Family Outbreak of Yersiniosis TOM MARTIN,L* GORDON F

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Family Outbreak of Yersiniosis TOM MARTIN,l* GORDON F. KASIAN,2 AND STANLEY STEAD3 Departments of Microbiology,t Pediatrics,2 and Social and Preventive Medicine,3 University of Saskatchewan, Saskatoon, Saskatchewan, Canada 57N OWO Received 1 February 1982/Accepted 24 June 1982 Yersinia enterocolitica biotype 1, serotype 0:21 was isolated from feces or rectal washings of three members of one family in northwestern Saskatchewan. The three isolates gave positive pathogenicity tests in guinea pigs with cultures grown at 22°C as inoculum. All three cases showed clinical symptoms consistent with yersiniosis. All three cases had symptoms of diarrhea and abdominal pain, and two cases had recorded fever. In two cases, appendicitis was initially suspect. One case with ileitis and peritonitis was fatal. The environmental source of the infection was not found, but river water, milk, and person-to-person spread are discussed as possible sources of the infections. The need for microbiology laboratories to culture stool specimens specifically for Y. enterocolitica, using cold-enrichment techniques is emphasized. This family outbreak of yersiniosis provides further evidence that certain biotype 1 strains of Y. enterocolitica are pathogenic.

Yersinia enterocolitica is a gram-negative ba- ferred by air ambulance to University Hospital, Saska- cillus now classified as a member of the family toon. Enterobacteriaceae. A particular characteristic Two stool specimens obtained on the second and this is its to survive and sixth days after his admission to University Hospital of organism ability were negative for ova and parasites and negative for multiply at low temperatures. virus particles by electron microscopy. An adenovirus Isolations of Y. enterocolitica in Canada have was isolated from both of these specimens. Cultures recently been reviewed by Toma and Lafleur for Salmonella spp., Shigella spp., and Campylobac- (25). These authors reviewed laboratory and ter spp. were negative. A moderate growth of Y. epidemiological findings for 1,485 strains from enterocolitica biotype 1, serotype 0:21 was obtained human sources and 495 strains from animal or by direct plating on salmonella-shigella agar of stool environmental sources isolated between 1966 specimens on March 6 and 9, 1981. and 1978. Most of the isolates from human Fluid and electrolyte balance was maintained during sources were children or adolescents in the boy's stay in the hospital. He recovered without from surgery and without antibiotics. Quebec and Ontario. Case 2. The 3-year-old sister of the boy described in The best established clinical manifestations of case 1 was admitted to the same hospital in northern infections with Y. enterocolitica are ileitis, mes- Saskatchewan on March 26, 1981. She had a high enteric lymphadenitis, and septicemia. The clini- fever, decreased appetite, and abdominal discomfort, cal aspects of infections with Y. enterocolitica but no evidence of localized abdominal tenderness. have been reviewed recently by several authors Two days after her admission to the local hospital, she (15, 28). developed watery brown diarrhea and vomiting. Intra- We report three cases of yersiniosis in mem- venous fluids were started. Four days after her admis- northwestern Sas- sion to the hospital, abdominal distension was evident, bers of a single family from and a nasogastric tube was inserted. The leukocyte katchewan due to Y. enterocolitica biotype 1, count ranged from 23,000 to 25,000, with a higher serotype 0:21. One case was fatal. proportion of neutrophilic leukocytes than normal. On March 31, 1981 the child was transferred to University CASE REPORTS Hospital, Saskatoon. Case 1. A 2-year-old boy from northwestern Sas- An abdominal X ray showed multiple, moderately katchewan developed fever, diarrhea, and vomiting. dilated, gas-filled loops of small bowel in the mid and Two days later on February 25, 1981, he was admitted upper abdomen. No gas was present in the colon or to a local hospital. His temperature on admission was rectum. Fluid appeared to be present within the perito- 40°C. His leukocyte count was 21,400 with 76% poly- neal cavity. morphonuclear