Pathology (1979), 11, pp. 67-70
ENTEROCOLITIS DUE TO YERSlNlA ENTEROCOLlTlCA IN SOUTH AUSTRALIA
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
T. W. STEELE AND S. N. MCDERMOTT Division of Clinical Microbiology, Institute of Medical and Veterinary Science, Adelaide
Summary From August 1976 to July 1977, all faecal specimens (3298) sent to the Enteric Department of the Institute of Medical and Veterinary Science, Adelaide were selectively cultured for Yersinia enterocolifica. Yersinia enterocolifica was isolated from three patients with diarrhoea, one of whom acquired her infection overseas. These organisms were not isolated from faecal or lymph node material collected from a limited number of sheep and pigs found to have enteritis at the time of slaughter. Enteric infection due to Yersinia enterocolitica does not appear to be common in Australia and selective culture methods using cold enrichment techniques do not appear to be justified especially in laboratories handling specimens derived mainly from adults.
Diarrhoea as a manifestation of intestinal infection due to Yersinia enterocolitica has been described in a number of European countries, in North America, Africa and Japan (MoIIaret, 1972). These organisms are seldom if ever isolated in clinical laboratories in Australia and the incidence of diarrhoea due to yersinia infection in this country is unknown. From 1 August 1976 to 31 July 1977, all faecal specimens (3298) received by the Division of Clinical Microbiology were cultured for yersinia. In addition, faeces and lymph nodes from 50 sheep and 50 pigs, with evidence of enteritis at the time of slaughter in the abattoir, were examined for these organisms. MATERIALS AND METHODS All specimens were routinely inoculated to MacConkey agar, desoxycholate citrate agar, selenite broth (43°C) and Rappaport broth for the isolation of Salmonella and Shigella. Selectiveculture for yersinia using nutrient broth and Rappaport broth with incubation at 4°C for 4 days (Eiss, 1975) and subculture to MacConkey agar with and without Tween 80 was performed. These plates were incubated at 22°C and examined at 24 and 48 h. Small colonies suggestive of yersinia were screened for urease and hydrogen sulphide production. Urease positive, H2S negative organisms were examined by further biochemical testing and identified as yersinia according to established criteria (Knapp & Thal, 1975; Bejot, et al., 1975).
68
Pathology (1979), 11, January
STEELE & MCDERMOTT
Isolate
31.2a.3X1
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
ONPG Motility 22 31 Om. decarb. Gas from glucose Sucrose Ara binose Xylose
+++ +-
112X,
403IX,
+ ++ +
-
+ +
+ + +-
-
-
RESULTS Yersiniu enterocolitica was isolated from faecal specimens of 3 patients with diarrhoea. Two patients, a 28-yr-old man and a 34-yr-old woman with diabetes mellitus and mild hepatic failure, acquired their infection in Australia. They were found to have a biotype 1 serogroup 12, phase type y and a biotype 3, serogroup 1, 2, 3, phage type XI respectively. The remaining patient, a 50-yr-old woman, developed diarrhoea while in Mauritius and diarrhoea persisted fcr 2 wk following her return to Australia. A biotype 4, serogroup 0:3, phage type IX, was isolated from this patient. Microscopic examination of faeces from these patients did not show inflammatory or red blood cells. Cold enrichment, as a primary isolation technique, was successful but in our hands did not prove to be superior to careful examination of the primary MacConkey plate incubated at 37°C for 48 h. All 3 isolates were readily detected as small non-lactose fermenting colonies on these plates. Yersinia were more easily found on MacConkey plates containing Tween 80 inoculated from the cold enrichment medium. However, this process required 6 days. Only one strain was recovered from Rappaport broth. The major biochemical characteristics of these yersinia strains are shown in Table 1. Yersinia can be readily distinguished from proteus by their inability to form phenylalanine deaminase. Identification of these strains was confirmed by Professor H. Mollaret at the Pasteur Institute who, in addition, provided the serological and phage typing information. No yersinia were isolated from the animal material collected at the abattoir. Salmonellae were isolated from 12 (24"/,) sheep and from 17 (34'4,) pigs. Infections with two or more strains of salmonella were more common in sheep. DISCUSSION
