detection and enumeration of indicator organisms in …aem.asm.org/content/9/4/295.full.pdf ·...

9
Detection and Enumeration of Fecal Indicator Organisms in Frozen Sea Foods1 2 II. Enterococci H. RAJ, W. J. WIEBE, AND J. LISTON College of Fisheries, University of Washington, Seattle, Washington Received for Publication October 3, 1960 ABSTRACT RAJ, H. (Univ. of Washington, Seattle), W. J. WIEBE, AND J. LISTON. Detection and enumeration of fecal indicator organisms in frozen sea foods. Appl. Microbiol. 9:295-303. 1961.-Consistently high recoveries of en- terococci as compared to the low numbers of coliforms obtained from the same samples of frozen sea foods are indirect evidence that enterococci are better indicators of contamination in such foods. The use of azide dextrose broth, modified by the in- corporation of bromthvmol blue, and of ethyl violet azide broth as presumptive and confirmation tests, re- spectively, were found to be highly specific for the detection and enumeration of enterococci in these samples. Tetrazolium agar medium, when used as a third step after the confirmation test, provides a reliable differentiation of Streptococcus faecalis types from other group D streptococci. A simple procedure is described for further identification of S. faecalis varieties and other enterococcal species. Incidence of biotypes within certain species is noted and relationships of these subgroups to the organisms described by other workers is discussed. The striking resistance of all group D streptococci to dihydrostreptomycin and polymyxin B seems to offer promise for evolving a new selective medium for these organisms. There is an increasing body of evidence indicating that the standard methods of evaluating the sanitary quality of water and milk, which have been applied somewhat indiscriminately to other foodstuffs, are unsatisfactory particularly in relation to frozen foods. In the first paper of this series (Raj and Liston, 1961), the unreliability of the EC test for fecal Escherichia coli, when applied to frozen sea foods, has been demon- strated. The estimation of enterococci as an index of con- tamination of frozen foods has been advocated in 1 This work was supported by National Institutes of Health grant no. RG-5932. 2 Contribution no. 88, College of Fisheries, University of Washington, Seattle, Wash. several recent publications (Larkin, Litsky, and Fuller, 1955, 1956; Zaborowski, Huber, and Rayman, 1958; Kereluk and Gunderson, 1959a). The greater resistance shown by these organisms, as compared with E. coli and other coliforms, to heating and freezing processes and to frozen storage has been cited as strong evidence supporting their use for this purpose (Larkin et al., 1955, 1956; Kereluk and Gunderson, 1959b). Moreover, some doubt has been cast recently on the value of E. coli as an index of fecal pollution by the observations of Buttiaux (1959) and Young et al. (1960) concerning the low recoveries of this organism from human feces. The enterococci appear to provide a good general index of fecal contamination and Streptococcus faecalis is be- lieved to be a specific index of humaii pollution (Cooper and Ramadan, 1955; Barnes, Ingram, and Ingram, 1956), though findings quoted by Buttiaux (1958) cast some doubt on this conclusion. There has been much work reported in recent years on the development of media and techniques for the detection and enumeration of enterococci in foodstuffs. A method has been evolved in which a presumptive count is made in azide dextrose (AD) broth or entero- cocci presumptive (EP) broth and confirmed by the ethyl violet azide (EVA) broth medium of Litsky, Mallman, and Fifield (1953). This method has been shown to provide a rapid and reportedly specific pro- cedure for counting enterococci in foodstuffs (Zaborow- ski et al., 1958). For differentiation of S. faecalis types from other enterococci, Barnes (1956a) has proposed an agar medium containing 2,3,5-triphenyltetrazolium chloride. The studies reported in this paper were designed to test the applicability and specificity of the EVA pro- cedure when applied to frozen sea foods, to evaluate the usefulness of the tetrazolium agar (TZA)3 medium of Barnes (1956a) for identification of S. faecalis types, and to evolve methods for the rapid identification of other group D streptococci isolated from frozen sea foods. 3Barnes has abbreviated her tetrazolium glucose agar medium as TG agar. Since we use the same abbreviation for tellurite glycine agar, we prefer to call tetrazolium glucose agar TZA. 295 on June 25, 2018 by guest http://aem.asm.org/ Downloaded from

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Page 1: Detection and Enumeration of Indicator Organisms in …aem.asm.org/content/9/4/295.full.pdf · Detection and Enumeration of Fecal Indicator Organisms in ... azide broth as presumptive

Detection and Enumeration of Fecal Indicator Organisms inFrozen Sea Foods1 2

II. Enterococci

H. RAJ, W. J. WIEBE, AND J. LISTON

College of Fisheries, University of Washington, Seattle, Washington

Received for Publication October 3, 1960

ABSTRACT

RAJ, H. (Univ. of Washington, Seattle), W. J. WIEBE,AND J. LISTON. Detection and enumeration of fecalindicator organisms in frozen sea foods. Appl. Microbiol.9:295-303. 1961.-Consistently high recoveries of en-terococci as compared to the low numbers of coliformsobtained from the same samples of frozen sea foods areindirect evidence that enterococci are better indicatorsof contamination in such foods.The use of azide dextrose broth, modified by the in-

corporation of bromthvmol blue, and of ethyl violetazide broth as presumptive and confirmation tests, re-spectively, were found to be highly specific for thedetection and enumeration of enterococci in thesesamples. Tetrazolium agar medium, when used as athird step after the confirmation test, provides a reliabledifferentiation of Streptococcus faecalis types fromother group D streptococci. A simple procedure isdescribed for further identification of S. faecalis varietiesand other enterococcal species.

Incidence of biotypes within certain species is notedand relationships of these subgroups to the organismsdescribed by other workers is discussed.The striking resistance of all group D streptococci to

dihydrostreptomycin and polymyxin B seems to offerpromise for evolving a new selective medium for theseorganisms.

