Zbl. Bakt. H yg. A 260, 57-64 (19 85)

Chemically Defined Media for Auxotyping of CampylobacterJeJum

NORBERT DI CK GIE SSER and DIETRI CH C ZY LW I K

Institut fur Hygiene und Medizinische Mikrobiologie der Fakul rat fiir Klinische MedizinMannheim der Universirar Heidelberg (Direktor: Prof. Dr. med. W. Wundt)

Received September 5, 1984 · Accepted December 20 , 1984

Abstract

A set of chemically defined media has been developed for the cult ivat ion of Campylobac­ter jejuni strains of human origin. A minimal medium, a comple te medium and 5 differentnutrient-deficient media (NDM I-NDM5) are described. Some of the strains investigatedrequired L-methionine (lacking in NDM1 ), L-cystine and L-cysteine (NDM2), K1HP04

(NDM 3), KH1P04 (NDM4) and NAD, thiamine and calcium pantothenate (NDM5).57.7 % of the strains invest igated requi red L-methionine. Th e strains grew at pH 6.6-7.7.The media described are not suita ble for C. intestinalis.

Zusammenfassung

Es werden chemisch defin ierte Nahrboden fur die Anzuchr von Campylobacter ieiun i­Stammen menschl icher Herkunft beschrieben. Diese N ahrboden bestehen aus einemMinirn alnahrboden, einem Voll- und 5 Mangelnahrboden (N DM I- NDM5). Die C. [etun iwerden danach beurreilt, o b sie L-Methionin (fehlr in NOM 1) benorigen, L-Cysrin und L­Cystein (NDM 2), K1 HP04 (NOM 3) KH1P04 (NDM 4) und NAO , Thi amin und Calcium ­pant orhenar (NDM 5). 57.7 % unserer Stamme benot igtcn L-Merh ionin . Die C. jejuniwachsen bei pH 6.6-7.7. Der Nahrboden eignet sich nichr fur die Auxotypisierung von C.intestinalis.

Introduction

Auxotyping is a method used to exa mine the nutritional requirements of bacteria inchemically defined media. Using this method it is possible to subdivide bacteria of th esa me species even if it is no t possible to differentiate between th ese strains by usin gmethods currently av ai la ble. The results ar e of epidemiologi cal interest if the auxotypesare determined to be chro mos o mally encoded, i.e. they a re suita ble, as wa s shown byCarifo (1) in N . gonorrhoeae,

At pre sent there ar e few descriptions of chemically def ined medi a for c. fetus ssp .jejuni (= C. jejuni). Fletcher (3) described a medium for Vibrio fetus, Miyamae (4 , 5)

50 mg100 mg50 mg80 mg50 mg10 ml

58 N. Dickgiesser and D. Czylwik

Table 1. Preparation of stock solutions

Solution 1NaCl 58.0 gK2S04 10.0 gMgCl 2 X 6H 20 4.1 gNH4Cl 2.2 gEDTA 0.037 g

Dissolved in 500 ml distilled water. Add 10 N NaOH to give pH 7.3. Then add distilledwater to give the final required volume of 100a m!.

Solution 2L-arginine-HCl 150 mgGlycine 25 mgLserine 50 mgDistilled water ad 10 ml

Solution 3L-leucine 90 mgL-isoleucine 30 mgL-valine 60 mgDistilled water ad 10 ml

Solution 4aK2HP04 3.48 gKH2P04 2.72 gDistilled water 200 ml

Solution 4bK2HP04 3.48 gDistilled water 2000 ml

Solution 4cKH2P04 2.72 gDistilled water 200 ml

L-cysteine-HCl . H 20 175 mg

Dissolved in a small volume of 1N HC!. Then add distilled water to give a final volume of 10m!.

Lcysrine 120 mg

Dissolved in a small volume of IN HC!. Then add distilled water to give a final volume of 10m!.

Solution 5L-prolineL-alanineL-Iysine-HCIL-tryptophaneL-threonineDistilled water ad

Solution 6L-phenylalanineL-asparagine . H20

Distilled water ad

50 mg50 mg10 ml

Chemically Defined Media for Auxotyping of Campylobacter jejuni 59

L-methionine

Dissolve in 2 ml distilled water.

