Growth Medium Affects the Cellular Fatty Acid Composition of Pasteurellaceae

Zent.bl. Bakteriol. 289, 9-17 (1999) © Urban & Fischer Verlag

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Growth Medium Affects the Cellular Fatty Acid Composition of Pasteurellaceae Ron Boot', Harrie C. W. Thuis', and Fra ns A. G. Reubsaet 2 I

2

Section of Laboratory Animal Microbiology and Section of Bacterial Determinations, Diagnostic Laboratory for Infectious Diseases and Perinata l Screening, Nationa l Institute of Public Health and the Envi ronment, PO Box 1,3720 BA Bilthoven, the Netherlands

Received July 25,1998· Revision received October 19, 1998· Accepted October 28, 1998

Summary We studied the cellular fatty acid composition of 10 Actinobacillus (A.) and Pasteurei·

ta (P.) reference strains grown on 2 types of agar by the MIDI Microb ial Identification System (MIS). A. capsulatus, A. equu/i, A.lig"ieresii, A. ureae, A. dagmatis, P. gallinar· um, P. haemolytica, P. multocida, P. pneum otropica biotypes Heyl and Jawetz were grown on GC agar supplemented with ascitic fluid and X and V factor (Levinthal's agar = LA agar) or GC agar supplemented with virox and hemoglobin (VH agar) on 3 to 7 and 7 [0 16 occasions respectively and fatry acid merhylcsrcr (FAME) profiles were submitted to principal component analysis (PCA ).

All Pasteurellaceae srrain s showed FAME profiles typical for the family. Maximum coefficients of variation of the percentage of the 3 major FAMEs 14 :0,16: 0, and 16: 'I cis were 0.03, 0.03 and 0.03 for fasteurellaceae strains grown on VH agar and 0.09, 0.17 aod 0.09 respectively fot strains grown on LA agar. PCA of FAl'v1E profiles ob· rained with growth from LA agar generally did nOt allow species separation of the Pas· teurelJaceae but most species were clearly discriminated by PCA when they were grown on VH agar. Our findings indicate that the growth medium had a significant effect on the reproducibility of fatty acid profiling in Pasteurellaeeae and that PCA of fatty acid data obtained under sra ndardized growrh conditions may discriminate Pasteurellaceae specIes.

0934·8840/99/28911-009

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10

R. Boot, H. C. W. Thuis, andA. G. Reubsaet

Introduction Pasteurellaeeae are important pathogens for many animal species including man (1, 10). The Pasteurellaeeae family has been established to accomodate species within the genera Actinobacillus, Haemophilus and Pasteurella (17 ). Various phenotypic and genotypic methods have been used to get more insight into the structure of the Pasteurellaeeae family (2 ) but the taxonomic position of various isolates rhat are considered to belong to the family is not yet clear. Cell wall lipid profiling of Pasteurellaeeae (4,12,13,21) led to the view that this chemotaxonomic method might be suitable to classify an isolate within the family but not suitable to separate members at the species level. Studies with other bacterial families however have shown that grouping based on this type of quantitative data matches very well the classification based on DNA hybridization studies and that separation of bacteria at the (sub)species level was possible after meticulous standardization (22). A study by Brandz et al. (5) on a small number of Pasteurellaeeae suggested tbat species separation might be further improved by multivariate analysis of quantitative data of the cell wall lipid composition and other phenotypic properties. We therefore determined the cell wall lipids of some Pasteurellaeeae reference strains grown on two types of agar and submitted our data to principal component analysis (PCA).

Materials and Methods Bacterial isolates, media and growth conditions All isolates studied are listed in Table 1. Part of the isolates were kindly supplied by Dr. R. Mutters, Zentrum fiir Hygiene und Medizinische Mikrobiologie, Klinikum der Philipps Universitat, Marburg, Germany (no's 1,2,3,9, & 10) and Dr. W. Frederik sen, Statens Serum Institut, Copenhagen, Denmark (no's 4 & 5). Isolate no 6 was obtained from the American Collection of Type Cultures (ATCC) and no's 7 and 8 were

from the National Collection of Type Cultures (NCTC). All isolates were phenotyp-

ically studied according to Mannheim et al. 1980 (14) and stored at -70 °C in Brain Heart Infusion (BHI) broth supplemented with 10 % (w/v) of glycerol until use.

