0095-1137/83/090486-05$02.00/0
Copyright ©1983,American SocietyforMicrobiology
Differentation
Between Virulent and Avirulent Yersinia
enterocolitica Isolates
by Using Congo Red Agar
J. KAYAPRPIC,ROY M. ROBINS-BROWNE,* ANDR. BRENT DAVEY Department of Microbiology, University of Melbourne, Parkville, Victoria, 3052, Australia
Received 28 February 1983/Accepted6 June 1983
Cultivation of clinical isolates ofYersinia enterocolitica of diversegeographical origin on amedium containing 5 ,ug ofCongo red perml disclosed two colony types. These were designated CR+ and CR- according to their ability to bind Congo red. CR+ strains bore plasmids of between 40 and 50 megadaltons and were positive in several testsofY.enterocolitica virulence, including autoagglu-tination, reduced growthonmagnesiumoxalateagar,resistancetothe bactericid-al effect of serum, and lethality for iron-overloaded mice. CR- strains were plasmidless and were negative in all these assays. The Congo red reaction providesasimple and efficientmeansofscreening Y. enterocolitica for virulence and is the best available method for identifying individual plasmid-bearing colonies.
Yersinia
enterocolitica has been
implicated
in
abroad
arrayof clinical
conditions, including
acutegastroenteritis,
mesentericadenitis,
septi-cemia, arthritis,
anderythema
nodosum(3).
Although the determinants
responsible
for the
pathogenicity
of
Y.enterocolitica
in thesedisor-ders have
notbeen
fully identified,
it has been
established
that virulence is associated with the presenceof
plasmids
with
massesof
40 to82
megadaltons
(Md)
(6, 11,
18,
26).
Properties determined by these
plasmids
in-clude
autoagglutination
(12), calcium
depen-dence
(5, 6),
production
of
Vand
Wantigens
(5),
detachment of tissue culture
monolayers (18),
pathogenicity
for mice
(21), alteration in
outermembrane
proteins (2, 18), and
serumresistance
(15).
Mostof these characteristics
are tempera-turedependent.
The
ability of
Yersiniapestis toabsorb hemin
and
Congo
redfrom
agarmedia is
correlatedwith virulence (8, 24). This correlation
has also been notedfor other bacteria(17). In this paper we report that the capacity to take up Congo redis
also associated withvirulence
of Y. enteroco-litica and that this property is plasmid mediated. TheCongo
red reaction, therefore, provides asimple indicator
of virulence in Y. enteroco-litica.MATERIALS AND METHODS
Bacteria.Seven strains of Y. enterocolitica isolated from patients in Belgium, Canada, South Africa, Swe-den,and the United States were examined (Table 1). Bacteriawerescreened for the presence of virulence-associated plasmids on magnesium oxalate (MOX) agar (6, 7), and the colonial variants thus obtained
were frozen at -20°C as thick suspensions in 34% glycerol-1% peptone.
Congoredagar. Stockculturesinitiallywereplated onto the Congo red agar described by Payne and Finkelstein (17).Althoughsomedifferential pigmenta-tion was evident, color differences were notgreatand became less pronounced with prolonged incubation. Several different base mediawere investigated. That which gave themost pronounced differential pigmen-tationwasCongoredacid-morpholinepropanesulfonic acidpigmentation (CRAMP)agar. CRAMP agar con-tained0.2%(wt/vol) galactose,0.2%CasaminoAcids, and51agofCongored per ml inabasalsalts solution (14)composed of 50mMNaCl, 40mM morpholinepro-panesulfonic acid, 10 mM NH4Cl, 2.5 mMNa2S203, 1.4mMK2HPO4,0.4mMMgSO4,and10 mMTricine (SigmaChemicalCo.,St. Louis,Mo.), pH 5.3 (unad-justed), and solidified with 1.4% agarose. On this medium,Y.enterocoliticadisplayedtwocolony types: intensely colored (CR+) colonies which bound Congo red andcolonies which remainednonpigmented (CR-) (Fig. 1).