There is an increasing body of evidence indicatingthat the standard methods of evaluating the sanitaryquality of water and milk, which have been appliedsomewhat indiscriminately to other foodstuffs, areunsatisfactory particularly in relation to frozen foods.In the first paper of this series (Raj and Liston, 1961),the unreliability of the EC test for fecal Escherichia coli,when applied to frozen sea foods, has been demon-strated.The estimation of enterococci as an index of con-

tamination of frozen foods has been advocated in

1 This work was supported by National Institutes of Healthgrant no. RG-5932.

2 Contribution no. 88, College of Fisheries, University ofWashington, Seattle, Wash.

several recent publications (Larkin, Litsky, and Fuller,1955, 1956; Zaborowski, Huber, and Rayman, 1958;Kereluk and Gunderson, 1959a). The greater resistanceshown by these organisms, as compared with E. coliand other coliforms, to heating and freezing processesand to frozen storage has been cited as strong evidencesupporting their use for this purpose (Larkin et al.,1955, 1956; Kereluk and Gunderson, 1959b). Moreover,some doubt has been cast recently on the value ofE. coli as an index of fecal pollution by the observationsof Buttiaux (1959) and Young et al. (1960) concerningthe low recoveries of this organism from human feces.The enterococci appear to provide a good general indexof fecal contamination and Streptococcus faecalis is be-lieved to be a specific index of humaii pollution (Cooperand Ramadan, 1955; Barnes, Ingram, and Ingram,1956), though findings quoted by Buttiaux (1958) castsome doubt on this conclusion.There has been much work reported in recent years

on the development of media and techniques for thedetection and enumeration of enterococci in foodstuffs.A method has been evolved in which a presumptivecount is made in azide dextrose (AD) broth or entero-cocci presumptive (EP) broth and confirmed by theethyl violet azide (EVA) broth medium of Litsky,Mallman, and Fifield (1953). This method has beenshown to provide a rapid and reportedly specific pro-cedure for counting enterococci in foodstuffs (Zaborow-ski et al., 1958). For differentiation of S. faecalis typesfrom other enterococci, Barnes (1956a) has proposed anagar medium containing 2,3,5-triphenyltetrazoliumchloride.The studies reported in this paper were designed to

test the applicability and specificity of the EVA pro-cedure when applied to frozen sea foods, to evaluatethe usefulness of the tetrazolium agar (TZA)3 mediumof Barnes (1956a) for identification of S. faecalis types,and to evolve methods for the rapid identification ofother group D streptococci isolated from frozen seafoods.

3Barnes has abbreviated her tetrazolium glucose agarmedium as TG agar. Since we use the same abbreviation fortellurite glycine agar, we prefer to call tetrazolium glucoseagar TZA.

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H. RAJ, W. J. WIEBE, AND J. LISTON

MATERIALS AND METHODSThe procedure for the preparation of samples of

frozen fish sticks (both in uncooked and precookedstate) obtained from a local frozen sea food processingplant was the same as described in the first paper

(Raj and Liston, 1961) of this series. In the preliminaryexperiments, three different media, AD broth,4 SFmedium,5 and EP broth,5 were inoculated with ap-

propriate decimal dilutions of fish sample and incubatedfor 48 hr at 37 C, 37 C, and 45 C, respectively. Subse-quently, AD broth modified slightly by the addition of0.003 % bromthymol blue was tested together with theoriginal AD broth and EP broth. In AD broth growthturbidity is the sole criterion of positive reaction andthus sometimes it is hard to tell a positive AD tubefrom a negative one. In AD modified (AD(M)) broth,there is a sharp color change from greenish blue tobright yellow when enterococci are present, whereas a

slight color change to pale green constitutes a negativereaction. In further experiments, the efficiency of ADbroth and AD(M) broth when incubated at 37 C and45 C was tested. Positive cultures in presumptive mediawere then transferred into EVA broth4 and incubatedfor 48 hr at 37 C. EVA broth cultures showing turbidityand/or a purple button (occasionally purplish-whitedeposit) constituted the confirmation test, and mostprobable number (MPN) counts were recorded ac-

cordingly.For further identification, 210 positive EVA cultures,

derived by the above confirmation test from frozen sea

food samples, and 5 ATCC enterococci cultures (S.faecalis (7080), S. faecalis var. liquefaciens (13398), S.faecalis var. zymogenes (6055), Streptococcus bovis (9809),and Streptococcus durans (6056)) were streaked on TZAmedium which was prepared as per the specificationsof Barnes (1956a) except for the following slight modi-fication: in place of peptone (Evans) and Lab-lemco,Bacto-peptone5 and Bacto-beef extract5 were used,respectively. In addition, prior to sterilization glucosewas added to the medium and pH adjusted to 6.0. Thestreaked plates were incubated at 37 C for 48 hr insteadof 24 hr as suggested by Barnes. Colony characteristicsas defined by the same author for enterococci were

followed. Well isolated colonies were transferred intobrain heart infusion broth5 and incubated at 37 C for20 to 24 hr. One hundred thirty-one EVA positive cul-tures and 5 type cultures were subjected to all thefollowing physiological and biochemical tests at thesame time (the remaining 84 isolates were tested forbiochemical reactions only).

Morphology. Each culture was Gram stained andexamined microscopically.Group precipitin test. The method used for preparing

the extracts of group antigen was that of Rantz and

4Baltimore Biological Laboratory, Inc., Baltimore, Md.5 Difco Laboratories, Inc., Detroit, Mich.

Randall (1955). The precipitin reaction was thencarried out by the capillary tube method using Bacto-streptococcal antiserum5 and the results read at inter-vals up to 30 min.

Hemolysis. Fifteen per cent human blood in bloodagar base5 was used for detection of hemolysis at 37 Cup to 96 hr.

Potassium tellurite tolerance. Blood agar (as above)containing sterile potassium tellurite in a concentrationof 1:2,500 was used as described by Ferraro and Apple-man (1957) and results read up to 96 hr at 37 C.

