INFECTION AND IMMUNITY, Jan. 1991, p. 162-1670019-9567/91/010162-06$02.00/0Copyright C) 1991, American Society for Microbiology

Pathogenicity of Yersinia kristensenii for MiceROY M. ROBINS-BROWNE,'* SAM CIANCIOSI,l ANNE-MARIE BORDUN,1

AND GEORGES WAUTERS2

Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia,1and Unite de Microbiologie, Universite de Louvain, Brussels, Belgium2

Received 31 July 1990/Accepted 8 October 1990

Forty-seven strains of Yersinia kristensenii from widely differing sources, representing all known 0

serogroups of this species, were investigated for virulence with a variety of animal and in vitro assays.

Twenty-four (51%) of the isolates were lethal for mice pretreated with iron dextran. Mouse-lethal strainsoccurred predominantly within 0 serogroups 0:11, 0:12,25, and 0:16. Virulent Y. kristensenii strainsgenerally did not express the virulence-associated phenotype (Ca2" dependence and binding of Congo red andcrystal violet) which characterizes virulent strains of Y. enterocolitica, nor did they carry the Yersinia virulenceplasmid. Although all strains hybridized with a DNA probe derived from the inv (invasin) gene of Y.enterocolitica, none was able to invade HEp-2 epithelial cell culture. Y. kristensenii strains were virulent onlywhen inoculated parenterally into iron-loaded mice. Animals infected in this way succumbed rapidly toinfection, generally within 24 h. This finding suggested that the pathogenicity of these bacteria may beattributable to a secreted toxin, but a search for such a substance and for other in vitro correlates ofpathogenicity was unsuccessful. These observations indicate that some strains of Y. kristensenii kill mice by a

mechanism not previously recognized in yersiniae.

The genus Yersinia includes three species, Y. pestis, Y.pseudotuberculosis, and Y. enterocolitica, which are patho-genic for mammals. The virulence mechanisms of thesebacteria have been extensively studied and exhaustivelyreviewed (8, 17, 21). Briefly, the pathogenicity of yersiniae isdetermined by both chromosomal and plasmid-borne genes.

Chromosomal genes encoding virulence include inv and ail,which are believed to specify initial attachment to andinvasion of host tissues (17). In addition, all virulent strainsof Yersinia carry a 70-kb virulence plasmid which contrib-utes to the survival and multiplication of bacteria in hosttissues (8, 21). These plasmids share a considerable degreeof DNA homology, irrespective of the Yersinia species inwhich they originate (8, 25).Nonpathogenic biotypes of Y. enterocolitica and other

Yersinia species, such as Y. frederiksenii, Y. intermedia, andY. kristensenii, carry chromosomal DNA homologous withthe inv determinant but generally lack the ail and plasmid-borne determinants of virulence (16, 25). Nonetheless, some

of these species are occasionally implicated in disease (3-5,11, 18). During a recent investigation of virulence of yersin-iae for mice, we discovered a strain of Y. kristensenii whichcaused a fatal infection in iron-loaded mice. This observationprompted us to examine a collection of Y. kristensenii strainsfor virulence attributes.

MATERIALS AND METHODS

Bacteria. The 47 strains of Y. kristensenii used in thisinvestigation are listed in Table 1. The bacteria were isolatedin widely dispersed areas from a variety of sources. A totalof 15 strains were of human origin, 14 were from animals, 14were from food (usually pork), and 3 were from water. Theorigin of one strain was unknown. Y. enterocolitica 30.42.67and A2635, virulent strains of serogroups 0:3 and 0:8,

* Corresponding author.

respectively, have been described previously (25, 26). Y.enterocolitica A2635c is a plasmidless derivative of A2635.For virulence studies, bacteria were cultivated on Trypti-

case soy agar (BBL, Cockeysville, Md.) at 28°C overnight.Bacteria were suspended in isotonic phosphate buffer (pH7.4) to an optical density equal to a McFarland no. 3 bariumsulfate nephelometric standard (19). This suspension (equiv-alent to approximately 2 x 109 CFU/ml) was used toinoculate mice by gavage. For screening of virulence byintraperitoneal (i.p.) inoculation, the suspension was diluted1 in 50 in phosphate buffer. The number of viable bacteria ineach inoculum was determined by plating 10-fold dilutionson duplicate Trypticase soy agar plates.

