Enumeration of virulent Yersinia enterocolitica colonies by DNA colony hybridization using alkaline treatment and paper filters

Molecular and Cellular Probes (1988) 2, 1 89-195

Enumeration of virulent Yersinia enterocolitica colonies by DNA colony hybridization using alkaline treatment and paper filters

James A . Jagow l ` and Walter E . Hil1 2 'Center for Microbiological Investigations, Food and Drug Administration, Minneapolis, MN 55401, USA and 2 Division of Microbiology, Food and Drug Administration, Washington, DC

20204,

USA

(Received 3 March 1988, Accepted 6 June 1988)

The efficiency of enumerating virulent Yersinia enterocolitica colonies was determined for 10 artificially contaminated foods by autoradiography after DNA colony hybridization using a 32 P - labelled genetic probe and paper filters . Enumeration efficiency, which ranged from 0-98% (average 25+31% standard deviation), improved (42-138%, average 83±27% standard deviation) after enrichment with 0 . 5% KOH-0-5% NaCl . These efficiencies are equivalent to those obtained with nitrocellulose filters . KEYWORDS : DNA hybridization, Yersinia enterocolitica, paper filters .

32 P-labelled

genetic probe,

INTRODUCTION Some recently developed DNA hybridization methods for determining the presence of pathogenic bacteria in foods' -' use nitrocellulose filters and radiolabelled DNA fragments obtained from plasmids or chromosomes . Several improvements include the use of paper filters (Whatman No . 541) 8 and oligodeoxyribonucleotides . 9 These modifications make the method less expensive and result in a stronger hybridization signal . We have described the use of DNA probes with nitrocellulose filters as a method for enumerating virulent Yersinia enterocolitica in a variety of foods' and have shown the probe used in this study to be specific for virulent strains . 4 This study evaluated the efficiency of using paper filters and a natural probe along with a KOH-NaCl pre-enrichment step to eliminate microbial background interference from indigenous microflora during the enumeration of virulent Y. enterocolitica cells seeded into foods similar to those used previously .' *Present address : Division of Microbiology, Food and Drug Administration, Washington, DC 20204, USA.

0890-8508/88/030189+07 $03 .00/0

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© 1988 Academic Press Limited



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J . A. Jagow and W . E . Hill MATERIALS AND METHODS

A culture of Y. enterocolitica 8081 10 " incubated overnight at 22-26° C in 10 ml of brain-heart infusion broth was used to artificially contaminate 10 different foods . A 50 g portion of each food was homogenized in 450 ml of Butterfield's buffer . 12 The aerobic plate count (APC) was determined by diluting and spread plating 0 . 1 ml of homogenate on plate count agar ;13 similarly, the gram-negative count was determined by spread plating 0 . 1 ml of homogenate on MacConkey agar (BBL Microbiology Systems, Cockeysville, Maryland) . This procedure was also used to determine the cfu titre of an overnight culture of Y . enterocolitica 8081, which, in addition, was added to two portions of the blended food (1 :10 homogenate) . One portion was homogenized for an additional 30 s ; 0 . 1 ml of the homogenate (yielding 102 Y . enterocolitica cells per millilitre) was spread-plated in duplicate on MacConkey agar . To the second, a 0. 5% KOH-0 . 5% NaCl pre-enrichment solution was added to produce a 1 :25 dilution . The food homogenate was mixed for 1-2 s, and 0 . 1 ml portions of the treated, seeded homogenate (yielding about 10 3 Y. enterocolitica cells per millilitre) were plated in duplicate on MacConkey agar . Inoculated MacConkey agar plates were incubated 24-48 h, depending on growth characteristics . From duplicate agar plates of each of the two preparation methods (i .e . with and without pre-enrichment treatment), Whatman No . 541 paper filters were prepared by using the method of Maas 8 as modified by Hill et a1. 9 Paper filters were prepared from cfu titre determination plates and from unseeded APC plates, which served as positive and negative controls . The hybridization protocol using a 32 P-labelled DNA probe was similar to that used previously,' except that the pre-incubation was eliminated . The enumeration efficiency of Y . enterocolitica 8081 seeded into each food with and without pre-enrichment was determined by comparing the number of spots on autoradiograms with the expected number of colonies as determined from the cfu titre (Fig . 1) .

