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Isolation of Yersinia enterocolitica and related species from river water
Zentralbl. Mikrobiol. 143 (1988),575-581 VEB Gustav Fischer Verlag Jena
[1) Istituto di Microbiologia Agraria e Tecnica, Universita di Bologna, Bologna (Italia), 2) Presidio Multizonale di Prevenzione, Settore Biotossicologico, Bologna (Italia), e 3) Dipartimento di Agrobiologia e Agrochimica, Settore di Microbiologia, Universita della Tuscia, Viterbo, (Italia)]
Isolation of Yersinia enterocolitica and Related Species from River Water S, MASSA'), DANIELA CESARONf), G. PODA') e L. D. TROVATELu3) With I Figure
Summary One hundred and twenty samples of river water collected from sampling sites in the period between December 1986 and December 1987 were examined for the presence of Yersinia enterocolitica and related species. Among a total of 26 Yersinia strains isolated the following species were recognized: Y. enterocolitica, 12 strains, Y. intermedia, 5 strains, Y. frederiksenii, 2 strains, Y. kristensenii and Y. aldovae, I strain each; 5 were atypical Yersinia strains (rhamnose+, citrate + , o:-methyl-D-glucoside+). Although the Yersinia isolates belong to the so-called "environmental" serotypes, which are usually considered as non-pathogenic for humans, Y. enterocolitica biotype I, serotype 0: 7.8, Iysotype Xo, which has previously been associated with gastro-intestinal infection in Italy, was found to be present. Statistical evaluation of our data showed that the presence of Yersinia spp. and the presence of total or fecal coliforms are unrelated.
Zusammenfassung Zwischen Dezember 1986 und Dezember 1987 wurden 120 FluBwasserproben entnommen und in Hinsicht auf das Vorkommen von Yersinia enterocolitica und verwandten Typen untersucht. Insgesamt wurden 26 Stiimme von Yersinia isoliert: 12 Y. enterocolitica, 5 Y. intermedia, 2 Y. jrederiksenii, 1 Y. kristensenii, 1 Y. aldovae und 5 atypische Yersinia spp. (Rhamnose +, Citrat+, Ci-methyl-D-Glukosid+). Obwohl aile isolierten Stamme den sogenannten Umwelt-Serotypen angehiirten, welche im allgemeinen fUr den Menschen als nicht pathogen angesehen werden, wurde Y. enterocolitica Biotyp 1, Serotyp 0:7.8, Lysotyp Xo isoliert, welche mit einem in Italien vorgekommenen Fall von Magendarminfektion verbunden worden ist. Es wurde kein statistisches Verhiiltnis zwischen dem Vorkommen von Yersinia spp. und Gesamt- oder Fiikalcoliformen gefunden.
Yersinia enterocolitica is receiving increased attention as a food- and waterborne pathogen in humans. Common clinical syndroms include gastroenteritis with pain that mimics appendicitis, arthritis, erythema nodosum, mesenteric lymphadenitis, and septicemia (BOTTONE 1977; BERCOVIER and MOLLARET 1984). Y. enterocolitica has been isolated from a large number of wild and domestical animals, a wide range of foodstuff and from water (SWANIMATHAM et al. 1982). The demonstration of the presence of Y. enterocolitica in water (HARVEY et al. 1976; HIGHSMITH et al. 1977; KAPPERUD 1977, SCHIEMAN 1978) has generated considerable interest in the aquatic environment as a possible reservoir and vehicle of transmission for yersiniosis (BARTLEY et al. 1982). Although many strains of Y. enterocolitica and related species isolated from water are non-pathogenic for man, there have been several cases in which the presence of these organisms has been implicated as cause of waterborne outbreaks (LASSEN 1972; KEET 1974; Eden et al. 1977; THOMPSON and GRAVEL 1986). In Italy, Yersinia spp. has been found in humans, animals, the environment, and water, but at present, it appears to be less frequent than in other European and non-European countries,
576
S. MASSA et al.
although, since 1978, the clinical cases seem to be of an increasing frequency (CHIESA et al. 1982; FANTASIA MAZZOTTI and GIRALDI 1983). In this investigation samples of river water were examined for the presence of Y. enterocolitica and Y. enterocolitica-like organisms together with the usual fecal indicator organisms.
Material and Methods Sampling sites 10 representive sampling sites, located in the upper part of the Reno river, were studied (Fig. 1): R I -R7 were situated on the river itself, whereas Stl-S13 were sited on tributaries at a distance of 500 m before the point where they joinded the Reno. The Reno river, between sites RJ and R7 , passes from a rural area to an increasingly urbanized and relatively industrialized area.
Collection of samples Water samples were collected in 21 sterile glass bottles, approximately 10 cm below the water surface, and tested within 4 h ofter collection. A total of 120 samples were analyzed between December, 1986 and December, 1987.
Coliform analysis The coliforms in each water sample were enumerated using the membrane filtration procedures as described in Standard Methods (APHA 1980). Total coliforms were determined on M-Endo medium at 35 DC and fecal coliforms on M-FC medium at 45 DC.
Isolation of Yersinia species 100 ml of Peptone water (Biolife), concentrated 10 times, were added to each water sample (1 I), together with bile salts (Difco) and sorbitol (C. Erba) to a final concentration of 0.15% and 1%, respectively (MEHLMAN et al. 1978). The samples were then stored at 4 DC. After 7, 14 or 21 days one loopful of cold enrichment was streaked onto cefsulodin-irgasan-novobiocin agar plates (Yersinia Selective Agar, Oxoid). The plates were incubated at 25 DC and examined after 24 and 48 h. Colonies showing typical "bull's eyes" morphology, after purification were subcultured onto K1iger-Iron-Agar (Biolife) slant and incubated for 48 hat 28 DC. Only those isolates that produced an acid butt and aIcaline slant without hydrogen sulphide or gas were Gram stained and tested for urease motility at 22 and 37 DC, growth on Simmons citrate, and lysine-arginine decarboxylase (WEAGANT 1983). Positiveness at these tests was held as indication of apartenance to the Yersinia genus. Additional tests for the identification of the strains (biotype, serogroup and lysotype) were carried out by the "Centro di Riferimento per la Yersinia" in Rome.
