Antonie van Leeuwenhoek 45 (1979) 4 6 5 4 7 7
465
Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica W . VAN PEE AND J. STRAGIER 1
Laboratory of Tropical Technology and Food Microbiology, University of Leuven, K. Mercierlaan 92, 3030 Heverlee, Belgium
VAN PEE, W. and STRAG~ER,J., 1979. Evaluation of cold enrichment and isolation media for the recovery of Yersinia enterocolitica. Antonie van Leeuwenhoek 45: 465477. An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4 °C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone - soya broth yielded better results than in - frequently u s e d - phosphate buffer, p H 7.6. While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, D H L agar was most satisfactory: after 20 h incubation at 29 °C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results. It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.
INTRODUCTION H u m a n infection with Yersinia enterocolitica (Y. enterocolitica) may result in a variety of clinical syndromes. These include enterocolitis, acute mesenteric lymph° adenitis, terminal ileitis, arthritis, erythema nodosum and septicaemia (Bottone, 1977). Though the first recognized description dates back to 1939 (Schleifstein and Coleman, 1939), only since the early 1960s this species gained more attention. Extensive studies in various fields resulted in placing Y. enterocolitica among the settled human enteric pathogens like Salmonella, Shigella and Vibrio cholerae. The main reason for its late recognition was the lack of proper isolation 1 Present address: The Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
466
W. VAN PEE AND J. STRAGIER
techniques, with consequent unawareness of the presence of Y. enterocolitica both in clinical material and foods. When using routine enteric media such as MacConkey or SS agar, colonies of Y. enterocolitica are often overlooked in the presence of an abundant and heterogenous flora: growth of the organism is slow and extremely temperature dependent, the appearance of the colonies is rather unspecific, and the recovery of the cells is often less than 100 ~. Especially the detection of a small number of Y. enterocolitica among larger quantities of other Enterobacteriaceae is difficult. In order to facilitate the recovery of Y. enterocolitica, more selective plating media need to be developed. An alternative solution is the use of a selective enrichment procedure followed by selective plating. A modified Rappaport broth (Wauters, 1970) is very suitable to enrich Y. enterocolitica (serotypes 0:3 and 0:9 mainly) from human faeces and pork tongues (Wauters, 1970, 1973; van Noyen, Vandepitte and Isebaert, 1970; Wauters and Janssens, 1976). However, a critical evaluation of the functioning of this broth (van Pee and Smet, unpublished data) revealed the inability of this medium to recover small inocula of Y. enterocolitica from a large accompanying flora; in addition, some strains fail to grow in the modified Rappaport broth (Lee, 1977a, b). An other enrichment procedure is the incubation of the suspect material at a low temperature in a suitable enrichment broth (Otsuki et al., 1973 ; Tsubokura, Otsuki and Itagaki, 1973; Zen-Yoji et al., 1974; Eiss, 1975; Greenwood et al., 1975 ; Inoue and Kurose, 1975 ; Leistner et al., 1975; Toma and Deidrick, 1975 ; Tsubokura et al., 1975; Barre et al., 1976; Feeley, Lee and Morris, 1976; Hanna et al., 1976; Pedersen, 1976; Tsubokura et al., 1976; Lee, 1977a, b). Y. enterocolitica is one of the few human pathogens able to grow at refrigeration temperature (0 to 4 °C), which results in a selective enrichment of this microorganism in the sample and a much higher isolation probability. This method is suitable to recover low cell concentrations of Y. enterocolitica and can be used both in food and clinical microbiology. The two main objectives of this study were: (i) a critical evaluation of several liquid media for their capacity to enrich Y. enterocolitica in a selective way at low temperature and, (ii) an examination of several isolation media on their ability to permit fast, specific and complete development of Y. enterocolitica. The strains of Y. enterocolitica used were the two " E u r o p e a n " human pathogen serotypes 0:3 and 0:9.
