctive REA group suggests that there is aspecific genetic
basis for thisphenotype.
Previous studies ofnosocomial outbreaks of
C.
difficile
have identified outbreaksinvolving
asingle
strain(10,
16,
38).
Inthisstudy,
we detected several distinct strains among the isolates onthe outbreakward,
indicating
thatmultiple
endemic strains canexist in thehospital simultaneously (30).
This observationis
compatible
with evidence that nosocomialacquisition
ofC.
difficile
is facilitatedby
itspersistence
on environmentalsur-faces and that it is
spread by personnel
andby
antibiotic pressure(9,
16,
18).
The presence ofseveral different strains in the samehospital
has also been demonstrated for otherendemic nosocomial
pathogens,
suchasmethicillin-resistantS.aureus
(22)
andLegionella
pneumoniae
(32). Reproducible,
highly discriminatory
typing
systems
suchasthose described inthis
study
are,therefore,
essential forreliably identifying
specific
nosocomial outbreaks andforaiding investigations
of theclinicalepidemiology
ofC.difficile-associated
diseases.ACKNOWLEDGMENTS
Thisworkwas
supported by
theFogarty
InternationalFellowship,National Institutes of Health
(M.K.),
and the Medical ResearchProgram,
Department
of VeteransAffairs(R.D.A.
andD.N.G.).We thank Connie Clabots and Lata Venkataraman for technical assistance.
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