Growth at pH 9.6. The medium suggested by Shattockand Hirsch (1947) was used. The pH of each tube wasadjusted aseptically to 9.6 just before inoculation.Growth observed after 24 and 48 hr at 37 C.

Growth in presence of 6.5% sodium chloride. Themedium suggested by Abd-El-Malek and Gibson (1948)was modified slightly. Instead of sterilizing each of theingredients separately, all of them were dissolved to-gether in distilled water and tubed in 10-ml aliquots.The tubes were then steamed for 30 min.

Starch hydrolysis. Each culture was streaked on anagar medium containing 0.2 % soluble starch, 1 %peptone, 0.5 % yeast extract, and 0.5 % sodium chloride.The plates were incubated at 37 C for 5 days whenhydrolysis of starch was tested by flooding with aniodine solution.

Antibiotic sensitivity test. High concentrationUnidisks5 for the antibiotics chlortetracycline (30,ug),penicillin (10 units), chloramphenicol (30 ,ug), polymyxinB (300 units), oxytetracycline (30 4g), erythromycin(15 jig), tetracycline (30 ,ug), and dihydrostreptomycin

TABLE. 1. Comparison of enterococci and coliform mostprobable number (MPN) counts in frozen precooked

fish sticks

Sample no. Enterococci Coliforms

MPN coun1/O00 g MPN count/100 g

1 86,000 62 18,600 193 86,000 04 46,000 3005 48,000 1506 46,000 287 46,000 1508 18,600 79 8,600 010 4,600 18611 4,600 18612 48,000 1,28013 8,600 4614 4,600 48015 48,000 24016 10,750 1,07517 10,750 17,00018 60,000 23,25019 10,750 2,275

Avg 32,339 2,457

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DETECTION OF ENTEROCOCCI IN FROZEN SEA FOODS

(10 jAg) were used on heart infusion agar5 plates andzones of inhibition read after 24 hr at 37 C. Sensitivity(S) was indicated by the presence of a distinct zone ofinhibition (at least 2 mm radius beyond the edge ofeach disc) and resistance (R) was shown by the absenceof such a zone. To compare the activity of any one anti-biotic to the others, the term "very sensitive (VS)"was used whenever the zone of inhibition was greaterthan 20 mm in diameter.Methods used for tests, such as gelatin liquefaction,

litmus milk reduction, growth in 0.1 % methylene bluemilk, and survival at 60 C for 30 min, were the same

as those used by Cooper and Ramadan (1955). Alsothe heated cultures were streaked on potassium telluriteblood agar plates for combined heat and tellurite re-

sistance as suggested by these authors.Fermentation reactions in sucrose, glycerol, mannitol,

sorbitol, raffinose, inulin, melibiose, melezitose, andarabinose were determined using purple broth base5

with a 1 % carbohydrate concentration. Acidity was

recorded every 24 hr up to 5 days.

RESULTS

Coliform counts were highly variable and often ex-

tremely low. Some of the typical coliform results to-gether with the corresponding enterococcus counts are

shown in Table 1. It will be noted that the enterococcuscount in these heat-treated and refrozen samples was

generally greater than 103 organism/100 g and usually104 organism/100 g.

Preliminary experiments using three different mediain the presumptive test for enterococci showed that SFbroth did not give as sharp a color change as EP broth.Although AD broth in almost all cases gave greaterconfirmed MPN counts than those given by the EP-EVA procedure (Table 2), the number of false positiveswas greater from AD broth. Modified AD broth con-

taining bromthymol blue was found to maintain as

high a percentage of true positives as normal AD broth

TABLE 2. Comparison of enterococci most probable number (MPN) counts obtained by three different procedures

Sea food examined

Breaded oystersBreaded oystersBreaded oystersBreaded oystersBreaded oystersPrecooked cod sticksRaw cod sticksRaw cod sticksRaw oystersBreaded oystersRaw halibut barsBreaded cod sticksPrecooked cod sticksPrecooked smeltRaw cod sticksRaw cod blocksRaw cod sticksBattered cod sticksBreaded cod sticksBreaded cod sticksBreaded salmon barsBreaded cod sticksBreaded halibut barsPrecooked cod sticksPrecooked cod sticksBreaded cod sticksPrecooked cod sticks

I

AD - EVA(37 C) (37 C)

IIAD(M) EVA(37 C) (37 C)

IIIEP -EVA(45C) (37C)

PoiiePositive Positive Positive MN'0 Positive PositiveAD tubes EVA tubes MPN/100 g tesM) | tubes MPN/100 g EP tubes EVA tubes MP1N/l00 g

_ _~~~~~~~~~~~~ue656566665646666666666666464

445360

36344534466666

6565451

5,250115,00023,25060,000

375,000<600

37,500275,000+

5,7509,75011,500

275,00060,00010,750

115,000275,000+275,000+275,000+275,000+275,000+60,00072,500275,000+

2,350115,000275,00010,750

646563454555645666664464445

54646225355562566666

44

64444

23,250115,00060,000115,000375,000

2,27518,750

275,0005,75023,250

275,000275,00060,0001,825

275,000275,000+275,000+275,000+275,000+275,000+115,000115,000275,000+10,750

115,000115,00010,750

433640

333114633434335642553

233540

333114633433333640

550

AD = azide dextrose; AD(M) = AD modified; EVA = ethyl violet azide; EP = enterococci presumptive.

1961] 297

1,82530,0005,750

23,250107,500<600

30,0005,7503,750

900900

10,75060,0005,7505,750

23,2505,7509,7505,7505,75018,75060,00010,750<600

23,25023,250<600

153 120 134 125 94 83

Percentage of false posi-tives 21.6% 6.7% 11.7%

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H. RAJ, W. J. WIEBE, AND J. LISTON

while reducing the number of false positives from22 to 7 %. This compares well with the false positiverate of 12 % in the case of EP. Both AD and AD(M)broths were found to give higher counts at 37 C than at45 C (Table 3).

