Relationship of Hela Cell Infectivity to Biochemical, Serological, and Virulence Characteristics of Yersinia Enterocolitica D

INFECTION AND IMMUNITY, Feb. 1982, p. 497-506 Vol. 35, No. 2 0019-9567/82/020497-10$02.00/0

Relationship of HeLa Cell Infectivity to Biochemical, Serological, and Virulence Characteristics of Yersinia enterocolitica D. A. SCHIEMANNt* AND J. A. DEVENISH Environmental Bacteriology Laboratory, Ontario Ministry ofHealth, Toronto, Ontario M5W IRS, Canada Received 26 May 1981/Accepted 13 October 1981 Examination of 69 strains of Yersinia enterocolitica which represented 20 serotypes and nontypable isolates for HeLa cell infectivity by a roller tube technique that provided a quantitative index of infection showed that infectivity (index, >3.50) was confined to strains of serotypes 0:8, 0:3, 0:5,27, 0:9, 0:1, 0:1,2,3, 0:2,3, 0:4,32, and 0:21. All strains that were HeLa positive were sucrose positive and negative for salicin, esculin, rhamnose, raffinose, melibiose, a-methylglucoside, and citrate. All HeLa-negative strains were either sucrose and salicin positive or were sucrose negative. Twenty-one strains were examined for virulence by the ability to produce guinea pig conjunctivitis and diarrhea in mice. Positive strains were limited to those that were HeLa positive and were autoagglutination positive and calcium dependent at 35°C. There was no associa- tion between virulence and the ability to produce enterotoxin measured by the infant mouse assay. Loss of autoagglutinability and calcium dependency was accompanied by loss of virulence, but HeLa cell infectivity was unchanged. The results suggest that at least two properties are necessary for virulence: the presence ofthe V and W antigens, mediated by the same plasmid as autoagglutination and calcium dependency, and an invasive factor demonstrated in vitro by HeLa cell infectivity. These virulence properties are found in only certain biotypes of Y. enterocolitica.

A number ofdifferent laboratory animals have that were negative in rhamnose, salicin, and been studied as potential models for evaluating esculin reactions. Une (32) found that HeLa cell the pathogenic potential and virulence of Yer- penetration by Y. enterocolitica correlated with sinia enterocolitica, including rabbits (19, 21, the ability to produce enterocolitis in rabbits. 31), mice (1, 18, 19, 26), guinea pigs (19, 25), and Une et al. (33) observed that HeLa cell infectiv- monkeys (17). It now is apparent that some of ity was limited to strains of Y. enterocolitica the earlier disagreements in results, such as the representing serotypes 0:3, 0:5B, 0:8, and 0:9. essentially negative findings by Mollaret and Pedersen et al. (22) also observed that HeLa cell Guillon (19) in their comprehensive study fol- invasiveness was limited to certain serotypes of lowed by positive results with mice reported by Y. enterocolitica that were always negative for Quan et al. (24) and Carter et al. (9) and with salicin and esculin, and that strains that were gerbils by Wetzler et al. (T. F. Wetzler, M. L. positive for these reactions or were sucrose French, and J. A. Tomas, Bacteriol. Proc. abstr. negative (serotypes 0:11 and 0:12) were nonin- M46, p. 73, 1968), were the result of working vasive. with different serotypes that also differed in Zink et al. (36) first reported that the invasive- pathogenic characteristics and were possibly ness of Y. enterocolitica, as demonstrated by the due to a fundamental property of virulence in Y. Sereny test, was dependent on the presence of a enterocolitica that is dependent on the presence 41-megadalton plasmid. Gemski et al. (12) sub- of a plasmid (36). sequently reported that virulence measured by In addition to laboratory animals, in vitro the Sereny test or mouse lethality after intraperi- tissue culture systems have been used for study- toneal or oral challenge was dependent on the ing the invasive capability of Y. enterocolitica. presence of a 42.2 + 1.1-megadalton plasmid. Lee et al. (16) reported that the ability to invade The same plasmid coded for a calcium require- HeLa cells was limited to certain clinical strains ment for growth at 37°C, which was determined t Present address: Department of Microbiology, Montana with the magnesium oxalate (MOX) agar devel- State University, Bozeman, MT 59717. oped by Higuchi and Smith for identifying viru- 497 498 SCHIEMANN AND DEVENISH INFECT. IMMUN. lent strains of Yersinia pestis (13). Carter et al. Kligler iron agar (alkaline over acid without gas or H2S), (10) showed that the virulence of Y. enterocoli- were positive for urease (Christensen's urea agar), tica in mice was related to the ability to produce ornithine decarboxylase, and fermentation of treha- V and W antigens which were immunologically lose, and were negative for arginine decarboxylase and identical to virulence antigens previously report- lysine decarboxylase. All strains were o-nitrophenyl- P-D-galactopyranoside positive (except one of sero- ed in Y. pestis and Yersinia pseudotuberculosis. type 0:8), fermented sorbitol, and fermented sorbose Laird and Cavanaugh (15) reported that viru- (except two nontypable strains). All strains gave a lence of Y. enterocolitica measured by lethality positive Voges-Proskauer test, except one culture or development of diarrhea (or both) in mice each of serotypes 0:4,32 and 0:4,33 and three cul- after oral challenge was related to their ability to tures, one each of serotypes 0:16, 0:12,26, and a autoagglutinate in a tissue culture medium at nontypable isolate. 