MATERIALS AND METHODS
Bacterial species The experiments were carried out with the following species: Y. enterocolitica 0 : 3 and 0: 9, Pseudomonasfluorescens (P. fluorescens), Escherichia coli (E. coli), Salmonella enteritidis ( S. enteritidis), Klebsiella pneumoniae (K. pneumoniae),
MEDIA F O R I S O L A T I O N OF Y. E N T E R O C O L I T I C A
467
Enterobacter cloacae ( E. cloacae) and Proteus mirabilis ( P. mirabiIis) . All strains were freshly isolated from river water, except.the two Y. enterocolitica strains which were of human origin and obtained from stools. The typing of all species was carried out according to the procedures described in the 8th ed. of Bergey's Manual (Buchanan and Gibbons, 1974). During all experiments, the bacteria were cultured in brain heart infusion broth (Institut Pasteur Production, Paris, France) at 29 °C; they were all in the logarithmic growth phase (i.e. grown for 24 h) before being inoculated in both a cold enrichment medium and an isolation medium. Cold enrichment Seven cold enrichment media were evaluated. They were all autoclaved for 15 min at 121°C. 1. Phosphate buffer M/15 pH 7.6 (Leistner et al., 1975); volume: 9.9 ml per tube of 20 ml. 2. Phosphate buffer M/15 plus 1 g sterile minced pork meat per 9.9 ml buffer. The meat was bought from a local butcher, added to an appropriate volume of buffer, mixed for 1 rain in a Waring Blendor, distributed into tubes and sterilized. 3. Phosphate buffer M/15 (5 ml) + Tryptone - Soy Broth 3 ~o (TSB, Oxoid, London, England) (1 ml). 4. TSB, 9.9 ml. 5. TSB + 1 g sterile minced pork meat per 9.9 ml TSB (see above under 2). 6. Nutrient broth as described by Eiss, 1975, 9.9 ml. 7. Phosphate buffer M/15 + beef extract (Institut Pasteur Production, Paris, France), 1 ~, 9.9 ml. Besides these cold enrichment media, the glucose broth used by Harvey et al. (1976) for the examination of water samples for tke presence of Y. enterocolitica was also evaluated. Since this medium is incubated at room temperature and under anaerobic conditions, its investigation was in fact beyond the scope of our study, but due to its successful application by Harvey et al., 1976, this medium was included in the experiments. Incubation of tubes containing 9.9 ml glucose broth was also performed as described by Harvey et al., 1976. After autoclaving, each enrichment broth was inoculated with 24-h-old pure cultures of the eight bacterial species using three different initial cell concentrations each time: 102, 106 and 10 s cells/ml medium. The cold enrichment media were placed in a refrigerator at + 4 °C under aerobic conditions. After 3, 6, 8, 10, 15 and 20 days of incubation, a 0.1 ml sample (diluted if necessary) was plated out in brain heart infusion agar (Difco, Detroit, USA), whereafter the dishes were placed at 29 °C until colonies were countable. Counting was performed at least in duplicate (generally five dishes per species and per medium were examined) : the listed cell numbers are the means of these counts.
468
W. VAN PEE AND J. STRAGIER
Isolation media The eight bacterial species were plated out as pure cultures in a number of about 150 cells per dish in the following media: 1. MacConkey-Tween 80 (MT) and deoxyribonuclease-sorbitol-Tween 80 (DST) agar (Lee, 1977a, b). 2. Polypectate medium (Lyle von Riesen, 1975). 3. SS agar (Difco, Detroit, USA) + 1.5 ~ deoxycholate (Merck, Darmstadt, Germany) + 1 ~o filtersterilized saccharose (Merck, Darmstadt, Germany) (SS-D(1.5)S) agar (Wauters, 1973). 4. D H L agar ("Eiken", Wellcome, Brussels, Belgium) and D H L agar + 1 deoxycholate (Leistner et al., 1975). 5. Ureum-glucose-novobiocin agar: peptone, 0.1 ~o; NaC1, 0.5 ~o; KHzPO4" HzO , 0.2 ~ ; glucose, 0.1 ~ ; phenol red, 0.0012 ~ ; filtersterilized urea, 20 ~ ; novobiocin (ampoules, Institut Pasteur Production, Paris, France), 0.002~. All incubations were carried out at 29 °C under aerobic conditions. Several other media, most of them slight modifications of those cited above, were also examined without yielding interesting results (van Pee and Stragier, unpublished data). A quantitative impression of the recovery of the cells inoculated in the media was obtained by plating out, in brain heart infusion agar, the same cell concentration that was inoculated in the isolation medium tested. The media were evaluated for their ability of permitting fast growth, a specific colonial aspect, and a full recovery of both Y. enterocolitica strains.
