Vol. 29, No. 7 JOURNALOFCLINICALMICROBIOLOGY,JUlY1991, P. 1303-1307
0095-1137/91/071303-05$02.00/0
Comparison of Ribotyping with Conventional
Methods for the
Type
Identification of Enterobacter cloacae
J. GARAIZAR,t M. E. KAUFMANN, ANDT. L. PITT*
Division of Hospital Infection, Central Public Health Laboratory, 61 ColindaleAvenue, London NW9
5HT,
UnitedKingdom
Received 9January1991/Accepted 2April1991
AnEscheyichiacolirRNA probewascomparedwithacombinationof0serotyping,phage susceptibility, and
biotypepattern forthetype identification ofstrains of Enterobactercloacae. Forty-five isolates ofE. cloacae from 36 patients in nine hospitalswereexamined. By conventional typing, only 26 (57.7%) could be assigned
toaspecific serotype and 6 (13.3%) wereautoagglutinating owingtorough lipopolysaccharide antigens. All isolates could be assignedtooneof threebiotypes,butmanyphagesensitivitypatternswereevident. Twenty-ninedistinctstrainswereidentifiedbycombined typing. Probing of EcoRI and BamHI digests of chromosomal
DNA withacDNAcopyofE. coli rRNAproved to be highlydiscriminatingbetween strains. Thirty different
ribotypesbasedon28bandswererecorded. Overall,agreementbetween theribotypingandcombinedtyping methodswasgood (84.4%), and discrepanciesweregenerallyconfinedtoserologicallyunclassifiablestrains and variability in biotype codes. Ribotypingwasreproducible, and five of six pairs of isolates from thesameand
differentpatientsgaveidenticalhybridization profilesonseparateoccasions. We conclude
tht4
ribotyping isahighly discriminatory and reproducible methodforthe typing of E. cloacae, but in most outbreaks it offers little increase in discriminationovertraditional methods.
In recent years Enterobacter cloacae has emerged as a
significantnosocomial pathogen, and outbreaks ofinfection
in surgical wards and intensive care units have been
de-scribed (2, 11). The organism is a normal member of the
gastrointestinal flora. Itisrelatively susceptible to antibiot-ics but isinvariably resistant toampicillin and first-genera-tion cephalosporins. Resistance often develops to newer
enzyme-stable beta-lactam antibiotics (18).
Many
typing
systems havebeen describedtoaidepidemi-ological
studies ofE. cloacae, including serological, phage, bacteriocin,andbiotypesystems(5).Noneofthesemethodsis satisfactorywhen used aloneforthetypingof E. cloacae due either toinadequate discrimination, poor reproducibil-ity,orlow typability. Whenusedincombinationthey allow
reasonablyaccurate identification of straintypes, butowing
totheir complexity, theyare notgenerallyavailable outside
referencecenters.
Grimont and Grimont (7) proposed the useofpatternsof
genesencodingrRNAlocalizedbyhybridization with radio-actively labeled Escherichia coli rRNA to identifythe gen-otypeof strains. Thisapproach has been used
successfully
todetect these genes in various bacterial species (hence the name ribotyping [21]) for both taxonomicand
epidemiolog-icalpurposes(9,13).As analternativetotheradioactiveendlabeling ofrRNA (7), a DNA strand complementary to the RNA sequence canbepreparedtofacilitate uniformlabeling with biotin (15). The genotype obtained is independent of
phenotypic and cultural variation, and differencesin hybrid-ization profiles are considered representative of species
and/or strains.
We havecompared ribotypingofE. cloacae with standard
referencetechniquesin avariety of outbreak situations. We report here thediscriminatory
potential
and reproducibility*Corresponding author.
tPresentaddress: Departamento de Microbiologia e Inmunolo-gia, Facultad de Medicina y Odontologia, Universidad del Pais Vasco, Apartado 699, Bilbao, Spain.
of the methods used and theirsuitabilityforepidemiological
investigations.