leukocytes and 17% lymphocytes. On April 1, 1981, a laparotomy was done because of No localized abdominal tenderness was detected on the suspect peritonitis. At surgery, the presence of his admission to the local hospital, and the initial acute ileitis with peritonitis was confirmed. The ap- diagnosis was gastroenteritis. After 3 days, an appen- pendix had a slight degree of inflammation and was diceal abscess was suspected, and the boy was trans- removed. The terminal ileum was very inflammed and 622 VOL. 16, 1982 YERSINIOSIS: A FAMILY OUTBREAK 623 edematous. Treatment was started with gentamicin (Difco), Christensen urea agar (Difco), Simmons ci- and clindamycin. trate agar (Difco), and tryptone broth (Difco). Hydrol- Microscopic examination of the appendix showed ysis of o-nitrophenyl-o-D-galactopyranoside, phenyl- mucosal ulceration and purulent exudate in the lumen. alanine deaminase production, and motility were test- Fibrinous exudate was present on the surface. The ed in the combined medium of Giammanco and Falci deeper layers of the appendix were edematous, con- containing o-nitrophenyl-3-D-galactopyranoside and gested, and infiltrated by polymorphonuclear neutro- phenylalanine (7). Cultures in these media were incu- phils and polymorphonuclear eosinophils. bated at 35°C. In addition, colonies were tested for Aerobic and anaerobic cultures of the peritoneal motility at room temperature in the semisolid medium fluid showed no growth. Culture of a biopsy of a lymph of Edwards and Bruner (BBL) and for the absence of node of the small bowel near the terminal ileum grew oxidase with 1% tetramethylparaphenylenediamine di- Escherichia coli. The strain was resistant to ampicillin hydrochloride (Marion Scientific, Kansas City, Mo.). but sensitive to gentamicin. Cold enrichment of the Nonlactose fermenting organisms were selected as lymph node tissue for 3 weeks failed to yield Y. suspect Y. enterocolitica on the basis of the following enterocolitica. Blood and spinal fluid cultures were characteristics: oxidase negative, H2S negative, 1- negative for Y. enterocolitica. galactosidase positive (or negative at 35°C), phenylala- Two days after surgery, rectal washings were ob- nine deaminase negative, urease positive or delayed tained for culture, since the child was no longer positive, citrate negative, and motile at room tempera- passing stool. After cold enrichment for 18 days in ture but not at 35°C. selenite F medium, Y. enterocolitica biotype 1, sero- Further biochemical characterization of suspect type 0:21 was isolated from this specimen. A serum strains of Y. enterocolitica was made with either the sample obtained on April 3, 1981, gave an agglutina- Enterobacteriaceae-plus Card of the AutoMicrobic tion titer of 1/100 with the homologous strain of Y. system (Vitek Corp., St. Louis, Mo.) or by the API enterocolitica serotype 0:21. 20E system (Analytab Products, Plainview, N.Y.), Between April 2, 1981, and her death on May 13, using the protocols described by the respective com- 1981, the child developed progressive multisystem panies. failure, including hepatic and renal failure, metabolic Biotyping by Wauter's method and serotyping of encephalopathy, thrombocytopenia, and severe gas- isolates of Y. enterocolitica was kindly done by S. trointestinal hemorrhaging. Toma, National Reference Service for Yersinia, To- An autopsy revealed a retroperitoneal hematoma, ronto, Canada. with intraperitoneal hemorrhage and cutaneous ecchy- Antimicrobial susceptibility tests were performed moses, indicating a degree of disseminated intravascu- by the Kirby-Bauer method. Minimal inhibitory con- lar coagulation. centrations were determined by using microdilution Case 3. A 21-year-old woman, the mother of the trays (Micro-Scan/Alkem, Calgary, Canada). children described in cases 1 and 2, came to University Pathogenicity tests. Pathogenicity testing was done Hospital, Saskatoon, to stay with her hospitalized by the Sertny method in guinea pigs (23). A loopful of daughter. Although she was asymptomatic, a stool a 48-h culture of the test strain grown on sheep blood specimen was obtained on April 3, 1981, for culture, agar (Trypticase soy base BBL) at room temperature because she stated she had intermittent diarrhea and was introduced into