Congoredbinding assay. Theability of bacteriato bindCongo red was tested byamodification of the method of Payne and Finkelstein (17). Strains were grown in a1% tryptone-0.25% yeast extract (TYE) broth withshakingat 25 or37°C for 30h. Cells were centrifuged, washed with phosphate-buffered saline, pH7.2, suspendedto aconcentration of 109 cells per ml in phosphate-buffered saline containing 30 ,g of Congo red per ml,andincubatedat4, 25, or 37°C with shaking for 12 h. At hourly intervals, samples were removed and centrifuged. The absorbance of the su-pernatantat488 nm wasmeasured, and the concentra-tion ofCongo red remaining was determined by com-parison withastandardcurve.Uptake of greater than 15
p.g
per109cells was scoredas apositive result.Calcium dependency. Virulent strains of Y. entero-colitica requirecalcium forgrowth at 37°C (6). Calci-umdependencywastested withMOX agar (7) which 486
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TABLE 1. Y. enterocolitica strains examined
Rapid Mouse Calcium Serum Auto- Plasmid
Strain
Cougru
Sero-or
Congored lethality depen- resis- aggluti-rens
sizegroup orgin
uptakea
(LD50)ab
dencea
tancea
nation' response (Md) WACR+C 08 UnitedStates + 50 + + + + 40
CR-c - >1.0 x 105 - - - - NDd
30.42.67
CR+ 03 Sweden + 2.0 x 105 + + + - 47
CR- - >5.0 x 108 - - - - ND
135
CR+ 03 South Africa + NT' + + + - 47
CR- - NT - - - - ND
518
CR+ 09 Belgium + 6.4x 106 + + + - 50
CR- >1.4 x108 - _ _ - ND
526
CR+ 03 Belgium + NT + + + - 50
CR- - NT - - - - ND
4209
CR+ 05 Canada + 7.4 x 103 + + + - 50
CR- - >2.5 x 108 - _ _ - ND
6003
CR+ 03 Canada + NT + + + - 44
CR- - NT - - - - ND
aSeetextforexplanation.
bLD50,
50%o
lethaldose.cCR+and CR- indicatecapacityof bacteriatobindpigmentornot tobind pigment, respectively, on CRAMP agar.
dND, Noplasmidsdetected. NT, Not tested.
contained 0.02 M sodium oxalate and 0.02 MMgCl2 in Columbia agar base(OxoidLtd., Basingstoke, Hamp-shire, England). Afterovernight incubation at 37°C, plasmid-bearingY.enterocolitica gave risetopinpoint colonies,whereasplasmidless strains yielded colonies 0.5to1mmin diameter.
Autoagglutination.
Strains were tested for autoag-glutination in tissue culture medium as previously described (12).It wasfound,however, that autoagglu-tinationcouldreadily be demonstrated in phosphate-buffered salinebyusingcells grown for 24h at37°Cin TYE broth, washingthem in phosphate-buffered sa-line, and suspending them to aconcentration of 109 cells per ml. Suspensions were incubated at 25 and 37°C with shaking. Autoagglutination was apparent after 1 h at 37°C. No autoagglutination occurred at250C.
Serumsensitivity. Resistance of strainstothe bacte-ricidal effects of normal humanserum was tested by
themethod of Pai andDeStephano(15),modified first byselectingCR+ and CR- variantsonCRAMP agar andsecondby growingthecells withshakingin TYE broth rather than onMOX agar. Under these condi-tions, serum-sensitive strains(CR-)gavenosurvivors after 1 h, whereas serum-resistant (CR+) bacteria survivedand begantomultiplyafter2 h.
Mouse lethality. With the exception of serogroup 08, Y. enterocolitica strains are avirulent for mice. However, iron-overloaded mice have been used to demonstrate virulence of serogroups 03 and09(13, 20, 23). Accordingly, strains investigated for mouse lethality were suspended in iron dextran before use. Cells grown for48 hon nutrient agar at 25°C were harvested in saline, washed, and suspended to a concentration of
108
cells per ml. Decimal dilutions wereprepared in 0.85% saline and mixed with equal volumes of 20% (vol/vol) iron dextran (Imferon; Fi-sonsPty. Ltd., Sydney,Australia)insaline. Determi-nations of the50%o
lethal dose(19)wereundertaken in groupsoffive BALB/c adult femalemice,whereeach mousereceivedanintraperitoneal injectionof 1 ml of a particular bacterial suspension. Controls included mice inoculated with thehighestconcentration ofcells without iron dextran and mice which received108
heat-killed bacteriawithiron.
Guineapigkeratoconjunctivitis (Sereny)test.Strains weregrownonnutrientagarat25 or37°Cfor48 h. A thick paste ofCR+ bacteria was inoculated into the
conjunctivalsacofoneeye ofanadult maleguinea
pig
(22). The other eye was inoculated with the CR-derivative of the same strain. Serogroupsother than 08werealso inoculated with10% iron dextran.
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FIG. 1. CR+andCR- colonies of Y. enterocolitica strain 30.42.67 on CRAMP agar after 48 h at 25°C. Dark (CR+) colonies were approximately 1 mm in diameter.
eapigswereobservedfor thedevelopmentofpurulent keratoconjunctivitisfor 7days.
Isolationand characterization ofplasmids. Plasmid DNA was isolated from overnight cultures in TYE brothat25°C bythe methodof Birnboim andDoly (1)
orPortnoyetal.(18).Molecularweightsof Y.
entero-coliticaplasmids weredeterminedby comparingtheir mobilitywiththoseof standardplasmidsfrom Esche-richia coli. StandardplasmidsusedwereR6K(26Md), R46(33 Md),RP4(36Md),TP114(41Md),andTP113 (57Md)(10).
