Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

FEMS MlerobmlogyLetters 66 (1990) 163-168 Pubhshed by Elsevier

163

FEMSLE03784

Enterotoxigenicity and frequency of Campylobacterjejuni, C. coli and C. laridis in human and animal stool isolates from different countries G.-B. L i n d b l o m 1, M. Johny 2, K. Khalil 3, K. Mazhar 3 G . M . Rulz-Palacios 4 and B. Kaijser 1 I Departmentof ChmcalBacterlolog},. Umversltr of Gotebor~S~eden. 2AmtrJ Hospital, Kuwait. ~Department of Paediatrws Mayo Hospttal. King EdwardMedical College. Lahore. Pak,slan. and ~Instuuto Nactonalde la Nutrwt6n. Me~acoDF. Me:~tco Roe.owed28 June 1989 Revisionrecetved20 July I989 Accepted 27 July 1989 Key words: Campylobacter jejunt, Campylobacter cob, Campylobacter lartdts, Enterotoxtgemclty

1. SUMMARY Campylobacler jejum and C colt strums were collected during three different years from adult patients with enterocohtis m Sweden (n = 372) from 49 pauents in Kuwmt. and Campylobacter strains from hens from Meraco, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C jejum was the predormnant spe~es m human and hen isolates, and C colt m pigs C, colt was slgmficantly more common m human isolates from Sweden, and more common m hen isolates from Pakistan, than m hens from Sweden and Mexico C lartdAs was only tsolated from ptgs (17,%) and was in no ease enterotoxlgenlc. Both m human and hen isolates, C, jejan# strains were more enterotoxigeme than C coh strains C jejuni stratus from Swedish hens were less enterotoxigenlc than those from pakistan and Mextco ( P < 0.001), and stratus from pigs were less enterotoxtgemc than those from hens ( P < 0.001) We conclude that C jejum are more often enterotoxigemc and pnsmbly more vtrulent than c

Correspondence to G-B Lmdblom. Department of Chmcal Bacteriology.Uluversayof Goteborg. Sweden

colt and C larJdJs The relauve frequency of C jejum and C colt m humans and ammals differs from one country to another. 2. INTRODUCTION Campylobacterjejum and Campylobacte. cob are recogmsed as one of the most common causes of enterms in man the world over [1-5]. C yejum and C colt are frequently found in the faeces of ammals, both domestic [6-8] and wild [91. Poultry would seem to be one of the major world-wide causes of Campylobacter enteritts [10-121 Campylobacter isolates from poultry. sheep and cattle are predominantly C .legato, whde m ptgs C colt ts the most common species [9I. Many studtes on the aehology of enteric infections m man make no dtstlnction between C jejum and C colt [5.13,1g]. Among studies where me two species have been dffferenttated, the reported tsolation rate of C cob has vaned from 3-44~ [15-18]. Georges-Courbot et aL found a higher frequency of C coh strains m chddren with Campylobacter diarrhoea than among healthy carnets [18]. while Taylor and coworkers reported that C cob was significantly less often assoctated

0378-1097/89/$03 50 © 1989FedcraBon of European Mtcroblolo~calSoc.etles

164 with symptomatic refections and wRh bloody diarrhoea than C jejunt [19] Enterotoxin production by C. jejum and C. cola has been compared in studies published recently [20-22] Johnson and Lior [20] found that C. colt strains isolated from humans with gastro-ententts were more often entvrotordgenic than C. jejum strains, wlule the reverse was true of stratus of non-human onglri, lq contrast to the Canathan study of human isolates, Belboun and M~graud found C jejum strains to be more often eaterotoxagenic than (7. coh stratus [21] which is in accord with the results of our Swedish study [22]. The aim of the present study was to mvesfigat¢ the enterotoxigeniclty and distribution of C. jejum and C coh in human isolates, and C. jejum, C. coh and C. landis in non-human isolates from different years and different parts of the world.