the right conjunctival sac of abdominal pain in the recent past and because the guinea pigs (400 to 450 g). The left eyes of the guinea family history indicated exposure to Y. enterocolitica. pigs were not inoculated and served as a negative Direct plating of the stool specimen yielded no control. The guidelines of the Canadian Council on pathogens, but Y. enterocolitica biotype 1, serotype Animal Care for the use and care of laboratory animals 0:21 was isolated after cold enrichment of the stool were followed. The degree of conjunctivitis in guinea specimen for 10 days in phosphate-buffered saline. A pigs was graded according to the method of Schiemann serum sample obtained on April 23, 1981, gave an and Devenish (20). agglutination titer of 1/100 with the homologous strain Serological tests. Tube agglutination tests with mi- of Y. enterocolitica serotype 0:21. crotiter plates for antibody to Y. enterocolitica were kindly done by M. T. Kennedy, National Reference MATERIALS AND METHODS Service for Yersinia, Toronto, Canada. Sera were tested with Y. enterocolitica serotypes 0:3, 0:5,27, Bacteriology. Freshly passed fecal specimens were and 0:8 and the homologous isolates of serotype 0:21. collected without preservative and inoculated directly In addition, sera were tested with a suspension of Y. on MacConkey agar (BBL Microbiology Systems, pseudotuberculosis serogroup III. Cockeysville, Md.) and SS agar (Difco Laboratories, Detroit, Mich.). Cultures were incubated aerobically at 35°C and examined after 24 and 48 h. After detection RESULTS of the initial case and after direct plating, fecal, lymph Bacteriology. Y. enterocolitica was isolated by node tissue, and environmental specimens were cold direct plating of the boy's stool on SS agar. After enriched in selenite F medium (Difco), phosphate 48 h of incubation at 35°C, the colonies on SS buffered saline (0.075 M, pH 7.6), or both at 4°C. Daily agar were colorless and 1.0 to 1.5 mm in diame- subcultures for 21 days were made from selenite F medium and phosphate-buffered saline to MacConkey ter. MacConkey agar and xylose-lysine-desoxy- and SS agar. Separate plates were incubated at room cholate agar inoculated with the same stool temperature (-22°C) and 35°C. specimens were overgrown with lactose-fer- Colorless colonies on MacConkey or SS agar were menting organisms. The strain from the girl was screened by inoculation of triple sugar iron agar isolated only after cold enrichment for 18 days in 624 MARTIN, KASIAN, AND STEAD J. CLIN. MICROBIOL. TABLE 1. Biochemical profiles of Y. enterocolitica TABLE 2. Sereny test with strains of Y. 0:21 isolates enterocolitica serotype 0:21" no. Degree of conjunctivitisb Source of Incubation Profile Source of strain isolate condition AMS" APIh Day 2 Day 4 Day 6 Son 350C 6100520144 0154523 Son + ++ ++ 220C (24 h) 1155523 Daughter + + + + + Mother + + Daughter 350C 6000520144 0154521 0154723 a Cultures were incubated at room temperature 220C (48 h) (-220C). Mother 350C 6101520144 0154723 b Symbols: + +, persistent, copious exudate, with 220C (48 h) 0154723 recovery of large numbers of bacteria by swabbing of the conjunctivae; +, mild progressive reaction, with a AMS, AutoMicrobic system, Enterobacteriaceae- recovery of moderate numbers of bacteria; +, very plus Card read after 8 h. weak transitory response, with few bacteria recov- b API, Analytab Products, 20E system. ered; and -, no response observed. selenite broth. Y. enterocolitica was isolated ples collected from six sites in the river and from the mother's stool after cold enrichment adjacent to the house where the outbreak oc- for 10 days in phosphate buffered saline. curred failed to grow any Yersinia strains either The biochemical profile numbers obtained for by direct plating or after cold enrichment for 4 the three isolates of Y. enterocolitica in the weeks. These samples were obtained approxi- AutoMicrobic system and the API 20E system mately 3 months after the initial case was detect- are given in Table 1. The strains were all sucrose ed. Water from the pump in the school yard was positive, but negative for rhamnose, raffinose, also negative on culture for Yersinia strains. melibiose, and citrate. Tests for salicin and The mean maximum temperature recorded at esculin, which are