Statistical analysis The relationship between the presence of Yersinia spp. and coliforms (total and fecal) was statistically evaluated using a corrected le-analysis.
Results A total of26 strains of Yersinia spp. were isolated from 120 samples of water collected from 10 sampling sites between December 1986 and December 1987, with a frequency of 21. 6 % (Table 1). Most strains (18 out of 26) were isolated only after 14-21 days of enrichment. Water samples were considered negative if yersinias were not isolated after an enrichment period of 21 days, since additional time is known not to produce any increase in the isolation frequency (NILEHN 1973; EISS 1975; WALKER and GRIMES 1985). Most of the Yersinia strains were isolated from the water samples from sampling site R2 and all during the cold months of the year (from November to March). The temperature of the river water at sampling time was in the range of 5 to 10 °C. The populations of fecal coliforms and total coliforms per 100 ml ranged from 1.6· 102 to 1.6.106 and 2.0' 10 to 4.5' 105 , respectively (Table 1). It is interesting to note that no strain of Yersinia spp. was isolated at any of the levels of total or fecal coliforms contamination at sampling sites St2 and St3 (Table 2). At the other sampling sites, the number of samples in which Yersinia spp. where isolated, when the total coliforms were less than 105 per 100 ml, was 15, whereas at a level of total coliforms between 105 and 106 per 100 ml the number of positive samples was 7. Yersinia spp. was found to be present in only one sample when the number of fecal coliforms was greater than 105
St3
Stz
R7 St]
0 0
2 5 2 5 3 2 2
12 12 12 12 12 12 12 12 12 12
R] Rz R3 R4 R5
~
No. of positive samples
No. of samples
Sampling site
3 7 2 5 3 2 2 2 0 0
No. of strains
9.2 5.8 9.0 2.0 3.0 8.3 8.3 1.9 2.7 1.8 X X X X X X
103 104 104 104 105 104 x lif x 104 X 103 X 104 6.0 8.5 1.8 3.5 2.5 1.8 8.0 8.0 1.6 9.0
102 103 104 103 104 103 x 103 X 102 X 102 X 102 X X X X X X
4.0 4.2 2.8 8.0 1.6 4.1 5.0 1.8 1.0 8.0
X X X X
x
X X X X X
105 105 104 104
1~
104 105 105 104 106
3.8 3.4 2.4 6.1 1.9 3.8 2.7 6.2 5.8 3.8
X X X X X X X X X X
Mean
Maximum
Mean
Minimum
Fecal
Total
Coliform bacteria per 100 ml
Table 1. Incidence of Yersinia spp. and coliform results for 120 water samples from Reno river
103 104 104 103 105 104 104 103 102 103
9.0 3.1 2.0 1.8 1.5 4.7 8.5 1.3 2.0 2.5
103 103 102 104 102 103 102 x 10 X 102 X X X X X X X
x 10
Minimum
2.0 1.0 6.0 2.2 4.5 1.8 8.0 6.0 1.5 1.6
X X X X X X X X X X
104 105 104 104 105 105 104 104 103 104
Maximum
OJ>
0
Vl
-.) -.)
.,
~.
;;: '""".., ,.., "" ~
~ .... '" S· E·
0
....
::>
o·
e;.
578
S.
MASSA
et al.
A
Scale 1: 150. 000
Fig. 1. Map of the Reno river showing the sampling sites.
per 100 ml; the number of samples which yielded Yersinia spp. at levels of fecal coliforms less than 105 per 100 ml was 21. A corrected x2-analysis was carried out to find a relationship between the presence of Yersinia spp. and coliforms (total and fecal). No correlation was found between the isolation of Yersinia spp. and the presence of coliforms at any of the levels. The results of identification of Yersinia strains are presented in Table 3. As shown, 12 strains were identified as Y. enterocolitica "sensu strictu" and 14 as Y. enterocolitica-like organisms according to BERKOVIER et al. (1980,1980a), BRENNER et aI. (1980), URSING et al. (1980) and BERCOVIER et al. (1984). All Y. enterocolitica strains were of biotype 1 and lysotype X o, but of different serogroups (autoagglutif!ated, 0 :4.32-10, 0: 7.8). The other strains were identified as: 2 Y.frederiksenii, 1 Y. kristensenii, 1 Y. aldovae, 5 atypical Yersinia spp. (rhamnose+ citrate+ and
579
Isolation of Yersinia fnlerocolitica Table 2. Relationships of Yersillia 'pp. isolation with coliforms population of water samples Range of bacteria
No. of samples positive for Yersillia spp.
All Total coliforms/ I00 ml 01,000 1,001 10,000 10,001- 100,000 100,00 1-1,000,000 Fecal coliformsllOO ml 0100 101- 1,000 1,001- 10,000 10.001-100,000 > 100,000
I 3
2
2 3
4 10 7
2
2
I
2
I 3
2
9 9
2
4
Table 3. Characteristics of Y. elllerocolitica and related species isolated from river water Species')
Biotype
Y. ellIerocolitica
Serogroup
Lysotype
No. of strains
AAGb)
Xo Xo Xo
9 I 2
0:4,32-10 0:7.8
Y. illlermedia
4 4 4
0: 10 0:36,37 0:35
AAG Y. frederiksellii
Nc)
Xo XO Xo Xo
0:4,33-10
Xo XO
Y. kristellsellii
0: 10. 34
Xo
Y. aldovae
AAG
XO
AAG
Xo Xo XO XO Xo
Atypical Yersillia Sppd)
0: 12, 26 0:48 0:21 0: 16,50,52
I
2
.) Results of "Centro di Riferimento per la Yersinia", Rome. b) AAG. autoagglutinated. 0) N, non agglutinable. d) Yersinia rhamose+, citrate+. a:-methyl-D-glucoside+.
