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Molecular Epidemiology: Application of


Contemporary Techniques to the Typing of
Microorganisms

Article in Clinical Infectious Diseases September 1993


DOI: 10.1093/clinids/17.2.153 Source: PubMed

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Molecular Epidemiology: Application of Contemporary Techniques to the Typing of
Microorganisms
Author(s): Joel N. Maslow, Maury Ellis Mulligan, Robert D. Arbeit
Reviewed work(s):
Source: Clinical Infectious Diseases, Vol. 17, No. 2 (Aug., 1993), pp. 153-162
Published by: Oxford University Press
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153

STATE-OF-THE-ARTCLINICALARTICLE

Molecular Epidemiology:Application of ContemporaryTechniques to the


Typingof Microorganisms
Joel N. Maslow, Maury Ellis Mulligan, From the Research Service, VeteransAffairs Medical Center, and the
and Robert D. Arbeit Section of Infectious Diseases, Departmentof Medicine, Boston
UniversitySchool of Medicine, Boston, Massachusetts;the Infectious
Diseases Service, VeteransAffairs Medical Center, Long Beach,
California;and the Department of Medicine, Universityof California,
Irvine, California

Introduction Theoreticalconsiderations. The basicpremiseinherentin


Fourpatientson a surgicalwarddeveloppostoperative any typing system is that epidemiologicallyrelated isolates
pneu- are derived from the clonal expansion of a single precursor
monia due to Klebsiellapneumoniae.Is this an outbreak?
and, consequently, share characteristicsthat differ from
A child recentlytreatedfor an Escherichiacoli urinarytract
those of epidemiologicallyunrelatedisolates.The utilityof a
infectionreturnswithanother.Is thisa relapsedue to theorigi-
nal organismor a new infectiondue to a differentstrain? particularcharacteristicfor typing is related to its stability
within a strainand its diversitywithin the species. The latter
Severalculturesof bloodfroma man witha prostheticheart
reflectsthe evolutionarygenetic divergencearisingfromran-
valveyield only Staphylococcusepidermidis.The isolateshave
dom, nonlethal mutationsthat accumulateover time. Such
differentantibioticsusceptibilities.Do they representa single mutations are detectable if they occur at sites that can be
infectingstrain or multiplecontaminants?
Each of these situationspresentsthe medical practitioner assayed (for example, in a gene encoding for a metabolic
with a problemthat involves reliablydifferentiatingmultiple enzyme or at a restrictionsite that determinesa DNA finger-
bacterialisolates or establishingthat the isolates are identi- printpattern).With the developmentof highlysensitivemo-
lecular techniques, the ability to detect more-subtlevaria-
cal. In recent years, investigations of epidemiology and
tions has increasedsubstantially.
pathogenesishave utilized a wide variety of techniques de-
rived from immunology, biochemistry,and genetics; these However,the pathogenicisolates of a species may exhibit
studiesareoften referredto collectivelyas molecularepidemi- appreciablyless genetic diversitythan do all of the clinical
and environmentalstrainsof that species. For example, the
ology. This commentarygives an overview of the systems
available for typing microorganisms and describes the genetic diversityamong invasive isolates of E. coli cultured
from patientswith pyelonephritisor bacteremiais relatively
strengthsand weaknesses of each. We focus almost exclu-
limited comparedto that of isolates colonizing the intestinal
sively on the analysis of bacteria,as molecular techniques
relatingto fungi arejust emergingand studies of virusesare tract [1]. The pathogenic isolates representgenetically re-
lated groups,referredto as lineages,that are characterizedby
generally restrictedto specific research settings. Although
virulencefactorsthat enhance colonization,persistence,and
typing systems can provide potentially useful information,
invasion at the infecting site; such virulence determinants
they are best used to supportor rejectclinical hypothesesand
thus augment decision makingratherthan to replaceclassic are absent from most commensal (i.e., fecal) isolates [2, 3].
clinical or epidemiologicalanalyses. As with any laboratory Antibiotic resistanceis another strong selection factor. Re-
determination,errorsin performanceof tests or interpreta- cent datasuggestthat isolatesof methicillin-resistantStaphy-
tion of results are possible, and a particularresult must al- lococcusaureus(MRSA) are derivedfrom one or a very few
ways be consideredwithin the overall clinical context. precursorstrains[4] and consequentlyrepresenta restricted
subsetof evolutionarylineagesin comparisonwith the diver-
sity among methicillin-sensitiveS. aureusisolates. Thus, the
most clinically relevant isolates-those with characteristics
Received 10 May 1993. that providefor increasedvirulenceor resistance-are often
Grant support:Supportedin partby the Medical ResearchProgram,De-
the most difficultto differentiateunambiguously.
partment of Veterans Affairs, and the National Institute of Allergy and
Infectious Diseases (grant no. IR01AI30373), National Institutes of Criteriafor evaluating typing systems. Several criteria
Health. shouldbe consideredin evaluatingtypingsystems.Typeabil-
Reprintsor correspondence:Dr. MauryEllis Mulligan, Medical Service
(111-1 D), Departmentof VeteransAffairsMedical Center, 5901 East 7th ity refers to the ability to obtain an unambiguous,positive
Street, Long Beach, California90822. result for each isolate analyzed; nontypeable isolates are
ClinicalInfectiousDiseases 1993;17:153-64 those for which typing yields either a null or noninterpret-
Thisarticleis in the publicdomain. able result. Reproducibilityrefers to the ability of a tech-
154 Maslow,Mulligan,and Arbeit CID 1993;17(August)