Infection due to Yersitzia enterocoliticu may take a variety of forms. Enterocolitis, regional ileitis, mesenteric adenitis, polyarthritis, erythema nodosum, septicaemia and abscess formation have been described. Enterocolitis is the most common type of infection accounting for more than two-thirds of all Yrrsinia enterocolitica infections. It is predominantly an infection of children below the age of 7 years (Mollaret, 1972) affecting mainly the 1-3 year age group (Lelorme et al., 1974; Bergstrand & Winblad, 1974). Between one-third and one-quarter of cases are found in adults. Abdominal pain with or without
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
YERSINIA ENTEROCOLITICA
69
diarrhoea is more common in older children and adults than in very young children who generally have mild gastroenteritis. As most faecal specimens received by our laboratory come from adults, the low isolation rate of yersinia of less than 0.1% may underestimate the true incidence of infection due to these organisms in Australia. However, our experience would suggest that it is not common. In view of this, it is difficult to justify the routine use of selective culture methods for yersinia. Cold enrichment with subsequent subculture to Tween 80 MacConkey agar requires 4 to 6 days. Laboratories which handle specimens from adults would be better advised to hold their primary MacConkey plates for 48 hours and screen all small non-lactose fermenting colonies for urease and hydrogen sulphide production. This was as effective and gave more rapid results than the use of cold enrichment. Yersinia did not usually produce detectable urease within 4 hours in our urea broth. It is relatively easy to miss these organisms on laboratory culture media. In addition, the results of biochemical tests are markedly influenced by the temperature of incubation (Niheln, 1967). Urease and hydrogen sulphide production can be demonstrated at 37°C but carbohydrate fermentation and acetoin formation are more variable at that temperature than below 33°C. Several of these strains fermented sugars at 30°C but not 37°C and 3/12a3/XI formed p galactosidase at 30°C or below but not at 37°C. Indole production, a feature of biotype 1 strains, was not formed by our 1/12/& strain. Laboratories which use the API system should incubate the test at 30°C and read the results at 48 hours. Citrate may be falsely positive in the API system. Yersinia enterocolitica has been isolated from water and from a wide variety of wild and domestic animals. Sheep and cattle seldom show signs of infection with Yersinia enterocolitica but asymptomatic carriage has been demonstrated in 2% of these animals in Europe. Clinical infection occurs in pigs and chinchillas. Our inability to detect yersinia in sheep and pigs at slaughter suggests that it is uncommon in these animals in South Australia and may explain the relatively low incidence of human infection in this State. The predominant human strains found in Europe, Africa, Canada and Japan belong to biotype 4, serogroup 0:3, while serogroup 0:8 is most common in the U.S.A. European and Japanese strains belong to phage type VIII, the Canadian strains to 1% and the South African strains to VIII or 1%. Biotype 4/0:3/1& p galactosidase negative strains have only been found in South Africa. Presumably its distribution now extends to Mauritius. Some serogroup/phage types are found exclusively among certain animals. Strains isolated from pigs frequently belong to serogroup 0:3 and bear a close relationship biochemically and by phage typing to human strains. This led to the suggestion (Winblad, 1973) that pigs may be an important source of human infection. The converse does not appear to have been considered. However, Mollaret (1976) has expressed the view that Yersinia enterocolitica infects both man and animals and that the major reservoir lies in the environment. Adaptation to a particular animal or to man appears to stabilize the various biochemical and serological characteristics of the strain. Nothing is known about the epidemiology or types of Yersinia enterocolitica found in Australia. A fourth strain referred by an interstate laboratory to the Salmonella Reference Laboratory for serotyping was subsequently identified as Yersinia enterocolitica 1/0:5/&. The strains which we have isolated and assume were acquired in Australia are uncommon human isolates in other parts of the world (Bercovier, 1976) though similar strains have been found in small mammals, in chinchillas and occasionally in man in Europe.