Discussion Y. enterocolitica have been isolated from water in many countries. On a probable account of different methods used, the isolation percentages reported by the different authors vary considerably (HARVEY et al. 1976; HIGHSMITH et al. 1977; MEADOWS and SNUDDEN 1982; BURRINI et al. 1986). In this investigation, Yersinia spp. were found in 18.3 % and Y. enterocolitica in 4.1 % of the samples tested. All the strains of Y. enterocolitica isolated belong to biotype I. WAUTERS (198 J) considers that Y. enterocolitica biotype 1 (except for the serotype 0: 8), and the related species, are ubiquitous and largely have no clinical significance.
580
S. MASSA et al.
One isolate of an atypical Yersinia species, which fennents L-rhamnose and not sorbose, cellobiose and melibiose, was identified on the basis of these characteristics as Y. aldovae, previously called Y. enterocolitica-like group X2 (BERCOVIER et al. 1984). This species is frequently isolated in the water sources together with Y. enterocolitica, but it has not been implicated in human infections. The number of samples positive for Y. enterocolitica and related species was greater in December and January, as observed by others authors (WEBER and KNAPP 1981; MEADOWS and SNUDDEN 1982; PIANETTI et al. 1985). No strain of the serogroups 0: 3 and 0: 9, most frequently associated with human yersiniosis in Europe, is found among the Yersinia isolates studied; the so-called "environmental" strains are represented instead. The question of the relationships of these strains with those involved in human infections is still unanswered; an increase has been noticed in the number of infections caused by nonhost adapted Y. enterocolitica, and atypical Y. enterocolitica-like organisms (BOTTONE 1978). One of the strains of Y. enterocolitica, isolated from the water of the Reno river, was the serotype 0:7.8; this type is not only widespread in the environment (FANTASIA MAZZOTTI and GlRALDI 1983; MARRANZANO et al. 1984), but it has also been associated with an outbreak of gastroenteritis among children at a nursery-school in Italy (GlRALDI et al. 1982). It was not possible to show a statistically significant relationship between Yersinia spp. and fecal indicator organisms. These observations lead to the conclusion that total or fecal colifonns cannot be used to predict the presence or absence of Y. enterocolitica and related species. Acknowledgements Warm thanks are due to Claudio Chiesa of the Third Pediatric Clinic of the University of Rome and the "Centro di Riferimento per la Yersinia" for the identification of the Y. enterocolitica strains.
References APHA: Standard Methods for the Examination of Water and Wastewater. 15th Edition. Washington, DC. American Public Health Association 1980. BARTLEY, T. D., QUAN, T. J., COLLINS, M. T., MORRISON, S. M.: Membrane filter technique for the isolation of Yersinia enterocolitica. Appl. Environ. Microbiol. 43 (1982),829-834. BERCOVIER, H., BRENNER, D. J., URSING, 1., STEIGERWALT, A. G., FANNING, G. R., ALONSO, 1. M., CARTER, G. P., MOLLARET, H. H.: Characterization Yersinia enterocolitica "sensu stricto". Curro Microbiol. 4 (1980),201-206. MOLLARET, H. H.: Yersinia. In Bergey's Manual of Systematic Bacteriology. (Edited by KRIEG, N. R.) Baltimore 1984, pp. 498-506. STEIGERWALT, A. G., GUIYOULE, A., HUNTLEy-CARTER, G., BRENNER, D. 1.: Yersinia aldovae (formerly Yersinia enterocolitica·like group X2): a new species of Enterobacteriaceae isolated from acquatic ecosystem. Int. J. Syst. Bacteriol. 34 (1984), 166-172. URSING, 1., BRENNER, D. 1., STEIGERWALT, A. G., FANNING, G. R., CARTER, G. P., MOLLARET, H. H.: Yersinia kristensenii a new species of Enterobacteriaceae composed of sucrose-negative strains (formerly called Yersinia enterocolitica-like). Curro Microbiol. 4 (l980a), 219-224. BRENNER, D. 1., BERCOVIER, H., URSING, 1., ALONSO, J. M., STEIGERWALT, A. G., FANNING, G. R., CARTER, G. P., MOLLARET, H. H.: Yersinia inlermedia: a new species of Enterobacteriaceae composed of rhamnose positive, melibiose positive, raffinose positive strains (formerly called Yersinia enterocolitica or Yersinia enterocolitica-like). Curro Microbiol. 4 (1980), 207-212. BOTTONE, E. 1.: YersilJia enterocolitica: A panoramic view of a charismatic microorganisms. Cril. Rev. Microbiol. 5 (\977), 211- 241. - Atypical Yersinia enterocolitica: clinical and epidemiological parameters. 1. Clin. Microbiol. 7 (1978), 562-567. BURRINI, D., GAMBASS1NI, L., NANNICINI, E., LANCIOTTI, E .: Presenza di Yersinia enterocolitica nell'acqua del Fiume Arno nel tratto fiorentino. L'lg. Mod. 85 (1986), 119-128.