RESULTS
Cold enrichment The enrichment quotients (i.e. theratio of cell number/ml of medium after 20 days of cold incubation to the initial cell number/ml) are shown for each microorganism in each medium in Table 1 (low initial cell number), Table 2 (medium initial cell number) and Table 3 (high initial cell number), respectively. Enrichment quotients in phosphate buffer M/15 + beef extract 1 ~o were only determined using a medium initial cell concentration (Table 2). Incubation was limited to 20 days since no further growth of Y. enterocolitica at low temperature is noticed after this period (Leistner et al., 1975 ; Toma and Deidrick, 1975). The growth of the different species at 4°C in TSB with various initial cell concentrations is shown in Figs. 1-3. The general impression is that both serotypes of Y. enterocolitica and P. fluorescens show distinct growth, whereas the other Enterobacteriaceae fail to grow at low temperature. The degree of enrichment is dependent on the initial cell concentration (compare Figs. 1, 2 and 3) and on the medium used (Tables 1
469
MEDIA FOR ISOLATION OF Y. ENTEROCOLITICA
log N
124 11109876543210-1 ~ -2-
; &1;
i;
i 2,
2o days
Fig. t. Growth curves in T S B at 4 °C with low initialcellconcentration. N: cell number/ml of enrichment broth; O, Y. e n t e r o c o l i t i e a 0 : 3 ; o, Y. enteroeolitica 0 : 9 ; x , P. f l u o r e s c e n s ; - - - , E. eoli; - - , S. enteritidis; - A
, E. cloacae;
, P . mirabilis; - +
, K. pneumoniae.
log N
.
8~
~
~
:2 6
8
1C)
15
i >
20 days
Fig. 2. Growth curves in TSB at 4°C with median inital cell concentration. Definition and legend: see Fig. 1.
470
W. VAN PEE AND J. STRAGIER log
~2_j 1110987654321-
.
.
.
.
.
.
.
.
.
.
.
0-1-2-
;
; 1;
1;
20 days
Fig. 3. Growth curves in TSB at 4 °C with high initial cell concentration. Definition and legend: see Fig. 1.
to 3). With a low initial cell number (Fig. 1) enrichment of Y. enterocolitica is better than with a medium or high initial concentration (Figs. 2 and 3). This is not surprising, considering the higher substrate concentration per cell at a low initial concentration of cells. Further, if cell numbers of 106/ml are present, direct plating will probably be successful, rendering enrichment less necessary. With each initial cell concentration, enrichment in pure phosphate buffer M/15 is rather slow, due to the low nutritional value of this solution. This is supported by the comparison of the enrichment quotients of both Y. enterocolitica strains in phosphate buffer M/15 + minced meat with the enrichment quotients in TSB (see e.g. Table 1), the last being higher than the first ones. This suggests that the diffusion of nutrients out of the suspect material (faeces, foods) into the buffer is insufficient to permit optimal growth of Y. enterocolitica at refrigeration temperature. Therefore, cold enrichment of Y. enterocolitica should be carried out in a medium with a high nutritional value (e.g. TSB, brain heart infusion broth, nutrient broth), rather than in the frequently used phosphate buffer M/15. TSB seems very suitable, since the enrichment of I1. enterocolitica in this broth is as high as in TSB + minced meat, which suggests that TSB itself can support the growth of Y. enterocolitica at 4°C. Isolation media On both M T and DST agar, serotypes 0: 3 and 0: 9 of Y. enterocolitica failed to hydrolyse Tween 80 or DNA. The presence of Tween 80- or DNA-hydrolysing enzymes is a variable characteristic among the various Y. enterocolitica strains
1.0 x 105 6.3 x 105 1.9 x 105 3.2 x 107
5.8 x 106 1.1 × 104 1.0 x 106
1.0 x 107
1.1 x 106
1.2 x 10 s 1.6 x 104 3.2 x 105
3.2 x 10 a
1.0 x 105
Pseud.