MATERIALS ANDMETHODS
Bacterial strains. Forty-five E. cloacae clinical isolates from 36 patients in nine different hospitals were selected
from isolates submittedtotheDivisionofHospitalInfection
forepidemiological typing. The
isolates
wererecoveredfrom avariety ofclinicalsituations, including urinarytractinfec-tion, bacteremia, and oral/rectal colonization. Reference strains ofE.cloacaeforserologicalandphage typingwereas
describedby Gaston andcoworkers (3, 6).
Biochemical identification. Isolates were screenedfor spe-cies identity in the API 20E system (API, Basingstoke,
United Kingdom) andby ashort set ofbiotypetests which included malonate, dulcitol, esculin, inositol, adonitol, and rhamnose(12).
Serotypeandphagetyping. The0serotypeofeach isolate was determined by agglutination tests with pooled and
specific 0 reference antisera (6), and phage typing was
performed asdescribed previously (4).
LPS profile. The lipopolysaccharides (LPS) of isolates were prepared byprotease Kdigestion (8). Following sepa-ration insodiumdodecylsulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) in 10% resolving gels (10), LPS
con-stituents weredetected bya modified silver stain(22).
Isolation of chromosomal DNA. The procedure followed
was as described previously(16) with the following modifi-cations. Isolates were grown, with shaking, in 20 ml of nutrient brothovernightat37°Candcentrifugedat8,000 xg for 20 min. After precipitation with ice-cold ethanol at
-20°C, the DNA was washed twice with 70%
(vol/vol)
ethanol-1.5 M ammonium acetateand once with70% etha-nol and dried in vacuo. Itwasredissolvedin 100
RI
of sterile distilled waterforuse.DNArestriction endonucleasedigestion. Theoptical densi-tiesat230, 260,and 280nm wereused to estimate the DNA 1303
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concentration and purity. DNA (5 p.g) was digested with 20 U of the restriction enzymes EcoRI, BamHI, and HindIII with the buffers and reaction conditions recommended by the manufacturer (BRL Laboratories). The digested DNA wassubjected to horizontal electrophoresis in 0.8% agarose
(Bio-Rad Laboratories) in 89 mM Tris-1 mM disodium EDTA-89mM boric acid (pH 8.3) for 16 h at a constant 30 V. DNA fragments were stained with ethidium bromide (1
,ug/ml [wt/vol]) for 20 min and examined under UV light.
Southernblotting to a nylon membrane (Hybond-N;
Amer-sham) was performed in a vacuum pump unit (Vacugen; Pharmacia-LKB), and the membranes were baked for 2 h at
800C.
rRNAprobe. A cDNA probe transcribed by reverse
tran-scriptase from highly polymerized rRNA from E. coli (BoehringerMannheim) was prepared by the random primer
method and labeled with biotin-16-dUTP as described
pre-viously(15).
Hybridization with probe. The membrane filters were
soaked inprehybridization solution for4h at420Cand then in hybridization solution (17), containing 200 ,ul of the
biotinylated probe, for 16 h at the same temperature.
Hy-bridization bands on the Southern blot membranes which contained digested Enterobacter DNA were detected with
the streptavidin-alkaline phosphatase system with the Blu-gene kit (Gibco-BRL), following the procedures of the
manufacturers. A commercial biotinylatedHindIIIdigest of bacteriophage lambdaDNA was included in all electropho-reticruns. Fragment size determinations utilized a computer programbased on regression analysis ofthe reciprocals of
standard fragments (gift from J. Hernandez, Universidad Politecnica, Valencia, Spain).