RESULTS Microbial loads of the 10 foods tested were similar to those previously found,' ranging from 1 . 0 X 101 to 4 . 0 X 108 cfu g -1 on plate count agar and from 0 to 2. 0 x 10 8 cfu g -1 on MacConkey agar (Table 1) . In 10 separate trials with each of the different foods, the recovery of Y . enterocolitica 8081 using DNA colony hybridization with paper filters was compared with the observed APC on MacConkey agar . The observed colonies were counted, paper filters were prepared, DNA colony hybridization was performed and autoradiograms were exposed . The efficiency of enumerating Y . enterocolitica 8081 using paper filters averaged 86±11% when compared with the observed plate counts (Table 2) . In five similar trials, including the pre-enrichment procedure, the efficiency of using paper filters was 91 ± 8% (Table 2) . At the 95o/o confidence level, there was no significant difference in the efficiency of enumerating Y. enterocolitica 8081 in these experiments with and without the pre-enrichment procedure .

Enumeration of Y. enterocolitica by DNA hybridization

1 91

Fig. 1 . Autoradiogram of Yersinia enterocolitica colonies from artificially contaminated tofu . Duplicate filters were prepared from alfalfa sprouts contaminated with : a, b, 10' cells ; c, d, 10' cells with pre-enrichment treatment . Controls : e, 10' Y. enterocolitica 8081 ; f, tofu before addition of Y . enterocolitica 8081 .

DISCUSSION AND CONCLUSIONS Autoradiograms of prepared paper filters were used to determine the efficiency of recovery of Y . enterocolitica 8081 from seeded food homogenates . Whatman No . 541 paper filters were prepared from the MacConkey agar plates inoculated with approximately 102 cells per millilitre of Y . enterocolitica 8081 and food homogenate untreated or treated with the 0t5% KOH-0 . 5% NaCI pre-enrichment solution .

Table 1 .

Aerobic plate count of foods tested

Food Alfalfa sprouts Brie cheese Bullhead fish Celery Lettuce Pasteurized milk Mung beans Pork Oysters Tofu

Plate count agar (cfu ml') 4 .0 X 10 8 1 . 5 X 10' 2 . 7 X 10' 1 . 1 X 108 1 .4 X 10 5 1 .0 X 10' 8 . 6 X 10' 2 . 1 X 106 1 . 7 X 10' 8 . 1 X 108

MacConkey agar (cfu ml - ') 2.0 7.7 1 .5 1.5 8.2

X

3.8 1 .1 2.0 1 .3

X

'Numbers in parentheses are percentages of plate count agar counts .

X X X X

X X X

108 (50)* 104 (0 . 5) 10' (56) 108 (136) 104 (57) <10 10' (44) 106 (52) 10' (118) 10' (2)



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Table 2.

J. A. Jagow and W. E . Hill

Cfu per millilitre of Yersinia enterocolitica on MacConkey agar Cfu titre (cells ml -1 ) Without pre-enrichment

Food

Observed*

With pre-enrichment

Autoradiogramt

Observed

62 (95)$ 167(97) 136(76) 73 (80) 96 (83) 82 (88) 47000) 53 (60) 59(88) 77(95) 86 f 11 sD¶

46 ND§ ND ND 50 76 ND 35 55 ND

Alfalfa sprouts 65 Brie cheese 173 Bullhead fish 180 Celery 91 Lettuce 116 Pasteurized milk 93 Mung beans 47 Pork 89 Oysters 67 Tofu 81 Average percentage observed

Autoradiogramt 46 (100)$

50000) 62 (82) 31 (89) 46(84) 91 3_ 8

sD

T

*One trial was conducted with each food-seeding experiment . tFrom hybridized, prepared paper filter . Numbers in parentheses are percentages of cfus observed . §ND, not done . ¶so, standard deviation .