29.8 mg

Spermine tetrahydrochloride: 87 mg. Dissolve in 5 ml distilled water.

NaHC0 1: 84 mg. Dissolve in 1 ml distilled water.

Sodium acetate: 3.4 g. Dissolve in 10 ml distilled water.

Solution 7Nicotinamide adenine dinucleot ide (NAD)Thiamine-HClCalcium pantothenateDistilled water

Solution 8Choline chlorideMyo-inositolDistilled water

I mgI mg1 mg1 ml

7 mg1.8 mgI ml

Biotin: saturated solution in 1 ml 50% (vol/vol) ethanol.

Cocarboxylase: 4.6 mg. Dissolve in 1 ml distilled water .

Agar: see table 2.

CaCll . 2H10 : 74 mg. Dissolve in 2 ml distilled water.

Fe(N0 1h . 9H10 : 8 mg. Dissolve in 2 ml distilled water.

for C. fetus ssp. intestinalis and ssp. venerealis. Smibert (6, 7) described a medium forVibrio fetus. In all these cases the strains used were isolated not onl y from clinicalspecimens but also from feces of cows, birds, pigs, sheep and chickens. The stra insfrom these an imals may have biochemical properties different from strains of humanorigin. Furthermore, it is not possible in each paper to recon struct the sub species ofVibrio fetus being examined. Th erefore it was desirable to develop a chemically defin edmedium for differentiat ing between C. jejuni strains of human origin . An observationimportant for the purposes of our study was that C. jejuni grew on a medium describedby Catlin (2) for N. gonorrboeae, the so-called NEDA medium. We developed a" minimal medium" (non e of th e strains grew on this medium), a complete medium (onwh ich all of our C. jejuni st rains grew within 2 days) and nutrient-deficient media. Thenutrient-deficient media were produced from the minimal medium to which chemicallydefined substances were added. Furthermore, we determined the optimum pH forgrowth.

Materials and Methods

Bacterial strains

52 Campylobaeter jejuni strains were isolated from human feces using Butzler's selectivemedium (Oxoid), The strains grew within 48 h at 3rc and 42 °C, but not at 25 °C in a

60 N.Dickgiesser and D. Czylwik

5 % O 2, 10 % COb 85 % N2 atm osphere. The y were ox idase-positive, catalase-positive,H 2S-positive and sensitive to nalid ixic acid. The y were of campylobacter-like morphologymicros cop ically. The strains were subcultivated on Srandard-l-Nutrient Agar (Merck) towhich hemoglobin (10 g/500 ml) (BBL) and Isovitalex (BBL) had been added.

Stock solutions

Chemicals of the highest grade obtainable were used . The following chemicals werepurchased from Sigma: NACl, EDTA, L-argin ine HCl, glycine, L-serine , L-leucine, L­isoleucine, L valine , L-cysteine -H CL, Lcystine, L-proline, L-alanin e, L-lysine-HCl , L-tryp-

Table 2. Final concentration of chemicals in comp lete medium

NaCIK1S0 4

MgCl2 • 6H 20NH 4Cl

EDTAL-arginine-HClglycineL-serineL leucineL-isoleucineL valineKzHP0 4

KH 2P0 4

I.-cysteine' HCI . H20L-cystineL-prolineL-alanineL-IysineL-tryptoph anL-threonineL-phenylalanineL-asparagine . H20L-meth ionineSpermine tetr ahydrochlorideNaHC 0 1

Sodium aceta teNicotinamide adenine dinucleotideThiamine-HCICalcium pan tothenateCholine chlorideMyo-inositolBiot inCocarboxylaseAgarCaC12 · 2H10

Fe(NO'lh . 9H20

gil; molecular weight uncerta in.

mgl100 ml

572 .7998 .7540.4821. 72

0.3614.792.474.938.882.965.92

343.33268.35

6.043.5 54.939.874.937.894.932.472.471.478.584.14

335.430.200.2 00.200. 140.0360.0730.045

986 .583.650.39

mAlol

98.755 .661.994.0 60.00960.700.320.460 .680.220.50

19.7119.7 J0.340.150.421.100.260380.4 10.150. 160. 10.250.50

24 .640.002 90.00590.0040.0 100.002O.OOy·0.00 1

0.250.0 1

Chemically Defined Media for Auxotyping of Campylobacter jejuni 61

tophane, L-threonine, L-phenylalanine, L-asparagine, L-methionine, spermine tetrahy­drochloride, sodium acetate, nicotinamide adenine dinucleotide (NAD), thiamine-HCl, cal­cium pantothenate, choline chloride, myo-inositol, biotin, cocarboxylase. The other chemi­cals shown in Table 1 were purchased from Merck.