6.3 x 10 - 3
0.004 0.100 0.100
0.100
0.400
E. coli
0.015
0.010 0.004 3.1 x 10 4
0.010
0.003
Salm.
1.2 x 10 . 3
0.002 1.2 × 10 -3 6.3 x I 0 3
0.002
0.013
Kleb.
0.013
0.015 2.5 x 10 3 0.001
0.010
0.010
Enter.
1.0 x 10 - 4
2.5 x 10 4 1.2 x 10 3 6.3 x 1 0 - s
6.3 x 10 - 4
0.008
Prot.
cloacae," Prot., P. mirabilis.
1 E n r i c h m e n t q u o t i e n t : T h e ratio o f the n u m b e r o f cells/ml e n r i c h m e n t b r o t h a f t e r 20 d a y s o f i n c u b a t i o n to the initial cell c o n c e n t r a t i o n . A b b r e v i a t i o n s : Y.e. 3, Y. enterocolitica 0 : 3 ; Y.e. 9, Y. enterocolitiea 0 : 9 ; Pseud, P. Jluorescens," Salm., S. enteritidis," Kleb., K. pneumonia," Enter., E.
1.0 x 106
5.8 x 103
+ meat Buffer M/15 + TSB TSB TSB+meat Nutrient broth (Eiss)
2.5 x 103
1.6 x 102
Y.e. 9
Buffer M/15 Buffer M/I5
Y.e. 32
T a b l e 1. E n r i c h m e n t quotients I a f t e r 20 d a y s o f i n c u b a t i o n at 4 ° C w i t h a l o w initial cell c o n c e n t r a t i o n .
--.,I
©
Z
0
©
>
1.6 × 103 3.2 x 102 2.5 x 10 z
1.0 x 10 a
1.0 x 103
3.2 x 102
1.6 x 103
1.0 x 103
Buffer M/15 + TBS
Buffer M/15 + beef extract
TSB
TSB + meat
Nutrient broth (Eiss)
1 F o r d e f i n i t i o n a n d a b b r e v i a t i o n s see u n d e r T a b l e 1.
1.0 × 103
3.2 × t02
1.2 x 102
1.2 x 10 z
Buffer M/15 + meat
0.50
0.19
Y.e. 9
Buffer M/15
Y.e. 3
1.6 × 103
1.0 x 104
6.3 x 103
1.2 x 103
3.2 x 103
1.2 × 103
58
Pseud.
0.063
0.063
0.063
0.063
0.063
0.031
0.022
E. coli
0.063
0.015
0.001
0.063
0.019
0.025
0.025
Salm.
1.2 × 10 - 3
0.001
1.6 x 10 4
1.2 x 10 3
6.3 x 10 - 4
0.005
0.010
Kleb.
T a b l e 2. E n r i c h m e n t q u o t i e n t s ~ a f t e r 20 d a y s o f i n c u b a t i o n a t 4 ° C w i t h m e d i u m initial cell c o n c e n t r a t i o n .
0.063
0.010
2.5 x 10 . 3
0.063
0.006
1.9 x 10 3
7.9 x 10 - 3
Enter.