36°C. We have previously reported that viru- Biotyping was done by the methods of Knapp and lence measured by gerbil lethality or guinea pig Thal (14), Nilehn (20), Wauters (G. Wauters, Ph.D. thesis, Vander, Louvain, Belgium) and Brenner et al. conjunctivitis was related to calcium dependen- (6). To describe all variations observed from the cy at 35°C (29). Schiemann et al. (30) described biotypes as originally defined, subtypes designated by the correlation of calcium dependency with au- a" in Table 1 were added. Reactions included in toagglutination (AA) and the ability to produce these schemes which were either not done (nitrate both guinea pig conjunctivitis (GPC) and mouse reduction) or were positive for all strains (trehalose, diarrhea (MD). These virulence characteristics ornithine decarboxylase) are not listed in Table 1. The were observed in some serotypes of Y. enteroco- results for the lactose OF test, which is used in the litica that are infrequently associated with ill- Nilehn and Wauters biotyping schemes, are also not ness in humans. shown because the reactions obtained were frequently contradictory with all other tests specified for a partic- We applied a new HeLa infectivity test proce- ular biotype. dure (11) to a group of Y. enterocolitica cultures The "observed" biotyping scheme presented in for the purpose of describing the correlation of Table 1 was developed to accommodate all of the this virulence indicator with serotype and bio- biochemical profiles represented among the 69 test type. A smaller group of strains was then select- strains, to group them according to common biochemi- ed for determining the correlations between var- cal profiles, and to divide the strains on the basis of ious in vitro and in vivo tests for virulence. virulence characteristics. HeLa cell infection. HeLa cells were maintained in MATERIALS AND METHODS minimal essential medium (Eagle) with Earle salts and Bacterial cultures. Cultures of Y. enterocolitica were L-glutamine plus 10% fetal bovine serum and 50 IU of obtained from these sources: (i) S. Toma, Canadian penicillin G and 50 ,ug of streptomycin per ml. Cell National Reference Service for Yersinia, Toronto, cultures were incubated at 35°C under 5% carbon Ontario; (ii) E. Christenson, State Laboratory of Hy- dioxide. Culture medium was replaced 24 h before giene, Madison, Wis.; (iii) M. Bissett, California State harvesting of the cells. Washed cells were recovered in Department of Health, Berkeley, Calif.; and (iv) T. antibiotic-free minimal essential medium (Eagle) with Wetzler, University of Washington, Seattle, Wash. A modified Earle salts and L-glutamine for suspension few of the cultures were isolated in our own labora- culture. The density of this suspension was deter- tory. All cultures were originally isolated from hu- mined by microscopic counting with a Fuchs-Rosen- mans, with the exception of one water isolate of thal chamber. The density was adjusted to 2.5 x 105 serotype 0:21. Serotypes were determined by slide cells per ml, and 2.0 ml of this suspension was added agglutination using 34 antisera prepared against somat- to plastic 16- by 125-mm tissue culture tubes. ic antigens in rabbits. The density of the bacterial suspension was estimat- Stock cultures were maintained in Trypticase soy ed turbidimetrically (Spectrophotometer 21; Bausch & agar (BBL Microbiology Systems) stabs without sub- Lomb, Inc., 540 nm) and then adjusted to provide 5 x culture. Cultures selected for study were prepared 106 cells in 0.25 ml added to each of three tissue from either stabs or a slant previously prepared from a culture tubes containing HeLa cells. This gave a single colony obtained by subculture of the Trypticase theoretical multiplicity (i.e., ratio of bacteria to HeLa soy agar stab. A single colony was used to prepare a cells) of 10. A colony count was completed on the cell suspension that was then used to inoculate the bacterial suspension to determine the true multiplicity. entire surface of a Trypticase soy agar-0.6% yeast The average multiplicity for all test strains using this extract (pH 7.6) plate. After incubation at 22°C for 48 h technique was 11.9 (n, 69; standard error, 0.35). the growth was washed off with phosphate-buffered The prepared tubes were placed on a roller appara- saline (pH 7.2), and this suspension was used for tus and incubated at 350C for 30 min. After this completing all further tests. infection period, 0.1 ml of gentamicin solution (2.5 mg/ All cultures were examined for 25 different biochem- ml) was added to each tube, and then the tubes were ical characteristics sufficient to confirm identification incubated for 1 h under the same conditions. After this and for the purpose of biotyping. Carbohydrates were period, which provided for destruction of extracellular observed for 7 days, and amino acids and citrate were bacteria, the tube was centrifuged (6,000 rpm, 10 min, observed for 4 days. Indole was measured after 1, 2, 4°C) and the supernatant was removed. A 1.0% solu- and 4 days of incubation at 30°C, and the Voges- tion of Tergitol 7 in phosphate-buffered saline (2.5 ml) Proskauer test was completed after 2 and 4 days of warmed to 37°C was added to each tube. The tubes incubation at 22°C. All test strains were typical on were sonicated (200 W, 30 s), and then a sample from VOL. 35, 1982 HeLa CELL INFECTIVITY OF Y. ENTEROCOLITICA 499 each of three tubes was composited. A series of suspension averaged 6.5 x 108 (standard error, 3.5 x dilutions of this composite were plated on triplicate 107). Two mice placed in separate cages were used for plates of Trypticase soy agar4.6% yeast extract each test organism. After 24 h the inoculated peptone which were incubated overnight at 32°C. The average solution was replaced with fresh drinking water. At 2, colony count was determined and used to calculate the 5, 9, and 14 days the mice were placed for about 2 h in density of viable bacteria in the composite from three a jar containing a screen supported over a piece of reaction tubes. The infectivity index was expressed as filter paper. The recovered feces were examined for the logarithm of this viable count. We have described evidence of diarrhea on the basis of consistency, in a previous paper the interpretation of the infectivity shape, color, and fluid and mucous content compared index with respect to gentamicin susceptibility of the with feces froin uninfected animals. Diarrhea was test organism and also presented data on the reproduc- recorded as present only when there were distinctive ibility of the roller tube system (11). changes