Stock solutions were prepared as described in Table 1. Solutions 1, 4a, 4b and 4c wereautoclaved and stored at room temperature. Solutions 2, 3, 5, 6, 7, 8, methionine, sperminetetrahydrochloride, NaHC03, sodium acetate, cocarboxylase, CaCl2 and Fe(N03h weresterilized by filtration and stored at -20°C. Autoclaving and filter sterilization were per­formed mainly to improve the stability of the stock solutions. If all the solutions were usedup quickly this is not required as another filter sterilization step is employed before the agaris added. L-cysteine and L-cystine were dissolved in a small volume of 1 N HCl, thensterilized distilled water was added to give the required final volume. L-cystine, L-cysteineand biotin were used the day they were prepared. The agar (Merck) was washed and spundown 3 times in 500 ml distilled water, then 2 times in 95 % ethanol and was dried at 37°Cfor several days. The agar was used at 2 %. In Table 2, concentrations of the substances incomplete medium are shown. The concentrations were calculated by using the amount ofstock solutions being added to the complete medium (see Table 3). The concentrations ofrequired substances are the same in both the minimal medium and the nutrient-deficientmedia.

Table 3. Composition of complete medium, minimal medium and nutrient deficient media

MM C NDMl NDM2 NDM3 NDM4 NDM5

Solution I 5.005 5.005 5.005 5.005 5.005 5.005 5.005Solution 2 0.5 0.5 0.5 0.5 0.5 0.5 0.5Solution 3 0.5 0.5 0.5 0.5 0.5 0.5 0.5Solution 4a 10.0 10.0 10.0 10.0Solution 4b 10.0Solution 4c 10.0Lcysteine 0.175 0.175L-cystine 0.15 0.15pH 1':-Solution 5 0.5 0.5 0.5 0.5 0.5 0.5 0.5Solution 6 0.25 0.25 0.25 0.25 0.25 0.25 0.25L-methionine 0.05Spermine tetra-HCl 0.25 0.25 0.25 0.25 0.25 0.25 0.25NaHCO j 0.025 0.D25 0.025 0.025 0.025 0.025 0.025Sodium acetate 0.5 0.5 0.5 0.5 0.5 0.5 0.5Solution 7 0.1 0.1 0.1 0.1 0.1Solution 8 0.01 0.01 0.01 0.01 0.01 0.01 0.01Biotin 0.05 0.05 0.05 0.05 0.05 0.05 0.05Cocarboxvlase 0.005 0.005 0.005 0.005 0.005 0.005 0.005Distilled ~ater 17.985 7.51 7.56 7.885 7.885 7.885 7.895pH 2':-Agar 25 25 25 25 25 25 25CaCl2 0.05 0.05 0.05 0.05 0.05 0.05 0.05Fe(NOJ)J 0.05 0.05 0.05 0.05 0.05 0.05 0.05

C = complete medium; MM = minimal medium; NDM = nutrient deficient medium. Allcompounds on the table are listed in m!' pH 1": add 10 N NaOH to give pH of 7.0. pH 2"":adjust medium to pH 7.45 +/- 0.05.

62 N.Oickgiesser and O. Czylwik

Preparation of minimal medium, complete medium and nutrient deficient media

The stock solutions were mixed in the order and volumes listed in Table 3 to give thebasal medium, complete medium and the 5 nutrient-deficient media (NOM I-NOM 5).Volumes were calculated to yield 2 Petri dishes of 17 em diameter, 50.68 ml altogether. Thesmall volume is proposed as the media should be prepared freshly and preparation in largeramounts is recommended only if the plates will not be stored very long. In Table 2, theconcentrations of the different substances in complete medium are listed.