nique to yield the same resultwhen the same strainis tested proachesare limited by the relativelylargefractionof strains
repeatedly;this criterion may be affected either by day-to- that appearphenotypicallynull and, consequently,are non-
day variation in the results of the particularmethod or by typeable.
variationin the stabilityof the bacterialcharacteristicbeing Biotyping. Biotypingrefersto establishingthe patternof
examined. Discriminatorypower refersto the ability to dif- activity of <20 cellular metabolic enzymes. Using auto-
ferentiateamong unrelatedstrains.Ideally,a typing method mated systemsdesignedfor speciesidentification,most clini-
will recognize each unrelatedisolate as unique; in practice, cal microbiology laboratoriesroutinely obtain such data.
the technique is considered statisticallyuseful if the most Biotypingis highly reliable for this purpose,and the detec-
common type it detects occurs in <5%of the population. tion of multipleisolatesof an unusualspeciescan effectively
Evaluatinga particularmethod accordingto these criteria identify an outbreak [5]. Although epidemic strains occa-
requiresinformationthat is often unavailable.Typing sys- sionally exhibit unique biotypes, in general the discrimina-
tems have been proposedor appliedwithoutadequateknowl- tory power of biotypingis poor; the techniquehas only lim-
edge of their effectiveness for a broad sample of isolates. ited ability to distinguish distinct strains within a given
There is currentlyno "gold standard"-that is, no definitive species.
typingsystemor even an authoritativelyvalidatedcollection Variationin gene expressionis the most common reason
of isolates-against which a new methodcan be evaluated.It that isolates of the same straindifferin termsof one or more
is clearlyinsufficientto evaluatea typing systemby compar- biochemical reactions;in addition, random mutations may
ing the results for a single, well-defined outbreak with a confound the interpretationof these data. For example, one
handfulof epidemiologicallyunrelated"control"strains.Al- of our laboratoriesrecently identifiedtwo colonies of Kleb-
most any system will have sufficientreproducibilityand dis- siella organismsthat were isolated fromthe same urine sam-
criminatorypower to indicate that the outbreakstrainsare ple and differedonly in their ability to produceindole from
more similarto each other than to a few other randomiso- tryptophan,a characteristicthat differentiatesKlebsiellaoxy-
lates. Rigorousevaluationof a method involves both analyz- toca from K. pneumoniae(the latteris indole-negative).The
ing adequatenumbersof epidemicand sporadicisolatesand two isolates were thereforedesignatedas differentspecies.
comparingthe resultsdirectlywith those of previouslywell- However, on detailed examination the isolates were other-
studied approaches.In determiningdiscriminatorypower, it wise phenotypicallyand genotypically identical, thus sug-
is particularlyinformative to test epidemiologically unre- gesting that they were simply variantsderivedfrom a single
lated isolates that have proved to be indistinguishableby clone [6]. This extremeexample indicatesnot only the lim-
other techniques.Such studies requireconsiderablecollabo- ited utility of biotyping in epidemiologicalstudies but also
rativeas well as technical effortand thus are not often real- the dangerof makingany identificationsystemcriticallyde-
ized. pendent on a single characteristic.
In addition to appropriateperformance-related character- Antimicrobialsusceptibility testing. Antimicrobialsuscep-
istics, ease of interpretationis a critical feature. Interpreta- is
tibility testing routinely performedby clinical microbiol-
tions are most reliableif they are basedon logical, objective, ogy laboratoriesin the evaluationof most bacterialisolates.
readily applied criteria. Finally, the ideal typing system Both manual and automated systems are widely available,
should be rapid,inexpensive,and technicallysimple.No one typicallyeasy to use, and relativelyinexpensive.The identifi-
system meets all of these criteria,and there is no preferred cation of a new or unusual pattern of antibiotic resistance
method for all clinical settingsor infecting species. among isolatesculturedfrommultiplepatientsmay raisethe
Classificationof typing methods. A convenient basis for suspicionof an outbreak.For example,a new endemic strain
classifyingtypingsystemsis to categorizethem as phenotypic of MRSA was recognizedby the detection of ciprofloxacin-
techniques(those that detect characteristicsexpressedby the resistantMRSA isolatesin a hospitalin which that patternof
microorganisms)and genotypic techniques (those that in- resistancehad not previouslyoccurred[7]; the identification
volve direct DNA-based analyses of chromosomalor extra- of the new strainwas subsequentlyconfirmedby immuno-
chromosomalgenetic elements [table 1]). blotting, a method discussedin detail below (figure 1).
Antibiotic susceptibilitytesting has relativelylimited util-
ity in epidemiologicalstudies because of phenotypic varia-
Phenotypic Techniques
tion and becauseantibioticresistanceis affectedby extraordi-
The classic techniquesfor differentiatingstrains-serotyp- nary selective pressurein contemporaryhospitals.There are
ing, biotyping,determiningantibioticsusceptibilitypatterns, multiplegenetic mechanismsby which resistancecan rapidly
and bacteriophagetyping-rely on phenotypic differences. evolve within a strain or be readily acquired from other
Such systemsare inherentlylimited by the capacityof bacte- strains. Of particularinterest is the fact that plasmids and
ria to alter unpredictablythe expressionof the characteristic other mobile genetic elements carryingantibioticresistance
being assessed.Thus, independentisolatesof the same strain genes may be exchangedbetween strains.Consequently,dif-
can vary phenotypically. In addition, some of these ap- ferent strains may develop similar resistancepatterns,and
CID 1993;17 (August) MolecularTechniquesin BacterialTyping 155