Preparation of culture filtrates, killed bacteria, and ho-mogenates. Bacteria were grown on Trypticase soy agar at 28or 37°C for 48 h. Bacterial cells were removed by centrifu-gation. Culture supernatants were sterilized by filtrationthrough 0.2-pum-pore-size cellulose acetate filters (Schleicherand Schuell, Dassell, Germany) and tested immediately inmice. For some experiments, bacteria were killed by beingheated at 60°C for 5 min or by being incubated in 1%formaldehyde or 1% glutaraldehyde at room temperature for2 h. Cells killed in this way were washed three times inphosphate buffer and resuspended at an optical density equalto a McFarland no. 3 standard. Homogenates of bacteriawere prepared by ballistic disintegration in an MSK homog-enizer (B. Braun, Melsungen, Germany) set at 4,000 rpmwith 0.1-mm-diameter glass beads for 1 min under liquidCO2. This treatment reduced the number of viable bacteriaby approximately 90%. The homogenates were sterilized byfiltration. The sterility of each of these preparations was

verified before they were inoculated into animals.Virulence testing. Unless otherwise stated, virulence stud-

ies were performed with 6- to 8-week-old female BALB/cmice in groups of five. Twenty-four hours before infection,mice were given an i.p. injection of 5 mg of iron as irondextran (Imferon; Fisons Pty. Ltd., Sydney, Australia)and/or 5 mg of desferrioxamine B mesylate (Ciba Geigy Ltd.,Sydney, Australia) (26).

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PATHOGENICITY OF YERSINIA KRISTENSENII 163

TABLE 1. Characteristics of Yersinia strains used in this study

Result ofv~: Hybridization with

Strain Serogroup Source Country of Lethality'~ Invasion' probe from:origin Chromosome

CAD CV CR HYD (iC Plasmid

CDC1 0:11 Food United StatesIP105 0:11 Human urine DenmarkIP1420 0:11 Fox SwitzerlandIP841 0:11 Human bile United StatesP1223 0:11 Human wound DenmarkWA122 0:11 Human urine CzechoslovakiaWA32a/89 0:11 Rat JapanWA336 0:11 Carrots FranceWA396 0:11 Pork United StatesWE89/79 0:11 Human feces BelgiumWS60/88 0:11 Human feces BelgiumIP490 0:12,25 Hare FranceIP6048 0:12,25 Not known Not knownP1494 0:12,25 Pork CzechoslovakiaWATe167 0:12,25 Monkey JapanWATe168 0:12,25 Monkey JapanWE17/90 0:12,25 Human feces BelgiumGK11047 0:12,26 Rodent NorwayIP103 0:12,26 Sheep Faroe IslandsWATe169 0:12,26 Monkey JapanWATe171 0:12,26 Pork JapanWATe172 0:12,26 Pork JapanWATe173 0: 12,26 Pork JapanWATe174 0:12,26 Pork JapanWS45/89 0:12,26 Human feces BelgiumCDC2 0:16 Food United StatesWA948 0:16 Human feces FinlandWA31a/89 0:16,29 Rat JapanWA758 0:16,29 Human feces FinlandWAT120 0:16,29 Pork CanadaWE211/80 0:16,29 Human feces BelgiumGK9036 0:28,50 Rodent NorwayGKG2 0:28,50 Water NorwayIP1474 0:28,50 Water NorwayWA596 0:28,50 Pork NorwayIP7230 0:46 Mouse United KingdomIP7229 0:50 Rodent CzechoslovakiaIP7209 0:52 Human feces BelgiumWA599 0:59 Pork NorwayWA936 0:61e Water GermanyWA139 NTf Pig NetherlandsWA17/87 NT Human feces NetherlandsWA30a/89 NT Rodent JapanWA584 NT Human feces FinlandWA590 NT Human feces FinlandWA595 NT Pork NorwayWA987 NT Meat Austria30.42.67g 0:3 Human feces SwedenA26359 0:8 Milk United StatesA2635cg 0:8 Laboratory

+++++++++++

++++++

++++

++

+++

9d - _

+ + +

a For mice treated with iron and desferrioxamine and inoculated i.p. +, Lethal; -, not lethal.b CAD, Calcium dependence; CR, binding of Congo red; CV, binding of crystal violet; HYD, production of hydroxamate.c Results are the percentage of a bacterial inoculum recovered from HEp-2 cells after killing of the extracellular bacteria with gentamicin (16). ND, Not

determined.d Results uninterpretable because bacteria produced large and small colonies on calcium-deficient media at both 28 and 37°C.Provisional serotype designation.

f NT, Nontypeable.g Y. enterocolitica strains (all others are Y. kristensenii).