Isolation of Yersinia enterocolitica
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CHIESA, C., MOLLARET, H. H., MIDULLA. M.: Epidcmiologia delle Yersiniosi in ltalia. Ann. Sclavo 24 (1982), 686-694. EDEN, K. V., ROSENBERG, G., STOOPLER, B., WOOD, A.. HIGHSMITH, A. K., SKALLY, P .. WELLS, J., FEELEY, J. C.: Waterborne gastroenteritis at a ski resort associated with the isolation of Yersinia enterocolitic:a. Public Health Rep. 92 (1977), 245-250. EISS, J.: Selective culturing of Yersinia enterocolitica at a low temperature. Scand. J. Infect. Dis. 7 (1975),249- 251. FANTASIA MAZZOTTI, M., GIRALDI, V.: Biosierotipi di Yersinia isolati in Italia dal 1979 al 1982. Nuovi Ann. Ig. Microbiol. 34 (1983), 437-446. GIRALD!, V., CAPRIOLl, A., FALBO, V., GENNARO, M., SALSOMASO, S.: Isolamento di Yersinia enterocolitica durante una epidemia di gastroenterite acuta infantile. Giorn. Mal. Inf. Paras. 34 (1982),595-596. HARVEY, S., GREENWOOD, J. R., PiCKETT, M. J., MAH, R. A.: Recovery of Yersinia enterocolitica from streams and lakes of California. App\. Environ. Microbiol. 32 (1976), 352- 354. HIGHSMITH, A. K., FEELEY, J. c., SKALLY, P., WELLS, J. G .. WOODS, B. T.: Isolation of Yersinia enterocolitica from well water and growth in distillated water. Appl. Environ. Microbiol. 34 (1977), 745-750. KAPPERUD, G.: Y. enterocolitica and Yersinia-like microbes from mammals and water in Norway and Denmark. Acta Pathol. Microbiol. Scand. B. 185 (1977),129-135. KEET, E. E.: Yersinia enterocolitica septicemia. N. Y. State J. Med. 74 (1974),2226-2230. LASSEN, J.: Yersinia enterocolitica in drinking water. Scand. J. Infect. Dis. 4 (1972), 125-127. MARRANZANO, M., URSINO, A.. AGOD!, A.: Presenza di Yersinia enterocolitica e di altri batteri apparentati in ortaggi del commercio. L'lg. Mod. 82 (1972),262-268. MEADOWS, C. A., SNUDDEN, B. H.: Prevalence of Yersillia enterocolitica in waters of the lower Chippewa River Basin, Wisconsin. Appl. Environ. Microbiol. 43 (1982), 953-954. MEHLMAN, I. J., AULISIO, C. C. G .. SANDERS, A. c.: Problems in the recovery and identification of Yersinia from food. J. Assoc. Off. Anal. Chem. 61 (1978), 761-771. NILEHN, D.: The relationship of incubation temperature to serum bactericidal effect, pathogenicity and in vivo survival of Yersinia enterocolirica. Contrib. Microbiol. Immunol. 2 (1973). 85-92. PiANETTI, A., BRAND!, G .. BRUSCOLlNI, F., BAFFONE, W., SALVAGGIO, L.. ALBANO, A.: Ricerca di Salmonelle e Yersinie nei liquami di fogna della citta di Urbino. Nuovi Ann. Ig. Microbiol. 36 (1985), 279-289. SCHIEMANN. D. A.: Isolation of Yersinia enterocolitica from surface and well water in Ontario. Can. J. Microbiol. 24 (1978), 1048-1052. SWAMINATHAN, B., HARMON, M. c.. MEHLMAN, I. J.: A review: Yersinia emerocolitica. J. Appl. Bacteriol. S2 (1982), 151-183. THOMPSON, J. S .. GRAWEL, M. J.: Family outbreak of gastroenteritis due to Yersinia elllerocolirica serotype 0: 3 from well water. Can'~ 1. Microbiol. 32 (1986). 700-701. URSING, J., BRENNER. D. H., BERCOVIER, H.. FANNING, G. R., STEIGERWALT, A. G., BRAULT, J., MOLLARET, H. H.: Yersillia frederiksenii: a new species of Emerobacreriaceae compm,ed of rhamnose positive strains (formerly called atypical Yersinia enterocolitica or Yersinia emerocolitica-like). CUlT. Microbiol. 4 (1980), 213-217. WALKER, P. J., GRIMES, D. J.: A note on Yersinia enterocolitica in a swine farm watershed. J. Appl. Bacteriol. S8 (1985), 139-143. WAUTERS, G.: The pathogenic significance of the different Yersinia groups in man. In "Psychrotrophic Microorganisms in Spoilage and Pathogenicity" (Edited by ROBERTS, T. A., HOBB, G., CRISTIAN, J. H. B., SCOVGAARD, N.). Academic Press, London 1981. pp. 401-403. WEAGANT. S. D.: Medium for presumptive identification of Yersinia enterocolitica. AppL Environ. Microbiol. 4S (1983),472-473. WEBER, A., KNAPP. W.: Seasonal isolation of Y. enterocolitica and Yersinia pseudotubercolosis from tonsils of healthy slaughter pigs. Zentralbl. Bakt., Abt. 1, 2S0 (1981), 78-83. Authors' adresses: Dr. S. MASSA, lstituto di Microbiologia Agraria e Tecnica, Universita di Bologna, Via Filippo Re, 6, 40126- Bologna (Italy). Dr. DANIELA CESARONI e Dr. G. PODA. Presidio MuitizonaJe di Prevenzione, Settore Biotossicologico, USL28, Via Triachini 17, 40138-Bologna (Italy). Prof. L. D. TROVATELLI. Dipartimento di Agrobiologia e Agrochimica, Universita della Tuscia, Loc. Riello. 10100- Viterbo (Italy).