Autoradiograms of these filters were used to determine the efficiency of enumeration by these procedures . Without pre-enrichment, 0-98% (average 25 ± 31 %) of input cells were enumerated ; with pre-enrichment, 42-138% (average 83 ± 27%) of input cells were enumerated in the 10 foods tested (Table 3). The ratio of added virulent Y. enterocolitica to indigenous gram-negative

Table 3.

Enumeration of Yersinia enterocolitica 8081 in artificially contaminated food No . of Y . enterocolitica colonies cfus added ml'

Food Alfalfa sprouts Brie cheese Bullhead fish Celery Lettuce Pasteurized milk Mung beans Pork Oysters Tofu Average percentage observed

65 173 180 91 116 93 47 89 67 81

determined from cfu titre . tDetermined by spot count on autoradiogram . $sD, standard deviation . *As

Observed (%)t without pre-enrichment

Observed (%)t with pre-enrichment

0 (0) 9 (5) 26 (14) 2 (2) 56 (48) 98(105) 0(0) 38 (43) 4 (6) 20 (25) 25 f 31 SDI

69 (106) 151 (87) 76(42) 73 (80) 71 (61) 90(97) 65 (138) 40 (45) 57 (85) 71 (88) 83 f 27 SDI

Enumeration of Y . enterocolitica by DNA hybridization

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microflora ranged from about 1 :1 to 1 :105 (Tables 1 and 3) . There was no regular relationship between the increase in recovery due to enrichment and the efficiency of detection . With no enrichment, detection efficiency dropped below 40% when the aerobic and gram-negative titres exceeded 106 cfu g -1 . Alkaline enrichment had little effect on enumeration efficiency when total titres dropped below 10 5 but allowed enumeration efficiencies to remain above 40% when titres reached 108 cfu g -1 . Multiple linear regressions were performed on this data, but no significant conclusions could be reached regarding the factors that contributed to the efficiency of detecting Y. enterocolitica under these conditions . Because the bacterial counts ranged over 10 8, a logarithmic transformation had to be performed which may have distorted the analysis . The efficiency of enumeration using preenrichment was significantly higher at the 95% confidence level . The use of DNA probes as screening devices for enumerating pathogenic organisms has already been described .' -'' 9 Association of human illness with the 14-17 consumption of food contaminated with Y . enterocolitica is well documented . A number of virulence tests have been used to evaluate the pathogenic potential of Y. enterocolitica isolates ."'--2 ' A DNA hybridization probe for a gene required for calcium-dependent growth reacts with a family of virulence-associated plasmids (41-48 MDa) . Use of this probe in a colony hybridization format provides a sensitive, specific method to detect the presence of pathogenic strains in many types of foods and eliminates the need for selective culture methods, which at best yield most probable number estimates and often require a cold enrichment step that may last a week or more . Other assays for virulent Y. enterocolitica use suckling mice and are not well suited for the screening of large numbers of samples or isolates . The usefulness of paper filters for enumerating Y . enterocolitica 8081 by this technique was demonstrated for a variety of foods with a wide range of microbial loads . This procedure required less time and was as efficient as the use of nitrocellulose filters when a pre-enrichment step was included . When colonial growth was observed, a suitable transfer of cells to filter paper was made by letting the filter remain in place for 20-30 min before removal . As previously reported," the paper filters are less cumbersome to handle than nitrocellulose filters and cost only about 10% as much as do nylon filters . They can easily be stored on the shelf for up to a year without appreciable decrease in the signal quality (unreported data) . With an efficient protocol and improvement in sensitivity, biotinylated or sulphonated probes may be more practical in a food screening laboratory . However, these probes are less sensitive when high levels of other bacterial species are present . When used to test samples containing unpurified materials (e .g . foods), nonisotopically labelled probes have high backgrounds even though they have shown promise in systems using purified DNA (D . Shook, Food and Drug Administration, unpubl . obs .) . Until low-background, high-sensitivity probes become available, the radiolabelled probe will remain the reagent of choice for use as a screening device . High microbial loads and possibly the type of food may affect the efficiency of enumeration without a pre-enrichment step . The average efficiency using this procedure without pre-enrichment was 25+31% . Thus, the method is not useful for enumeration as a precise quantitative assay under these conditions, but may still serve as a screening method . Use of the 0 . 5% KOH-0E5% NaCl pre-enrichment solution in conjunction with the 1 :10 homogenate resulted in a dilution change which improved the efficiency of enumerating virulent Y . enterocolitica more than