70 STEELE & MCDERMOTT Pathology (1979), 11, January ACKNOWI.EDGEMENTS We wish to thank Professor H. Mollaret of the Centre National des Yersinia, Institut Pasteur for his valuable assistance in identifying, serotyping and phage typing these strains and for providing type cultures and reference literature. We are most grateful to Dr Gordon Pevie for arranging collection and forwarding the specimens from animals at the abattoir. Address f h r rcyrinr requc’src; T.W.S.. Institute of Medical and Veterinary Science, Box 14 Rundle Street P.O., Adelaide, South Australia 5000
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
References BEJOT,J.. ALONSO,J . M. & MOI.I.ARET.H. H. (1975): Bacteriologic diagnosis of human yersiniasis (Fr.). .1/PJ. Y I MU/.Ii!frc’/.4, 233-236. BEKCOVIER. H. (1976): Contribution to the epidemiology of Yersinia mrerocoliricu infections (Pi-.). :M5d et Mal. h f e c r . 6, 425-433. C . G , Br WIXBLAD. S. (1974): Clinical HERCSTRAXD. manifestations of infection with Yer.Pinia cvifrrocoiitica in children. d i ~ r a Pmvliatr. . Scatld. 63, 875 877 E m . J . (1975): Selective culturing of 1i.rsinia entcrocoliticu at a low temperature. Scutzd. J . l t i f i w . Di.5 7, 249--251 . KNAPP.W. & THAL.E. (1975): Differentiation of Ytwinici erztcrocolirica by biochemical reactions. Cowrrib. Microhiol. Itnniriiiol. 2, 10- 16
LELORSIE,J.. LAVERDIERE, M., MARTINEAU, B. & LAFLEUR, L. (1 974): Yersiniosis in children. Can. Mecl. Acsoc. J . 110, 281-284. MOLLARET, H. H. ( 1972): Yersinia entcrocolitica infection: a new problem in pathology. Ann. Biol. Cliii. / P a r i s / . 30, 1-6. MOLLAR~T. H. H. (1976): Contributions to the epidemiology of Yersiniu cwrerocolitica infections (Fr.). McU el Mul. Infecr. 6, 442448. N I H ~ L NB.. (1967): Bacteriological studies on 80 strains of Yer.siniu enterocolitica of human origin. hiernaiional Syniposiuni on P.reudotuberculosi.~, Pari.s 1967, Series Irnniunobiol. Standard, Vol. 9. Karger. Basel, New York. pp. 327-336. WINBLAD,S. (1973): Studies o n the 0-serotypes of Yersinia enrerocolirica. Contrib. Microbial. Imniunol. 2. 27-37.
ENTEROCOLITIS DUE TO YERSlNlA ENTEROCOLlTlCA IN SOUTH AUSTRALIA
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
T. W. STEELE AND S. N. MCDERMOTT Division of Clinical Microbiology, Institute of Medical and Veterinary Science, Adelaide
Summary From August 1976 to July 1977, all faecal specimens (3298) sent to the Enteric Department of the Institute of Medical and Veterinary Science, Adelaide were selectively cultured for Yersinia enterocolifica. Yersinia enterocolifica was isolated from three patients with diarrhoea, one of whom acquired her infection overseas. These organisms were not isolated from faecal or lymph node material collected from a limited number of sheep and pigs found to have enteritis at the time of slaughter. Enteric infection due to Yersinia enterocolitica does not appear to be common in Australia and selective culture methods using cold enrichment techniques do not appear to be justified especially in laboratories handling specimens derived mainly from adults.
Diarrhoea as a manifestation of intestinal infection due to Yersinia enterocolitica has been described in a number of European countries, in North America, Africa and Japan (MoIIaret, 1972). These organisms are seldom if ever isolated in clinical laboratories in Australia and the incidence of diarrhoea due to yersinia infection in this country is unknown. From 1 August 1976 to 31 July 1977, all faecal specimens (3298) received by the Division of Clinical Microbiology were cultured for yersinia. In addition, faeces and lymph nodes from 50 sheep and 50 pigs, with evidence of enteritis at the time of slaughter in the abattoir, were examined for these organisms. MATERIALS AND METHODS All specimens were routinely inoculated to MacConkey agar, desoxycholate citrate agar, selenite broth (43°C) and Rappaport broth for the isolation of Salmonella and Shigella. Selectiveculture for yersinia using nutrient broth and Rappaport broth with incubation at 4°C for 4 days (Eiss, 1975) and subculture to MacConkey agar with and without Tween 80 was performed. These plates were incubated at 22°C and examined at 24 and 48 h. Small colonies suggestive of yersinia were screened for urease and hydrogen sulphide production. Urease positive, H2S negative organisms were examined by further biochemical testing and identified as yersinia according to established criteria (Knapp & Thal, 1975; Bejot, et al., 1975).