Media used were a) Levinthal's agar (LA agar) consisting of GC agar base (Dileo ) supplemented with x factor (lysed horse blood) and v factor (yeast extract) and b) GC agar supplemented with vitox (Oxoid) and hemoglobin (Dileo) (VH agar). Pasteurellaceae strains were 3-7x grown on LA agar (totalling 46 cultures ) and 7-16x on VH agar (totalling 110 cultures). All plates were incubated under 5-10% CO 2 for 18 ± 1.0 hrs at 37 ± 0.5 °C, and Pasteurellaceae were subsequently analyzed for their fany acids. As we collected our data over an extended period of time we used various

lots of both types of agar. Fatty acid analysis Whole-cell fatty acids were extracted and analyzed as fatry acid methylesters (FAMEs) as previously described (1S ). Recognition of fatty acid profiles was performed with a Microbial Identification System (MIS, MicrobiallD, Newark, DE, USA) consisting of

Medium effect on FAME composition of Pasteurellaceae

II

Table 1. Fatty acid composition of 10 PasteurelJaceae strains cultured on 2 types of agar

Bacterial species

LA agar n= 14:0

16: 1

VH agar 16 :0

cis9

n= 14:0

16: I cis 9

16:0

x* sd cv

4

20.50 .>5.91 27.66 0.49 2.26 0.48 0.02 0.06 0.02

12

13.92 37.80 31.33 0.4 0.93 0.58 0.03 0.02 0.02

x

7

23.34 38.25 21.62 2.18 1.21 3.67 0.17 0.09 0.Q3

7

16.02 39.83 28.93 0.23 0.67 0.60 0.01 0.02 0.02

x sd cv

6

28,75 30,71 25,11 2.56 1.53 0.79 o,m 0,09 0,05

12

A. ureae

x

4

sd cv

25.52 32,94 26,11 1.42 1.16 0,71 0,05 0,02 0,05

12

NCTc;T

17.42 37.33 32.64 0.44 1.24 1.06 om 0,03 0.03

5

22.92 31.18 26.42 0,89 0,63 0,70 0,03 0.04 0,02

15

17,57 34,65 33,99 0,56 0.31 0.55 0.02 0,02 0,02

6

20.46 33.68 32.40 3,21 1.76 1.01 0,09 0,03 0.10

10

16,87 30.42 36,94 0.37 0,61 0,57 0,02 0,02 0,02

3

17.57 32.59 29.77 0.45 2.23 1.08 0,03 0.07 0,04

7

11.04 37.37 30,74 0,17 0.34 0.38 0,02 0.01 0.01

4

18.47 33,93 30.44 0,85 2,96 0.46 0,05 0,09 0,02

16

12.33 36.13 35,25 0,34 0,91 0.40 0.03 0.03 0.01

3

30,29 26.77 30.80 1.17 0,83 0,90 0,04 0,03 0,03

8

19,74 30,50 36,66 0.26 0.35 0,79 0,01 0,01 0,02

4

30,06 26.03 30,96 0,93 0,81 1.36 0,04 0.03 0.05

11

19.68 31.12 36.33 0,63 0.60 0,90 0,03 0,02 0,02

A. capsulatus NCTC 11408 T

A. equuli NCTC 8529" A. lignieresii

NCTC 4189T

P. dagrnatis NCTC" P. gal/ina rum

ATCC 13361 T P. haemolylica

NCTC 9380T P. multocida

NCTC 10223 T

sd cv

x

sd cv x

sd cv x

sd cv x

sd cv

P. pneumotropica

x

ATCC 12555

sd cv

P. pneumotropica NCTC 8141 T

x

sd cv

16,62 37,11 0,3 0.42 0,02 0,01

32.29 0,23

om

x: mean; sd: standard deviation of meanj cv: variation coefficient calculated as (sdlx) :(- 100; n: number of cultures tested. T type strain.

12

R. Boot, H. C. W. Thuis, and A. G. Reubsaet

a Hewlett-Packard (HP) 5890 gas chromatograph equiped with a 5 % phenylmethyl silicone capillary column (0.2 mm by 25 m, Chrompack CP SIL 5), a flame ionization detector, a model HP 3392 A integrator, a model HP 7673 A automatic sampler, and a model Vectra VL2 4151 computer. System-controlled operating parameters were as follows: injector temperature 250°C, detector temperature 300 °C oven temperature programmed to cqulibrate at 170°C for 3 min at the beginning of the cycle and then

raised from 170'C

to

270'C at 5'Omin and from 270"C to 310'C ar 30'Omin.