Table 1. Characteristicsof bacterialtyping systems.

Proportionof Discriminatory
Typing system strainstypeable Reproducibility power

Phenotypicmethods
Biotyping All Fair Poor
Antimicrobialsusceptibilitytesting All Fair Poor
Serotyping Most Good Fair
Bacteriophagetyping Most Fair Poor
Immunoblotting All Excellent Good
Multilocusenzyme electrophoresis All Excellent Good
Genotypic methods
Plasmidprofileanalysis Most Fair Fair
Restrictionendonucleaseanalysis All Good Fair
Ribotyping All Excellent Fair
Pulsed field gel electrophoresis All Excellent Excellent
Polymerasechain reaction
restrictiondigests All Excellent Good
Arbitrarilyprimedpolymerase
chain reaction All Good Good
Nucleotide sequence analysis All Excellent Excellent

NOTE. These judgments representthe views of the authors;many systems remainincompletely evaluated,
and characteristicsmay vary when the systemsare applied to differentspecies. See the text for details.

sequential clinical isolates of the same strain may exhibit species for which a number of lytic bacteriophages(i.e., vi-
differentantibiotic susceptibilitypatterns(figure 1). ruses capable of infecting and lysing bacterial cells) have
Serotyping. For a centuryit has been recognizedthat mi- been identified, such as S. aureus and Salmonellaspecies,
croorganismsof the same species can differin terms of the phage typing has been the mainstayof straindiscrimination
expressionofantigenic determinantson the cell surface.Sero- [9]. In this technique, isolatesare characterizedby their sus-
typing-the detectionof such differencesby meansof immu- ceptibilityor resistanceto lysis by each memberof a panel of
nologic techniques-has been one of the classic tools for bacteriophages.Because of the need to maintain stocks of
epidemiologicalstudies of gram-negativebacteriasuch as E. biologicallyactive phagesand controlstrains,phagetypingis
coli, Haemophilusinfluenzae,and Neisseriameningitidisand available only at reference laboratories.Even for experi-
some gram-positivebacteriasuch as Streptococcuspneumo- enced workers,phage typing is technically very demanding
niae. Some distinctvirulencefactorsand relatedclinical syn- and is subjectto considerableexperimentalas well as biologi-
dromes are linked to particularserotypeswithin a species. cal variability.In addition, since many strainsare nontype-
For example, E. coli 01 57:H7 organismsare associatedwith able with the available bacteriophagepanels, other phages
the hemolytic-uremicsyndrome. often need to be consideredfor experimentation.Bacteriocin
For evaluationof certainorganisms,e.g., salmonellaeand typing depends on susceptibilityof the test organismto tox-
shigellae, serotyping is a rapid, simple, readily available ins producedby other bacteria;the method has limitations
method. However,as a generaltool for detailedclinicalanal- similarto those describedfor phage typing.
yses, serotypingis subjectto severalcriticallimitations.Spe- Electrophoretic protein typing and immunoblotting.
cific typing reagents, including both polyclonal sera and Variationsin the structureof bacterialproteins can be de-
monoclonal antibodies, are often difficultand expensive to tected by a varietyof methods. Electrophoreticprotein typ-
develop and characterizeand are not availablein most clini- ing is performed by isolating whole-cell or cell-surface
cal microbiologylaboratories.Even for bacterialspecieswith proteins, separatingthem by SDS-PAGE, and staining the
large numbers of antigenic variants, serotyping often has gel to determinethe resultingpattern.Alternatively,the pro-
poor discriminatorypowerbecausemany of the strainseither teins can be radiolabelledduring isolation and the pattern
are nontypeableor compriseonly a few serotypes.For exam- detected by autoradiography[10].
ple, the serotypesof 75%of clinical isolates of Mycobacte- Immunoblottingis performedby transferringthe sepa-
rium aviumrepresentonly four of 11 defined serotypes,and rated bacterialproducts to a nitrocellulose membraneand
10%-15%of M. avium strainsare nontypeablebecause they then detecting them with use of antisera to specific type
fail to react with any available reagentsor they autoaggluti- strains or with pooled human sera as a source of broadly
nate [8]. reactiveantibodies[ 11]. The use of antibodyfor detectionin
Bacteriophageand bacteriocintyping. Among bacterial the immunoblottingtechnique permitsassessmentof addi-
156 Maslow, Mulligan, and Arbeit CID 1993; 17 (August)