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three-fold (to 83±27%) . This result is similar to the 86±12% efficiency observed when a more labour-intensive method incorporating nitrocellulose filters' was used and was not significantly different at the 95% confidence level . The presence of a food homogenate provided some protection for Y. enterocolitica 8081 during exposure to the pre-enrichment solution, i .e . cfu titres on MacConkey agar were reduced for pre-enrichment-treated cells (Table 2) . However, the pre-enrichment effectively eliminated most or all of the background contamination, making virulent Y. enterocolitica enumeration more precise .

ACKNOWLEDGEMENTS We thank D . A . Portnoy, Rockefeller University, New York, for providing Y . enterocolitica 8081 ; and William L. Payne, Division of Microbiology, Food and Drug Administration (FDA), Washington, DC, for technical assistance . We also thank Albert Schwab, Center for Microbiological Investigations, FDA, Minneapolis, Minnesota, for his support of this project .

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13 . Messer, J . W ., Peeler, J . T . & Gilchrist, J . E . (1984) . Aerobic plate count, p . 4 .01-4 .10 . In Bacteriological Analytical Manual, 6th edn . Arlington, Virginia : Association of Official Analytical Chemists . 14 . Aulisio, C . C . G ., Lanier, J . M . & Chappel, M . A . (1982) . Yersinia enterocolitica 0 :13 associated with outbreaks in three Southern states . journal of Food Protection 45, 1263 . 15 . Aulisio, C . C . G ., Starfield, j . T ., Weagent, S . D . & Hill, W . E . (1983) . Yersiniosis associated with tofu consumption : serological, biochemical and pathogenicity studies of Yersinia enterocolitica isolates . Journal of Food Protection 46, 226-30, 234 . 16. Centers for Disease Control . 1977 . Yersinia enterocolitica outbreak-New York . Morbidity and Mortality Weekly Report 20, 53-4 . 17 . Shayegani, M., Morse, D ., DeForge, I ., Root, T., Parsons, L . M . & Maupin, P . S . (1983) . Microbiology of a major foodborne outbreak of gastroenteritis caused by Yersinia enterocolitica serogroup 0:8 . journal of Clinical Microbiology 17, 35-40 . 18 . Aulisio, C . C . G ., Hill, W . E ., Stanfield, J . T . & Sellers, R . L., Jr . (1983) . Evaluation of virulence factor testing and characteristics of pathogenicity in Yersinia enterocolitica . Infection and Immunity, 40, 330-5 . 19 . Aulisio, C . C . G., Hill, W . E ., Stanfield, J . T . & Morris, J . A . (1983) . Pathogenicity of Yersinia enterocolitica demonstrated in the suckling mouse . journal of Food Protection 46, 856-60, 863 . 20 . Gemski, P ., Lazere, J . R. & Casey, T . (1980) . Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica . Infection and Immunity 27, 682-5 . 21 . Laird, W . J . & Cavanaugh, D . C . (1980). Correlation of autoagglutination and virulence of Yersinia . Journal of Clinical Microbiology 11, 430-2 .