At pH 1 the medium was neutralized to pH 7.0 and at pH 2, pH was adjusted finally topH 7.45 ± 0.05 (see Table 3). Then the medium was sterilized by filtration and the auto­cIaved agar (50-60°C) was added. CaClz and Fe(N03h had to be added quickly as themedium would solidify very fast. The plates were dried overnight at room temperaturebefore use for auxotyping.

Performance of auxotyping

Freshly grown C. jejuni were suspended in sterile 0.9 % NaCl to give a visible turbidity.Using the same loop, the minimal, complete and nutrient-deficient media were inoculated.These cultures were incubated at 37°C in a 5 % 0z, 10 % COz, 85 % Nz atmosphere for 48h. Then bacterial growth was scored.

Determination of the optimal pH value to grow the bacteria

The complete medium was prepared as described above (Table 3). At pH 2 the pH wasadjusted to give: pH 6.4; 6.6; 6.8; 7.0; 7.2; 7.4; 7.6; 7.7. The media were inoculated andincubated as described above. The amount of growth was examined after 48 h using 7 C.[eiuni strains.

Results

The growth of 52 C. jejuni strains on the chemically defined media is shown in Table4. There was no growth on the minimal medium but all the strains grew on thecomplete medium. The nutrients of importance for differentiation between the strainsare L-methionine, L-cysteine, L-cystine, KzHP04, KH zP04, thiaminehydrochloride,NAD and calcium panthorhenate. 22 (= 42.3 %) of the strains grew on NDMl andhave been classified as methionine-positive strains. 8 of these 22 strains, i.e. 15.4 % ofall our strains, grew on NDM 2; these strains were totally independent of all the

Table 4. Growth of Campylobacter [ejuni on chemically defined media

Medium No . of strains grown percentage of strains grown

Minimal medium 0Complete medium 52NOMI 22NOM2 8NOM3 7NOM4 4NOMS 3

NOM = nutrient-deficient medium

o10042.315.413.5

7.75.8

Chemically Defined Media for Auxotyping of Campylobacter jejuni 63

sulphur-containing amino acids methionine , cystine and cysteine. These strains can befurther subdivided according to their needs of KHzP04, K1HP04 and NAD, thiamine­HCl and calcium pantothenate. 7 of these 8 strains, i.e. 13.5 % of all the strains tested,grew on NDM 3, indicating that K1HP04 for growth was needed in only one of thesestrains. 4 of the 8 C. jejuni growing on NDM 2 grew on NDM 4 (= 7.7 % of all thestrains) showing that in 4 of these strains KH1P04 for growth is essential. 3 of these 8strains grew on NDM 5. These 3 strains did not require any of the chemicals insolution 7 (NAD, thiamine-HCl, calcium pantothenate) or for L-cystine, L-cysteine ormethionine.

By using the media described we were able to classify our 52 strains in 6 groups .

Growth on complete medium with different pH value

All the stra ins grew at pH 6.6 up to pH 7.7. Growth was reduced at pH 6.4.

Discussion

Smibert (6) and Miyamae (5) have shown methionine to be essential for a smallnumber of Campylobacter strains . In the strains tested, it was of great importance as57.7 % of the C. jejuni strains needed this amino acid to grow. The significance of thetwo other sulphur-containing amino acids, cystine and cysteine has been described bySmibert (6, 7), Fletcher (3) and Miyamae (5). Smibert used cystine as a criterion fordifferentiation among his strains. Fletcher did the same with cysteine, Miyamae de­scribed cystine to be of importance. We cannot confirm this for all of our strains as15.4 % grew without L-cystine and L-cysteine. In further studies we tested whetherstrains could be subdivided in further groups if L-cystine or L-cysteine alone werewithdrawn. This was not the case. This is why both amino acids were usually added oreliminated together.

Fletcher (3) differentiated between Campylobacter strains using K1HP04 andKH1P04. We can confirm his results. We could find neither glutamic acid nor asparticacid (5, 6) nor L-tyrosine (6) to play an essential role or to have a growth-promotinginfluence. This is why these amino acids were not added to our media. We cannotrecommend adding glucose as its influence was more growth-retarding than growth­promoting. We do believe that differences between our results and the ones of the otherauthors are easily explained as these authors examined Campylobacter strains fromanimals as well as humans . Furthermore they checked strains other than C. jejuni .