1 2 3 4 with use of specificsubstrates[ 13]. Variationsin electropho-


retic mobility typically reflect amino acid substitutionsthat
alterthe chargeof the proteinand therebyidentifyvariations
- 130K in the chromosomalgenes encoding the enzyme. Although
individualenzymes may be absent (null) fromparticulariso-
lates, the evaluationof multiple metabolic enzymes ensures
75 K that all isolates are typeable.
Multilocus enzyme electrophoresisis technicallyvery de-
mandingand, in general,only moderatelydiscriminatoryfor
clinical isolates. For example, use of this technique to ana-
450K lyze E. coli cultured from patients with pyelonephritishas
3- shown that such virulent,invasive strainsare representedby
a limited number of closely related genetic lineages [1, 3].
Consequently,this method has had relativelylimited appli-
cation to epidemiologicalstudies;however, it has proved to
be uniquely useful in providingquantitativedata regarding
--- 39 K. the populationgenetics of pathogenicspecies.

Genotypic Techniques
27 K
Becauseproblemswith typeability,reproducibility,or dis-
criminatorypower have been associatedwith many pheno-
typic techniques,numeroussystemswith use of DNA-based
Figure 1. Immunoblot analysis of four isolates of MRSA. Iso- methods have been developed. Initially these techniques
lates 1 and 2, which differed in resistance to ciprofloxacin,were were used only in a few researchlaboratories,but they are
cultured 1 week apartfrom a single patient. Immunoblotanalysis rapidly becoming more widely available for use in clinical
demonstratedthat these isolates were distinct; the antibiotic and
immunoblot patterns of isolate 1 were typical of the endemic practice. In addition to the technical intricacies of these
MRSA isolates priorto the introductionof an epidemic strain(iso- methods, the substantialcomplexitiesinvolved in effectively
late 2). Isolates 3 and 4 are two additional ciprofloxacin-resistant interpretingthe resultsare becoming increasinglyapparent.
isolates, culturedfrom differentpatients,that differedin resistance Plasmid profile analysis. The first DNA-based tech-
to rifampinand were analyzedas partof the same outbreak.Immu- niquesappliedto epidemiologicalstudiesinvolved the analy-
noblot analysis determined that the two isolates (3 and 4) were sis of plasmids. In the most basic system, plasmids are
identical to each other and had an immunoblot patternsimilarto
the priorciprofloxacin-resistantstrain(2) and thus representedthe isolated fromeach isolate and then separatedelectrophoreti-
epidemicstrainratherthan a thirdstrainof MRSA. K = molecular cally in an agarosegel to determine their numberand size.
weight in kilodaltons. Additional information can be obtained by digesting the
plasmidswith a restrictionendonuclease(discussedin detail
below) and then comparingthe number and size of the re-
tional bacterialproductssuch as polysaccharidesand glyco- sulting restrictionfragments.The discriminatorypower of
lipids. When pooled or individualhumanseraare employed, these approachesis poor for those strainsthat lack plasmids
immunoblottingoffersinsightsinto the diversityof bacterial or possess only one or two. Technically, plasmid analysisis
productsthat elicit host responses. the simplest of the DNA-based methods and can be effi-
Virtuallyall strainsare typeable by these techniques.The ciently performedwith basic electrophoreticequipmentand
resultsof immunoblottinghave correlatedwell with those of commerciallyavailable reagents.
other typing systems,and the method has provedeffectivein There are, however, notable potential problems in plas-
investigationsof a varietyof species, includingS. aureus(fig- mid analysesthat resultfromthe fact that plasmidsareextra-
ure 1) and Clostridiumdifficile[11, 12]. Because individual chromosomalelements. Plasmidscan be lost spontaneously
electrophoreticpatternscan include largenumbersof bands, fromor acquiredreadilyby a host strain.Moreover,because
interpretationof the resultsmay be difficultand potentially of the strongselective pressurefor nosocomial organismsto
subjective. develop antibiotic resistanceand the frequencywith which
Multilocus enzyme electrophoresis. Multilocus enzyme plasmids carry resistancedeterminants,such plasmidsmay
electrophoresisdetects differencesin the electrophoreticmo- spreadrapidlyfromone strainto the next and can persistfor
bilities of individualsoluble metabolicenzymes.The cellular prolonged periodswithin an institution [14]. Plasmidsmay
proteins of the microorganismsare separatedby starch gel also be involved in the spreadof virulence factorsand may
electrophoresis, and the individual enzymes are detected be selected for on that basis. For example,aerobactin,which
CID 1993;17 (August) Molecular Techniques in Bacterial Typing 157