in the feces as compared with normal feces. AA. The test for AA was completed by inoculating The feces were transferred to a tared tube containing 5 duplicate tissue culture tubes containing 2 ml of mini- ml of phosphate-buffered saline, and appropriate dilu- mal essential medium with Earle salts and L-gluta- tions were prepared for a colony count on CIN agar. mine, plus 10%o fetal bovine serum with approximately Identification of recovered organisms was confirmed 106 cells of the test organism. One tube was incubated by typical colonial appearance on CIN agar and agglu- at 35°C, and the other was incubated at 22°C, for about tination by specific antiserum for typable strains. In 20 h. A positive test (AA') consisted of agglutinated most cases the diarrhea was very obvious, and many bacteria along the lower wall and bottom of the tube times it was so severe that the feces consisted only of a incubated at 35°C with a nearly clear medium above in watery, light-colored mucoid material. contrast to a nearly uniform turbidity in the tube at Infant mouse assay. The ability to produce entero- 22°C. In some instances the tube at 22°C also showed toxin was determined by inoculating 25 ml of Trypti- some cell agglutination but not to the same degree case soy-0.6% yeast extract broth contained in a observed at 35°C and with less clearing of the medium. 125-ml baffle flask with an initial cell density of 103 to A few cultures produced clumped growth at 35°C that 104 per ml. After incubation at 22°C for 48 h on a was clearly distinguishable in nature from true AA. rotary shaker (240 rpm) the broth was centrifuged Gentle shaking of the tubes was useful in distinguish- (4°C) at 10,000 rpm for 30 min. The supernatant was ing AA from clumped or settled cells. passed through a 0.45-,um membrane filter and frozen Calcium dependency. Culture suspensions were di- at -20°C until testing. luted to provide about 100 cells in the inoculum added Two drops of 2% Evans blue was added to 1 ml of to duplicate plates of MOX agar (13). The absence of culture filtrate, and 0.1 ml of this mixture was injected growth on MOX agar after incubation at 35°C for about into the stomach of each of three or four mice which 20 h and growth on Trypticase soy agar-0.6% yeast were 2 to 4 days old. After 4 h at 22°C the mice were extract control plates inoculated in the same manner sacrificed by chloroform exposure, and the intestines and incubated at 22°C for 48 h (MOX-) was interpret- were removed. The infant mouse ratio was calculated ed as evidence of calcium dependence. Occasionally from the weight of the combined intestines to the the MOX agar plate would show the presence of a few weight of the combined carcasses. A ratio greater than fully developed colonies or barely visible pinpoint 0.083 was taken as a positive test for enterotoxin. colonies, which was still interpreted as evidence of calcium dependency for the test organism. Cultures which were not calcium dependent at 35°C produced RESULTS large colonies equivalent in number to those obtained on Trypticase soy agar-0.6% yeast extract control The distribution of HeLa cell infectivity in- plates. dexes for each serotype represented among 69 GPC. Ten microliters of the initial bacterial suspen- test strains is shown in Fig. 1. There is a clear sion was added to one eye of two different guinea pigs separation at the index value of 3.5. Conse- which were 6 to 7 weeks old. This volume of the quently, this index was chosen as the dividing bacterial suspension provided an average dose of 1.3 value between HeLa-positive (HeLa+) and x 109 (standard error = 6.9 x 107) cells. The animals HeLa- cultures. Figure 1 also shows that for were observed for 7 days for signs of inflammation and swelling of the conjunctivae, eyeball depression, fluid most serotypes all the test strains were either and mucous accumulation, and clouding of the cornea HeLa+ or were HeLa-, but that two serotypes, determined by examination of the pupil and iris. If 0:4,32 and 0:21, included both HeLa+ and symptoms were present at 2 days or thereafter the eye HeLa- strains. was cultured by swabbing. A positive reaction (i.e., Tables 2 and 3 present a description of the 69 conjunctivitis; GPC+) was reported only when there test strains examined for HeLa cell infectivity were obvious symptoms which persisted after the first according to their serotype and biotype. The day and when a significant number of the infecting data demonstrate how the strains that represent- organism could be recovered at 2 days or thereafter. ed a particular serotype divided All recovered organisms were identified by appear- themselves by ance on CIN agar (27) and agglutination with specific biotype and HeLa cell infectivity. For example, antiserum if available. five strains of serotype 0:8 were examined, and MD. A 0.1-ml sample of the bacterial suspension all gave positive HeLa results. Biochemically was added to 100 ml of a 0.1% peptone solution which these strains represented Knapp and Thal type was then provided to a mouse who had been deprived 2, Nilehn types 2 (4 strains) and 2a (1 strain, a- of drinking water for 24 h. The cell density in this nitrophenyl-p-D-galactopyranoside negative), 500 SCHIEMANN AND DEVENISH INFECT. IMMUN. TABLE 1. Biotypes of Knapp and Thal (14) Nilehn (20) Reaction (°C) 1 la 2 2a 3 3a 1 la 2 2a 3 3a 4 Indole (30) - - + + + + + + + + - - - Xylose (22) vd + + + + + + + + + + + - Salicin (35) - - - v + + + + - - - - - Esculin (22) - - - - + + + v - - - - - Sorbitol (35) + v + + + + + Voges-Proskauer (22) + v + v + - + ONPG' (35 or 22) + + + v + + + Sucrose (22) + - + v + + + v + v + v + Sorbose (35) + + + + + v + Lecithinase (22) Rhamnose (22) - - - - - + Raffinose (22) Mellibiose (22) a-Methylglucoside (22) Citrate (22) a Types identified with "a" represent varieties observed that differ from original description. See text regarding reactions which are not shown. b ( ), Delayed 2 to 4 days. C +, Most strains positive; +, most strains negative. d V, + or - reactions. e ONPG, o-Nitrophenyl-,-D-galactopyranoside.