For most experiments, mice were inoculated i.p. with 0.5 mice were given 10-fold serial dilutions of bacteria. Theml of a bacterial suspension and observed for 14 days. In LD50 was calculated according to the method of Reed andsome experiments, bacteria were inoculated perorally (by Muench (23). The keratoconjunctivitis (Sereny) test wasgavage; 0.5 ml), subcutaneously (0.5 ml), or intravenously performed with guinea pigs pretreated with 50 mg of iron and(0.2 ml). For determination of the 50% lethal dose (LD50), 50 mg of desferrioxamine as described previously (26).

- 0.003- 0.005- 0.004- 0.002- 0.004- 0.001- 0.004- 0.003- 0.006- 0.002- 0.009- 0.007- 0.005- 0.007- 0.003- 0.003- 0.003- 0.010- 0.009- 0.002- 0.007- 0.004- 0.003- 0.006- 0.001+ 0.009+ 0.004+ 0.005+ 0.003+ 0.005- 0.001- 0.002- 0.005- 0.002- 0.004+ 0.003- 0.003+ 0.002- 0.005- 0.002- 0.007- 0.003- 0.004- 0.004- 0.005- 0.008- 0.004- 6.5- ND- 14.0

+

+++

++

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164 ROBINS-BROWNE ET AL.

Assay for enterotoxin. Bacteria were grown in Trypticasesoy broth supplemented with 0.6% (wt/vol) yeast extract(BBL) at 26°C for 48 h with constant shaking. Bacteria wereremoved by centrifugation, and 0.1 ml of the culture super-

natant was inoculated by gavage into each of three 3- to4-day-old infant mice. The presence of enterotoxic activitywas determined as described previously (24).Mouse protection studies. Mice in groups of six or more

were inoculated i.p. with various doses of live or killedbacteria. Three weeks or more later, one mouse from eachgroup was sacrificed by cervical dislocation. Cardiac blood,the entire spleen, and approximately half of the liver werecollected aseptically. The organs were homogenized in 5 mlof Trypticase soy broth, which was incubated at 28°C for 7days and then subcultured onto Trypticase soy agar plates.These studies were performed to ensure that no viablebacteria remained in the mice at the time of challenge. Theremaining animals in each group were inoculated i.p. with 5mg of iron dextran and desferrioxamine B. The next dayeach mouse was inoculated with approximately 100 LD50s ofa virulent Yersinia strain. The animals were observed for upto 14 days.

In vitro studies. 32P-labeled single-stranded DNA probeswere prepared from the cloned inv and ail chromosomalgenes of Y. enterocolitica 8081 (serogroup 0:8) and from thevirulence plasmid of Y. enterocolitica A2635 as describedpreviously (25). These probes were used to examine coloniesof Y. kristensenii for homologous DNA sequences at highstringency (25). The ability of bacteria to invade HEp-2 cellswas determined by the method of Miller et al. (16). Tests forcalcium dependence and binding of Congo red and crystalviolet were performed as described previously (22, 25). Theability of bacteria to produce hydroxamate siderophores wasdetermined by the hydroxylamine assay as described previ-ously (29).

RESULTS

Virulence of Y. kristensenii strains for mice. In a prelimi-nary study, we showed that the type strain of Y. kristensenii,IP105 (serogroup 0:11), was lethal when injected i.p. intomice pretreated with iron and desferrioxamine B. We sub-sequently investigated the pathogenicity of a wider range ofstrains in this animal model. The results showed that all 11serogroup 0:11 isolates in our collection were lethal for mice(Table 1). Other serogroups with a large proportion ofvirulent strains were 0:12,25 (five of six strains), 0:16 (twoof two strains), 0:16,29 (two of four strains), 0:59 (onestrain), and 0:61 (one strain) (Table 1). By contrast, 0

serogroups 0:12,26 (one of eight strains) and 0:28,50 (noneof four strains) were typically avirulent. Of seven strainswhose serogroup could not be determined, only one wasvirulent. The LD50s of i.p. inoculated Y. kristensenii for micegiven iron and desferrioxamine B were determined for threestrains of different serotypes. The values obtained rangedfrom 2 x 106 CFU for strain IP105 (serogroup 0:11) to 8 x106 for WE17/90 (0:12,25) and 6 x 107 for WA948 (0:16).There was no significant correlation between virulence formice and the origin of the bacteria. Of 15 strains isolatedfrom humans, 9 (60%) were virulent for mice, as were 6(43%) of 14 strains from animals and 8 (47%) of 17 strainsfrom food or water.Some inbred strains of mice are inherently susceptible to

infection with yersiniae (12). In case the virulence of Y.kristensenii was confined to BALB/c mice, we examined thesusceptibilities of two other mouse strains, CBA and C57BL/