39
ZentralbL MikrobioL, Bd. 143
[1) Istituto di Microbiologia Agraria e Tecnica, Universita di Bologna, Bologna (Italia), 2) Presidio Multizonale di Prevenzione, Settore Biotossicologico, Bologna (Italia), e 3) Dipartimento di Agrobiologia e Agrochimica, Settore di Microbiologia, Universita della Tuscia, Viterbo, (Italia)]
Isolation of Yersinia enterocolitica and Related Species from River Water S, MASSA'), DANIELA CESARONf), G. PODA') e L. D. TROVATELu3) With I Figure
Summary One hundred and twenty samples of river water collected from sampling sites in the period between December 1986 and December 1987 were examined for the presence of Yersinia enterocolitica and related species. Among a total of 26 Yersinia strains isolated the following species were recognized: Y. enterocolitica, 12 strains, Y. intermedia, 5 strains, Y. frederiksenii, 2 strains, Y. kristensenii and Y. aldovae, I strain each; 5 were atypical Yersinia strains (rhamnose+, citrate + , o:-methyl-D-glucoside+). Although the Yersinia isolates belong to the so-called "environmental" serotypes, which are usually considered as non-pathogenic for humans, Y. enterocolitica biotype I, serotype 0: 7.8, Iysotype Xo, which has previously been associated with gastro-intestinal infection in Italy, was found to be present. Statistical evaluation of our data showed that the presence of Yersinia spp. and the presence of total or fecal coliforms are unrelated.
Zusammenfassung Zwischen Dezember 1986 und Dezember 1987 wurden 120 FluBwasserproben entnommen und in Hinsicht auf das Vorkommen von Yersinia enterocolitica und verwandten Typen untersucht. Insgesamt wurden 26 Stiimme von Yersinia isoliert: 12 Y. enterocolitica, 5 Y. intermedia, 2 Y. jrederiksenii, 1 Y. kristensenii, 1 Y. aldovae und 5 atypische Yersinia spp. (Rhamnose +, Citrat+, Ci-methyl-D-Glukosid+). Obwohl aile isolierten Stamme den sogenannten Umwelt-Serotypen angehiirten, welche im allgemeinen fUr den Menschen als nicht pathogen angesehen werden, wurde Y. enterocolitica Biotyp 1, Serotyp 0:7.8, Lysotyp Xo isoliert, welche mit einem in Italien vorgekommenen Fall von Magendarminfektion verbunden worden ist. Es wurde kein statistisches Verhiiltnis zwischen dem Vorkommen von Yersinia spp. und Gesamt- oder Fiikalcoliformen gefunden.
Yersinia enterocolitica is receiving increased attention as a food- and waterborne pathogen in humans. Common clinical syndroms include gastroenteritis with pain that mimics appendicitis, arthritis, erythema nodosum, mesenteric lymphadenitis, and septicemia (BOTTONE 1977; BERCOVIER and MOLLARET 1984). Y. enterocolitica has been isolated from a large number of wild and domestical animals, a wide range of foodstuff and from water (SWANIMATHAM et al. 1982). The demonstration of the presence of Y. enterocolitica in water (HARVEY et al. 1976; HIGHSMITH et al. 1977; KAPPERUD 1977, SCHIEMAN 1978) has generated considerable interest in the aquatic environment as a possible reservoir and vehicle of transmission for yersiniosis (BARTLEY et al. 1982). Although many strains of Y. enterocolitica and related species isolated from water are non-pathogenic for man, there have been several cases in which the presence of these organisms has been implicated as cause of waterborne outbreaks (LASSEN 1972; KEET 1974; Eden et al. 1977; THOMPSON and GRAVEL 1986). In Italy, Yersinia spp. has been found in humans, animals, the environment, and water, but at present, it appears to be less frequent than in other European and non-European countries,
576
S. MASSA et al.
although, since 1978, the clinical cases seem to be of an increasing frequency (CHIESA et al. 1982; FANTASIA MAZZOTTI and GIRALDI 1983). In this investigation samples of river water were examined for the presence of Y. enterocolitica and Y. enterocolitica-like organisms together with the usual fecal indicator organisms.
Material and Methods Sampling sites 10 representive sampling sites, located in the upper part of the Reno river, were studied (Fig. 1): R I -R7 were situated on the river itself, whereas Stl-S13 were sited on tributaries at a distance of 500 m before the point where they joinded the Reno. The Reno river, between sites RJ and R7 , passes from a rural area to an increasingly urbanized and relatively industrialized area.
Collection of samples Water samples were collected in 21 sterile glass bottles, approximately 10 cm below the water surface, and tested within 4 h ofter collection. A total of 120 samples were analyzed between December, 1986 and December, 1987.
Coliform analysis The coliforms in each water sample were enumerated using the membrane filtration procedures as described in Standard Methods (APHA 1980). Total coliforms were determined on M-Endo medium at 35 DC and fecal coliforms on M-FC medium at 45 DC.
Isolation of Yersinia species 100 ml of Peptone water (Biolife), concentrated 10 times, were added to each water sample (1 I), together with bile salts (Difco) and sorbitol (C. Erba) to a final concentration of 0.15% and 1%, respectively (MEHLMAN et al. 1978). The samples were then stored at 4 DC. After 7, 14 or 21 days one loopful of cold enrichment was streaked onto cefsulodin-irgasan-novobiocin agar plates (Yersinia Selective Agar, Oxoid). The plates were incubated at 25 DC and examined after 24 and 48 h. Colonies showing typical "bull's eyes" morphology, after purification were subcultured onto K1iger-Iron-Agar (Biolife) slant and incubated for 48 hat 28 DC. Only those isolates that produced an acid butt and aIcaline slant without hydrogen sulphide or gas were Gram stained and tested for urease motility at 22 and 37 DC, growth on Simmons citrate, and lysine-arginine decarboxylase (WEAGANT 1983). Positiveness at these tests was held as indication of apartenance to the Yersinia genus. Additional tests for the identification of the strains (biotype, serogroup and lysotype) were carried out by the "Centro di Riferimento per la Yersinia" in Rome.