Wauters types 1 (4 strains) and la (1 strain, c- of Wauter's six biotypes. HeLa- cultures were nitrophenyl-3-D-galactopyranoside negative), predominantly biotype 1, but were also found in Brenner et al. type 1, and observed type I. biotypes la, 2, 3, and 3a. All HeLa+ cultures HeLa-positive strains were confined to sero- were Brenner et al. biotype 1, but so were the types 0:8, 0:3, 0:5,27, 0:9, 0:1, 0:1,2,3, majority of the HeLa- strains. Consequently, 0:2,3, 0:4,32, and 0:21. none of these four biotyping schemes provided a All cultures examined for HeLa infectivity basis for predicting HeLa infectivity. were also tested for AA. AA+ strains were The observed biotypes were based on a larger found only among HeLa+ strains, however, combination of biochemical tests that are com- some HeLa+ strains were AA-. Isogenic pairs mon to other typing schemes. These biotypes for AA were selected from one strain of serotype were developed primarily for separation of viru- 0:8. The AA- culture was MOX+ at 35°C, but lent and avirulent strains. The definition of these the AA+ culture was not, indicating that the biotypes also considered the most common pro- plasmid that codes for calcium dependency at files that were presented, especially when the 35°C (12) also codes for AA, and that this profile was stable within a certain serotype. For plasmid was present in one and absent in the example, all strains of serotype 0:8 are biotype other culture of this isogenic pair. Both cultures I, all strains of serotype 0:3 are biotype II, and were tested for HeLa cell infectivity; one gave all strains of serotype 0:5,27 are biotype III. an index of 4.61, and the other gave an index of Strains of biotype III resemble II, except some 4.73. This demonstrated that HeLa cell infectiv- show delayed indole production and are xylose ity is not altered with loss of the plasmid coding positive. Biochemical variation is rare among for AA and calcium dependency. The data in strains within any of these three serotypes, Table 4 further support this finding in that especially when isolated from humans. In con- HeLa+ cultures of serotypes 0:8, 0:3, and trast, two serotypes included strains which dif- 0:5,27 are sometimes AA- MOX+ and avirulent fered in biotype (0:4,32 and 0:21). Some strains and at other times are AA+ MOX- and virulent. of serotype 0:4,32 are biotype I (HeLa+), and All HeLa+ cultures were biochemically de- others are biotype IV (HeLa-). Some strains of scribed by Knapp and Thal biotype 1 or 2. serotype 0:21 are biotype III (HeLa+), and However, four other biotype 1 cultures were others are biotype IV (HeLa-). Nontypable HeLa-. All cultures of Knapp and Thal biotypes strains presented a variety of biotypes all falling la, 2a, 3, and 3a were HeLa-. HeLa+ cultures in groups IV, V, or VI. There were some sero- were found in Nilehn biotypes 2, 2a, 3, and 4. types that presented a uniform biochemical pro- Nilehn biotype 2a also included HeLa- strains. file and were consistently HeLa-, for example, Nilehn biotype 1 and la strains were always eight cultures of serotype 0:5 (Knapp and Thal HeLa-. HeLa+ cultures were found among all '3, Nilehn 1, Wauters 1, Brenner 1, observed V), VOL. 35, 1982 HeLa CELL INFECTIVITY OF Y. ENTEROCOLITICA 501 Y. enterocoliticaa Wauters (Ph.D. thesis) Brenner et al. (6) Observed 1 la 2 3 3a 4 1 la 2 3 3a 4 I II III IV V VI + + (+)b - _ - + - -(+) + + +c + + + + + - + - + + + + _ _ - + + + _ _ -_ + ± _

+ V + + + + + + + + + - + + + + + _