2.5 x 10 - 3
0.004
0.001
0.063
1.9 x 10 - 3
1.2 x 10 - 3
2.5 x 10 - 3
Prot.
m
Z
465
Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica W . VAN PEE AND J. STRAGIER 1
Laboratory of Tropical Technology and Food Microbiology, University of Leuven, K. Mercierlaan 92, 3030 Heverlee, Belgium
VAN PEE, W. and STRAG~ER,J., 1979. Evaluation of cold enrichment and isolation media for the recovery of Yersinia enterocolitica. Antonie van Leeuwenhoek 45: 465477. An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4 °C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone - soya broth yielded better results than in - frequently u s e d - phosphate buffer, p H 7.6. While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, D H L agar was most satisfactory: after 20 h incubation at 29 °C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results. It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.
INTRODUCTION H u m a n infection with Yersinia enterocolitica (Y. enterocolitica) may result in a variety of clinical syndromes. These include enterocolitis, acute mesenteric lymph° adenitis, terminal ileitis, arthritis, erythema nodosum and septicaemia (Bottone, 1977). Though the first recognized description dates back to 1939 (Schleifstein and Coleman, 1939), only since the early 1960s this species gained more attention. Extensive studies in various fields resulted in placing Y. enterocolitica among the settled human enteric pathogens like Salmonella, Shigella and Vibrio cholerae. The main reason for its late recognition was the lack of proper isolation 1 Present address: The Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
466
W. VAN PEE AND J. STRAGIER
techniques, with consequent unawareness of the presence of Y. enterocolitica both in clinical material and foods. When using routine enteric media such as MacConkey or SS agar, colonies of Y. enterocolitica are often overlooked in the presence of an abundant and heterogenous flora: growth of the organism is slow and extremely temperature dependent, the appearance of the colonies is rather unspecific, and the recovery of the cells is often less than 100 ~. Especially the detection of a small number of Y. enterocolitica among larger quantities of other Enterobacteriaceae is difficult. In order to facilitate the recovery of Y. enterocolitica, more selective plating media need to be developed. An alternative solution is the use of a selective enrichment procedure followed by selective plating. A modified Rappaport broth (Wauters, 1970) is very suitable to enrich Y. enterocolitica (serotypes 0:3 and 0:9 mainly) from human faeces and pork tongues (Wauters, 1970, 1973; van Noyen, Vandepitte and Isebaert, 1970; Wauters and Janssens, 1976). However, a critical evaluation of the functioning of this broth (van Pee and Smet, unpublished data) revealed the inability of this medium to recover small inocula of Y. enterocolitica from a large accompanying flora; in addition, some strains fail to grow in the modified Rappaport broth (Lee, 1977a, b). An other enrichment procedure is the incubation of the suspect material at a low temperature in a suitable enrichment broth (Otsuki et al., 1973 ; Tsubokura, Otsuki and Itagaki, 1973; Zen-Yoji et al., 1974; Eiss, 1975; Greenwood et al., 1975 ; Inoue and Kurose, 1975 ; Leistner et al., 1975; Toma and Deidrick, 1975 ; Tsubokura et al., 1975; Barre et al., 1976; Feeley, Lee and Morris, 1976; Hanna et al., 1976; Pedersen, 1976; Tsubokura et al., 1976; Lee, 1977a, b). Y. enterocolitica is one of the few human pathogens able to grow at refrigeration temperature (0 to 4 °C), which results in a selective enrichment of this microorganism in the sample and a much higher isolation probability. This method is suitable to recover low cell concentrations of Y. enterocolitica and can be used both in food and clinical microbiology. The two main objectives of this study were: (i) a critical evaluation of several liquid media for their capacity to enrich Y. enterocolitica in a selective way at low temperature and, (ii) an examination of several isolation media on their ability to permit fast, specific and complete development of Y. enterocolitica. The strains of Y. enterocolitica used were the two " E u r o p e a n " human pathogen serotypes 0:3 and 0:9.