RESULTS
Isolates of E. cloacaefrom nine separatehospitals where
incidents of infection occurredwereexamined. An incident comprised two to nine isolates recovered from agroup of patients within 1 to 3 weeks (Table 1). All isolates were
typed first by serotype, biotype, and phage type, and the
distinct strains in each incident were assigned a roman
numeral. By thecombinedtyping methods, isolates from the
same incident were considered representatives ofa single strainifthey hadthe same 0serotypereactionsandbiotype and did notdifferbytwo or moremajor reactions inphage sensitivitypattern.Forribotyping,isolates which differedby
one or more bands in their hybridization
patterns
weredenoted asdistinctstrains byan arabicnumeral.
Combined typing methods. Twenty-six
(57.7%)
of the 45isolatescould beassignedto aspecificserotype, and 9 were
agglutinated by polyvalent serabut failedto giveaspecific reactiononfurthertesting. Six isolateswere
autoagglutinat-ing in saline, and four did not react with any of the sera. Only
three biotype patterns werefound, andtype 22 (rhamnose and maltose positive) was the most common. Many phage sensitivity patterns were found among the 45 isolates, but
distinctphagetypes could not be definedowingtotheknown lack of reproducibility of the method (4). Therefore, the
phage codes listed in Table 1 refer only to phage patterns
within an incident and were not compared between
inci-dents. The combinedtyping methods provided ameansby
which eachofthe 45isolates could be characterized as one of 29distincttypes.
Ribotyping. Preliminary experimentswith several restric-tion endonucleases and a panel ofthe serotype reference strains showed thatEcoRI,
BamHI,
and HindIII were theTABLE 1. Correlation of combinedsero-bio-phagetypesof E.cloacae fromincidents of infection with rRNA gene
hybridization patterns(ribotype)
Inci- Isolate Patient Type
dent no. no.
1 1 1 AA-62-1
2 3 4 5 6 7 8 9 10 2 11 12 13 1 1 2 3 3 4 4 5 6 1 2 3 AA-62-1 AA-62-2 19-62-3 19-62-3 19-62-3 19-62-3 19-62-3 AA-62-3 AA-22-NT 3-22-1 13-22-1 13-22-1
Code'a Ribo-type
I 1 I 1 II 2 III 1 III 3 III 1 III 1 III 4 I 1 IV 5 V 6 VI 7 VI 7
3 14 1 10-22-NT VII
15 2 15-62-1 VIII
4 16 1 12-26-1
17 2 3-62-2
18 3 AA-22-3
19 4 3-22-2
20 5 8-22-NT
21 6 3-22-NT
22 7 3-22-NT
23 8 NT-22-4
24 9 13-22-5
25 8 NT-22-4
5 26 1 3-22-NT
27 2 2-26-NT
28 3 11-62-1
29 4 IV-62-2
6 30 1 NT-22-NT
31 1 NT-22-NT
32 2 11-22-1
33 2 11-22-1
34 2 11-22-1
35 3 11-22-ND
7 36 1 8-22-NT
37 2 8-22-NT
38 3 8-22-NT
8 39 1 11-62-1
40 1 II-62-1
9 41 1 13-ND-ND
42 2 29-ND-ND
43 3 3-ND-ND
44 4 VI-ND-ND
45 5 2-ND-ND
IX X XI XII XIII XII XII XIV XV XIV XVI XVII XVIII XIX XX XX XXI XXI XXI XXII XXIII XXIII XXIII XXIV XXIV XXV XXVI XXVII XXVIII XXIX 8 9 10 11 12 12 13 12 14 15 16 15 17 18 19 20 21 21 22 22 22 23 24 24 24 25 25 26 27 28 29 30 Agree-mentb + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
aStraincodeallocatedfrom results ofserology,biotypepattern,andphage susceptibility, listed inthatorderandjoined by hyphens. AA,
autoagglutin-able; NT,nottypable; ND,notdetermined.
b Agreement of conventionaltyping methods withribotypingin
distinguish-ingbetween strains: +,agreement;-, no agreement.
mostdiscriminating, asjudged bythenumber and
variety
of bandpositions after hybridization with E. coli rRNA (datanot shown). These enzymes wereused in the
ribotyping
of theisolatesunderinvestigation,
but itwasevidentthateach gave similar levels of discrimination. Figure 1 shows theon April 12, 2020 by guest
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RIBOTYPING FOR IDENTIFICATION OF E. CLOACAE 1305
Eco RI
Bam Hi
Incidents
8
9
7
4ib ~ ~
i4*~~~~~~~~~~~~4
L
A
.