Statistical analysis The relationship between the presence of Yersinia spp. and coliforms (total and fecal) was statistically evaluated using a corrected le-analysis.
Results A total of26 strains of Yersinia spp. were isolated from 120 samples of water collected from 10 sampling sites between December 1986 and December 1987, with a frequency of 21. 6 % (Table 1). Most strains (18 out of 26) were isolated only after 14-21 days of enrichment. Water samples were considered negative if yersinias were not isolated after an enrichment period of 21 days, since additional time is known not to produce any increase in the isolation frequency (NILEHN 1973; EISS 1975; WALKER and GRIMES 1985). Most of the Yersinia strains were isolated from the water samples from sampling site R2 and all during the cold months of the year (from November to March). The temperature of the river water at sampling time was in the range of 5 to 10 °C. The populations of fecal coliforms and total coliforms per 100 ml ranged from 1.6· 102 to 1.6.106 and 2.0' 10 to 4.5' 105 , respectively (Table 1). It is interesting to note that no strain of Yersinia spp. was isolated at any of the levels of total or fecal coliforms contamination at sampling sites St2 and St3 (Table 2). At the other sampling sites, the number of samples in which Yersinia spp. where isolated, when the total coliforms were less than 105 per 100 ml, was 15, whereas at a level of total coliforms between 105 and 106 per 100 ml the number of positive samples was 7. Yersinia spp. was found to be present in only one sample when the number of fecal coliforms was greater than 105
St3
Stz
R7 St]
0 0
2 5 2 5 3 2 2
12 12 12 12 12 12 12 12 12 12
R] Rz R3 R4 R5
~
No. of positive samples
No. of samples
Sampling site
3 7 2 5 3 2 2 2 0 0
No. of strains
9.2 5.8 9.0 2.0 3.0 8.3 8.3 1.9 2.7 1.8 X X X X X X
103 104 104 104 105 104 x lif x 104 X 103 X 104 6.0 8.5 1.8 3.5 2.5 1.8 8.0 8.0 1.6 9.0
102 103 104 103 104 103 x 103 X 102 X 102 X 102 X X X X X X
4.0 4.2 2.8 8.0 1.6 4.1 5.0 1.8 1.0 8.0
X X X X
x
X X X X X
105 105 104 104
1~
104 105 105 104 106
3.8 3.4 2.4 6.1 1.9 3.8 2.7 6.2 5.8 3.8
X X X X X X X X X X
Mean
Maximum
Mean
Minimum
Fecal
Total
Coliform bacteria per 100 ml
Table 1. Incidence of Yersinia spp. and coliform results for 120 water samples from Reno river
103 104 104 103 105 104 104 103 102 103
9.0 3.1 2.0 1.8 1.5 4.7 8.5 1.3 2.0 2.5
103 103 102 104 102 103 102 x 10 X 102 X X X X X X X
x 10
Minimum
2.0 1.0 6.0 2.2 4.5 1.8 8.0 6.0 1.5 1.6
X X X X X X X X X X
104 105 104 104 105 105 104 104 103 104
Maximum
OJ>
0
Vl
-.) -.)
.,
~.
;;: '""".., ,.., "" ~
~ .... '" S· E·
0
....
::>
o·
e;.
578
S.
MASSA
et al.
A
Scale 1: 150. 000
Fig. 1. Map of the Reno river showing the sampling sites.
per 100 ml; the number of samples which yielded Yersinia spp. at levels of fecal coliforms less than 105 per 100 ml was 21. A corrected x2-analysis was carried out to find a relationship between the presence of Yersinia spp. and coliforms (total and fecal). No correlation was found between the isolation of Yersinia spp. and the presence of coliforms at any of the levels. The results of identification of Yersinia strains are presented in Table 3. As shown, 12 strains were identified as Y. enterocolitica "sensu strictu" and 14 as Y. enterocolitica-like organisms according to BERKOVIER et al. (1980,1980a), BRENNER et aI. (1980), URSING et al. (1980) and BERCOVIER et al. (1984). All Y. enterocolitica strains were of biotype 1 and lysotype X o, but of different serogroups (autoagglutif!ated, 0 :4.32-10, 0: 7.8). The other strains were identified as: 2 Y.frederiksenii, 1 Y. kristensenii, 1 Y. aldovae, 5 atypical Yersinia spp. (rhamnose+ citrate+ and
579
Isolation of Yersinia fnlerocolitica Table 2. Relationships of Yersillia 'pp. isolation with coliforms population of water samples Range of bacteria
No. of samples positive for Yersillia spp.
All Total coliforms/ I00 ml 01,000 1,001 10,000 10,001- 100,000 100,00 1-1,000,000 Fecal coliformsllOO ml 0100 101- 1,000 1,001- 10,000 10.001-100,000 > 100,000
I 3
2
2 3
4 10 7
2
2
I
2
I 3
2
9 9
2
4
Table 3. Characteristics of Y. elllerocolitica and related species isolated from river water Species')
Biotype
Y. ellIerocolitica
Serogroup
Lysotype
No. of strains
AAGb)
Xo Xo Xo
9 I 2
0:4,32-10 0:7.8
Y. illlermedia
4 4 4
0: 10 0:36,37 0:35
AAG Y. frederiksellii
Nc)
Xo XO Xo Xo
0:4,33-10
Xo XO
Y. kristellsellii
0: 10. 34
Xo
Y. aldovae
AAG
XO
AAG
Xo Xo XO XO Xo
Atypical Yersillia Sppd)
0: 12, 26 0:48 0:21 0: 16,50,52
I
2
.) Results of "Centro di Riferimento per la Yersinia", Rome. b) AAG. autoagglutinated. 0) N, non agglutinable. d) Yersinia rhamose+, citrate+. a:-methyl-D-glucoside+.