RESULTS
Pigmentation on
Congo
red media. In initial experiments with CRAMP agaradjusted to pH 7.0, all Y. enterocolitica strains examined dis-playedtwocolony
typesafter 72 hat25°C.
CR+ variants were an intense red, whereas CF-colonies ranged from colorless to pale pink.Subsequently,
itwasfoundthat when the pHof the mediumwasleftunadjusted (pH5.3),
differ-ential pigmentation was more pronounced and unequivocal:CR+
colonies became dark violet after 48 h at 25°C, whereas CR- colonies re-mained colorless and translucent (Fig. 1). CRAMPagarmodifiedbyomitting
galactose or bysubstituting
galactosewithglucose, glycerol,
lactose,
sucrose, or mannitol was also investi-gated. In eachcase, either acid productionwas so excessive that differential pigmentation was obscured or growth was unacceptably scanty. Similarfindings
have beenreported for Y.pestis (4).The
stability
oftheCongo
red reaction of Y. enterocolitica wasinvestigated
bysubculturing
CR+ strains on CRAMP agar. In theseexperi-ments, CR+ colonies
always
yielded a mixture ofCR+
and CR-colonies,
with the proportionof
CR+ colonies
ranging
between
70 and97%.
In contrast, CR-colonies remained
nonpigmented.
The
behavior
ofCR4
and CR-colonies
wasfurther examined
in arapid Congo
redbinding
assay.
CR+
cells grown at25°C
bound
Congo
red
after
6h,
but
only
when the assay wasperformed
at37°C. The samebacteria
grown at37°C,
however,
demonstrated
rapid uptake
ofdye (greater
than
15[tg/109
cells in 1h)
at4,
25,
and
37°C. CR-
cells,
on theother
hand,
bound
very
little
dye,
even after 12h, irrespective
of
growth
and assaytemperatures.
Correlation between
pigmentation
and viru-lence. CR+ and CR-pairs
derived from
each strainwereexaminedfor various
virulence-asso-ciated
characteristics.
In every case,pigmenta-tionwascorrelated with
virulence,
asshown
by
lethality
for
iron-overloaded
mice, resistance
tothe
bactericidal effects
ofhuman
serum,autoag-glutination
at37°C,
andreduced growth
onMOX
agar at37°C
(Table 1).
Although
theSereny
testhasbeen
usedas atestforvirulence
of Y.
enterocolitica
(6, 26),
wefound
thatonly
CR+
derivatives
ofthe serogroup 08 strainWA
evoked
clear-cut
keratoconjunctivitis.
To
confirm
thecorrelation
between
ability
tobind
Congo
red and othervirulence
characteris-tics,
wedetermined
theCongo
redreaction
of Y.enterocolitica
strains whichexpressed these
characteristics.
Surviving
colonies from
serumkilling
experiments
always
contained
anover-whelming majority
ofCR+
cells,
asdidautoagg-lutinating
strains.
Pinpoint
colonies
onMOX
agaralso contained
CR+
cells(usually about
4to20%),
whereas
large
colonies
werealmost
exclu-sively
CR-.
Correlation
betweenpigmentation
andplasmidcarriage.
Asshown
in Table 1 and Fig. 2, all CR+ strainsharbored
plasmids between
40 and 50Md.
Plasmids
were notdetected
inCR-derivatives.
Furthermore,
in all strainsexam-ined,
loss ofplasmid
resulted in loss ofCongo
red
binding,
as well as othervirulence
proper-ties.
DISCUSSION
Experimental
procedures todetermine
Y.en-terocolitica pathogenicity
arecostly, unreliable, orboth.
Although
the virulence of certain sero-groups canbedemonstrated
effectively in labo-ratoryanimals (4, 16, 18, 21, 23), such tests aregenerally
notsuitable
for routine diagnostic use. Invitro
methods such as serumresistance
(15) and autoagglutination (12) are laborious, and thewidely
used test forcalciumdependence
has thedisadvantage
that virulent strains growpoorly,
if atall,
onMOX agar (6, 15).Ourresults
indicate thatpigmentation
onCRAMP agar differentiatessimply
andrapidly between
virulent andaviru-lent
strains of Y. enterocolitica.J. CLIN. MICROBIOL.
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FIG. 2. Agarose gel electrophoresis o DNA fromsevenCR+strains of Y.enteroco lowerband in each track is linear DNA(pred chromosomal fragments). Standards (St( TP114(41 Md)and TP113 (57 Md).Molecu assignments in Table 1 were confirmed aga tional standardscoveringabroaderrangeof weights (datanotshown).