encodes for iron uptake and may thereby confer a growth analysismore widely available.The discriminatorypowerof
advantage to E. coli strains, is typically plasmid-associated the analysis is directlyrelatedto the numberand variability
among clinical isolates. Overall,plasmidanalysisis most ef- of the fragmentsdetected;the most effectiveprobesarethose
fectivein studiesthat are restrictedin termsof time and place that detect multiple bands simultaneously.For example, an
(for example, those involvingacute outbreakswithina single outbreak-associatedstrainof multidrug-resistant Mycobacte-
hospital). rium tuberculosiswas readilydifferentiatedfrom other, spo-
Restrictionendonucleaseanalysis of chromosomalDNA. radic multidrug-resistantstrainsof the species by means of
Eachrestrictionendonucleaseenzymaticallycuts ("digests") Southernblot analysisof a DNA sequence presentin multi-
DNA at a particular("restricted")nucleotiderecognitionse- ple copies within the M. tuberculosisgenome [19]. Southern
quence. The number and size of the restrictionfragments blot analyses of genes encoding for metabolic functions or
generated by digestion of a given piece of DNA reflect the virulence factorshave been used to type isolates, but since
frequencyand distributionof such restrictionsites. In restric- there is typically only a single chromosomalcopy of such
tion enzyme analysis (REA), endonucleaseswith relatively genes, it is often necessaryto performsequentialanalysisof
frequent restriction sites are used to digest the bacterial multiple, differentgenes to achieve straindifferentiation.
DNA, thereby generating hundreds of fragments ranging Ribotyping refersto the use of Southernblot analysis to
from -0.5 kb to 50 kb in length. Such fragmentscan be detect polymorphisms associated with the ribosomal
separatedby size with use of constant-fieldagarosegel elec- operon(s) [20]. Ribosomal sequences are highly conserved,
trophoresis,and the patterncan be detected by stainingthe and a probederivedfromthe E. coli ribosomaloperoncan be
gel with ethidiumbromideand photographingit underultra- used with a wide rangeof bacterialspecies.All bacteriacarry
violet light (figure2). Differentstrainsof the same bacterial these operonsand thereforearetypeable.For organismswith
species have differentREA profilesbecause of variationsin multiple (5-7) ribosomaloperons, such as E. coli, Klebsiella,
their DNA sequencesthat alterthe distributionof restriction Haemophilus,and Staphylococcus,ribotype patterns typi-
sites. All isolatesare typeableby REA, and the approachhas cally have 10-15 bands and provide moderateto good dis-
been applied successfully to many species, in particular criminatorypower (figure2). For mycobacteria,which have
streptococciand C. difficile[15, 16]. only a single ribosomaloperon, ribotypingdetects only one
The major limitation of this technique is the difficultyof or two bandsand, consequently,is of limited utility. In gen-
interpretingthe complex profiles,which consist of hundreds eral, ribotypesare a relativelystable characteristicwithin a
of bands that may be unresolvedand overlapping.Further- species, and epidemiologicallyunrelatedisolates sometimes
more, REA patternsmay be confounded by the presenceof demonstratethe same pattern,therebylimitingthe discrimi-
plasmids, whose DNA can readily contaminate genomic natory power of the method. Conversely,however, isolates
DNA preparations.The restrictionfragmentsderived from of an outbreak-associated straintypicallyhave identicalribo-
the plasmidscan detectablyalter the REA profileand cause types;thus, the technique is useful in some respects.
isolates that differonly in their plasmidcontent to be desig- Pulsed field gel electrophoresisof chromosomal DNA.
nated as different strains. While analysis of independent Pulsed field gel electrophoresis(PFGE) is a variationof aga-
plasmid preparationscoupled with computer-assistedsub- rose gel electrophoresisthat permits analysis of bacterial
tractiontechniquescan compensatefor this effect [17], such DNA fragmentsover an orderof magnitudelargerthan that
laboriousproceduresare rarelyimplemented. with conventional REA. Chromosomal DNA is digested
Southernblot analysis of chromosomalDNA. Following with restrictionenzymesthat have few restrictionsites,yield-
agarose gel electrophoresis,the separated restriction frag- ing 5-20 fragmentsrangingfrom - 10 kb to 800 kb in length
ments can be transferredonto a nitrocellulose or nylon [21, 22]. Theoretically,all bacterialisolates are typeable by
membrane. The result is called a Southern blot, after the PFGE. While the process is technically more demanding
investigatorwho first describedthe technique [18]. Using a than REA and requiresmore expensive, specializedequip-
labeled fragment of DNA as a probe, one can detect the ment, PFGE providesa highly reproduciblerestrictionpro-
restrictionfragment(s)containing sequences (loci) homolo- file that typicallyshowsdistinct,well-resolvedfragmentsrep-
gous to the probe.Variationsin the numberand size of these resentingthe entire bacterialchromosomein a single gel.
fragmentsare referredto as restrictionfragmentlength poly- For analysisof severalcommon bacterialpathogens,such
morphisms(RFLPs) and reflect variationsin both the num- as E. coli, Enterococcus,Staphylococcus,and M. avium [22-
ber of loci that are homologousto the probeand the location 25], PFGE has provedto be highly discriminatoryand com-
of restrictionsites within or flanking those loci. All strains parableor superiorto other available techniques(figure 2).
carryingloci homologous to the probe are typeable,and the Particularlyfor E. coli and M. avium,PFGE appearsto be a
resultsare highly reproducible.The procedurerequiressome nearly optimal typing method; that is, each epidemiologi-
technical expertiseand has classicallyutilized radioisotopes cally unrelated isolate has a unique profile. However, for
to label the probe; however, the development of reliable pathogenssuch as MRSA or H. infiuenzaetype b that repre-
nonradioactivedetection systems is making Southern blot sent genetically restrictedsubsets of strainswithin a species
158 Maslow, Mulligan, and Arbeit CID 1993;17 (August)