3. MATERIALS A N D M E T H O D S

3.1. Human stratus The Swedish material comprises 372 Campylo. bacter 1.~olates from as many adult patients seekm g medical care for diarrhoea at the Hospital for Infectious Diseases, G6teborg The isolates were collected during three different years: 202 from 1978, 95 from 1984 and 75 from 1987. Those from 1978 and 1984 were analysed after lyophthsatton. Those from 1987 were analysed directly after isolation from the patient (referred to here as 'fresh' isolates). The material from Kuwait consisted of 49 Campylobacter isolates from as many patients seeking medical attention at Anuri Hospital, Kuwmt. Although the patients' ages ranged from 3 months to 60 yeats, they were mamly young children (72.6~). All isolates had been lyophilised before despatch to G~teborg. 3.2, Ammal stratus 3 2 1. Hens Campylobaeter isolates from hens aged 7-30 weeks (1 isolate/hen) were collected from three different countries, Mexico, Pakistan and Sweden The strains from Pakistan (n = 60) came from two different commercial breeders, newly culti-

rated and stored m deep agar dunng transportation to Gdteborg. The isolates from Mexico (n = 10) were collected from hens raised on three family farms. The strains were lyophihsed before use. The Swedish hen strains (n = 37) came from five different commercial breeders and were lyoplulised before use. 3.2.2. Pigs Forty-seven "fresh" Campylobacter isolates collected from as many pigs (aged 1 week-24 months) were obtamed from one commercial breeder in Sweden.

3.3. Bacterial growth and identtfrcatwn All tests were done on strains grown on bloodagar plates in microaerobm atmosphere condiUons (5~ 02, 10~ CO2, 85~ N2) for 24 h at 42°C, and were identified by the following cnteria: eatalase and oxtdase production, Oram-statmng, susceptlhihty to nalidlxlc acid (30/Lg/drskL and ability to hydrolyse hippurate. 3.3.1. fltppurate hydrolysis test. The luppurat¢ hydrolysis test was done as described by Hwang and Ederer, using a heavy inoculum [23]. All negative samples were redone. Three reference strains were used as controls: C. dejum biotype I N C T C 11168, C. jejum biotype II N C T C 11392 and C. colt N C T C 11353. 3.3.2. Enterotoxin assay, Chinese hamster ovary (CHO-K1) cells were used for detection of enterotoxin using a supematant of a strain grown over night in Gc-medium (Oxotd). One hundred cells per well were counted and a strain was considered to be enterotoxin productive if > 50~ of the C H O cells were elongated. All tests were porformed in duphcate and positwe and negative controls were included [22]. Statistical analysis: Differences of probabilities wero tested with the chi-squared test.

4. RESULTS 4. l Human strums Campylot~acter, isolates from Swedish and Kuwaiti patients were predomlnately C. jejum, comprising 88.4~ and 71,4~ of these groups respectively (Table la, h).

165 Table la Frequency and enterotoxJgentclty of C je)um and C colt stratus from adult humans with acute entetoenhtts m Sweden from 3 different yca~

Samphog

No of

~ of isolates

year

patients

C jejum

C cob

CHO-pos C 2eJunt

CHO-pos C colt

CHO-pos Total

1978 1984 1987 Total

202 95 75 372

M2 937 93 3 884

15 8 a 63 ~ 67" 11 6 r

M1 753 50 0 486 ©

21.9 0 20 0 186 ~

32.2 be 705 bd 48 0 c,e 4~2 s

a b " d c

X2 = X2 = X~ = X2 = X2 =

7 9; P < 001 (more C coh m 1978 than m 1984 or 1997), 38 5, P < 0001 (more CHO pos strmns m 1984 than in 1978), 5 9, P < 002 (more CHO pos stratus m 1987 than In 1978), 8.9, P < 0 01 (more CHO pos stratus m 1984 than m 1987), 13.8, P < 0 001 (more C l i O pos C jejum than C colt )

Table l h Frequent3' and enterotoxtgemclty of C jejum and C coil strmns from 49 patients with acute entcrocohtts tn Kuwast Samphng year

No of patients

% of isolates C jejum

C col:

CHO-po~ C jejum

CHO-pos C ~h

CHO-pos Total

1986

49

71 4

28.6 I

85 7

57 1

77 6 B

r X2 = 10 71, P g 0 0! (more C coil in Kuwait than m Sweden) x 2 = 18 2, P < 0,001 (more CHO pos strmns m Kuwait than In Sweden)

Tablc 2 Frequency and enterotoxagern~ty of C jejum. C col: and C /andJs so hen stratus from Pakistan (n = 60), Sweden (n = 37) and Mexico (n = 10). as compared with that in strains from Swedish pigs (n = 47) Ammal

NO of

~ of,solates

count~

samples

C 2ejum

C col:

C lar:d:s

CHO pos C jejun:

CHO pof. C colt

CHO pos C lan~s

CHO pos total

Hens Pakistan

60

65 0

35 0 a

0

g7 2 b c

23 8 b

0

65 0

Hcns Sweden

37

89 2

10 8 ~

0

39 4 ~

25 0

0

37 8

Hens Meraco

10

|000

0

0

1000 g

0

O

lfl00

107

76.6

23 4

0

69.3

24,0

0

58 9 a

47

0

83 0

17 0

0

30 8

0

25 5 a

Hens total Pigs Sweden

X 2 = 7 O, P < 00l (more C colt m Paklstam hens than m Swedish hens)

b X2 = 24.1, P < 0001 (more CHO pos C jejum than CHO pos C coil m Pakistan) X2 - 18 0, P < 0.001 (Palostan), X2 = 1L3, P < 0 fl01 (Mexaen) (more CHO pos C jeflmJ strmns m Paluomn and Mexw,o than m Sweden) d X 2 = 14 5 P < 0 001 (more CHO pcs strmns among cluckcn than mnong pig strc.~g)

166 Among the Swedish stratus, the frequency of C cob was greater m specimens from 1978 than in those from 1984 or 1987 ( P < 0 . 0 1 ) ; and the frequency of enterotox~genic strains was lower among lyophihsed strains from 1978 than among those from 1984 ( P < 0.001) or among fresh stratus from 1987 ( P < 0.02). In the Swedtsh matertal C jejum strmns were generally more frequently enterotomgemc than C coh strmns ( P < 0.001) (Table la). Strmns from Kuwmt were classified as C coh m 28.6% of cases, which is more common than m the Swedtsh material ( P < 0.01). The proportion of enterotoxlgenic strams was greater in the Kuwatt material than in the Swedtsh material ( P < 0.001) (Table lb). 4.2. Ammal stratus The frequency of C. cob strmns was higher among hens from Pakastan than among hens from Sweden ( P < 0.01). Among strmns from Pakistan C jejum were more often enterotoxigenic than were C. colt ( P < 0.001) (Table 2). All hen stratus from Mera¢o were enterotordganic C, jejum. O f the C. jejum strains, those from hens in Pakistan and Mexico were more enterotox~gemc than were Swe&sh ones ( p < 0.001 respectively). Strains from Swedish pigs conststed of 83.0% C. colt and 17.0% C. larMis. N o C. lartdls strata was enterotoxigeaie. Pig stratus were much less enterotoxtgenie than strmns from hens ( P < 0.001). No C. jejum strain was found in pigs, and no C lar:dls strain in hens (Table 2).

5. DISCUSSION The disertmination of different Campylobacter species has been successively extended since the involvement of Campylobacter in enterocofitls was first discovexed. We now tdenttfy 14 Campylobacter species [24]. Those most commonly related to enterocolitls tn humans are C jejum and C cob, The reported frequency of C. jejuni in re|alma to C coli in enterocolitis patients differs widely from one country to another, at least partly perhaps due to

differences in methodology. The Inppurate test used here was recently found to be susceptible to variaUons in its performance; tf the moculauon of the test tubes ts insufficient, results may be inaccurate (Lindblom, G-B., m manusenpt). Reports on the enterotoxigenicity of C. 7ejum and C cob have been sparse. Belboun and M6grand found more enterotoxigemc C jejum than C col~ stra:ns from children in Algeria [21], which Is in accord voth our fmdmgs. In the present study, we found C. cob to be significantly less enterotoxJgenlc than C. jejum, both in human and ammai isolates. In contrast to Johnson and Liar [20], we found no enterutOxdgenic C lartdts. In view of the importance of toxan producUon as a virulence factor, these results are tn accord with the suggestion that C jejunt ts more wrulent than C. coll. These result> are m good agreement with ehnlcal reports from Hongkong, for example, where asymptomaUc Campylobacter colonisauon ts common as well as a high frequency of C coh [25], and from Thailand where C colt was found to be less often a~oclated wtth symptomattc infections and bloody diarrhoea than was C. jejum [19]. In relatively few invc~tigauons has coterotordgenicity been studied over a penod of several years. The same applies to the relative frequency of tsolatton of C jejum and C. col*. In our studtes, we found differences in these aspects over time, aspe~tally vis-/L-vm enterotoxln production. We behere tlns to represent a true vanation, as the methodology was Identical at all Umes, and analysing fresh (first cnltwatton) strains instead of lyophilised did not noticeably promote enterotoxln production. The main source of Campylobacter infectton throughout the world Is the cincken. In our stodtes as well as in earlier invesugattons [26] it has been found that the relative frequencies of C. je)um and C. cob in chickens are very similar to those m humans with enterocolitis. This is in contrast to other sources, such as pigs where C cob is found in 80-100% [27]. The question of whether C. jejmu and C. colt differ in virulence remains to be answered. Further strains are needed both from animals and humans, both with and without symptoms. The indication of a virulence difference seen in our study empha-

167 sis the s~gmhcance of f u r t h e r e x t e n d i n g Campylobacter species d e t e r r m n a t i o n o n a r o u t i n e ba s i s m patic~ats v a t h enterocolitts.