Peaks were automatically integrated and fatty acids were identified on basis of equiv-

aleor chain length and percentages were calculated (19, 20). The reproducibility of the

gas chromatographic technique determined by repeated analysis of th e standard quantitative FAME mi xture (MicrobiallD Inc., Newark, Del. ), The coefficient of variation was calculated for each peak in the chromatogram.

Analysis of fatty acid data Differences in the amounts of major FAMEs of Pasteurellaceae obtained on the two agar types wert: ana lysed by the paired t-test using the SYSTAT statistical package run on a personal computer. The level of significance was presented at p < 0.01. The quantitative data obtained from the FAME profiles were also used as the basis for numerica l analysis. Peak area va lues for each FAME were calculated as percentage of the tota l peak area to eliminate the effect of inocu lum size variation. The data set was submitted to principal component analysis (peA) using the program provided in the HP Library Generation Softwa re (MicrobiaI 1D, Newark, DE, USA.) and the results were ploued graphically in two dimensions.

Results Effect of growth medium on cellular fatty acids The 3 major FAMEs in all 10 Pasteurellaceae strains detected (Table 1) were unbranched tetradecanoic acid 14: 0 and the hexadecanoic acids 16: 0 and 16: 1 cis 9. All strains contained lower amounts of summed feature (sf) 3 which included 3-hydroxytetradecanoic acid 14: 0 3-0H and 16: 1 iso I (isoI-hexadecenoic acid). Various other FAMEs were detected in trace amounts in (nearly) all or some of the Pasteurellaceae, namely dodecanoic acid 12: 0, 3hydroxydodecanoic acidl2: 0 3-0H, an unknown acid at retention time 14.5, pentadecanoic acid 15: 0, octadecanoic acid 18: 0, cis-9-octadecenoic acid 18:1 cis 9, eicosanoic acid 20:0, cis-20-trans-ll-eicosanoic acid 20: 1 tr 11, cis-5-eicosanoic acid 20:4 cis 5 and some other FAMEs included in sf 6 (cis9,12 octodecadienoic acid 18:2 cis 9, 12 ) and sf 7 (cis-11 octa decenoic acid 18: 1 cis 11 ). No systematic qualitative and quantitative differences were observed in the FAMEs between species belonging to the genus Pasteurella sensu stricto (P. dagmatis, P. gallinarum and P .multocida) and the remaining species that are all assigned to or related to the genus Actinobacillus. The mean percentage of 14: 0 of Pasteurellaceae grown on VH aga r was lower (range 11-20%) than when strains were grown on LA agar (range 18-30 %). Within the exeption of P. gallinarum the Pasteurellaceae strains o n VH agar contained more of 16 : 1cis 9 (range 30-40 %) than strains grown on LA agar

Medium effect on FAME composition of Pasteurellaceae

13

(range 26-38 %). Also the amounts of 16: 0 were higher for strains cultured on VH agar (range 29-37 %) than for strains cultured on LA agar (range 22-32 %). Growth on the two types of agar gave for these three FAMEs statis· tically significant differences (t-test, p < 0.01). The amounts of FAMEs making up sf 3 did not show systematic differences that could be related to the agar medium used and differences were not statistically significant (t-test, p < 0.1). Maximum coefficients of variation (sci/mean) of the percentages of the 3 major FAME's 14: 0, 16: 0 and 16: 1 cis were 0.03, 0.03 and 0.03 for Pasteurellaceae strains grown on VH agar and 0.09, 0.17 and 0.09 respectively for strains grown on LA agar. The variation observed in sf 3 was in most strains grown on VH agar lower than in strains grown on LA agar, the exceptions being P. dagmatis and P. pneumotropica ATCC 12555. Discrimination of Pasteurellaceae strains Principle component analysis (PCA) of the profiles of the Pasteurellaceae grown on LA agar did not gave a good separation of the strains (Fig. la, b). PCA of the fatty acid profiles of the same strains grown on VH agar (Fig. 1c, d ) generally showed distinction of the species: so e. g. P. haemolytica NCTC 9380T was clearly distinct from P. multocida NCTC 103231". The P. pneumotropica strains NCTC 8141 T (Jawerz biotype) and ATCC 12555 (Heyl biotype) were distinct from all other Pasteurellaceae strains but both P. pneumotropica strains were not distinct from each other on either type of agar. A. ureae NCTC 10219T , A./ignieresii NCTC 4189 T and P. dagmatis NCTC 11617T were distinct from other Pasteurellaceae strains, but A. ureae NCTC 10219T appeared to be an intermediate of both other strains. Examination of individual FAMEs of the 3 strains revealed that A. lignieresii NCTC 4289T grown on VH agar contained about twice the amount of 18: 0 (2.6 %) in comparison to both A.ureae NCTC 10219 T (1.3% ) and P.dagmatis NCTC 1161 7T (1.2 %) (data not shown).