TABLE 2. Effect of pretreatment with iron dextran anddesferrioxamine B on the susceptibility of mice to infection with

Yersinia species

LD50 (CFU) of:Pretreatment" Y. kristensenii Y. enterocolitica

IP105 30.42.67

None >5x 108 >5x 108Iron 1x107 3x107Desferrioxamine B >5 x 108 2 x 101Iron and desferrioxamine B 2 x 106 <10

a Mice were inoculated i.p. with 5 mg of iron as iron dextran and/or 5 mg ofdesferrioxamine B 24 h before i.p. inoculation with bacteria.

10, to infection with Y. kristensenii IP105 (0:11) and CDC2(0:16). The results showed that all three mouse strains wereequally susceptible to infection with these bacteria (data notshown).

Effect of pretreating mice with iron and desferrioxamine B.We have demonstrated previously that the susceptibility ofmice to infection with Y. enterocolitica is enhanced ifanimals are preinoculated with iron or, especially, with thehydroxamate siderophore desferrioxamine B (26). In a sim-ilar fashion, only mice preinoculated with iron dextran weresusceptible to infection with Y. kristensenii. Mice given bothiron and desferrioxamine were more susceptible to infectionthan those given iron alone, but the differences were not aspronounced as those noted with respect to Y. enterocolitica(Table 2). Mice pretreated with 20 mg of dextran 40 (Phar-macia AB, Uppsala, Sweden) and those given desferrioxam-ine B alone were resistant to the lethal effects of infection.

Influence of the route of infection. Une and Brubaker haveshown that the susceptibility of mice to infection withattenuated mutants of Y. pestis and Y. pseudotuberculosisvaries according to the route of infection (30). In the presentstudy, mice were susceptible to infection with Y. kristenseniiIP105 only when bacteria were administered parenterally.Animals infected by gavage were entirely resistant to infec-tion even when pretreated with iron and desferrioxamine B.LD50s (in CFU) for routes of infection were as follows:peroral (by gavage), >2 x 109; i.p., 3 x 106; intravenous, 8x 106; and subcutaneous, 5 x 108. Animals not pretreatedwith iron were resistant to the lethal effects of Y. kristenseniiregardless of the route by which the bacteria were adminis-tered (data not shown).

Kinetics of infection. During the course of these studies,we noted that mice which survived infection with Y. kris-tensenii beyond 2 days almost always recovered completely.This appeared to differ from what we had previously ob-served with respect to Y. enterocolitica. We therefore un-dertook a study in which mice pretreated with iron dextranwere inoculated i.p. with approximately five LD50s of Y.kristensenii IP105 or Y. enterocolitica 30.42.67 organisms.The results demonstrated that mice given a lethal dose of Y.kristensenii succumbed more rapidly to infection than thosegiven an equivalent number of Y. enterocolitica organisms(Fig. 1).

Effects of culture filtrates, killed bacteria, and bacterialhomogenates. Twenty-four hours after receiving an i.p. inoc-ulation of a sterile culture filtrate, bacterial homogenate, or akilled preparation of Y. kristensenii, mice were hunched,lethargic, and reluctant to feed. These signs gradually im-proved over the next 72 h, by which time the animalsappeared to have recovered completely. The effects resem-

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PATHOGENICITY OF YERSINIA KRISTENSENII 165

100 -

80 -

X 60-0E

0

. 40 -

E

20

0

FIG. 1. Cumuenii and Y. enterwith 5 mg of ironi.p. with approxi0:11) or Y. enterwere observed ft

bled those in ncharide, and eflmouse-lethal sithose obtainedstrains (IP1474filtrates were ufor 5 min and Mwere heated atmice died as a

preparations of killed bacteria contained as much as theequivalent of 200 times the LD50 of living yersiniae.