Discussion Y. enterocolitica have been isolated from water in many countries. On a probable account of different methods used, the isolation percentages reported by the different authors vary considerably (HARVEY et al. 1976; HIGHSMITH et al. 1977; MEADOWS and SNUDDEN 1982; BURRINI et al. 1986). In this investigation, Yersinia spp. were found in 18.3 % and Y. enterocolitica in 4.1 % of the samples tested. All the strains of Y. enterocolitica isolated belong to biotype I. WAUTERS (198 J) considers that Y. enterocolitica biotype 1 (except for the serotype 0: 8), and the related species, are ubiquitous and largely have no clinical significance.
580
S. MASSA et al.
One isolate of an atypical Yersinia species, which fennents L-rhamnose and not sorbose, cellobiose and melibiose, was identified on the basis of these characteristics as Y. aldovae, previously called Y. enterocolitica-like group X2 (BERCOVIER et al. 1984). This species is frequently isolated in the water sources together with Y. enterocolitica, but it has not been implicated in human infections. The number of samples positive for Y. enterocolitica and related species was greater in December and January, as observed by others authors (WEBER and KNAPP 1981; MEADOWS and SNUDDEN 1982; PIANETTI et al. 1985). No strain of the serogroups 0: 3 and 0: 9, most frequently associated with human yersiniosis in Europe, is found among the Yersinia isolates studied; the so-called "environmental" strains are represented instead. The question of the relationships of these strains with those involved in human infections is still unanswered; an increase has been noticed in the number of infections caused by nonhost adapted Y. enterocolitica, and atypical Y. enterocolitica-like organisms (BOTTONE 1978). One of the strains of Y. enterocolitica, isolated from the water of the Reno river, was the serotype 0:7.8; this type is not only widespread in the environment (FANTASIA MAZZOTTI and GlRALDI 1983; MARRANZANO et al. 1984), but it has also been associated with an outbreak of gastroenteritis among children at a nursery-school in Italy (GlRALDI et al. 1982). It was not possible to show a statistically significant relationship between Yersinia spp. and fecal indicator organisms. These observations lead to the conclusion that total or fecal colifonns cannot be used to predict the presence or absence of Y. enterocolitica and related species. Acknowledgements Warm thanks are due to Claudio Chiesa of the Third Pediatric Clinic of the University of Rome and the "Centro di Riferimento per la Yersinia" for the identification of the Y. enterocolitica strains.
References APHA: Standard Methods for the Examination of Water and Wastewater. 15th Edition. Washington, DC. American Public Health Association 1980. BARTLEY, T. D., QUAN, T. J., COLLINS, M. T., MORRISON, S. M.: Membrane filter technique for the isolation of Yersinia enterocolitica. Appl. Environ. Microbiol. 43 (1982),829-834. BERCOVIER, H., BRENNER, D. J., URSING, 1., STEIGERWALT, A. G., FANNING, G. R., ALONSO, 1. M., CARTER, G. P., MOLLARET, H. H.: Characterization Yersinia enterocolitica "sensu stricto". Curro Microbiol. 4 (1980),201-206. MOLLARET, H. H.: Yersinia. In Bergey's Manual of Systematic Bacteriology. (Edited by KRIEG, N. R.) Baltimore 1984, pp. 498-506. STEIGERWALT, A. G., GUIYOULE, A., HUNTLEy-CARTER, G., BRENNER, D. 1.: Yersinia aldovae (formerly Yersinia enterocolitica·like group X2): a new species of Enterobacteriaceae isolated from acquatic ecosystem. Int. J. Syst. Bacteriol. 34 (1984), 166-172. URSING, 1., BRENNER, D. 1., STEIGERWALT, A. G., FANNING, G. R., CARTER, G. P., MOLLARET, H. H.: Yersinia kristensenii a new species of Enterobacteriaceae composed of sucrose-negative strains (formerly called Yersinia enterocolitica-like). Curro Microbiol. 4 (l980a), 219-224. BRENNER, D. 1., BERCOVIER, H., URSING, 1., ALONSO, J. M., STEIGERWALT, A. G., FANNING, G. R., CARTER, G. P., MOLLARET, H. H.: Yersinia inlermedia: a new species of Enterobacteriaceae composed of rhamnose positive, melibiose positive, raffinose positive strains (formerly called Yersinia enterocolitica or Yersinia enterocolitica-like). Curro Microbiol. 4 (1980), 207-212. BOTTONE, E. 1.: YersilJia enterocolitica: A panoramic view of a charismatic microorganisms. Cril. Rev. Microbiol. 5 (\977), 211- 241. - Atypical Yersinia enterocolitica: clinical and epidemiological parameters. 1. Clin. Microbiol. 7 (1978), 562-567. BURRINI, D., GAMBASS1NI, L., NANNICINI, E., LANCIOTTI, E .: Presenza di Yersinia enterocolitica nell'acqua del Fiume Arno nel tratto fiorentino. L'lg. Mod. 85 (1986), 119-128.