Discussion Effect of growth medium on cellular fatty acids The FAME profiles obtained with the Pasteurellaceae in our study qualitatively agree wirh the outcome of previous studies in which tetradecanoic acid 14: 0, 3·hydroxytetradecanoic acid 14: 0 3·0H, and both hexadecanoic acids 16:0 and 16: 1 cis 9 were found typical for this bacterial family (12,13 ). The relative percentages of individual FAMEs found with a given Pasteurellaceae species (e.g. A. equuli) differed between previous studies (6, 8, 9, 12, 13,16,21 ). Such quantitative differences have been found with species from all the 3 genera (Actinobacillus, Haemophilus and Pasteurella) that have been incorporated into the Pasteurellaeeae. Quantitative differences may be explained by differences in culrure conditions and in the processing of bacterial

14

R. Boot, H. C. W. Thuis, and A. G. Reubsaet

growth. For instance some base hydrolysis procedures do not quantitatively liberate all amide-linked hydroxy acids resulting in a relatively low percentage of 3-0H 14: (21) compared with quantities found in other studies (6, 8, 9,12,13,16). As the processing of bacterial growth was similar in most studies, differences in culture conditions were most likely the cause of quantitative differences in cell wall lipid composition. Our data on the relative percentages of the ·m ajor FAMEs in the 10 Pasteurellaceae examined indeed show a considerable influence of the type of agar used (Table 1). The effect of type of medium on the reproducibility and the discriminative power of fatty acid profiling has been shown to be important in various groups of bacteria (7, 11). Our study shows that also the degree of variation in the percentages of the major FAMEs (14:0, 16:0 and 16: 1 cis) of all Pasteurellaceae examined was influenced by the culture medium (Table 1). Strains gtown on LA agar showed for these FAMEs 3-5 times higher maximum coefficients of variation (sd/mean) than strains grown on VH agar. The degree of variation for the FAMEs observed with Pasteurellaceae grown on VH agar (less than 3 % ) appeared much lower than the variations that can be estimated from data given by Schlater et al. (21) for various Pasteurellaceae (7-33 % for 14: 0, 6-35 % for 16: and 6-35 % for 16: 1 cis 9). Schlater's data however are based on examinations of different isolates of a Pasteurellaceae species whereas our data are based on repeated examination of individual strains. Most other studies gave ranges (9, 12, 13, 16) or mean values (18) for the FAMEs found with different Pasteurellaceae species but did not show data on the repeatability of the assays. The fact that LA agat (containing ascitic fluid) gave much more variation for the major FAMEs than the agar type without ascitic fluid (VH agar) (Table 1), suggests that media without body fluids should be used for maximal standardization. This is supported by Calhoon et a!. (6) who used a medium without serum or blood and reported that in various Pasteurellaceae the moles percent values varied no more than 2 % of the average value for each major peak. In most other mldies media supplemented with various body fluid have been used to grow Pasteurellaceae (8, 9,12,13,21).

°

°

Discrimination of Pasteurellaceae strains Most authors concluded from their studies that fatty acid composition is of little value in differentiating among genera and species of Pasteurellaceae. This seems to be supported by the outcome of principal component analysis (PCA) of FAME profiles of Pasteurellaceae grown on LA agar (Fig. la, b). However PCA of profiles obtained with growth from VH agar gave a much better discrimination of strains (Fig. Ie, d), presumably due to the lower variability of FAME profiles on successive test occasions and to the fact that PCA assigns much statistical weight to minor peaks. PCA and other multivariate statistical techniques performed on phenotypic and genetic data have been succesfully used to separate bacteria, including some Pasteurellaceae onto the (sub)species level (5).