The optimum growth temperature c terocolitica is around 25°C, and des
elevated body temperature of host
strains are more virulent when grown
(4). Congo red uptake on solid media
servedat 25 and37°C, although otherv associatedproperties,suchasautoagglu serum resistance, and calcium depi were evident at 37°C only. The finc uptake ofCongo red in the rapid bindi
was morepronouncedwhen cellswere 37°Cis ofinterest. Thereasonsfor the
discrepancy between growth and C( uptakefrom solidandliquidmediaare X butit isimportant tonote that the bind involves cells suspended in buffer ral
growing in culture medium.
Ability to bind Congo red appear( encoded by the virulence-associated
p]
Y. enterocolitica: all CR+ (andno CRreported here,andmany othersexamin
quently,harboredaplasmidandexpres virulence attributes that have been re] be plasmiddetermined. Although virul
plasmid mediated in all strains
exami
plasmids involved differedin moleculaThus, in epidemiological studies, it is cient to search for plasmids of a I
molecular weight as indicators of Y. i
litica virulence, especially since virulei ciatedplasmidsin Y. enterocolitica spa rangeof molecularweights (6, 11, 18,
The correlation betweenabilityto bii red and virulencewas most clearlydei edby thefindingthat only CR+ strain less ofserogroup,were lethal for mice
Congo
red
reactivity
wasthe
criterion used
for
selecting
strains
tobe tested. It has been
difficult
todate
todemonstrate
virulence of
Y.enteroco-litica in animals.
Only
serogroup08 is lethal for
mice
orinduces
apositive
Sereny
reaction. Our
results
demonstrated, however,
that
iron-over-loaded mice
maybe
used
toexamine virulence
of
serogroups03,
05, 08,
and 09.
Our data
indicate that
twoconditions
mustbe
-:
metfor
mousevirulence of these
serogroups.These
conditions
are:readily
available
iron and
the
presenceof
aplasmid
which encodes
binding
of
Congo
red. Virulence
of
Y.pestis
has also
been shown
tobe
critically dependent
oniron
(9),
and
in that
organism,
binding
of
Congo
red
If
plasmid
corresponds
totheability
totake
uphemin
(24).
litica.The The
CR+
Y. enterocolitica strains studied herelominantly
also
become
pigmented
onthe
hemin
agarde-ds.)
werescribed
by
Jackson
and Burrows
(8)
(data
notilst
adight
shown).
SinceCongo
reduptake parallels
heminmolecular
uptake,
itis
possible
that theability
to bindCongo
red reflects
aplasmid-determined
system
for
assimilating iron,
which is similar
tothat
specified by
ColV
plasmids
in
E.coli
(25).
f Y. en-
The
relationship
between
Congo
red
binding,
spite
the
iron
metabolism,
and the virulence-associated
animals,
plasmid of Y. enterocolitica is
currently
under
at 250Cinvestigation.
Regardless
of the
natureof this
was
ob-
relationship,
it is evident that
pigmentation
onrirulence-
solid
growth
media
containing
Congo
red
(or
itination,
hemin)
provides
asimple
and
rapid
indication of
lendence,
probable virulence in Y. enterocolitica.
Further-ling
that
more,unlike MOX
agar,which inhibits the
ing
assaygrowth
of, and therefore tends
toselect
against,
grownat
plasmid-bearing
Y.
enterocolitica,
CRAMP
agarapparent
permits the identification of bacterial colonies
rngo
red
containing
alarge
proportion
of
plasmid-bearing
not
clear,
cells.
ing
assayther than ACKNOWLEDGMENTS
Weare indebtedto I. Juhlin(UniversityofLund, Malmo, ed to be Sweden), H. J. Koornhof (Universityof the Witwatersrand, lasmid of Johannesburg, South Africa), S. Toma (Ontario Ministry of Health, Toronto, Ontario, Canada), K. Wachsmuth(Centers )
strains
for DiseaseControl, Atlanta,Ga.),andG. Wauters (Universi-edsubse- ty ofLouvain, Brussels,Belgium)forgenerouslyprovidingsed other the bacteria used in these investigations.
This research was supported in partby agrant from the
ported
to Australian National Health and MedicalResearch
Council.
encewas J. K.P. is the recipient of an Australian Public Service
ined,
the Postgraduate StudyAward. rweight.
not suffi- LITERATURECITED
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Differentiation
Between
Virulent
and
Avirulent
Yersinia
enterocolitica Isolates
by
Using Congo
Red
Agar
J. KAYAPRPIC, ROYM. ROBINS-BROWNE, ANDR. BRENTDAVEY Department ofMicrobiology, Universityof Melbourne, Parkville, Victoria,3052, Australia
Volume 18, no. 3, p. 486, abstract, line 2, and column 2, line 13: ". . . 5 ,g ofCongo red per ml . . ." should read ". . . 50 ,ug of