+ V - - + -+ - + + + + _ _ + + + _ -_ ± _ + + _ ± - _ _ +...... _-_ + _- _ - + _ + _ - + - - ±. and three cultures of serotype 0:6,30 (Knapp type I, II, or III, and all HeLa- cultures were and Thal 3, Nilehn 1, Wauters 1, Brenner 1, either biotype IV, V, or VI. Biotype I, II, and III observed V). strains are sucrose positive and negative for All HeLIa+ cultures were either observed bio- salicin, esculin, rhamnose, raffinose, melibiose, a-methylglucoside, ~~~~~~~~~~~~~~~~~~~~~~~~and citrate. Biotype IV and INFECTIVITY INDEX V strains are also sucrose positive, but always positive for salicin as well. Biotype VI was _,S,;, ^ @ o provided to accommodate the sucrose-negative b° ° bo b 0 strains, which were found among nontypable 17 and serotype 0:16 and 0:12,26 cultures. Biotype VI strains were also negative for salicin and 16 *6- * esculin but differed further from types I, II, and 14 III in being negative for the Voges-Proskauer 12 test. All sucrose-negative (biotype VI) strains were AA- HeLa-. 6 In addition to the 69 cultures examined for 5 HeLa cell infectivity, 43 more strains of Y. 13,7 **0- * enterocolitica were tested for AA and biotyped 12,26 .,8 (data not included). Twelve AA' strains were en 6,31 found, and they were always among the sero- m types represented by HeLa+ strains. All AA+ o NT so * * '0 *-0 strains were either biotype I, II, or III. -< 6,30 * ,-0A group of 21 from the 69 cultures of Y. m 2,3 enterocolitica, representing 12 serotypes plus nontypable strains, were examined for six po- 1,2,,3 tential indicators of virulence. All determina- 4,33 tions were completed simultaneously by using 4,32 .0.0 the same cell suspension prepared by subculture of a single colony. The results presented in Table 4 indicate that only strains that were AA+ 21 ** MOX- HeLa+ were capable of producing GPC 3 * * ~ and MD. MD was accompanied by fecal excre- 5,27 * .. tion of the infecting organism in high numbers. The only HeLa+ cultures that were AA- GPC- 8 MD- represented serotypes that also included 9 * strains that were AA+ GPC+ MD+. All strains FIG. 1. Distribution of HeLa cell infectivity index- that were HeLa- were always AA- GPC- es for each serotype represented among 69 test strains. MD. Because of the clear separation at 3.5, this index was On occasion some of the infected mice chosen as the dividing value between HeLa+ and showed symptoms of diarrhea, but without fec.a' HeLa- culltures (see text). .._. excretion of the organism (cultures E690, E633 502 SCHIEMANN AND DEVENISH INFECT. IMMUN. TABLE 2. Relationship of serotype and biotype of Y. enterocolitica with HeLa cell infectivity No. of HeLa positive (negative) strains of biotype:

Serotype 0: No.no.positive/tested ______Knapp and Thai Nilehn 1 la 2 2a 3 3a 1 la 2 2a 3 3a 4 8 5/5 5 4 1 3 4/4 4 4 5,27 3/3 3 3 9 1/1 1 1 1 1/1 1 1 1,2,3 3/3 3 1 2 2,3 1/1 1 1 4,32 2/3 2 (1) (1) 1 1 21 2/3 1 1 (1) (1) 1 1 NTa 0/9 (1) (2) (2) (4) (6) (2) (1) 6 0/2 (2) (2) 12 0/1 (1) (1) 14 0/1 (1) (1) 17 0/1 (1) (1) 4,33 0/4 (4) (4) 16 0/4 (1) (3) (3) (1) 6,30 0/4 (4) (4) 12,26 0/3 (3) (3) 7,13 0/5 (1) (4) (4) (1) 6,31 0/3 (3) (3) 5 0/8 (8) (8) Totals 22/69 10 (4) (2) 12 (6) (25) (14) (39) (3) 9 2 (3) 5 (2) 6 a NT, Nontypable.

and E696). In one case, one of the two infected DISCUSSION mice showed some fecal excretion, but without symptoms of diarrhea (E667 serotype 0:6,30). The studies reported here suggest that not With these few exceptions, diarrheal symptoms only are certain virulence factors in Y. enteroco- were always accompanied by excretion of the litica related, but also that biotype, and to some organism in high numbers in the feces, and those extent serotype, can be used to predict viru- animals not shedding the organism remained lence. Only certain serotypes, some of which healthy. Excretion in the feces usually persisted always present the same biotype, possess viru- beyond the time required for recovery from lence factors. Some serotypes present variable diarrhea. biotypes (for example, 0:4,32, and 0:21), but GPC correlated very well with the MD model only those strains presenting a certain biotype as well as the tests for AA, MOX, and HeLa cell will demonstrate virulence. Virulent strains are infectivity. AA- MOX+ HeLa+ cultures were always sucrose positive and negative for salicin, always GPC-. Reactions in the guinea pig eye esculin, rhamnose, raffinose, melibiose, a-meth- varied considerabl.y