*
4'
* *Incidents
8
AL-At
£41-Isolates
Isolates
36
37
38 39 40 41 42 43 44 45 36 37 38 39 40
41 42 43 44
FIG. 1. Ribotyping ofE. cloacae from threeincidents of infection.Fororigin of isolates, seeTable 1.
complementary discrimination achieved by EcoRI and BamHIbandingpatternsof the isolates from three incidents. In practice, the EcoRI patterns were often easier to comparebecause of the size range andnumber (8 to 13) of bands. Twenty-eight patterns of different molecular size
were revealed by the EcoRI digests of the 45 isolates, and
BamHIdifferentiatedtwoisolates whichwere
indistinguish-able by EcoRI. Nevertheless, most patterns were clearly
distinguishable by the presence orabsence ofavariety of
bands.Thebandsranged in size from 1.3to 27 kb.
Aformalassessmentof the intertest variation of ribotyp-ing was made by comparing duplicates of six isolates, two from the same patient and four from different patients, on separate occasions. Onlyone minor differencewasfound in
theposition of bands inonepair of EcoRI digests.
Agreement of methods within incidents. Overall, there was
goodagreement (38 of 45 [84.4%]) between ribotyping and thecombinedconventional methods, and discrepancieswere
foundinincidents 1 and 4(Table 1).
In thefirstoutbreak (incident 1),anincreaseinE. cloacae isolations was associated with oral/rectal colonization of
babies who were receiving antibiotic therapy for other
infections. Five of 10 isolates wereserologically
autoagglu-tinating, andtheremainderwereserotype019. SDS-PAGE showed that the autoagglutinating isolates lacked
high-mo-lecular-weight repeating units in their LPS. Phage typing distinguished three strains, andthere was an exact correla-tionbetween thephage sensitivitypatternandserotype019. One of the autoagglutinating isolates was insensitive to phages, and two patterns were observed for the other autoagglutinating isolates. All butoneof the isolates hadthe
same biotype. Five different rRNA hybridization patterns were identified, but only 50% agreement between this method and the combined methods was apparent. The
discrepancies were confined to those isolates coded III (Table 1) by the combined methods which were clearly divisibleby ribotyping.
In incident4, nine babies intwo specialcareunits inthe same hospital were colonized with E. cloacae (throat and
rectum), and typing evidence was required to establish whether these infections were sporadic orfrom acommon source. Three of the 10 isolates were serologically non-typable orautoagglutinating, andtheabsenceof0
polysac-charide was confirmed by SDS-PAGE. Four isolates were
serotype3,andtwoofthem had thesamephagepattern. The
7
9
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1306 GARAIZAR ET AL.
'':
f- 00' '
Table 1);EcoRI digests. X, lambda DNA size markers.
biotype code was unhelpful in discriminating between the strains.
Seven distinct strains were identified by both ribotyping
(Fig. 2) and the combined methods, but two different ri-botype profiles were found for isolates 21 and 22, which
could notbe differentiatedby the combined methods.
Simi-larly, isolates18and 19wereidentifiedas distinct strainsby thetraditional methods butwere indivisible byribotyping.
It shouldbe noted that isolates 19 and 21 were allocated the samestrain codedespiteadifference inphage typability. Strain 21 was lysed by a single phage, and the loss of this reaction in strain 19 was not considered sufficient for the
definitionofa different strain.
The remainder of the outbreaks all showed complete agreementbetweenthetwotypingmethods.