Assay for enterotoxin. Some strains of Y. kristenseniiproduce a heat-stable enterotoxin which is reactive in infantmice (13). Because this substance may conceivably be avirulence determinant of Y. kristensenii, we examined the

0f>' ability of 10 strains (5 mouse lethal and 5 nonlethal, from a

range of serogroups) to produce this substance. The resultsshowed that only one strain, GK9036 (0:28,50), produced anenterotoxin which was reactive in infant mice. This strain isnot virulent in adult iron-treated mice. By contrast, none ofthe five mouse-lethal strains investigated (CDC1, CDC2,IP105, WA758, and WE17/90) was positive in this assay.Mouse protection experiments. In order to determine

whether mouse-lethal strains of Y. kristensenii possessed* Y. kristensenii common protective antigens, we inoculated mice with livingo Y. enterocolitica or killed Y. kristensenii organisms according to various

schedules and examined their susceptibilities to subsequentinfection with Y. kristensenii of homologous and heterolo-gous serogroups and to Y. enterocolitica. The results

' ' ' ' ' showed that prior inoculation with Y. kristensenii conferred0 1 2 3 4 5 6 7 solid protection against infection with bacteria of the same or

Days after inoculation a related 0 serogroup (Table 3). Prior infection with a strainof an unrelated serogroup (irrespective of virulence) pro-

rliative mortality mice infected with Y. kristens- vided partial protection against lethal infection with Y.

idextran. Micefollowingroups were injecultedi kristensenii. Similar degrees of protection were conferred byl dextran. The following day, they were inoculated

mately 5 LD50s ofY. kristensenii*P105 (serogroup sterile culture filtrates of virulent or avirulent Y. kristensenii*ocolitica 30.42.67 (serogroup 0:3) organisms. Mice and by 25 p.g of purified lipopolysaccharide derived fromDr 14 days, but no deaths occurred after day 6. Escherichia coli (Sigma Chemical Co., St. Louis, Mo.) (data

not shown). Infection with Y. kristensenii even afforded micepartial protection against infection with Y. enterocolitica(Table 3).

nice given sublethal amounts of lipopolysac- Guinea pig keratoconjunctivitis test. Four mouse-virulentFects obtained with preparations derived from strains of Y. kristensenii of different serogroups were exam-trains (IP105 and WE17/90) were similar to ined in this assay. None gave a positive reaction.I with preparations derived from nonlethal In vitro studies. The results of these investigations areand WS45/89). The effects of the culture summarized in Table 1. Almost all of the Y. kristensenii

indiminished when preparations were boiled isolates were negative in the assays for calcium dependencevere only partially reduced when preparations and binding of Congo red and crystal violet. The few strains121°C for 5 min (data not shown). None of the which did give a positive reaction in one of these assaysresult of these experiments, even though the included both mouse-virulent and avirulent varieties.

TABLE 3. Effect of prior exposure to Y. kristensenii on the susceptibility of mice to subsequent infection with Yersinia species

Strain (serogroup) No. of Intervalc Strain (serogroup) Potectiveused for initial CFU/doseb doses (wk) used for subsequentexposureadss(k infection' efficacy (%)

IP105 (0:11) 5 x 107 (1) 1 3 IP105 (0:11) 100IP105 (0:11) 5 x 105 (1) 1 12 IP105 (0:11) 100IP105 (0:11) 5 x 107 (1) 1 3 IP841 (0:11) 100IP105 (0:11) 5 x 107 (1) 1 3 WE17/90 (0:12,25) 40IP105 (0:11) 5 x 107 (1) 1 3 30.42.67 (0:3}f 0IP105 (0:11) 5 x 107 (1) 39 3 30.42.67 (0:3)f 60IP105 (0:11) 5 x 107 (1 ) 3 3 WE17/90 (0:12,25) 60IP105 (0:11) 2 x 108 (g) 1 3 IP105 (0:11) 100IP105 (0:11) 2 x 108 (h) 1 3 IP105 (0:11) 100WATe167 (0:12,25) 3 x 107 (1) 1 3 WE17/90 (0:12,25) 100WS45/89 (0:12,26) 4 x 107 (1) 1 3 WE17/90 (0:12,25) 100WS45/89 (0:12,26) 4 x 107 (1 ) 3 3 IP105 (0:11) 40

Given i.p. to mice not pretreated with iron or desferrioxamine B.b 1, Living bacteria; g, glutaraldehyde-killed; h, heat-killed.c Between the final dose of the initial exposure and subsequent challenge.d Approximately 100 LD50s were given i.p. to mice pretreated with iron and desferrioxamine B.' Determined from the following equation: [(mortality rate in control mice - mortality rate in test mice)/mortality rate in control mice] x 100.f Virulent strain of Y. enterocolitica.g Given at weekly intervals.