TABLE 3. Comparison of enterococci MPN counts obtainedat two different temperatures

Sea foodexamined AD - EVA AD - EVA AD (M) - EVA AD (M) - EVA

(37 C) (37 C) (45 C) (37 C) (37 C) (37 C) (45 C) (37 C)

MPN count/100 g sample MPN count/lOO g sample

Precooked <600 <600 2,275 <600cod sticks

Raw cod 275,000+ 3,750 275,000 5,750sticks

Breaded 9,750 750 23,250 5,750oysters

Raw halibut 11,50 900 275,000 <600blocks

Breaded cod 275,000 115,000 275,000 60,000sticks

Raw cod 115,000 23,250 275,000 10,750sticks

AD = azide dextrose; AD(M) = AD modified; EVA =ethyl violet azide; EP = enterococci presumptive.

Microscopic examination of all cultures derived frompositive EVA tubes revealed typical enterococcal forms.No false positive EVA tubes were encountered exceptin the case of two tubes containing a Bacillus sp. whichpassed through the test schedule. In this case the tubesshowed a slight turbidity and a flaky, purplish sedimentwhich, however, could be distinguished clearly fromthe typical purple button produced by enterococci.Negative tubes always showed a clear supernatant andoccasionally a slight deposit.

All positive EVA tubes were streaked on TZA mediumand colony characteristics noted. Colonies showing an

intensely deep red center and a white periphery (oc-casionally some colonies had only a small pink centerwith white peripheral zone) were classified here as typeI, and white or a very pale pink colony with no periph-eral differentiation as type II. The Bacillus sp. notedabove when subcultured on TZA produced deep redconvex colonies without any peripheral zone, whichseemed similar to those described by Barnes for groupN streptococci.The physiological and biochemical reactions of the

210 EVA positive cultures and the 5 ATCC enterococcicultures tested according to the procedure describedin Materials and Methods are shown in Tables 4 and 5.

TABLE 4. Physiological and biochemical characters of enterococci

Streptococcus faccalisStreptococcus Streptococcus Streptococcus Unclassified

bovis faecium durans typeTypical var. liquefaciens var.

zymogenes

126 EVA isolates .................................. 970 0 10 3 22 125 ATCC cultures*.1 1 1 1 1

Lancefield group D D D D D D DHemolysis _ ,a 7* -*, a 4 -a ac 7, * a,-5Tetrazolium reduction + + + _ _ _0.04% Potassium tellurite tolerance + + + _ _ _Growth in pH 9.6 broth + + + + + + +Growth in 6.5%o NaCl broth + +*, -3 + -*, +3 + + +Growth in 0.1% methylene blue milk + + + +*-1 -, +1 -, +8* +, -3Strong litmus milk reduction + +*, -3 + +*, -1 +, -1 -, +6* +, -5Survival at 60 C for 30 min + + + +, -3* + + +Survival at 60 C for 30 min plus 0.04% _*, +2 +*, -9 + _*, +1 - _potassium tellurite tolerance

Starch hydrolysis ... ... ... +1 - _Gelatin liquefaction _ + - - _ _Fermentation of:

Sucrose A*,-1 *,A 25 A A A * A 1 AGlycerol A*, -5 A A - - _Mannitol A A A A*, -1 A - ASorbitol A A A A*,-1 - ARaffinose - _ - A - _Inulin - _ - A*,-6 - -

Melibiose - - - A A ,A 11* AMelezitose A*, -1 *, A 23 A - - -

Arabinose ... .. ... A A - A

Numbers with certain reactions indicate the number of strains showing variation from the reaction given by the majority. Ais for acid only in fermentation reactions at 37 C up to 5 days. *Sign over certain reactions indicates the particular reactions givenby the ATCC cultures. EVA = ethyl violet azide.

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DETECTION OF ENTEROCOCCI IN FROZEN SEA FOODS

The three type cultures of S. faecalis and its varietiesliquefaciens and zymogenes gave type I colonies on TZAand the type cultures of S. durans and S. bovis gave typeII colonies. All positive EVA cultures from sea foodswhich gave type I colonies on TZA were found to betypical S. faecalis or one of its two varieties. None ofthe cultures giving rise to type II colonies was foundon examination to belong to the S. faecalis group.Notably, cultures of S. bovis and S. durans types alwaysgave white colonies on TZA, which is in agreement withthe low percentages of reduction of tetrazolium toinsoluble red formazan worked out by Barnes (1956b).It may be noted from Tables 4 and 5 that all the 215

TABLE 5. Biochemical reactions of enterococci

84 EVA isolates ........

1. Lancefieldgroup

2. Tetrazoliumreduction

3. Gelatin lique-faction

4. Sucrose5. Glycerol6. Mannitol7. Sorbitol8. Raffinose9. Inulin

10. Melibiose11. Melezitose12. Arabinose

Strceptococcusfaecalis

Typical var. lique-

10

D

A, -1AAA

A, -1.*..

30

D

,AAAA

, A.. .

11

5

Strepto- Strepto- Strepto- Unclas-coccus COCC 5 COCCUS sifiedboris faecium durans type

10

D

A

AA, -3A

A, -2A

A

D

A

A

A

A

27

D

-, A 1

A, -3

Numbers with certain reactions indicate the number ofstrains showing variation from the reaction given by the ma-

jority. A is for acid only in fermentation reactions at 37 C uso

to 5 days. EVA = ethyl violet azide.