between serotypes, with ylglucoside, and citrate. Other serotypes are strains of serotypes 0:8 and 0:21 producing the also biochemically stable and never demonstrate most severe infections. virulence. These avirulent types are either su- Of the 21 cultures examined for production of crose and salicin positive or are sucrose nega- heat-stable enterotoxin, 4 were negative by the tive. Biotyping by the scheme proposed herein infant mouse assay. One of these cultures was appears more useful than serotyping for identifi- serotype 0:3 (E674) and was AA- HeLa+; cation of virulent strains of Y. enterocolitica. consequently, it was avirulent in mice. A second Our studies indicate that the properties of heat-stable enterotoxin-negative culture was se- autoagglutination and calcium dependency are rotype 0:5,27 (E654), and it was positive for all mediated by the same plasmid. Gemski et al. other tests for virulence. Many of the strains (12) previously reported the presence of a plas- with no evidence of other virulence factors mid associated with calcium dependency, and produced the heat-stable enterotoxin. Laird and Cavanaugh (15) described isogenic VOL. 35, 1982 HeLa CELL INFECTIVITY OF Y. ENTEROCOLITICA 503

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0 z 0. C=- enC Clf)U C cC0'C m'-4 00 en i O T4 senN" E o " rN ^c o -- Cd W) "Ic4-:>"Z - - 4-.6"r-s 0 Z 2 z (A,C) 504 SCHIEMANN AND DEVENISH INFECT. IMMUN. TABLE 4. Correlation of six potential indicators of virulence in 21 strains of Y. enterocolitica [Sero- 1Bo- Infant Symptoms/fecal count per g in mice on dayd: Culture typeBio AA MOXaI HeLaI mouse GPKC 1 1 no. [F jtype [ratio_ _ 2 5 9 14 E660 8 I + 4.86 0.112 -I- -/8 X 104 -/2 x 102 -/3X 102 -/<1 x 102 -/<9 x 102 -/<2 x 102 -/<2 x 102 -/1 x 10l E661 8 I + 4.73 0.121 +I+ +/1 x 109 Dead +/4 x 109 Dead E674 3 II + 4.14 0.080 -/<1 X 103 -/<3 x 102 -/<1 10xX -/<1 x 10' +I+ -/<1 X 103 -/<1 X 103 -/<2 x 103 -/<1 x 10' E675 3 II 3.70 0.107 +/4 x 101 +/8 x 107 +/1 x 108 -/2 x 106 + +I+ +/1 x 1011 +/6 x 108 +/2 x 107 -/2 x 107 E654 5,27 III 4.20 0.068 +/3 x 108 +/8 x 107 +/2 x 107 -/3 X 106 +I+ +/2 x 1010 +/3 X 107 -/8 X 106 -/2 x 103 E657 5,27 III 4.61 0.094 +15 X 108 -/l X 107 -/l X 107 -/9 x 105 +/1 x 109 +/8 x 107 +/3 X 107 -/6 x 107 E659 5,27 III + 4.75 0.085 +I+ -/<1 x 102 -/<9 x 10l -/<6 x 101 -/<1 x 102 -/<1 x 102 -/<2 x 102 -/<2 x 102 -/<2 x 102 E705 9 III 4.89 0.091 +I+ +/6 x 108 ±/4 X 107 ±/1 x 107 -/3 X 107 +/3 x 108 ±/2 X 107 ±/8 x 106 -/5 x 106 E029 1 III + 4.50 0.087 +I+ +/1 x 108 +/4 X 107 +/1 X107 -/4 x 104 +/3 X 108 +/6 X 107 +/4 X 106 -/<6 x 102 E766 1,2,3 II 4.20 0.100 +15 x 108 +/2 x 108 +15 x 107 ±/1 x 108 +I+ +/4 X 108 +/8 x 107 +/2 x 107 ±/4 x 107 E562 4,32 IV + 2.72 0.053 -/<2 x 102 -/<4 x 102 -/<8 x 101 -/<1 x 102 +I+ -/<1 x 102 -/<1 X 103 -/<8 x 101 -/<2 x 102 E701 4,32 I 3.89 0.087 -/1 X 107 +/6 x 106 -/1 X 106 -/8 x 103 + -I- +n7 x 107 Ill Dead E736 21 III 4.31 0.130 +/9 x 108 Dead +/1 x 109 Dead E690 NT IV + 1.12 0.060 ±/<1 X 103 +/<3 X 102 +/<7 x 10' -/<2 x 102 ±/<1 X 103 -/<4 x 103 ±/<1 x 102 +/<3 x 102 E764 8 I + 4.58 0.096 I+/+ +/1 x 109 Dead +/1 x 109 Dead E667 6,30 V + 2.78 0.087 -I- -/<1 x 102 -/<1 x 102 -/<7 x 101 -/<2 x 10' -/2 x 102 ±/3 X 105 -/8 x 105 -/1 x 105 E673 6,30 V + 3.06 0.105 -I- -/<7 x 102 -/<2 x 103 -/<2 x 103 -/<2 x 10' -/<4 x 102 -/<4 x 103 -/<1 x 103 -/<6 x 102 E633 12,26 VI + 2.28 0.095 -I- -/<3 X 102 -/<1 x 102 -/3 x 102 -/<1 x 102 +/<4 x 104 +/<2 x 102 -/<1 x 102 -/<6 x 101 E696 12,26 VI + 2.29 0.131 -I- -/<2 x 102 -/<6 x 102 -/<4 x 102 -/<2 x 102 -/3 x 102 -/<4 x 101 ±1<5 x 101 +/<7 x 101 E114 7,13 V + 2.15 0.104 -I- -/<2 x 102 -/<2 x 102 -/<1 x 102 -/<1 x 102 +/<4 x 102 -/<4 x 101 -/<1 x 102 -/<8 x 101 E699 S V + 2.51 0.091 -I- -/2 x 102 -/3 X 102 -/<8 x 101 -/<2 x 102 -/<4 x 102 -/2 X 102 -/3 x102 -/<2 x 102 a Growth on MOX agar. b HeLa cell infectivity index. C GPC in animal no. 1/animal no. 2. d Data are given for animals no. 1 and 2.