68
Pathology (1979), 11, January
STEELE & MCDERMOTT
Isolate
31.2a.3X1
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
ONPG Motility 22 31 Om. decarb. Gas from glucose Sucrose Ara binose Xylose
+++ +-
112X,
403IX,
+ ++ +
-
+ +
+ + +-
-
-
RESULTS Yersiniu enterocolitica was isolated from faecal specimens of 3 patients with diarrhoea. Two patients, a 28-yr-old man and a 34-yr-old woman with diabetes mellitus and mild hepatic failure, acquired their infection in Australia. They were found to have a biotype 1 serogroup 12, phase type y and a biotype 3, serogroup 1, 2, 3, phage type XI respectively. The remaining patient, a 50-yr-old woman, developed diarrhoea while in Mauritius and diarrhoea persisted fcr 2 wk following her return to Australia. A biotype 4, serogroup 0:3, phage type IX, was isolated from this patient. Microscopic examination of faeces from these patients did not show inflammatory or red blood cells. Cold enrichment, as a primary isolation technique, was successful but in our hands did not prove to be superior to careful examination of the primary MacConkey plate incubated at 37°C for 48 h. All 3 isolates were readily detected as small non-lactose fermenting colonies on these plates. Yersinia were more easily found on MacConkey plates containing Tween 80 inoculated from the cold enrichment medium. However, this process required 6 days. Only one strain was recovered from Rappaport broth. The major biochemical characteristics of these yersinia strains are shown in Table 1. Yersinia can be readily distinguished from proteus by their inability to form phenylalanine deaminase. Identification of these strains was confirmed by Professor H. Mollaret at the Pasteur Institute who, in addition, provided the serological and phage typing information. No yersinia were isolated from the animal material collected at the abattoir. Salmonellae were isolated from 12 (24"/,) sheep and from 17 (34'4,) pigs. Infections with two or more strains of salmonella were more common in sheep. DISCUSSION
Infection due to Yersitzia enterocoliticu may take a variety of forms. Enterocolitis, regional ileitis, mesenteric adenitis, polyarthritis, erythema nodosum, septicaemia and abscess formation have been described. Enterocolitis is the most common type of infection accounting for more than two-thirds of all Yrrsinia enterocolitica infections. It is predominantly an infection of children below the age of 7 years (Mollaret, 1972) affecting mainly the 1-3 year age group (Lelorme et al., 1974; Bergstrand & Winblad, 1974). Between one-third and one-quarter of cases are found in adults. Abdominal pain with or without
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
YERSINIA ENTEROCOLITICA
69
diarrhoea is more common in older children and adults than in very young children who generally have mild gastroenteritis. As most faecal specimens received by our laboratory come from adults, the low isolation rate of yersinia of less than 0.1% may underestimate the true incidence of infection due to these organisms in Australia. However, our experience would suggest that it is not common. In view of this, it is difficult to justify the routine use of selective culture methods for yersinia. Cold enrichment with subsequent subculture to Tween 80 MacConkey agar requires 4 to 6 days. Laboratories which handle specimens from adults would be better advised to hold their primary MacConkey plates for 48 hours and screen all small non-lactose fermenting colonies for urease and hydrogen sulphide production. This was as effective and gave more rapid results than the use of cold enrichment. Yersinia did not usually produce detectable urease within 4 hours in our urea broth. It is relatively easy to miss these organisms on laboratory culture media. In addition, the results of biochemical tests are markedly influenced by the temperature of incubation (Niheln, 1967). Urease and hydrogen sulphide production can be demonstrated at 37°C but carbohydrate fermentation and acetoin formation are more variable at that temperature than below 33°C. Several of these strains fermented sugars at 30°C but not 37°C and 3/12a3/XI formed p galactosidase at 30°C or below but not at 37°C. Indole production, a feature of biotype 1 strains, was not formed by our 1/12/& strain. Laboratories which use the API system should incubate the