REA Ribotype PFGE


Ribotype B.2 B.2 A AC B.2 B.2 A AC B.2 B.2 A AC
O serotype 4 4 6 85 4 4 6 85 4 4 6 85
Strain no. L62 B40 L5 B22 L62 B40 L5 B22 L62 B40 L5 B22

674 kb

12 kb1- .. .... . .

361 kb

5 kb- 208 kb
... .:....

Figure 2. Analysisofbloodstream isolatesof E. coliculturedfromfourepidemiologically unrelatedpatientsfromLongBeach,California


(isolatesL62 and L5), and Boston,Massachusetts (isolatesB40 and B22), performed withthreephenotypicand threegenotypictyping
systems.IsolatesL62andB40,fromtwogeographically diversesites,wereidenticalby REAafterEcoRIdigestionandribotypeanalysisof
thesamedigest,buttheyweredistinctby PFGEafterXbaIdigestion.IsolateL5wassimilarto theseby REAandribotyping butdifferedby
PFGE,whileisolateB22wasdistinctbyall techniques.Theisolateswerealsoanalyzedphenotypically. Biotypingwithuseof 30 biochemi-
cal tests(Vitek,Hazelwood,MO)indicatedonlyfourtestresultsdifferedamongtheisolates,withthreedifferentoverallpatternsdetected.
Twoisolates
Two isolates(L5
(L5and L62)had
and L62) an identical
had an biotype;the
identical biotype; the biotypes of strains
biotypesof strainsB40 andB22
B40and B22differed
differedfrom
fromthat biotypein
thatbiotype in two
twoand
andthree
threetests,
tests,
respectively. Anevaluationof 18antibioticsrevealedthatisolatesB22andL62wereresistantonlyto tetracycline, whereasisolatesB40and
L5 weresensitiveto all antibioticstested.The O serotypesare listedaboveeachstrainin the figure(serotypingwas performed by Dr.
RichardWilson,Pennsylvania StateUniversity,StateCollege,PA).

[4, 26], less variationis detectedamong unrelatedisolatesby tems, several variationshave been developed. In the most
PFGE as well as by other typing methods. direct modification,the PCR product is digested with a re-
Polymerasechain reaction. The polymerase chain reac- striction endonuclease, and the resulting restriction frag-
tion (PCR) has proven useful for the detection of infectious ments are analyzed by electrophoresis.This approach has
agents [27]. The essential feature of PCR is the ability to been successfully used to type strains of Cryptococcus neofor-
replicate("amplify")rapidlyand exponentiallya particular mans and S. aureus and appears to be highly reliable and
DNA sequence (the "template,"typically0.5 kb to 2.0 kb in moderatelydiscriminatory[28, 29].
length). The reaction requiresa DNA polymerase, only a Another variation,referredto as arbitrarilyprimedPCR,
minute amount of template(theoretically,as little as a single employs a single, short (typically 10 base pairs in length)
copy), and two small oligonucleotides("primers,"typically primer, whose nucleotide sequence is not directed at a
18-20 base pairs in length) correspondingto sequences at known genetic locus [30, 31]. Such arbitraryprimerswill
opposite ends of the template. Under suitable conditions, a resultin amplificationof one or more unpredictableloci, and
readilydetectedamount of productis generatedin less than a the PCR reactionwill generatea set of fragments.The num-
few hours.A majordifficultyin performingPCR is preparing ber and size of these fragmentsis the basis for typing the
suitablesamples, since even the slightestcontaminationwill isolate. This approachappearsto combine eleganceand sim-
result in a false-positiveresult. plicity; however, identifyinga suitable primerthat provides
As originally described, PCR providedonly a positive or consistent, reproducibleresultsmay be difficult,and the dis-
negative result. To provide for epidemiologicaltyping sys- criminatorypower of the technique is uncertain.
CID 1993;17 (August) MolecularTechniquesin BacterialTyping 159