Ribotyping
wasparticularlyuseful incorroboratingthatminor differences in
biotype
were significant (e.g., isolates9 and 10and isolates17and19).Onlyserotypedatawereavailable forthe isolates
fromincident9,whichwere all multiple-antibioticresistant,
andribotypingsupported the serological tests which
distin-guished between the strains.
DISCUSSION
A combination ofserological and phage typing methods
hasbeenused with some successfor the type identification
of E. cloacae (2, 4). However, some strains either lack
0-specific LPS or, because ofsubtle alterations ofantigen
epitopes,
react nonspecifically inagglutination tests with 0antisera. Furthermore,asLPS oftenserve asbacteriophage
receptors,changesin theformer may affect the sensitivityof
strains to phages. Even in 0-typable strains, phage lytic patterns often overlap, and owing to the inherently poor
reproducibility of the method, at least two major reaction
differences in lytic pattern should be allowedbefore strains fromthe sameincident and of the same serological typecan
beconsidered truly distinct (4). Itwasclear, therefore,that more discriminatory and reproducible methods were
re-quiredtoimprove the typeidentification ofstrains in epide-miological studies.
Our results show that indexingthevariationof rRNAgene loci with a cDNA copy ofE. coli RNA provides such a
method. In some species, rRNA gene restriction patterns may show little heterogeneity (23) because of the low
num-berofcopies of rRNA genes in their genome, but in others they may provide a basis for strain identification (21). We found the restriction endonucleases EcoRI, BamHI, and HindIll to be highlydiscriminating, but inpractice, EcoRI patterns were sufficient to distinguish between the great majority of strains. Anadditional advantage ofthisenzyme was the wide size range and large number of fragments
generatedthroughout the length of the blot.
Variation in the fingerprints generated by different
en-zymes has been found with other species, e.g., Salmonella
typhi (1)andProvidencia stuartii (14), and thus thechoiceof enzyme for ribotyping may be critical to thedivergence of patterns produced.
A minor but important technical point was the use ofa
vacuum system for the transfer offragments to the
mem-brane. In preliminary experiments we used the traditional
capillarity transfer method of Southern (20) and
usually
obtained diffuse bands which were difficult to resolve. Vac-uum-assisted transfer of fragments often revealed the pres-ence of more than one band withinpreviously dense single
bands produced by capillary blotting.
Other studies on ribotyping have commented onthe high
degreeofreproducibility afforded by thistechnique (19). For E. cloacae, we can confirm the stability of the patterns
obtained, and the overall agreementbetweenribotyping and the reference method provided supportive evidence of the reproducibility of the method. Discrepancies between the two typing systems were invariably confined to strains with aberrant 0 serotypes or with overlapping phage patterns.
Cross-infectionbetweenpatients,asindicatedby isolation
of the same strain from two or more patients in a
single
hospital, was evident in four of the nine incidents and involved only 13 of the 36 patients whose isolates were
typed. This finding supports the view of Gaston (5) that
cross-infection withE. cloacae isinfrequent and shows that the majority of patients with E. cloacae in an apparent outbreak harbor unique strains. Three patients, all in the sameincident(number 1),appearedtobe colonizedby more than one strain, but two of them yielded
serologically
typable and autoagglutinating strains. The different ri-botypes found in these patients may reflect genotypic vari-ationdue tomutation or mayindicatethat these strainswere
indeeddistinct.
Overall, ribotyping offered little significant advantage, in terms ofdiscrimination, over the combined reference meth-ods for E. cloacae; but when equivocal results were ob-tained, ribotyping provided conclusive evidence of strain
relatedness. Themethod iscomparativelysimple,althoughit does require considerably more technical expertise than conventional typing techniques. Further studies ofthe sta-bility of the ribotypes of strain populations that exhibit
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RIBOTYPING FOR IDENTIFICATION OF E. CLOACAE 1307 marked divergence in phenotype are warranted, but the
technique isto be recommended for definitive and
compar-ative typing ofE. cloacae inepidemiological studies.
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