ACKNOWLEDGEMENTS T h i s i n v e s t t g a t i o n w a s s u p p o r t e d b y the S w e d i s h M e d i c a l R e sear ch C o u n c d ( g r a n t no. 7169), a n d the S w e d t s h C o u n o l for F o r e s t r y a n d A g r i c u l t u r a l R e s e a r c h ( g r a n t no. 598). W e t h a n k Leif O ~ r a n s son. a n a g r o n o r m s t at Sveriges S w n - C e n t e r . SvaliSv, S w e d e n , [or p r o w d i n g the p i g s a m p l e s , a n d secretary A n n e - B e l l E k for slulfui t y p i n g of the m a n u script.

REFERENCES [11 Svedhem, A, and Katlser, B (19801 J Infect Das 142, 353-359 [2} Blaser. M J and Relier. L B 09811 New Engl J Med 305.1444-1452 [3] 0o~enheuser. V D . Rtchardson. N J. Bqmer. J H . Roux, D.J. Schutte. A.V., Kornhof, HJ.. Freiman. I. and Hanman, E (19791 J Chn Mtcroblol 9. 227-232 [4] (?alva. J J . Rutz-palacms, G M . Lopez-Vlda]) A B . Ramos. A and BojahL R (1988) Lancet 1 No 8584, 503-506 [5} M61bak. K . HOjhng. N and Gaar~lev. K (1988) Eptdem lnf 100, 227-237 [6] Svedhem./~ and Kaijser, B (19811J Infect Dm 3. 37-40 [7] Lmdblom. G-B. Sj6rgen. E and Katjser, B (19861J Hyg Camb 96. 385-391 [8] RasnnauL L , Suthlenkul, O , Echevema, P, Taylor, D, Senwatana, J, Bangtrakulnouth, A and Lexomboon, U (19~8) Trop Me.d Hyg 39. 97-102

[9] Manser. PA and Dalael, RW (1985)J Hyg. Camb 95. 15-21 [I0] Norkrans. G and Svcdh~m, ~ (1982) J Hyg. Camb 89, 163-170 [111 Soto, A, Flgueroa, G and Urcelay. S (198613 Vet Med Ser B 33, 676-684 1121 Dennng, M S, Tauxe, R V and Blake, P A (19871 Am J Epldem 126, 526-534 [13} Lassen. J and Kapperud. G (1984) J Chn Microblol 19. 153-156, [14] Agatha Am, E, Takahaslu, T and Shonekan. R.A.O (19881 J Chn Mlcroblol 26, 605-606 [151 Sktrrow, M B and Benjamin, J. (19801 J Clm Pathol 33, 1122 [16] Karmah. M A, Penner, J L, Flenumng, P C , Wdhams. A and Hcnnessy, J N (1983) J Infect. Dis 147. 243-246 [17] Kapperud. G . Lassen. J , Lauwers, S and RoseL O (1984) J Chn MtcrOblol. 19.15%160. [lg} Georges-Courbot, M C . Baya. C . Beraud. A M. Meumer, MY and Georges, A J (19861 J Clm Mlcroblol 23, 592-594 [19] Taylor. D N . Echevema. P. Plttarangsl. C . Senwalan,-L J . Bodhldatta, L and Blaser, M J (1988) J Chn Microblol 26, 863-868 [20] Johnson, W M and Llor, H (1986) J Chn Mlcroblol 24, 275-281 [21] Relhoun. A and M~graud, F (1988) FEMS Microblol Lett 51 25-28 122] Lmdhlom, G - B . Kauscr, B and S]ogren, E (19891 .I Chn Mlcrobml 27,1272-1276. [23] Hwang, M -N and Ederer, G M (19751 J Chn Microbtol 1,114-115 [24] Penner. J (19881 Chn Mlcrohtol Rcv 1,157-172 [25] Ho, B S W and Wong. W T (1985) J Hyg. Camb 94. 55-60 [26] Oostcrom, J , Banffer, J R J , Lau'.vcrs, S and Busschbach, A E (19851 Antoine Leeuwenhoek, J Mtcroblo| 51, 65-70 [271 Walder, M, Sandstedt, K and Ursmg, J 09831 Curt Mlaroblol 9, 291-296