cultures gave a positive group D precipitin test, grew

at pH 9.6, and survived (except for 3 strains of S. bovis)60 C for 30 min. The only test, other than colony ap-

pearance on TZA, which differentiated S. faecalis typesfrom other enterococci appeared to be potassiumtellurite tolerance. Some of the tests, such as growth in0.1 % methylene blue milk, litmus milk reduction,hemolysis, growth in the presence of 6.5 % sodiumchloride, and resistance to potassium tellurite (0.04 %)after heating the cultures at 60 C for 30 min, gave

variable results. Antibiotic sensitivity tests showed thatall the enterococci cultures examined here were quitesensitive to chlortetracycline, penicillin, chlorampheni-col, oxytetracyline, erythromycin, and tetracycline,whereas with few exceptions they were highly resistantto polymyxin B and dihydrostreptomycin (Table 6).An analysis of the results of all the tests shown in Tables4 and 5 revealed that a limited number of the testscould be used to provide a rapid and satisfactorydifferentiation of the various enterococcal species.A comprehensive scheme of identification based on

such tests is outlined in Table 7. Initially, the or-

ganisms are identified on the basis of growth at 37 Cin AD(M) broth followed by confirmation in EVAbroth. Then using the TZA medium, the enterococciare classified into type I corresponding to S. faecalisincluding its varieties and type II including all othergroup D streptococci. Subdivision of type I enterococciis effected by four simple tests: gelatin liquefaction,hemolysis, and fermentation of melibiose and sorbitol.The breakdown of type II organisms is made by thefermentation reactions in arabinose, glycerol, melezi-tose, raffinose, and sorbitol.

DISCUSSIONThe consistently high recoveries of enterococci from

frozen sea foods and the low and erratic recoveries ofcoliforms from the same samples are indirect evidence

TABLE 6. Antibiotic sensitivities of enterococci

Streptococcus faecatis

Streptococcus bovis Streptococcus Sire tococcus Unclassified typevar. zy- ~~~~faciumn aurans

Typical var. liquefaciens mogenes

210 EVA isolates ...................... 19 100 0 20 4 49 185 ATCC cultures ..................... 1 1 1 1 0 1 0

Chlortetracycline (30 IAg) VS, S 7* VS*, S 43 VS VS*) S 2 VS, S 2 VS*, S 3 VS, S 2Chloramphenicol (30,g) VS*, S 1 VS*, S 44, R 1 VS VS*, S 2 VS, S 2 VS*, S 4 VSDihydrostreptomycin (10,ug) R R*, S 2 R R*, S 2 R R*, S 2 RErythromycin (15 jg) VS*, S 6 VS*, S 27 VS VS*, S 5 VS, S 1 VS*, S 2 VS, S 4Penicillin (10 units) S St, R 3 S VS*, S 7, R 1 S S, VS 11* VS, S 5, R 1Polymyxin B (300 units) R R*, S 3 R R*, S 2 R R*, S 4 ROxytetracycline (30 Mg) Vs, S 2* VS*, S 34 VS VS*, S 1, R 1 VS VS*, S 6 VS, S 2Tetracycline (30 Mg) VS*, s 1 VS*, S 24 VS vs*, R 1 VS, S 1 VS*, S 3 VS, R 1

VS = very sensitive; S = sensitive; R = resistant. Numbers with certain reactions indicate the number of strains showing vari-ation from the reaction given by the majority. * Sign over certain reactions shows the reactions given by the type cultures. EVA =ethyl violet azide.

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H. RAJ, W. J. WIEBE, AND J. LISTON

of the value of enterococci as better indicators of con-tamination in sea foods. These findings are in goodagreement with those reported by other workers (Larkinet al., 1955, 1956; Zaborowski et al., 1958; Kerelukand Gunderson, 1959a).The procedure, presumptive test in AD(M) broth,

confirmation in EVA broth and completed test on TZAmedium, seems to provide a satisfactory and sufficientlyrapid method for enumerating and grouping enterococcifrom sea foods and possibly other foods in terms of theirpresumed human or animal origin. Where this appearsdesirable, a further classification of enterococci may bemade easily by following the abbreviated scheme out-lined in Table 7.For the presumptive test, modified AD broth at 37 C

was found to be more satisfactory than the original ADmedium at 37 C, because it eliminated most of the largenumber of false positives given by the latter as shownin Table 2. The low MPN counts obtained by theEP-EVA procedure might be attributed to the high(0.04 %) sodium azide content, high pH (8.4) of EPbroth, and its incubation temperature (45 C). The ADbroth contains only 0.02 % sodium azide, has pH 7.2,and is incubated at 37 C. The superiority of AD brothover EP and SF broths for presumptive testing hasalso been shown by Zaborowski et al. (1958). In agree-ment with those authors, we have found EVA mediumto be highly specific for enterococci. The reliability ofthis method is in marked contrast to the variability ofresults obtained with the EC test for fecal E. colireported elsewhere (Raj and Liston, 1961).When the TZA medium of Barnes (1956a) is added

as a third step after the EVA procedure, a method isobtained for S. faecalis (and its varieties) which is

analogous to the EMB step in the completed test forE. coli. A rapid and accurate estimation of human fecalcontamination may thus be obtained. However, it maybe noted that TZA medium by itself is not selective forenterococci and, in the absence of the presumptive(e.g., AD broth) and confirmatory (EVA broth) steps,organisms other than enterococci can grow and producecolonies. It is well known that many gram-negativebacteria will grow in the presence of concentrations oftetrazolium even greater than 0.1 % (Weinburg, 1953).Gyllenberg, Niemala, and Sormunen (1960) have grownLactobacillus bifidus in the presence of 0.01 % tetra-zolium, and the growth of a Bacillus sp. on TZA hasalready been noted above. Thus the use of TZA mediumfor direct counting of enterococci in a sample would beunsatisfactory and erratic, unless it is made selective byadding some inhibitory agent such as thallous acetate(Barnes, 1956a). Hartman (1960) appears to haveused Barnes' medium without thallous acetate fordirect plate counting and recorded only red coloniesas enterococci. It is possible that these counts includedred colonies produced by organisms other than entero-cocci, and that white or pale pink colonies of S. durans,S. bovis, and others were thus excluded.