Nucleotidesequenceanalysis. Given that a DNA segment cally linked isolates of a rare species or with a distinctive
can be amplified rapidlywith PCR and that techniques for resistancedeterminantmay not need more microbiological
nucleotide sequencinghave improved,it is technicallypossi- data.However,when the organismsor resistancepatternsare
ble to compare multiple isolates by sequencing the same common ones or the relationshipsamong the isolates are
locus fromeach one. This approachhas alreadybeen applied unclear, the question of typing is more difficult and addi-
in studiesanalyzingnaturalvariationwithinbacterialpopula- tional molecularanalysesmaybe valuable.In a similarexam-
tions and clearly provideshighly reliableand objective data ple (figure 1), immunoblotting effectively confirmed both
for subsequentquantitativeanalyses.A particularlydifficult the introductionof a new strainof MRSA with ciprofloxacin
question relatingto the transmissionof human immunodefi- resistanceas well as the appearanceof resistanceto rifampin
ciency virus (HIV) from a dentist to his patients was an- in a previouslydetected strain.
sweredby this approach,which obviously requiresconsider- In a classic epidemiologicalstudy, isolates from multiple
able technical effortand expertise[32]. patients are examined to determine if the infections are
PCR-basedsequence analysisof bacterialribosomalRNA linked or associated;isolatesof the same type areconsidered
has been used to detect pathogens that cannot currentlybe to be of a single bacterialstrain,presumablytransmittedby a
recoveredfrom infected tissues by culturetechniquesand to common source of exposureor from patient to patient. The
identify them at the species level. This method has success- strengthof the typing result depends on the discriminatory
fully detectedbacteriaidentifiedas an unusualgram-positive powerof the methodused;that is, the conclusionis relatively
actinomycete in the tissues of patients with Whipple's dis- tentativeif the methodassignsmany epidemiologicallyunre-
ease [33] and a new Mycobacteriumspecies causingdissemi- lated isolates to the same type, as is seen with serotyping,
nated disease in patients with AIDS [34]. It is likely that multilocus enzyme electrophoresis,and ribotyping.Regard-
specificquestionsrelatingto bacterialepidemiologycould be less of the techniqueemployed,the analysisof the isolatesis
addressedsimilarly;critical, unresolved issues include how likely to be more complex as an outbreak becomes more
to identify the most suitable loci for such an analysis and extensive either in durationor scope. Under these circum-
how large a fragmentto sequence in order to obtain suffi- stances, there is more opportunity for the outbreak-asso-
cient data for straindifferentiation. ciated isolates to exhibit differencesin type. Such differences
may reflectphenotypicvariationsor limitationsin the repro-
Discussion ducibilityof the technique(as have been observedwith elec-
trophoreticprotein typing) or actual genotypic variations
In clinical practice, bacterialtyping systems are applied (for example, changes in plasmid or phage content or se-
primarilyto addressone fundamentalissue: are two isolates quence mutations altering restrictionsites). In addition, as
the "same"or "different"?This issue arisesin multiplecon- increasing numbers of strains are assayed, there is an in-
texts, includingepidemiologicalinvestigations,patientman- creased chance that epidemiologicallyunrelated strains of
agement, and studies of the pathogenesisof bacterialinfec- the same type will be included in the analysis.
tions. Although this question appears basic (almost Interpretingand applying typing results are particularly
elementary), it contains numerous complexities relatingto difficult when two isolates are typed as "similar"-that is,
the particulartyping methods, the interpretationof the typ- they differin only one or a few of the multiplecharacteristics
ing data, and the basic biology of microorganisms.To ad- under consideration.Our experiencesuggeststhat detection
dressthe problemat hand constructivelyand with the appro- of a single differenceby any typing system (e.g., a difference
priate technique requires an active collaboration between in lactose fermentationor the shift of a single band on an
the clinician or infection control practitioner,who formu- electrophoreticgel) is not a reliablebasisfor concludingthat
lates the question based on clinical or epidemiologicalevi- two isolates that are epidemiologicallylinked representdif-
dence, and the microbiologylaboratory,which strivesto pro- ferent strains [21]. On the contrary,in systems in which a
vide additionaldata. single differencecan be directlyexplainedas a single genetic
Fourpatientson a surgicalwarddeveloppostoperative pneu- event-a descriptionthat includes many phenotypicand es-
monia due to Klebsiellapneumoniae.Is this an outbreak? sentiallyall genotypicmethods-such variationcan occurin
If this examplehad involveda distinctlyuncommonorgan- vivo among epidemiologicallyrelatedstrains.We have occa-
ism, simple biotyping would be a sufficienttyping method. sionally detected such genetic events when using Southern
In 1971 an outbreakof Enterobacteragglomeranssepticemia blottingand PFGEto analyzemultipleisolatesfromsequen-
was detected and found to be due to contaminatedintrave- tial cultures of blood from the same patient. Increasingly
nous fluidson the basisof isolationof an organismthat previ- discriminatorytechniques are, by definition, able to detect
ously had been considered a plant pathogen [5]. Similarly, smallerand less frequentvariations;the methods now avail-
only antimicrobialsusceptibilitytesting is requiredto iden- able are able to detect the small genetic variationsthat can
tify a cluster of MRSA in a hospital where that organismis occur duringthe courseof an outbreakor infection or during
not endemic. Thus, the clinician encounteringepidemiologi- the culturingprocess.These observationsdirectlychallenge
160 Maslow, Mulligan, and Arbeit CID 1993; 17 (August)