Smibert (7) added thiamine hydrochloride, nicotinamide adenine dinucleotide andcalcium pantothenate to his minimal medium to grow C. fetus. He did not indicatewhether any of his strains were able to grow without these substances. We also testedmedia lacking thiamine hydrochloride, nicotinamide adenine dinucleotide, and calciumpantothenate. However, based on these tests it was not possible to further subdivideour strains. We found no strains where the amino acids, L-aspartic acid, L-glutamicacid, Lryrosine and L-glutamine were essential.

Within the 6.6-7.7 range, the pH value was not as important as expected. Sm ibert(6) recommended pH 6.8-7.2. Fletcher (3) pH 8.0, Miyamae (4) pH 7.2 or 6.4. On ourmedia with pH 6.4, the strains grew poorl y.

The medium described is suitable to auxotype C. jejuni of human origin. We alsowere interested in evaluation of the properties of C. fetus ssp. intestinalis (= C. intes-

64 N. Dickgiesser and D. Czylwik

tinalis). However, C. intestinalis is not able to grow on our medium. According torecommendations by other authors (2, 3,4,5,6,7), we added to our complete mediumfurther substances to give two media, called medium I and medium II. These mediawere composed as follows (in parenthesis: concentrations of the chemicals in themedia; concentrations in mMol if not marked otherwise); Medium I: completemedium (see Tables 1 and 2) + L-tyrosine (0.38) + uracil (0.07) + hypoxanthine(0.022) + oxalacetic acid (1.50) + L-aspartic acid (3.70) + L-glutamic acid (8.72) + L­glutamine (0.33) + L-histidine (0.11) + glutathione (0.15) + glucose (27.38). MediumII: Medium I + nicotinic acid (8.12) + taurocholic acid (0.35) + ribose (3.99) +pimelic acid (3.74) + glycogen (0.6 gIl) + thioglycolic acid (5.42) + adenine (0.15) +cytosine (0.18) + guanine (0.13) + thymine (0.16) + (NH 4hHP04 (8.68) + sodiumlactate (1.3) + glycerin (10.0) + polyvinyl alcohol (0.005 gIl) + Tween 80 (0.025 gIl)+ hemin (0.003) + L-histidine (0.013) + 2,2',2" nitrilotriethanol (0.54).

The 2 Campylobacter intestinalis strains tested grew neither on medium I nor onmedium II.

Acknowledgement. I thank Dr. S. Projan, Department of Plasmid Biology, Public HealthResearch Institute of the City of New York, New York, USAfor assisting in the translationof the manuscript.

References

1. Carifo, K. and B. W. Cat/in: Neisseria gonorrhoeae auxotyping: differentiation of clinicalisolates based on growth responses on chemically defined media. Appl. Microbiol. 26(1973) 223-230

2. Cat/in, B. W.: Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis, andNeisseria /actamiea in chemically defined media and the use of growth requirements forgonococcal typing. J. infect. Dis. 128 (1973) 178-194

3. F/eteher, R. D. and W. N. P/astridge: Chemically defined medium for some micro­aerophilic vibrios. J. Bact. 85 (1963) 992-995

4. Miyamae, T.: Studies on the growth conditions of Campy/obaeter fetus. I. Some physicalfactors affecting growth in static cultivation. Jap. J. Microbial. 18 (1974) 57-64

5. Miyamae, T.: Studies on the growth conditions of Campy/obaeter fetus. II. The manifes­tation of nutritional requirements by conditional static cultivation. Jap. J. Microbial. 18(1974) 65-78

6. Smibert, R. M.: Nutrition of Vibrio fetus. J. Bact. 85 (1963) 394--3987. Smibert, R. M.: The genus Campy/obaeter. Ann. Rev. Microbial. 32 (1978) 673-709

Prof. Dr. med. N. Diekgiesser, Institut fur Hygiene und Med. Mikrobiologie, Klinikumder Stadt Mannheim, Postf. 23, D-6800 Mannheim

Present address: The Public Health Research Institute of the City of New York, Depart­ment of Plasmid Biology, 455 First Avenue, New York, NY 10016, USA