Medium effect on FAME composition of Pasteurellaeeae y...... PM"a""l t om ~ _l MEM·I -U6Ii MEAII·l: I.5n

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so-x'

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c..." ...,tl

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SO-I: 4.m SO·l: Uti

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A. capsulatus NeTe 114001 A. equuli NeTe 8529 1 A. lignlere$ii NeTe 4189 T CJ A. ureae NeTe 102H T _ P. dagmatis NeTe 11611 T

I .•.

u;;;;;'i

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,... " .l

'- ,- --- ---,. ---

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P. gall inarum

-_ ... . -- --

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Alec 13336T

P.haemolylica NeTe 9380 1 P.multocida NClC 103231 P .pneumotropica AlCC 12555 (HByl biotype) P .pneumolropica NCTC 8184 1 (Jawelz biotype )

Fig.1a- d. Two-dimensional plot of principle components (pes ) 1 and 2, pes 1 and 3 based on the mean fatty acid methyl ester (FAME) composition of 10 Pasteurellaceae type and reference stra ins cultured o n LA- and VH-agar, respectivel y.

16

R.Boot, H. C. W. Thuis, and A. G. Reubsaet

Surprisingly both P. pneumotropica strains were not discriminated: strain NCTC 8141 T is biotype Jawetz and strain ATCC 12555 is biotype Heyl and it is agreed that both biotypes stand for two distinct species without genetic intermediates (17). Both P. pneumotropiea strains were initially isolated from mice and studies on the hemagglutinating properties of Pasteurellaeeae (notably P. pneumotropica) from various rodent species indicated that within the P. pneumotropica complex test results might be related to host species of origin (3). In agreement with such a host relationship is the close similarity of A. ureae and P. dagmatis (both of human origin). The finding however that the bovine strains A.lignieresii and P. multocida appeared very distinct (Fig. 1c, d ) disagrees with a host-lipid profile relationship but does not exclude that for other bacteria. We conclude that the growth medium had a significant effect on the reproducibility of fatty acid profiling in Pasteurellaeeae and that PCA of fatty acid data obtained under standardized growth conditions may discriminate Pasteurellaeeae species. However further studies are needed to elucidate the full chemotaxonomic power of PCA of fatty acid data of Pasteurellaceae. Acknowledgements. We thank Dr. R. Mutters (Zentrum fur Hygiene und Mcdizinischc Mikrobiologie, Klinikum def Philipps Universitat, Marburg, Germany) and Dr. W. Frederiksen (Statens Serum Institute, Copenhagen, Denmark) for supplying strains in this study.

References 1. Bisgaard, M.: Ecology and significance of Pasteurellaceae in animals. Zbl. Bakt. 279

(1993) 7-26 2. Bisgaard, M.: Taxonomy of the family Pasteurellaceac Poh11981. In Haemophilus, Actinobacillus and Pasteurella (w. Donachie, F. A. Lainson and]. C. Hodgson eds), pp. 1-7. Plenum Press, New York (1995) 3. Boot, R., H. Thuis, and]. S. Teppema: Hemagglutination by Pasteurellaceae isolat-

ed from rodents. Zbl. Bakt. 279 (1993) 259-273

4. Braunthal, S. D., S. C. Holt, A. C. R. Tanner, and S. S. Socransky: Cellular fatty acid composition of Actinobacillus actinomycetemcomitants and Haemophilus aphrophilus.]. Clin. Microbiol. 11 (1980) 625-630 5. Brondz, [., [, Olsen, and M, Sjostrom: Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, HaemophiIus and Pasteurella spp.]. Gen. Microbial. 136 (1990) 507-513 6. Calhoon, D. A., Iv. R. Mayberry, and}. Slots: Cellular fatty acid and soluble protein composition of Actinobacilllus actinomycetemcomitans and related organisms. J.