Practically all of the physiological and biochemicaltests shown in Tables 4 and 5, except for the potassiumtellurite tolerance test before and after heating theculture at 60 C for 30 min and fermentation reactions inmelibiose and melezitose, were first specified bySherman (1937) for isolation and differentiation ofenterococci. Chesbro and Evans (1959) have suggestedthat some inhibition may occur in the high pH (9.6)medium of Shattock and Hirsch (1947) because of thesmall amount of glycine (0.006 M) present as buffer.

TABLE 7. Schematic diagram for rapid identification of enterococci

AD(M) broth*

EVA brotht

TZA plate$

TYPE I COLONY

(Intensely deep red colony with white periphery)1

1. Gelatin - +2. Hemolysis -, a -, a

3. Melibiose - -

4. Sorbitol A A

Strepto-coccus

faecalis

S. faecalisvar. lique-faciens

A

S. faecalisvar. zymo-genes

TYPE II COLONY

(White or pale pink colony without any periphery)4,

1. Arabinose - A V2. Glycerol - -

3. Melezitose - -

4. Raffinose - - A5. Sorbitol - - V

Strepto-coccus

durans

Strepto-coccus

faecium

Strepto-coccus

bovis

A

AUnclassi-fied type

* Azide dextrose broth (Difco) modified by adding 0.003% bromthymol blue; incubated at 37 C for 48 hr.t Ethyl violet azide broth (BBL) at 37 C for 48 hr.I Tetrazolium glucose agar medium (Barnes, 1956a) at 37 C for 48 hr.Fermentation reactions at 37 C for 72 hr; A for acid only, V for variable, and - for no reaction.

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DETECTION OF ENTEROCOCCI IN FROZEN SEA FOODS

Very good growth was observed in this medium within48 hr at 37 C in the case of all the culture examinedduring this study. It seems, therefore, that any inhibi-tion due to such low concentrations of glycine isnegligible and does not interfere with the test. Theusefulness of some of the tests for differentiation ofenterococci seems doubtful due to the variable response

to them by organisms of the same species. These testsinclude: growth in 0.1 % methylene blue milk, litmusmilk reduction, hemolysis, and resistance to potassiumtellurite (0.04%) after heating the culture at 60 C for30 min. Shattock and Mattick (1943) had also observedthat some of these tests vary in their reliability. It maybe noted here that Barnes et al. (1956) omitted themethylene blue reduction test in their identification ofenterococci. Cooper and Ramadan (1955) found Janusgreen a better indicator than litmus which they foundto change in color readily with pH changes of the milkcultures. Shattock (1955) reported that hemolysisshould not be relied upon for identification of entero-cocci. However, only S. faecalis var. zymogenes has beenshown to give a consistent (3-type hemolysis whichdifferentiates this variety from the typical S. faecalistypes. Lake, Diebel, and Niven (1957) reported that a

#-hemolytic culture of S. durans may easily lose thisproperty. The variability in the resistance to potassiumtellurite (0.04 %) by the heat-treated cultures, as notedhere, is in contrast to the findings of Cooper andRIamadan (1955), who utilized this characteristic as a

means of classifying enterococci into six habitat groups.

It is evident from the results (Table 4) that some strainsof typical S. faecalis and its varieties, which, prior toheat treatment, were resistant to 0.04 % potassiumtellurite, lost this property of resistance as a conse-

quence of heat treatment. However, there was a directcorrelation between the resistance of unheated culturesto 0.04% potassium tellurite and tetrazolium reductionon TZA medium. All type I colonies were resistant,

whereas type II colonies were sensitive to potassiumtellurite.A careful analysis of the biochemical reactions of all

the isolates tested here indicated the existence of atleast three biotypes under each of the following species:S. faecalis (typical), S. faecalis var. liquefaciens, S.bovis, and S. durans, as shown in Table 8. These bio-types differ from one another within the species in

only one or two properties. Each of the reactions dis-tinguishing the biotypes was tested at least three timesunder identical conditions with each organism involved,and found to be a stable characteristic. Certain strainsof S. faecalis (typical) designated here as biotype b,did not utilize glycerol and these have already beenreported by Frost and Engelbrecht (1940). Strainsshown under biotype c failed to ferment sucrose whichhas been reported to be a variable reaction for S.faecalis in Bergey's Manual of Determinative Bacteriology(Breed, Murray, and Smith, 1957). Most of the strainsof S. faecalis var. liquefaciens, including the ATCC cul-ture, fell into biotype d and did not ferment sucrose

although most published descriptions of this varietyreport positive reaction for sucrose except againBergey's Manual where the reaction is listed as variable.The sucrose fermenting types have been subdividedinto biotypes e and f according to their ability to utilizemelezitose. Since melezitose is a trisaccharide with a

sucrose moiety, strains breaking down this trisaccharidecan also utilize sucrose, but those able to ferment su-

crose may not necessarily utilize melezitose. Most ofthe S. bovis strains including the ATCC culture fer-mented mannitol and sorbitol, as shown under biotypeu. Many reports in the literature show negative reac-

tions for mannitol and sorbitol for S. bovis but Bergey'sManual and Sherman (1937) report variable reactionsfor these carbohydrates, whereas Frost and Engelbrecht(1940) observed positive reaction for mannitol. Thebiotypes v and w in this group have thus been separated

TABLE 8. Biotypes of some species of enterococci

Streptococcus faecalisStreptococczus bovis Streptococcus durans

Typical var. liquefaciens

Biotype ........................ a b c d e f u v w x y z

No. of isolates 13* 5 2 65* 8 28 17* 3 1 35* 13 2

Gelatin liquefaction - - - + + + - - - - -

Sucrose A A _ _ A A A A A _ _ AGlycerol A _ A A A A _ - - _ _Mannitol A A A A A A A A _ _ _Sorbitol A A A A A A A _ _ _ _Raffinose _ - _ _ _ _ A A A _ _Melibiose _ _ - _ _ _ A A A A _Melezitose A A _ _ _ A _ _ _ _ _Arabinose .. .. .. .. A A A -_ _

* Number includes ATCC culture.