test at 30°C and read the results at 48 hours. Citrate may be falsely positive in the API system. Yersinia enterocolitica has been isolated from water and from a wide variety of wild and domestic animals. Sheep and cattle seldom show signs of infection with Yersinia enterocolitica but asymptomatic carriage has been demonstrated in 2% of these animals in Europe. Clinical infection occurs in pigs and chinchillas. Our inability to detect yersinia in sheep and pigs at slaughter suggests that it is uncommon in these animals in South Australia and may explain the relatively low incidence of human infection in this State. The predominant human strains found in Europe, Africa, Canada and Japan belong to biotype 4, serogroup 0:3, while serogroup 0:8 is most common in the U.S.A. European and Japanese strains belong to phage type VIII, the Canadian strains to 1% and the South African strains to VIII or 1%. Biotype 4/0:3/1& p galactosidase negative strains have only been found in South Africa. Presumably its distribution now extends to Mauritius. Some serogroup/phage types are found exclusively among certain animals. Strains isolated from pigs frequently belong to serogroup 0:3 and bear a close relationship biochemically and by phage typing to human strains. This led to the suggestion (Winblad, 1973) that pigs may be an important source of human infection. The converse does not appear to have been considered. However, Mollaret (1976) has expressed the view that Yersinia enterocolitica infects both man and animals and that the major reservoir lies in the environment. Adaptation to a particular animal or to man appears to stabilize the various biochemical and serological characteristics of the strain. Nothing is known about the epidemiology or types of Yersinia enterocolitica found in Australia. A fourth strain referred by an interstate laboratory to the Salmonella Reference Laboratory for serotyping was subsequently identified as Yersinia enterocolitica 1/0:5/&. The strains which we have isolated and assume were acquired in Australia are uncommon human isolates in other parts of the world (Bercovier, 1976) though similar strains have been found in small mammals, in chinchillas and occasionally in man in Europe.
70 STEELE & MCDERMOTT Pathology (1979), 11, January ACKNOWI.EDGEMENTS We wish to thank Professor H. Mollaret of the Centre National des Yersinia, Institut Pasteur for his valuable assistance in identifying, serotyping and phage typing these strains and for providing type cultures and reference literature. We are most grateful to Dr Gordon Pevie for arranging collection and forwarding the specimens from animals at the abattoir. Address f h r rcyrinr requc’src; T.W.S.. Institute of Medical and Veterinary Science, Box 14 Rundle Street P.O., Adelaide, South Australia 5000
Pathology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.
References BEJOT,J.. ALONSO,J . M. & MOI.I.ARET.H. H. (1975): Bacteriologic diagnosis of human yersiniasis (Fr.). .1/PJ. Y I MU/.Ii!frc’/.4, 233-236. BEKCOVIER. H. (1976): Contribution to the epidemiology of Yersinia mrerocoliricu infections (Pi-.). :M5d et Mal. h f e c r . 6, 425-433. C . G , Br WIXBLAD. S. (1974): Clinical HERCSTRAXD. manifestations of infection with Yer.Pinia cvifrrocoiitica in children. d i ~ r a Pmvliatr. . Scatld. 63, 875 877 E m . J . (1975): Selective culturing of 1i.rsinia entcrocoliticu at a low temperature. Scutzd. J . l t i f i w . Di.5 7, 249--251 . KNAPP.W. & THAL.E. (1975): Differentiation of Ytwinici erztcrocolirica by biochemical reactions. Cowrrib. Microhiol. Itnniriiiol. 2, 10- 16
LELORSIE,J.. LAVERDIERE, M., MARTINEAU, B. & LAFLEUR, L. (1 974): Yersiniosis in children. Can. Mecl. Acsoc. J . 110, 281-284. MOLLARET, H. H. ( 1972): Yersinia entcrocolitica infection: a new problem in pathology. Ann. Biol. Cliii. / P a r i s / . 30, 1-6. MOLLAR~T. H. H. (1976): Contributions to the epidemiology of Yersiniu cwrerocolitica infections (Fr.). McU el Mul. Infecr. 6, 442448. N I H ~ L NB.. (1967): Bacteriological studies on 80 strains of Yer.siniu enterocolitica of human origin. hiernaiional Syniposiuni on P.reudotuberculosi.~, Pari.s 1967, Series Irnniunobiol. Standard, Vol. 9. Karger. Basel, New York. pp. 327-336. WINBLAD,S. (1973): Studies o n the 0-serotypes of Yersinia enrerocolirica. Contrib. Microbial. Imniunol. 2. 27-37.
