Reliable identification ofPrevotella andButyrivibrio spp. from rumen by fatty acid methyl ester profiles

Folia Microbiol. 46 (1), 57-59 (2001)

h t t p : //www. b i o m e d , c a s . c z / m b u / f o l i a /

Reliable Identification of Prevotella and Butyrivibrio spp. from Rumen by Fatty Acid Methyl Ester Profiles R. MAR~EK LOGARa, M. ZOREC a, J. KOPE~N9 b aZootechnical Department, Biotechnical Faculty, University of Ljubljana, 1230 Don#ale, Slovenia bInstitute of Animal Physiology and Genetics Academy of Sciences of the Czech Republic, 10400 Prague I0, Czechia Received 20 December 2000 ABSTRACT. Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera, Prevotella and Butyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzed Prevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The

strains of Prevotella could be well identified on species level by

the characteristicratios amongmajor fatty acids and by acids unique fatty for each species. The 45 Butyrivibrio strains were grouped into4 majorand 2 minorgroups accordingto FAMEs profiles. The major fatty acids for the bulk of the Butyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups correspondedto thosebasedon 16S rDNAsequences.

Rumen bacteria are usually identified by morphological and biochemical characteristics in combination with molecular biology approaches (cf. Avgu~tin et al. 2001; Kopeen~ et al. 2001; Lipoglav~ek and Avgu~tin 2001; Tep~ie and Avgu~tin 2001). The majority of the biochemical identification techniques, however, are cumbersome and time consuming. For many years, analysis of short-chain fatty acids has been routinely used in identification of anaerobic bacteria. With the advent of fused silica GC capillary columns (which allow the recovery of hydroxy acids and resolution of many isomers) it has become practical to use gas chromatography of whole-cell fatty-acid methyl esters (FAMEs) as a fingerprinting technique to identify or classify a wide range of bacteria (Moore et al. 1994). Composition of cellular fatty acids is a genetically controlled and highly stable trait (Stoakes 1991). FAMEs profiling is a relatively simple, rapid and reliable method for bacterial identification and taxonomy, especially when automated and combined with molecular biology techniques such as PCR-RFLP and cloning and DNA sequencing (Stead 1995; Ritchie et al. 2000). The genus Prevotella has recently undergone a taxonomic reclassification based on comparisons of 16S rDNA sequences of culturable strains. The species P. ruminicola was redefined and an elevation of three different groups of strains to species level (P. bryantii, P. albensis and P. brevis) was proposed (Avgu~tin et al. 1997). Successive studies further confirmed the vast diversity of rumen Prevotella species (Ram~ak et al. 2000). The genus Butyrivibrio is currently represented only by one ruminal species B. fibrisolvens although its phenotypic and genetic heterogeneity promises an impending reclassification into several new species (Forster et al. 1996; Kope~n2~2000; Marin~ek Logar 2000). The MIDI databases (Newark, USA) for the automatic identification of bacteria by FAMEs currently include some clinically important prevotellas and only one rumen Prevotella species (a type strain of Prevotella ruminicola ssp. ruminicola) according to the previous classification of prevotellas ( h t t p : / / ~ r a w . m i d i - i n c . c o m / m o o r e body.htm; 2000-11-05). Only the type strain of B. fibrisolvens ATCC 19171, which is not closely connected to other Butyrivibrio strains, is included in the MIDI database (http : / / w w w . midi-inc, corn/moore_body, htm; 2000-11-05). Here we report on the creation of the databases of FAMEs profiles for two predominant ruminal genera, viz. Prevotella and Butyrivibrio.

MATERIALS AND METHODS

Bacterial strains. The majority of 23 analyzed strains of rumen prevotellas (a gitt from The Rowett Research Institute, Aberdeen, UK) were previously classified as one of the 4 Prevotblla species - P. ruminicola (6 strains), P. bryantii (6), P. albensis (3) or P. brevis (3) (Avgu~tin et al. 1997). We analyzed the FAMEs profiles of 45 Butyrivibrio strains of both domestic and wild ruminants including the type strain B. fibrisolvens ATCC 19171 and 6 strains from Deutsche Sammlung von Mikroorganismen (DSM) culture collection. The isolates were collected from three European countries (Czechia,

Slovenia and UK), from Africa, Australia and Canada.