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166 ROBINS-BROWNE ET AL.

We have previously shown that Y. kristensenii may pro-duce the hydroxamate siderophore aerobactin (29). In casethe production of such substances was associated withvirulence, we examined all the bacteria for their ability tosynthesize hydroxamates under iron-limiting conditions.The results showed that production of these compounds waslimited to bacteria in serogroups 0:16, 0:16,29, 0:46, and0:52 (Table 1). Thus, the production of hydroxamates wasnot associated with virulence.The DNA hybridization studies showed that the Inv probe

of Y. enterocolitica recognized all the bacteria in this study(data not shown). This result was not unexpected, becausethis probe detects all members of the genus Yersinia, regard-less of virulence (20, 25). The HEp-2 invasion assay, how-ever, showed that none of the Y. kristensenii strains pro-duced active invasin, as they all invaded HEp-2 cells to anegligible extent compared with strains of Y. enterocoliticawhich are known to express this gene (Table 1). Only onestrain (IP7229) hybridized with the Ail probe of Y. entero-colitica, but this strain did not invade HEp-2 cells and wasavirulent for mice. None of the Y. kristensenii strains exam-ined hybridized with any of three DNA probes (BamHIfragments 2, 6, and 7) prepared from widely dispersedregions of the virulence plasmid of Y. enterocolitica A2635(25).

DISCUSSION

Before 1980, Y. kristensenii was considered to be part of abroad Y. enterocolitica complex. It is distinguishable fromY. enterocolitica sensu stricto and other yersiniae, however,on the bases ofDNA relatedness and biochemical reactivity,being positive for trehalose and ornithine decarboxylase andnegative for sucrose and rhamnose (1, 2).

Little is known of the epidemiology of infections with thisorganism. In surveys of fecal carriage of yersiniae in hu-mans, Y. kristensenii is found infrequently compared withother yersiniae (6, 10, 14, 28, 32). Nonetheless, when it hasbeen recovered from human material, it generally has beenregarded to be of clinical significance (3-5, 11, 18). As far ascan be ascertained, these strains usually belong to those 0serogroups which are virulent for mice (3-5). The isolation ofY. kristensenii from the feces of a young woman with acuteenteritis, in conjunction with detectable serum agglutinins,prompted Bottone to speculate that Y. kristensenii is inter-mediate in virulence between Y. enterocolitica on the onehand and Y. frederiksenii and Y. intermedia on the other (4).This suggestion has been borne out by the present study, inwhich the virulence of Y. kristensenii for animals has beenshown for the first time.Although Y. kristensenii shares few of the phenotypic or

genetic characteristics associated with virulence in Y. en-terocolitica or other Yersinia species (Table 1), the lethaleffect of Y. kristensenii for mice was specific. This wasevidenced by the findings that only certain serogroups werevirulent in this model and that solid serogroup-specificimmunity was induced after a single exposure to a sublethaldose of a homologous strain (Table 3). Moreover, we havepreviously examined several hundred isolates of Yersiniaspecies, including tissue culture-invasive, plasmid-curedstrains of Y. enterocolitica and Y. pseudotuberculosis, iniron- and desferrioxamine-treated mice and, until now, havefound none that gave a false-positive result (22, 25).The mechanisms underlying the pathogenicity of Y. kris-

tensenii for mice are not clear. The short time course of theillness compared with that caused by Y. enterocolitica