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H. RAJ, W. J. WIEBE, AND J. LISTON

on the basis of their reactions in mannitol and sorbitol.The carbohydrate reactions for S. durans are, in general,in agreement with the published descriptions of thisspecies. Most strains including the type culture fer-mented only melibiose as shown under biotype x. Theother strains which did not utilize melibiose have beenseparated into biotypes y and z on the basis of sucrosefermentation which has been reported to be a variablecharacteristic for S. durans by Breed et al. (1957),Wilson and Miles (1955), and Cooper and Ramadan(1955). The incidence of biotypes as distinct subgroupswithin a species thus explains some of the variablereactions reported in the literature for certain species ofenterococci. It would be interesting to determinewhether these biotypes could be established as sero-types too.

Considering only those physiological and biochemicalreactions which were consistently specific for the dif-ferentiation of enterococci, a comprehensive scheme(Table 7) has been evolved for their rapid identificationas already described under Results. The classification ofKereluk (1959) for identification of enterococcal speciesseems to be incomplete as it excludes S. bovis species andunclassifiable types. For example, 54 isolates classifiedby Kereluk as S. faecium because of fermenting arabi-nose and melibose only could have been S. bovis or un-classifiable types. Cooper and Ramadan (1955) have re-ported five so-called atypical faecalis types which theyhave differentiated from the typical types. The atypicaltype I reported by these authors was not encounteredin our study, however, their atypical type II strainsseem to be identical with those reported by us as un-classified type (Tables 4 and 5). Incidence of suchstrains as a separate entity is therefore confirmed. Theatypical types III and IV reported by the above authorsseem to belong to the S. bovis group since all utilize raf-finose. Cooper and Ramadan distinguish atypical typesIII, IV, and S. bovis by differences in growth at pH 9.6and in the presence of 6.5 % sodium chloride. In ourstudies, we found that three S. bovis-like strains wereable to grow in the presence of 6.5 % sodium chloride,and thus resembled Cooper and Ramadan's atypicaltype III or IV. However, this test has been found by usto give variable results even with some strains of S.faecalis var. liquefaciens (Table 4). Shattock andMattick (1943) also noted that, though all strains of S.faecalis and other group D streptococci grew in 6.5 %sodium chloride broth, in some cases growth was verypoor. These authors have also commented on the unre-liability of the pH 9.6 test as a differential criterion. Weare, therefore, inclined to put all those strains similar toatypical types III and IV of Cooper and Ramadan intothe S. bovis group. Also the atypical type V reported bythese workers seems to be identical with the biotype zof S. durans (Tables 4 and 8), since the type culture ofS. durans and 22 isolates in this group grew in pH 9.6

broth under our conditions. By the same token, 45strains reported as unclassified type I by Barnes (1956a)could also belong to the S. durans group.The striking resistance of all enterococci to dihydro-

streptomycin and polymyxin B is quite interesting.This property may possibly be explored further formaking a new selective broth or agar medium for thesegroup D organisms in the same way as bacitracin hasbeen used for group A streptococci (Maxted, 1953;Levinson and Frank, 1955).

LITERATURE CITED

ABD-EL-MALEK, Y., AND T. GIBSON. 1948. Studies in thebacteriology of milk. I. The streptococci of milk. J.Dairy Research 15:233-248.

BARNES, E. M. 1956a. Methods for the isolation of faecalstreptococci (Lancefield group D) from bacon factories.J. Appl. Bacteriol. 19:193-203.

BARNES, E. M. 1956b. Tetrazolium reduction as a means ofdifferentiating Streptococcus faecalis from Streptococcusfaecium. J. Gen. Microbiol. 14:57-68.

BARNES, E. M., M. INGRAM, AND G. C. INGRAM. 1956. Thedistribution and significance of different species of faecalstreptococci in bacon factories. J. Appl. Bacteriol. 19:204-211.

BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH. 1957.Bergey's manual of determinative bacteriology. 7th ed.The Williams & Wilkins Co., Baltimore. 1094 p.

BUTTIAUX, R. 1958. Les Streptocoques f6caux des intestinehumains et animaux. Ann. inst. Pasteur. 94:778-782.

BUTTIAUX, R. 1959. The value of the association of Es-cherichieae-group D streptococci in the diagnosis of con-tamination in foods. J. Appl. Bacteriol. 22:153-158.

CHESBRO, W. R., AND J. B. EVANS. 1959. Factors affectingthe growth of enterococci in highly alkaline media. J.Bacteriol. 78:858-862.

COOPER, K. E., AND F. M. RAMADAN. 1955. Studies in thedifferentiation between human and animal pollution bymeans of faecal streptococci. J. Gen. Microbiol. 12:180-190.

FERRARO, F. M., AND M. D. APPLEMAN. 1957. Microbiologyof frozen orange concentrate. IV. Further studies ofenterococci in frozen orange concentrate. AppI. Micro-biol. 5:300-303.

FROST, W. D., AND M. A. ENGELBRECHT. 1940. The strepto-cocci. Their descriptions, classification and distribution,with special reference to those in milk. Willdof Book Co.,Madison, Wisc. 172 p.

GYLLENBERG, H., S. NIEMALX, AND T. SORMUNEN. 1960.Survival of bifid bacteria in water as compared with thatof coliform bacteria and enterococci. Appl. Microbiol.8:20-22.

HARTMAN, P. A. 1960. Enterococcus-coliform ratios infrozen chicken pies. Appl. Microbiol. 8:114-116.

KERELUK, K. 1959. Studies on the bacteriological quality offrozen meat pies. III. Identification of enterococci isolatedfrom frozen meat pies. Appl. Microbiol. 7:324-326.

KERELUK, K., AND M. F. GUNDERSON. 1959a. Studies on thebacteriological quality of frozen meat pies. I. Bacteriolog-ical survey of some commercially frozen meat pies. Appl.Microbiol. 7:320-323.