not incorporated in either of the nearest Environment Canada weather sta- the above identification systems, were per- tion during February 1981 was -5.8°C. The formed separately. The isolates were salicin and mean minimum temperature was -17.6°C. The esculin negative. All three isolates of Y. entero- highest temperature in February was +10.4°C. colitica were biotype 1, serotype 0:21. All three The lowest temperature in February was -42°C. isolates were sensitive to ampicillin, gentamicin, The index case in this outbreak was first admit- tetracycline, chloramphenicol, and trimetho- ted to hospital on February 25, 1981. prim-sulfamethoxazole. Pathogenicity tests. The results of the Sereny tests are summarized in Table 2. All three iso- DISCUSSION lates produced conjunctivitis in guinea pigs Only three isolations in Saskatchewan of Y. when inoculated with cultures grown at 220C. enterocolitica from human sources have been Epidemiology. Three people in a four-member recorded by Toma and Lafleur between 1966 family were involved in this outbreak of yersin- and 1978 (25). Two of the isolates were serotype iosis. The father remained asymptomatic. Stool 0:8. One of these strains was isolated from pus cultures from the father were not done, but (24). paired serum samples were negative for anti- In Ontario and Quebec, the predominant sero- body to Y. enterocolitica. type of Y. enteroolitica is 0:3. Among the The family lives at St. George's Hill on the relatively few isolates from human sources in Dillon River in west-central Saskatchewan (lati- western Canada, the most frequent serotypes tude 55.80 N; longitude 1090 W). Access to this are 0:8, 0:5,27, and 0:4,32 (25). community of about 500 people is usually by air, Before 1978, only seven isolations of serotype although road access is possible in the winter. 0:21 were reported from human sources in The 2-year-old boy often stayed at his grand- Canada (25). The same serotype was isolated in mother's house on the adjacent Indian reserva- Canada from water on seven occasions and tion. His grandmother kept a cow and a calf. three times from food products, including raw The family admitted to drinking unboiled wa- milk (19, 22, 25). ter obtained directly from the river beside their Isolations of Y. enterocolitica or Yersinia-like home. There is no running water in the house. organisms which agglutinated in antisera for Although the family had been advised by public more than one serotype, but including serotype health personnel not to drink water from the 0:21, have been reported from water and brown river, they found this more convenient than trout (Salmo trutta L) in Norway and from using the pump in the school yard farther away. lemmings (Lemmus lemmus) in Norway and Mud and centrifuged deposits of water sam- Denmark (12, 13). VOL. 16, 1982 YERSINIOSIS: A FAMILY OUTBREAK 625 Y. enterocolitica and Yersinia-like organisms summer may decrease the survival time of Yer- have often been isolated from water (6, 9, 10, 12, sinia spp. in water. 14, 16, 19, 25). There are, however, only a few The importance of cold-enrichment tech- well-documented reports linking water as the niques for isolation of Yersinia spp. from fecal source of human infections with Y. enterocoli- specimens is emphasized by the diagnosis of our tica (14, 16). We were unable to verify water as cases. Only one of the three isolates was ob- the source of our cases, although the epidemio- tained by direct plating of stool or rectal wash logical history suggested river water was a possi- specimens. The report of Weissfeld and Sonnen- ble source. The isolation of the same biotype wirth suggests that cold-enrichment techniques and serotype of Y. enterocolitica from the three are particularly important in the isolation of cases in the family suggests a common source biotype 1 strains of Y. enterocolitica (27). for the organism, but the sequential occurrence Low-level serological titers were detectable of at least two of our cases may indicate that against the homologous isolate in two of our person-to-person spread was also involved. It is three cases. Serum was not available from the possible that the boy was infected by milk or boy. Although a fourfold rise in titer was not fecal matter from the cow or calf at his grand- demonstrated