pairs selected from the same culture that differed lost through subculture. Consequently, the in ability to autoagglutinate and produce MD. HeLa cell test becomes the only in vitro system When the plasmid coding for these two proper- for identifying strains of Y. enterocolitica that ties is lost, so is virulence. However, HeLa cell were fully virulent at some earlier time in that infectivity is unchanged with loss of the plasmid. they possessed the Vwa plasmid (10) or are Portnoy et al. (23) recently reported that inva- potentially virulent in that they are able to siveness of HEp-2 cells by Y. enterocolitica was receive this plasmid by transfer from a Vwa+ not plasmid mediated. The identification of viru- strain, a process that has not yet been demon- lent strains of Y. enterocolitica by the AA or strated. Since the test for HeLa cell infectivity is MOX tests is not always dependable, since the a bit cumbersome for routine screening pur- plasmid coding for these properties can easily be poses, the use of biotyping as the first approach VOL. 35, 1982 HeLa CELL INFECTIVITY OF Y. ENTEROCOLITICA 505 to identification of virulent strains becomes even Yersinia kristensenii: a new species of Enterobacteriaceae more attractive. composed of sucrose-negative strains (formerly called atypical Yersinia enterocolitica or Yersinia enterocolitica- The results of our studies indicate that the like). Curr. Microbiol. 4:219-224. heat-stable enterotoxin produced by many 4. Brenner, D. J. 1979. Speciation in Yersinia. Contrib. strains of Y. enterocolitica plays no role in Microbiol. Immunol. 5:33-43. pathogenesis as described by the MD model. 5. Brenner, D. J., H. Bercovier, J. Ursing, J. M. Alonso, A. G. Steigerwalt, G. R. Fanning, G. P. Carter, and H. H. Studies previously reported with a strain of MoUaret. 1980. Yersinia intermedia: a new species of serotype 0:3 isolated from pork that was heat- Enterobacteriaceae composed of rhamnose-positive, mel- stable enterotoxin negative found that this cul- libiose-positive, raffinose-positive strains (formerly called ture was AA' MOX- HeLa+ and capable of Yersinia enterocolitica or Yersinia enterocolitica-like). Curr. Microbiol. 4:207-212. producing MD (28). 6. Brenner, D. J., J. J. Farmer III, F. W. Hickman, M. A. It has become very clear that the organism Asbury, and A. G. Steigerwalt. 1977. Taxonomic and presently defined as Y. enterocolitica is, in fact, Nomenclature Changes in Enterobacteriaceae, HEW a large biochemically heterogeneous group of publication no. (CDC) 78-8356. U.S. Department of Health, Education, and Welfare, Atlanta, Ga. bacteria. The biochemical variability within this 7. Brenner, D. J., A. G. Steigerwalt, D. P. Fakcao, R. E. group encouraged the development of a number Weaver, and G. R. Fanning. 1976. Characterization of of different biotyping schemes (6, 14, 20, 35; Yersinia enterocolitica and Yersinia pseudotuberculosis Wauters, Ph.D. thesis). A scheme proposed by by deoxyribonucleic acid hybridization and by biochemi- Brenner et al. is on cal reactions. Int. J. Syst. Bacteriol. 26:180-194. (7) based DNA relatedness 8. Brenner, D. J., J. Ursing, H. Bercovier, A. G. Steigerwalt, and forms the basis of a recent recommendation G. R. Fanning, J. M. Alonso, and H. H. Mollaret. 1980. for subdividing this group into four different Deoxyribonucleic acid relatedness in Yersinia enterocoli- species (4, 8). Only strains of relatedness group tica and Yersinia enterocolitica-like organisms. Curr. Mi- crobiol. 4:195-200. 1 would be called Y. enterocolitica. Relatedness 9. Carter, P. B., C. F. Varga, and E. E. Keet. 1973. New group 2 would be designated Yersinia interme- strain of Yersinia enterocolitica pathogenic for rodents. dia, and relatedness group 3 would be called Appl. Microbiol. 26:1016-1018. Yersinia frederiksenii. Sucrose-negative strains 10. Carter, P. B., R. J. Zahorchak, and R. R. Brubaker. 1980. Plague virulence antigens from Yersinia enterocolitica. in relatedness group 4 would be referred to as Infect. Immun. 28:638-640. Yersinia kristensenii. The justification for this 11. Devenish, J. A., and D. A. Schiemann. 1981. HeLa cell proposal and a detailed description of the bio- infection by Yersinia enterocolitica: evidence for lack of chemical criteria for each species have been intracellular multiplication and development of a new procedure for quantitative expression of infectivity. In- presented in a series of recent papers (2, 3, 5, fect. Immun. 32:48-55. 34). Our studies indicate that only the species Y. 12. Gemski, P., J. R. Lazere, and T. Casey. 1980. Plasmid enterocolitica would include virulent forms. Our associated with pathogenicity and calcium dependency of further indicate that virulent strains of Yersinia enterocolitica. Infect. Immun. 27:682-685. findings 13. Higuchi, K., and J. L. Smith. 1%1. Studies on the this species could be identified by a positive nutrition and physiology of Pasteurella pestis. IV. A sucrose and negative salicin and esculin reac- differential plating medium for the estimation of the tions (i.e., biotypes I, II, and III). A previous mutation rate to avirulence. J. Bacteriol. 