KERELUK, K., AND M. F. GUNDERSON. 1959b. Studies on thebacteriological quality of frozen meat pies. IV. Longevitystudies on the coliform bacteria and enterococci at lowtemperature. Appl. Microbiol. 7:327-328.

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1MEDIA FOR ENUMERATING ENTEROCOCCI IN FROZEN VEGETABLES

LAKE, D. E., R. H. DIEBEL, AND C. F. NIVEN. 1957. Theidentity of Streptococcus faecium. Bacteriol. Proc.1957:13.

LARKIN, E. P., W. LITSKY, AND J. E. FULLER. 1955. Fecalstreptococci in frozen foods. II. Effect of freezing storageon Escherichia coli and some fecal streptococci inoculatedinto green beans. III. Effect of freezing storage onEscherichia coli, Streptococcus faecalis and Streptococcusliquefaciens inoculated into orange concentrate. Appl.Microbiol. 3:102-106.

LARKIN, E. P., W. LITSKY, AND J. E. FULLER. 1956. Inci-dence of fecal streptococci and coliform bacteria in frozenfish products. Am. J. Public Health 4:464-468.

LEVINSON, M. L., AND P. F. FRANK. 1954. Differentiation ofgroup A from other ,-hemolytic streptococci with baci-tracin. J. Bacteriol. 69:284-287.

LITSKY, W., W. L. MALLMAN, AND C. W. FIFIELD. 1953. Anew medium for the detection of enterococci in water.Am. J. Public Health 43:873-879.

MAXTED, W. R. 1953. The use of bacitracin for identifyinggroup A haemolytic streptococci. J. Clin. Pathol. 6:224-226.

RAJ, H., AND J. LISTON. 1961. Detection and enumerationof fecal indicator organisms in frozen sea foods. I. Es-cherichia coli. Appl. Microbiol. 9:171-174.

RANTz, L. A., AND E. RANDALL. 1955. Use of autoclaved ex-

tracts of hemolytic streptococci for serological grouping.Stanford Med. Bull. 13:290-291.

SHATTOCK, P. M. F. 1955. The identification and classifica-tion of Streptococcus faecalis and some associated strepto-cocci. Ann. inst. Pasteur (Lille), 7:95-100.

SHATTOCK, P. M. F., AND A. T. R. MATTICK. 1943. The sero-logical grouping of Streptococcus lactis (group N) and itsrelationship to Streptococcus faecalis. J. Hyg. 43:173-188.

SHATTOCK, P. M. F., AND A. HIRSCH. 1947. A liquid mediumbuffered at pH 9.6 for the differentiation of Streptococcusfaecalis from S. lactis. J. Pathol. Bacteriol. 59:495-497.

SHERMAN, J. M. 1937. The streptococci. Bacteriol Rev.1:1-97.

WEINBURG, E. D. 1953. Selective inhibition of microbialgrowth by the incorporation of triphenyl tetrazoliumchloride in culture media. J. Bacteriol. 66:240-242.

WILSON, G. S., AND A. A. MILES. 1955. Topley and Wilson'sprinciples of bacteriology and immunity. 4th ed. vol. 1.The Williams & Wilkins Co., Baltimore. 1106 p.

YOUNG, V. M., H. C. GILLEM, E. D. MASSEY, ANDW. C. BRANCHE, JR. 1960. Observations on Escherichiacoli as a component of the normal intestinal flora: Aberrantfindings in certain individuals. Bacteriol. Proc. 1960:134.

ZABOROWSKI, H., D. A. HUBER, AND M. M. RAYMAN. 1958.Evaluation of microbiological methods for the examinationof precooked frozen foods. Appl. Microbiol. 6:97-104.

Studies on Media for Enumerating Enterococci in Frozen Vegetables'D. F. SPLITTSTOESSER, R. WRIGHT, AND G. J. HUCKER

New York State Agricultural Experiment Station, Cornell University, Geneva, New York

Received for publication October 10, 1960

ABSTRACT

SPLITTSTOESSER, D. F. (Cornell University, Geneva,N. Y.), R. WRIGHT, AND G. J. HUCKER. Studies on

media for enumerating enterococci in frozen vegetables.Appl. Microbiol. 9:303-308. 1961.-A study was madeof the relative sensitivity and specificity of presumptiveand confirmatory media for the most probable numberenumeration of enterococci in frozen vegetables. Azidedextrose broth yielded the highest numbers of con-

firmable enterococci and its sensitivity was shown tobe comparable to nonselective media. The use of ethylviolet azide broth as a confirmatory medium resulted ina significant number of false positive tests. Growth inbroth containing 6.5% sodium chloride incubated at45 C for 2 days was found to be a more specific con-

firmatory test for enterococci.

There is a divergence of opinion with regard to thesignificance of the presence of enterococci in frozen

I Approved by the Director of the New York State Agricul-tutral Experiment Station for publication as Journal paperno. 1237, October 4, 1960.

foods. To clarify the question of their sanitary implica-tions there is need for further information on the ac-curacy of the various methods used for theirenumeration.A number of procedures have been used for deter-

mining the most probable number (MPN) of enterococciin frozen foods. In general, the methods are based onthe same principle as those used to demonstrate thepresence of the coliform types, i.e., growth in a pre-sumptive medium followed by confirmatory tests. Forthe presumptive tests most workers have used a mediumcontaining sodium azide as the selective agent. Con-firmation procedures have been based either on theso-called "Sherman tests" (Sherman, 1937) or growthin ethyl violet azide (EVA) broth (Litsky, Mallmann,and Fifield, 1955).The suitability of the various media for determining

the MPN of enterococci in precooked frozen foods hasbeen evaluated by Zaborowski, Huber, and Rayman(1958), and Fanelli and Ayres (1959). Zaborowski et al.(1958) found that azide dextrose (AD) broth as a pre-sumptive medium yielded the highest most probablenumber of counts. Also they reported 100 % confirma-

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