MATERIALS AND METHODS
Bacterial species The experiments were carried out with the following species: Y. enterocolitica 0 : 3 and 0: 9, Pseudomonasfluorescens (P. fluorescens), Escherichia coli (E. coli), Salmonella enteritidis ( S. enteritidis), Klebsiella pneumoniae (K. pneumoniae),
MEDIA F O R I S O L A T I O N OF Y. E N T E R O C O L I T I C A
467
Enterobacter cloacae ( E. cloacae) and Proteus mirabilis ( P. mirabiIis) . All strains were freshly isolated from river water, except.the two Y. enterocolitica strains which were of human origin and obtained from stools. The typing of all species was carried out according to the procedures described in the 8th ed. of Bergey's Manual (Buchanan and Gibbons, 1974). During all experiments, the bacteria were cultured in brain heart infusion broth (Institut Pasteur Production, Paris, France) at 29 °C; they were all in the logarithmic growth phase (i.e. grown for 24 h) before being inoculated in both a cold enrichment medium and an isolation medium. Cold enrichment Seven cold enrichment media were evaluated. They were all autoclaved for 15 min at 121°C. 1. Phosphate buffer M/15 pH 7.6 (Leistner et al., 1975); volume: 9.9 ml per tube of 20 ml. 2. Phosphate buffer M/15 plus 1 g sterile minced pork meat per 9.9 ml buffer. The meat was bought from a local butcher, added to an appropriate volume of buffer, mixed for 1 rain in a Waring Blendor, distributed into tubes and sterilized. 3. Phosphate buffer M/15 (5 ml) + Tryptone - Soy Broth 3 ~o (TSB, Oxoid, London, England) (1 ml). 4. TSB, 9.9 ml. 5. TSB + 1 g sterile minced pork meat per 9.9 ml TSB (see above under 2). 6. Nutrient broth as described by Eiss, 1975, 9.9 ml. 7. Phosphate buffer M/15 + beef extract (Institut Pasteur Production, Paris, France), 1 ~, 9.9 ml. Besides these cold enrichment media, the glucose broth used by Harvey et al. (1976) for the examination of water samples for tke presence of Y. enterocolitica was also evaluated. Since this medium is incubated at room temperature and under anaerobic conditions, its investigation was in fact beyond the scope of our study, but due to its successful application by Harvey et al., 1976, this medium was included in the experiments. Incubation of tubes containing 9.9 ml glucose broth was also performed as described by Harvey et al., 1976. After autoclaving, each enrichment broth was inoculated with 24-h-old pure cultures of the eight bacterial species using three different initial cell concentrations each time: 102, 106 and 10 s cells/ml medium. The cold enrichment media were placed in a refrigerator at + 4 °C under aerobic conditions. After 3, 6, 8, 10, 15 and 20 days of incubation, a 0.1 ml sample (diluted if necessary) was plated out in brain heart infusion agar (Difco, Detroit, USA), whereafter the dishes were placed at 29 °C until colonies were countable. Counting was performed at least in duplicate (generally five dishes per species and per medium were examined) : the listed cell numbers are the means of these counts.
468
W. VAN PEE AND J. STRAGIER
Isolation media The eight bacterial species were plated out as pure cultures in a number of about 150 cells per dish in the following media: 1. MacConkey-Tween 80 (MT) and deoxyribonuclease-sorbitol-Tween 80 (DST) agar (Lee, 1977a, b). 2. Polypectate medium (Lyle von Riesen, 1975). 3. SS agar (Difco, Detroit, USA) + 1.5 ~ deoxycholate (Merck, Darmstadt, Germany) + 1 ~o filtersterilized saccharose (Merck, Darmstadt, Germany) (SS-D(1.5)S) agar (Wauters, 1973). 4. D H L agar ("Eiken", Wellcome, Brussels, Belgium) and D H L agar + 1 deoxycholate (Leistner et al., 1975). 5. Ureum-glucose-novobiocin agar: peptone, 0.1 ~o; NaC1, 0.5 ~o; KHzPO4" HzO , 0.2 ~ ; glucose, 0.1 ~ ; phenol red, 0.0012 ~ ; filtersterilized urea, 20 ~ ; novobiocin (ampoules, Institut Pasteur Production, Paris, France), 0.002~. All incubations were carried out at 29 °C under aerobic conditions. Several other media, most of them slight modifications of those cited above, were also examined without yielding interesting results (van Pee and Stragier, unpublished data). A quantitative impression of the recovery of the cells inoculated in the media was obtained by plating out, in brain heart infusion agar, the same cell concentration that was inoculated in the isolation medium tested. The media were evaluated for their ability of permitting fast growth, a specific colonial aspect, and a full recovery of both Y. enterocolitica strains.