Clin. Microbiol. 14 (1981) 376-382

7. Chung, A. P., O. C. Nunes, B.}. Tindall, and M. S. Da Costa: The effect of the growth medium composition on the fatty acids of Rhodothermus marinus and

'Thermus thermophil us' HB-8. FEMS Microbiol.Lett.112 (1993) 13-18

8. Dees, S. B., J. Powell, C. Iv. Moss, D. C. Hollis, and R. E. Weaver: Cellular fatty acid composition of organisms frequently associated with human infections resulting

Medium effect on FAME composition of Pasteureliaceae

17

from dog bites: Pasteurelia multocida and groups EF-4, lij, M-5 and DF-2. J.Clin. Microbiol.14 (1981) 612-616 9. Engelhard, E., R. M. Kroppellsted, R. Mutters, and W Mannheim: Carbohydrate patterns, cellular lipoquinones, fatty acids and phospholipids of the genus Pasteurella sensus striero. Med. Microbiol. lmmunol. 180 (1991) 79-92 10. Frederiksen, w,: Ecology and significam:e of Pasteurellaceae in man - an update, Zbl. Bakt. 279 (1993) 27-34 11. Huys, G., P. Kampfer. M. Van Canneyt, R. Coopman, P.Janssen, and K. Kersters: Effect of the growth medium on the cellular fatty acid composition of aeromonads: consequences for the chemoraxonomic differentiation of DNA hybridization groups in the genus Aeromollas. J. Microbio!. Methods 28 (1997) 89-97 12.Jantzen, E., B. P. Berdal, and T. Omlalld: Cellular fatty acid composition of Haemophilz45 species, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Haemophilus vagina lis (Corynebacterium vaginale). Acta, Pathol. Microbiol. Scand. Sect. B 88 (1980) 89-93 13.Jalmen. E., B. P. Herdal, and T. Omland, Cellular fatty acid taxonomy of Haemophilus, Pasteurella and Actinobacillus. In Haemophilus, Pasteurella and Actinobacillus 1M. Kilian, W Frederiksen and E. L. Biberstein eds. ), pp. 197-203. London: Academic Press (1981) 14. Manllheim, W, S. Pohl und R. Hollander: Zur Systematik von Actinobacillus, Haemophilus und Pasteurella: Basenzusammensetzung der DNS, Atmungschinone und kulrurell-biochcmischc Eigcnschaftcn reprascntativer Sammlungsstammc. Zbl, Bakt.246 (1980 ) 512-540 15. Miller, L. and T. Berger: Bacteria identification by gas chromatography of whole cell fatty acids. Hewlett-Packard application note 228-241. Hewlett-Packard Co., Avondale, Pa., USA (1985) 16. Mouahid, M., K. H. Hinz, E. Engelhard, R. Mutters, and W. Mannheim: Characterization of Haemophilus paragalJinarum by analysis of whole cell carbohydrates, fatty acids and phospholipids. Avian Parho!. 21 (1992) 127-136 17. Mutters, R., W. Mannheim, and M. Bisgaard: Taxonomy of the group. In: Pasteurella and pasteurellosis (c. Ad/am and J. M. Rutter eds.), pp. 3 - 34. Academic Press, 1.ondon (1989) 18. Mutters, R., M. Mouahid, E. Engelhard, and W. Mannheinz: Characterization of the family Pasteurellaceae on the basis of cellular lipids and carbohydrates. Zbl. llakt.279 (1993) 104-113 19. Osterhout, G. L, V. H. Shull, and J. D. Dick: Identification of clinical isolates of gram-negative nonfennemative bacteria by an automated cellular fatty acid identification system. J. Clin. Microbia!. 29 (1991 ) 1822-1830 20. Sasser, M.: Identification of hacteria through farty acid analysis. In: Methods of phytobacterio/ogy (Z. Klement, K. Rudolph and D. C. Sands eds.), pp.199-204. Akademiai Kiado, Rudapest (1990) 21. Schlater, L. K., D.}. Brenner, A. C. Steigerwalt, C. W Moss, M. A. Lambert, and R. A. Packer: Pasteurella cahalli, a new species from equine clinical specimens. j. Clin. Microbia!. 27 (1989) 2169-2174 22. Welch, D. F.: Applications of cellular fatty acid analysis. Clin. Microbiol. Rev. 4 (1991) 422-438

Corresponding author: R. Boot, Section of Laboratory Animal Microbiology, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Rilthoven, the Netherlands, Tel.: + 313 02 74 24 32, Fax: + 3 1302744418, E-mail: [email protected] 2

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