Isolation of Yersinia enterocolitica
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CHIESA, C., MOLLARET, H. H., MIDULLA. M.: Epidcmiologia delle Yersiniosi in ltalia. Ann. Sclavo 24 (1982), 686-694. EDEN, K. V., ROSENBERG, G., STOOPLER, B., WOOD, A.. HIGHSMITH, A. K., SKALLY, P .. WELLS, J., FEELEY, J. C.: Waterborne gastroenteritis at a ski resort associated with the isolation of Yersinia enterocolitic:a. Public Health Rep. 92 (1977), 245-250. EISS, J.: Selective culturing of Yersinia enterocolitica at a low temperature. Scand. J. Infect. Dis. 7 (1975),249- 251. FANTASIA MAZZOTTI, M., GIRALDI, V.: Biosierotipi di Yersinia isolati in Italia dal 1979 al 1982. Nuovi Ann. Ig. Microbiol. 34 (1983), 437-446. GIRALD!, V., CAPRIOLl, A., FALBO, V., GENNARO, M., SALSOMASO, S.: Isolamento di Yersinia enterocolitica durante una epidemia di gastroenterite acuta infantile. Giorn. Mal. Inf. Paras. 34 (1982),595-596. HARVEY, S., GREENWOOD, J. R., PiCKETT, M. J., MAH, R. A.: Recovery of Yersinia enterocolitica from streams and lakes of California. App\. Environ. Microbiol. 32 (1976), 352- 354. HIGHSMITH, A. K., FEELEY, J. c., SKALLY, P., WELLS, J. G .. WOODS, B. T.: Isolation of Yersinia enterocolitica from well water and growth in distillated water. Appl. Environ. Microbiol. 34 (1977), 745-750. KAPPERUD, G.: Y. enterocolitica and Yersinia-like microbes from mammals and water in Norway and Denmark. Acta Pathol. Microbiol. Scand. B. 185 (1977),129-135. KEET, E. E.: Yersinia enterocolitica septicemia. N. Y. State J. Med. 74 (1974),2226-2230. LASSEN, J.: Yersinia enterocolitica in drinking water. Scand. J. Infect. Dis. 4 (1972), 125-127. MARRANZANO, M., URSINO, A.. AGOD!, A.: Presenza di Yersinia enterocolitica e di altri batteri apparentati in ortaggi del commercio. L'lg. Mod. 82 (1972),262-268. MEADOWS, C. A., SNUDDEN, B. H.: Prevalence of Yersillia enterocolitica in waters of the lower Chippewa River Basin, Wisconsin. Appl. Environ. Microbiol. 43 (1982), 953-954. MEHLMAN, I. J., AULISIO, C. C. G .. SANDERS, A. c.: Problems in the recovery and identification of Yersinia from food. J. Assoc. Off. Anal. Chem. 61 (1978), 761-771. NILEHN, D.: The relationship of incubation temperature to serum bactericidal effect, pathogenicity and in vivo survival of Yersinia enterocolirica. Contrib. Microbiol. Immunol. 2 (1973). 85-92. PiANETTI, A., BRAND!, G .. BRUSCOLlNI, F., BAFFONE, W., SALVAGGIO, L.. ALBANO, A.: Ricerca di Salmonelle e Yersinie nei liquami di fogna della citta di Urbino. Nuovi Ann. Ig. Microbiol. 36 (1985), 279-289. SCHIEMANN. D. A.: Isolation of Yersinia enterocolitica from surface and well water in Ontario. Can. J. Microbiol. 24 (1978), 1048-1052. SWAMINATHAN, B., HARMON, M. c.. MEHLMAN, I. J.: A review: Yersinia emerocolitica. J. Appl. Bacteriol. S2 (1982), 151-183. THOMPSON, J. S .. GRAWEL, M. J.: Family outbreak of gastroenteritis due to Yersinia elllerocolirica serotype 0: 3 from well water. Can'~ 1. Microbiol. 32 (1986). 700-701. URSING, J., BRENNER. D. H., BERCOVIER, H.. FANNING, G. R., STEIGERWALT, A. G., BRAULT, J., MOLLARET, H. H.: Yersillia frederiksenii: a new species of Emerobacreriaceae compm,ed of rhamnose positive strains (formerly called atypical Yersinia enterocolitica or Yersinia emerocolitica-like). CUlT. Microbiol. 4 (1980), 213-217. WALKER, P. J., GRIMES, D. J.: A note on Yersinia enterocolitica in a swine farm watershed. J. Appl. Bacteriol. S8 (1985), 139-143. WAUTERS, G.: The pathogenic significance of the different Yersinia groups in man. In "Psychrotrophic Microorganisms in Spoilage and Pathogenicity" (Edited by ROBERTS, T. A., HOBB, G., CRISTIAN, J. H. B., SCOVGAARD, N.). Academic Press, London 1981. pp. 401-403. WEAGANT. S. D.: Medium for presumptive identification of Yersinia enterocolitica. AppL Environ. Microbiol. 4S (1983),472-473. WEBER, A., KNAPP. W.: Seasonal isolation of Y. enterocolitica and Yersinia pseudotubercolosis from tonsils of healthy slaughter pigs. Zentralbl. Bakt., Abt. 1, 2S0 (1981), 78-83. Authors' adresses: Dr. S. MASSA, lstituto di Microbiologia Agraria e Tecnica, Universita di Bologna, Via Filippo Re, 6, 40126- Bologna (Italy). Dr. DANIELA CESARONI e Dr. G. PODA. Presidio MuitizonaJe di Prevenzione, Settore Biotossicologico, USL28, Via Triachini 17, 40138-Bologna (Italy). Prof. L. D. TROVATELLI. Dipartimento di Agrobiologia e Agrochimica, Universita della Tuscia, Loc. Riello. 10100- Viterbo (Italy).
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ZentralbL MikrobioL, Bd. 143