RESULTS
Cold enrichment The enrichment quotients (i.e. theratio of cell number/ml of medium after 20 days of cold incubation to the initial cell number/ml) are shown for each microorganism in each medium in Table 1 (low initial cell number), Table 2 (medium initial cell number) and Table 3 (high initial cell number), respectively. Enrichment quotients in phosphate buffer M/15 + beef extract 1 ~o were only determined using a medium initial cell concentration (Table 2). Incubation was limited to 20 days since no further growth of Y. enterocolitica at low temperature is noticed after this period (Leistner et al., 1975 ; Toma and Deidrick, 1975). The growth of the different species at 4°C in TSB with various initial cell concentrations is shown in Figs. 1-3. The general impression is that both serotypes of Y. enterocolitica and P. fluorescens show distinct growth, whereas the other Enterobacteriaceae fail to grow at low temperature. The degree of enrichment is dependent on the initial cell concentration (compare Figs. 1, 2 and 3) and on the medium used (Tables 1
469
MEDIA FOR ISOLATION OF Y. ENTEROCOLITICA
log N
124 11109876543210-1 ~ -2-
; &1;
i;
i 2,
2o days
Fig. t. Growth curves in T S B at 4 °C with low initialcellconcentration. N: cell number/ml of enrichment broth; O, Y. e n t e r o c o l i t i e a 0 : 3 ; o, Y. enteroeolitica 0 : 9 ; x , P. f l u o r e s c e n s ; - - - , E. eoli; - - , S. enteritidis; - A
, E. cloacae;
, P . mirabilis; - +
, K. pneumoniae.
log N
.
8~
~
~
:2 6
8
1C)
15
i >
20 days
Fig. 2. Growth curves in TSB at 4°C with median inital cell concentration. Definition and legend: see Fig. 1.
470
W. VAN PEE AND J. STRAGIER log
~2_j 1110987654321-
.
.
.
.
.
.
.
.
.
.
.
0-1-2-
;
; 1;
1;
20 days
Fig. 3. Growth curves in TSB at 4 °C with high initial cell concentration. Definition and legend: see Fig. 1.
to 3). With a low initial cell number (Fig. 1) enrichment of Y. enterocolitica is better than with a medium or high initial concentration (Figs. 2 and 3). This is not surprising, considering the higher substrate concentration per cell at a low initial concentration of cells. Further, if cell numbers of 106/ml are present, direct plating will probably be successful, rendering enrichment less necessary. With each initial cell concentration, enrichment in pure phosphate buffer M/15 is rather slow, due to the low nutritional value of this solution. This is supported by the comparison of the enrichment quotients of both Y. enterocolitica strains in phosphate buffer M/15 + minced meat with the enrichment quotients in TSB (see e.g. Table 1), the last being higher than the first ones. This suggests that the diffusion of nutrients out of the suspect material (faeces, foods) into the buffer is insufficient to permit optimal growth of Y. enterocolitica at refrigeration temperature. Therefore, cold enrichment of Y. enterocolitica should be carried out in a medium with a high nutritional value (e.g. TSB, brain heart infusion broth, nutrient broth), rather than in the frequently used phosphate buffer M/15. TSB seems very suitable, since the enrichment of I1. enterocolitica in this broth is as high as in TSB + minced meat, which suggests that TSB itself can support the growth of Y. enterocolitica at 4°C. Isolation media On both M T and DST agar, serotypes 0: 3 and 0: 9 of Y. enterocolitica failed to hydrolyse Tween 80 or DNA. The presence of Tween 80- or DNA-hydrolysing enzymes is a variable characteristic among the various Y. enterocolitica strains
1.0 x 105 6.3 x 105 1.9 x 105 3.2 x 107
5.8 x 106 1.1 × 104 1.0 x 106
1.0 x 107
1.1 x 106
1.2 x 10 s 1.6 x 104 3.2 x 105
3.2 x 10 a
1.0 x 105
Pseud.