in these cases, we believe that the mother's house and that person-to-person isolated strains of Y. enterocolitica biotype 1 spread occurred to other members of the family. were responsible for the clinical symptoms in Several familial outbreaks of yersiniosis have the boy and for at least the initial symptoms in been reported previously (1, 2, 4, 8, 17, 18). the girl. The strain isolated from their mother Person-to-person spread was postulated in sev- was assumed to represent a carrier state possi- eral of these reports (1, 8, 17). Zoonotic aspects bly subsequent to clinical yersiniosis. of Y. enterocolitica infections have recently Although Van Noyen et al. (26) consider that been reviewed by Hurvell (11). Hurvell also lists most biotype 1 strains of Y. enterocolitica isolat- reported isolations of Y. enterocolitica from ed in Belgium are nonpathogenic for humans, a cattle. recent report by Schiemann and Devenish (21) Mollaret found that the incidence of human concludes that all strains of Y. enterocolitica disease caused by Y. enterocolitica in Europe is with certain biochemical characteristics possess highest during the cold months of the year (18). an invasive factor demonstrable by HeLa cell Marks et al., however, found that yersiniosis in infectivity. The HeLa cell-positive strains re- children in Quebec tended to be more common ported by these authors were all sucrose posi- in the summer months (17). Whether the low tive, but negative for salicin, rhamnose, raffi- environmental temperature was a factor in the nose, melibiose, esculin, and citrate. Our iso- pathogenicity of the strain involved in the out- lates had these same biochemical characteristics break we describe is not clear. Carter and Col- and were invasive when tested by the Sereny lins report a particular strain of Y. enterocolitica method in the guinea pig conjunctival sac. We (ATCC 27729) which showed an initial lowering regard this as additional evidence for the viru- of virulence for mice when cultured at 37°C lence of these isolates of Y. enterocolitica bio- compared with the same strain cultured at 25°C type 1, serotype 0:21. (3). We compared the pathogenicity of one of our isolates by the Sereny method, using cul- ACKNOWLEDGMENTS tures incubated at 22 and 35°C. Only the culture We are indebted to B. Goertz, L. Rossi, J. Pinilla, F. incubated at 22°C produced conjunctivitis in Murphy, D. Moola, P. Jensen, and M. Majid, all of whom guinea were involved in the diagnosis or management of these cases. pigs. We also thank R. W. J. Naismith and D. Bauer for epidemio- There is some evidence that Y. enterocolitica logical and environmental information. may survive longer in the environment during the colder months of the year. Dominowska and LITERATURE CITED Malottke in Poland found that Y. pseudotuber- 1. Ahvonen, P. 1972. Human yersiniosis in Finland. II. culosis and Y. enterocolitica, when introduced Clinical features. Ann. Clin. Res. 4:39-48. into samples of lake and river water, survived 2. Ahvonen, P., and T. Rossi. 1970. Familial occurrence of Yersinia enterocolitica infection and acute arthritis. Acta for much longer periods in the samples collected Paediatr. Scand. Suppl. 206:121-122. during autumn and winter than in samples ob- 3. Carter, P. B., and F. M. Collins. 1974. Experimental tained in spring and summer (5). These authors Yersinia enterocolitica infection in mice: kinetics of also found that Y. enterocolitica at environmen- growth. Infect. Immun. 9:851-857. tal 4. Delorme, J., M. Laverdiere, B. Martineau, and L. Lafleur. temperatures survived in unfiltered surface 1974. Yersiniosis in children. Can. Med. Assoc. J. water obtained in the summer for only 7 days, 110:281-284. whereas the same water after being filtered 5. Dominowska, C., and R. Malottke. 1971. Survival of allowed the survival of Y. enterocolitica for 184 Yersinia in water samples originating from various sources. Biul. Inst. Marine Trop. Med. 22:173-182. days. This suggests that the presence of larger 6. Eden, K. V., M. L. Rosenberg, M. Stoopler, B. T. Wood, quantities of other flora in surface waters in the A. K. Highsmith, P. Skaliy, J. G. Wells, and J. C. Feeley. 626 MARTIN, KASIAN, AND STEAD J. CLIN. MICROBIOL.

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