81:605-608. report by Schiemann et al. (30) also noted that 14. Knapp, W., and E. Thal. 1973. Differentiation of Yersinia enterocolitica by biochemical reactions. Contrib. Micro- virulent strains of Y. enterocolitica were always biol. Immunol. 2:10-16. negative for salicin, esculin, rhamnose, and cit- 15. Laird, W. J., and D. C. Cavanaugh. 1980. Correlation of rate reactions. It appears, therefore, that it is autoagglutination and virulence in Yersiniae. J. Clin. now feasible to separate with very few and Microbiol. 11:430-432. tests virulent 16. Lee, W. H., P. P. McGrath, P. H. Carter, and E. L. Elde. simple from avirulent forms of Y. 1977. The ability of some Yersinia enterocolitica strains to enterocolitica. invade HeLa cells. Can. J. Microbiol. 23:1714-1722. 17. Maruyama, T. 1973. Studies on biological characteristics ACKNOWLEDGMENTS and pathogenicity of Yersinia enterocolitica. 2. Experi- mental infection in monkeys. Jpn. J. Bacteriol. 28:413- All serotyping was completed by S. Toma and V. Deidrick 422. (In Japanese.) of the Canadian National Reference Service for Yersinia. 18. Maruyama, T., T. Une, and H. Zen-YojI. 1979. Observa- Financial support for this study was provided in part by tions on the correlation between pathogenicity and sero- Research Grant PR-742 from the Ontario Ministry of Health. vars of Yersinia enterocolitica by the assay applying cell culture system and experimental mouse infection. Con- LITERATURE CITED trib. Microbiol. Immunol. 5:317-323. 19. Mollaret, H. H., and J. C. Guillon. 1965. Contribuion a 1. Alonso, J. M., H. Bercovier, P. Destombes, and H. H. l'etude d'un nouveau groupe de germes (Yersinia entero- Mollaret. 1975. Pouvoir pathogene experimental de Yer- colitica) proches du bacille de malassez et vignal. 2. sinia enterocolitica chez la souris athymique (Nude). Pouvoir pathogene experimental. Ann. Inst. Pasteur Ann. Inst. Pasteur 126:187-199. 109:608-613. 2. Bercovier, H., D. J. Brenner, J. Ursing, A. G. Steigerwalt, 20. Nilehn, B. 1969. Studies on Yersinia enterocolitica with G. R. Fanning, J. M. Alonso, G. P. Carter, and H. H. special reference to bacterial diagnosis and occurrence in MoUaret. 1980. Characterization of Yersinia enterocolitica human acute enteric disease. Acta Pathol. Microbiol. sensu stricto. Curr. Microbiol. 4:201-206. Scand. 206:1-48. 3. Bercovier, H., J. Ursing, D. J. Brenner, A. G. Steigerwalt, 21. Pai, C. H., V. Mors, and T. A. Seemayer. 1980. Experi- G. R. Fanning, G. P. Carter, and H. H. Mollaret. 1980. mental Yersinia enterocolitica enteritis in rabbits. Infect. 506 SCHIEMANN AND DEVENISH INFECT. IMMUN.

Immun. 28:238-244. lian gerbils and by the Sereny test. Infect. Immun. 29:500- 22. Pedersen, K. B., S. Winblad, and V. Bitsch. 1979. Studies 506. on the interaction between different 0-serotypes of Yer- 30. Schiemann, D. A., J. A. Devenish, and S. Toma. 1981. sinia enterocolitica and HeLa cells. Acta Pathol. Micro- Characteristics of virulence in human isolates of Yersinia biol. Scand. 87:141-145. enterocolitica. Infect. Immun. 32:400-403. 23. Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981. 31. Une, T. 1977. Studies on the pathogenicity of Yersinia Characterization of plasmids and plasmid-associated de- enterocolitica. I. Experimental infection in rabbits. Mi- terminants of Yersinia enterocolitica pathogenesis. Infect. crobiol. Immunol. 21:349-363. Immun. 31:775-782. 32. Une, T. 1977. Studies on the pathogenicity of Yersinia 24. Quan, T. J., J. L. Meek, K. R. Tsuchiya, B. W. Hudson, enterocolitica. II. Interaction with cultured cells in vitro. and A. M. Barnes. 1974. Experimental pathogenicity of Microbiol. Immunol. 21:365-377. recent North American Yersinia enterocolitica isolates. J. 33. Une, T., H. Zen-Yoji, T. Maruyama, and Y. Yanagawa. Infect. Dis. 129:341-344. 1977. Correlation between epithelial cell infectivity in 25. Rakovsky, J. 1973. Experimental pathogenicity of Yer- vitro and 0-antigen groups of Yersinia enterocolitica. sinia enterocolitica serotype 3. Contrib. Microbiol. Im- Microbiol. Immunol. 21:727-729. munol. 2:81-84. 34. Ursing, J., D. J. Brenner, H. Bercovier, G. R. Fanning, A. 26. Ricclard, J. D., A. D. Pearson, W. G. Suckling, and C. G. Steigerwalt, J. Brault, and H. H. Mollaret. 1980. Klein. 1978. Long-term fecal excretion and resistance Yersinia frederiksenii: a new species of Enterobacteria- induced in mice infected with Yersinia enterocolitica. ceae composed of rhamnose-positive strains (formerly Infect. Immun. 21:342-344. called atypical Yersinia enterocolitica or Yersinia entero- 27. Schlemann, D. A. 1979. Synthesis of a selective agar colitica-like). Curr. Microbiol. 4:213-217. medium for Yersinia enterocolitica. Can. J. Microbiol. 35. Winblad, S. 1979. Differentiation of Yersinia enterocoli- 25:1298-1304. tica strains in subgroups after biochemistry and serology. 28. Schlemann, D. A. 1981. An enterotoxin-negative strain of Contrib. Microbiol. Immunol. 5:44-49. Yersinia enterocolitica serotype 0:3 is capable of produc- 36. Zink, D. L., J. C. Feeley, J. G. WeUs, C. Vanderzant, J. C. ing diarrhea in mice. Infect. Immun. 32:571-574. Vickery, and G. A. O'Donovan. 1978. Possible plasmid- 29. Schlemann, D. A., and J. A. Devenish. 1980. Virulence of mediated virulence in Yersinia enterocolitica. Trans. Gulf Yersinia enterocolitica determined by lethality in Mongo- Coast Mol. Biol. Conf. 3:155-163.