the concept of what constitutesa clone and supportthe sug- 15A 1513 2A 21B
gestion by Eisensteinthat clonality is more usefully consid-
ered as a relativeratherthan absolute concept [35]. 674 kb
In most typing methods this problemof classifying"simi-
lar" isolates is implicitly recognized by the designation of
types and subtypes. In each system, types differ from each
other in some specifiedmanner, although, with the notable
exception of multilocus enzyme electrophoresis,the differ-
ence is rarelyquantitated.Isolatesthat differonly minimally 361 kb
from a given type are typically designatedas membersof a
subtype, although which is determined to be dominant is
obviously subjective. For complex reasons, this process is
logically confounded (for example, by the same criteria,an
isolate could be designatedas belonging to a subtype of ei-
ther of two types), and as a result, relationshipsamong iso-
lates can be obscuredin the processof data compilation.We
anticipatethatas more discriminatorytechniquesareapplied
to largercollections of isolates, this issue will be encountered 208 kb
more frequently. From the perspective of the practitioner
who attemptsto apply such determinations,it is criticalthat
the criteriaused for assigning labels be explicitly defined.
Furthermore,information regarding accumulated knowl-
edge of the clinical and epidemiologicalsignificanceof those
criteriais directlyrelevantand potentiallycriticalto the ap-
propriateapplicationof the typing results. 80 kb
A childrecentlytreatedfor an E. coli urinarytractinfection
returnswithanother.Is this a relapsedue to theoriginalorgan-
ism or a new infection due to a different strain?
Typing systems can be used to examine multiple isolates
recoveredsequentiallyfrom an individualpatient, and such
analyses may help define the pathophysiologyof the infec-
tion. In the example illustrated(figure3), relapsinginfection Figure 3. E. coli strainscausingrecurrenturinarytractinfections
in two children, as analyzed by PFGE after XbaI restrictiondiges-
wasclinicallysuspectedfor both patients,but one child had a tion. For patient 15 the pretreatment(15A) and posttreatment
new infection due to a different strain. The occurrence of (15B) isolates have identical restrictionprofiles and representre-
reinfectionmay suggest that a patient is predisposedto the lapse with the same strain, whereas for patient 2 the two isolates
particularinfection, possibly because of a host defense de- (2A and 2B) have distinct restrictionprofilesand representreinfec-
fect. Recoveringthe same strain from a patient on different tion with a new strain.
occasions suggestsa relapsinginfection (figure 3), possibly
arisingfroma residualfocus of infectionor a site of persistent
colonization. nating among strains within such genetically restricted
In general, two isolates that are typed as distinctly differ- groups is difficult.
ent can be reasonablyjudged to representdifferentstrains. Several cultures of bloodfrom a man with a prosthetic heart
(Plasmid analyses can be a notable exception to this guide- valve yield only Staphylococcus epidermidis. The isolates have
line, since a given straincan readily lose and acquire plas- different antibiotic susceptibilities. Do they represent a single
mids). The conclusionthat two isolatesareidenticaldepends infecting strain or multiple contaminants?
both on the discriminatorypower of the method used, as Examination of multiple isolates of the same species ob-
consideredabove, and on the genetic diversityof the class of tained during a single episode of infection may be useful in
isolates being examined. Two examples include MRSA, establishing a diagnosis and in investigating pathogenesis. In
which appearsto have evolved fromone or very few transfers the example presented above, a reliable, discriminatory anal-
of the methicillin-resistancegene into S. aureus [4] and H. ysis indicating that all the isolates represent the same strain
influenzae type b; for the latter, the capsular polysaccharide is would suggest a true bacteremia, whereas the detection of
a criticalvirulence factorfound among only a limited subset multiple strains would be more compatible with the presence
of the genetic lineages[26]. Withall typingsystems,discrimi- of independent skin contaminants [36]. These interpreta-
CID 1993; 17 (August) Molecular Techniques in Bacterial Typing 161

Strain no. oratorytest, the results are most effectively used to supple-
ment rather than replace hypotheses and questions
62.1 62.2 135 136 thoughtfully developed by the clinician or epidemiologist.
Ideally,typingis performedindependentlyby the laboratory
to avoid bias, but the results are applied collaborativelyto
674 kb
ensure that both the potential insightsand the unavoidable
ambiguitiespresentedby the resultsare clearly appreciated.
The mannerin which the typing resultsare interpretedand
361 kb
applied is as importantas the particulartyping method em-
ployed.

208 kb Acknowledgments

The authorsare indebtedto numerouscollaboratorsfor iso-


lates, unpublisheddata, and invaluableconversations.Their
as is the outstandingtechnical
opennessis sincerelyappreciated,
supportfromRobertaDunn, KennethS. Adams,MaureenBar-
low, and RichardY. Y. Kwok.

8O kb
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