6.3 x 10 - 3
0.004 0.100 0.100
0.100
0.400
E. coli
0.015
0.010 0.004 3.1 x 10 4
0.010
0.003
Salm.
1.2 x 10 . 3
0.002 1.2 × 10 -3 6.3 x I 0 3
0.002
0.013
Kleb.
0.013
0.015 2.5 x 10 3 0.001
0.010
0.010
Enter.
1.0 x 10 - 4
2.5 x 10 4 1.2 x 10 3 6.3 x 1 0 - s
6.3 x 10 - 4
0.008
Prot.
cloacae," Prot., P. mirabilis.
1 E n r i c h m e n t q u o t i e n t : T h e ratio o f the n u m b e r o f cells/ml e n r i c h m e n t b r o t h a f t e r 20 d a y s o f i n c u b a t i o n to the initial cell c o n c e n t r a t i o n . A b b r e v i a t i o n s : Y.e. 3, Y. enterocolitica 0 : 3 ; Y.e. 9, Y. enterocolitiea 0 : 9 ; Pseud, P. Jluorescens," Salm., S. enteritidis," Kleb., K. pneumonia," Enter., E.
1.0 x 106
5.8 x 103
+ meat Buffer M/15 + TSB TSB TSB+meat Nutrient broth (Eiss)
2.5 x 103
1.6 x 102
Y.e. 9
Buffer M/15 Buffer M/I5
Y.e. 32
T a b l e 1. E n r i c h m e n t quotients I a f t e r 20 d a y s o f i n c u b a t i o n at 4 ° C w i t h a l o w initial cell c o n c e n t r a t i o n .
--.,I
©
Z
0
©
>
1.6 × 103 3.2 x 102 2.5 x 10 z
1.0 x 10 a
1.0 x 103
3.2 x 102
1.6 x 103
1.0 x 103
Buffer M/15 + TBS
Buffer M/15 + beef extract
TSB
TSB + meat
Nutrient broth (Eiss)
1 F o r d e f i n i t i o n a n d a b b r e v i a t i o n s see u n d e r T a b l e 1.
1.0 × 103
3.2 × t02
1.2 x 102
1.2 x 10 z
Buffer M/15 + meat
0.50
0.19
Y.e. 9
Buffer M/15
Y.e. 3
1.6 × 103
1.0 x 104
6.3 x 103
1.2 x 103
3.2 x 103
1.2 × 103
58
Pseud.
0.063
0.063
0.063
0.063
0.063
0.031
0.022
E. coli
0.063
0.015
0.001
0.063
0.019
0.025
0.025
Salm.
1.2 × 10 - 3
0.001
1.6 x 10 4
1.2 x 10 3
6.3 x 10 - 4
0.005
0.010
Kleb.
T a b l e 2. E n r i c h m e n t q u o t i e n t s ~ a f t e r 20 d a y s o f i n c u b a t i o n a t 4 ° C w i t h m e d i u m initial cell c o n c e n t r a t i o n .
0.063
0.010
2.5 x 10 . 3
0.063
0.006
1.9 x 10 3
7.9 x 10 - 3
Enter.
2.5 x 10 - 3
0.004
0.001
0.063
1.9 x 10 - 3
1.2 x 10 - 3
2.5 x 10 - 3
Prot.
m
Z