suggested that a bacterial toxin may be involved. In order toexamine this possibility, we inoculated bacterial lysates,culture filtrates, and whole killed organisms into mice. Noneof these preparations was lethal, suggesting that bacterialmultiplication in vivo was required for Y. kristensenii toexpress virulence. Additional support for this suggestioncame from animal protection experiments in which priorexposure to Y. kristensenii WS45/89, a nonlethal serogroup0:12,26 strain which was presumed to lack putative viru-lence determinants, conferred solid immunity against infec-tion with WE17/90, a virulent strain of serogroup 0:12,25(Table 3). The immunity in this instance seemed likely to bemediated by antibodies to the shared 0:12 antigen. On theother hand, exposure to a virulent or avirulent strain of Y.kristensenii afforded only partial protection against subse-quent infection with a virulent strain of an unrelated sero-group (Table 3). Similar degrees of protection were con-ferred by prior exposure to sterile culture filtrates of Y.kristensenii, heat-treated filtrates, and purified endotoxinfrom E. coli (data not shown), indicating that this nonspecificimmunity was likely to be mediated by lipopolysaccharide.Nonspecific endotoxin-mediated immunity of this type to avariety of infectious agents is well documented (7, 31).New evidence that Y. kristensenii may share a unique

virulence determinant with Y. enterocolitica has emergedfrom a recent study by Delor et al., who used a DNA probederived from the chromosomal gene for the heat-stableenterotoxin of Y. enterocolitica to examine a heterogeneouscollection of Yersinia species (9). They found that the probehybridized specifically with DNA from virulent strains of Y.enterocolitica and selected Y. kristensenii isolates but notwith DNA from other strains (9). In the present study,however, there was no correlation between the ability of Y.kristensenii strains to produce enterotoxin and their viru-lence for iron-stressed mice.Although Y. kristensenii is frequently isolated from ani-

mals, it does not appear to be a natural pathogen of mice.Peroral administration of large numbers of bacteria to iron-and desferrioxamine-treated mice caused no ill effects, pre-sumably because the bacteria were unable to penetrate theintestinal mucosa. This suggestion was corroborated byexperiments which demonstrated the inability of Y. kristens-enii strains to invade HEp-2 cell culture (Table 1) or theconjunctival epithelium of guinea pigs. By contrast, Y.enterocolitica strains, which are virulent for mice by the oralroute, readily invade HEp-2 monolayers. The observationthat Y. kristensenii strains do not invade cell culture despitebearing DNA homologous to the inv locus of Y. enteroco-litica indicates that the inv gene in these bacteria is nonfunc-tional, as is the case with nonpathogenic strains of Y.enterocolitica (20). One strain in this study, IP7229, evi-dently also carried a nonfunctional ail gene.

Y. kristensenii was pathogenic for mice only when admin-istered parenterally, suggesting that under normal circum-stances blood-sucking arthropods, such as fleas, may trans-mit these bacteria. Two factors argue against this possibility,however: (i) Y. kristensenii has seldom been isolated fromarthropods, and (ii) Y. kristensenii caused no illness and didnot persist in the tissues of mice not pretreated with iron(data not shown).The precise mechanism whereby treatment with iron

increases the susceptibility of mice to infection with yersin-iae is unresolved. Possible explanations include the supplyof an essential, growth-limiting nutrient to the bacteria or theimpairment of immune responsiveness of the host (27). Afinding analogous to that reported here is the observation

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PATHOGENICITY OF YERSINIA KRISTENSENII 167

that certain mutants of Y. pestis are lethal for iron-stressedmice only when given by i.p. injection (15). These findingssuggest that iron overload may interfere with a critical hostdefense mechanism, such as macrophage effectiveness, inthe peritoneal cavity.

In terms of iron responsiveness, Y. kristensenii behavedsimilarly to Y. enterocolitica (Table 2). An important differ-ence between these two species, however, concerned theeffect of desferrioxamine B, which markedly enhanced thesusceptibility of mice to infection with Y. enterocolitica buthad only a moderate effect on the pathogenicity of Y.kristensenii, and then only when administered together withiron (Table 2). Of interest in this regard is the finding thatalthough some strains of Y. kristensenii produced an iron-regulated hydroxamate siderophore, this property did notcorrelate with virulence (Table 1).

In summary, this study has shown that Y. kristensenii ispathogenic for iron-treated mice. The mechanisms of viru-lence of this species are obscure but are clearly differentfrom those which operate in other species of Yersinia.Further studies are required to characterize the virulencedeterminants of Y. kristensenii.

ACKNOWLEDGMENTS

We are grateful to Vicki Bennett-Wood for assistance with cellculture; to Stanley Falkow and Virginia Miller, Stanford University,for providing the Inv and Ail probes; and to Simon Stuart, LaTrobeUniversity, for helpful discussion.

This study was supported by a research grant from the AustralianNational Health and Medical Research Council.

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