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Culturing conditions. All strains were grown in a medium without rumen fluid (DSM, medium no. 330). Cultures were incubated at 37 ~ for 1 d in a 100 % CO2 atmosphere in Hungate tubes. Fatty acid methylation and extraction. The cells were harvested by centrifugation (50 Hz, 10 min, 4 ~ The cell pellets were washed twice in sodium phosphate buffer (50 mmol/L, pH 6.5). The preparation of FAMEs by saponification, methylation, extraction into hexane and sample cleanup strictly followed the guidelines of M1DI Inc. (Newark, USA) for their Microbial Identification System (Hewlett-Packard 1987; Sasser 1990). Gas chromatography. Two laL of each FAMEs extract was chromatographed on a fused-silica capillary column (cross-linked 5 % methyl phenyl siloxane, Hewlet-Packard part no. 19091B-102, length 25 m, ID 0.2 ram, film thickness 0.33 ~tm) with a Shimadzu GC-14A chromatograph equipped with flame ionization detector and a C-R6A integrator. Helium was used as carrier gas (40 mL/min), hydrogen (40 mL/min) and air (400 mL/min) as detector gases. A temperature program 170-270 ~ was used for the analysis. The injector and detector temperatures were 280 and 330 ~ respectively. The analysis was finished in 30 min. Identification of FAMEs and comparison of FAMEs profiles. The saturated FAMEs were identified by their retention times according to the known peaks in the standard solution chromatogram (MIS Calibration Standard; MIDI Inc., part no. 1200-A). The chromatographic data for FAMEs were loaded into MS Excel spreadsheets. The equivalent chain length (ECL) factors were calculated for unknown peaks and they were further identified by their ECLs from the Peak Naming List (Hewlett-Packard 1987). The response factors and relative concentrations for individual fatty acids (FAs) were calculated. The presence or absence of individual FAs and the ratios between major FAs for each strain provided the basic datafor grouping of the Prevotella and Butyrivibrio strains by NTSYS-PC program (Computer Package EXETER Software, New York, USA).

RESULTS AND DISCUSSION

Major long-chain cellular FAs found in the 23 analyzed Prevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. Branched chain FAs prevailed; this confirmed the previous results of Miyagawa (1982) for the type strain of Bacteroides ruminicola (later P. ruminicola) although his profiles were influenced by rumen fluid in the cultivation medium. The strains of Prevotella could be successfully identified on species level by the characteristic ratios between major FAs and by the unique FAs for each species (Table I). For the subspecies level identification all the FAs in the profiles should be qualitatively and quantitatively compared. The FAMEs profiling made it possible to identify the Prevotella strains 223/M2/7A, TF 2-5, TC 2-24 and 2202 on the genus level but not on the species level. Stoakes (1991) reported that prevotellas were the most frequently misidentified among Gram-negative anaerobic bacilli, but could be arranged into groups according to FAMEs profiles. Table 1. Characteristic ratios between major and unique fatty acids of rumen Prevotella speciesa Ratios between major FAsb Species

Unique FAs 15:0 (anteiso)

P. rummicola P. bryantii P. brevis P, albens~s

1 1 1 1

15:0

15:0 (iso)

16:0

0.37 039 0.39 0.20

0.35 0.17 0.40 0.10

0.27 0.30 0.36 0.22

18:1 cts-9 10:0 (2OH) 10:0 (iso)

aSee Materials andMethods for the number of strains analyzed for each species. bCalculated for the major FA from the mean percentages of the total FA.

The FAMEs profiles were much more heterogeneous for Butyrivibrio strains than for Prevotella strains; this reflected the high variability of Butyrivibrio isolates and confirmed similar observations of Miyagawa (1982). Forty five Butyrivibrio strains were grouped into 4 major and 2 minor groups based on the FAMEs profiles. FAMEs profiles of 5 strains were unique and did not show any close similarity to the rest of the strains, the type strain ATCC 19 171 was among them. The major fatty acids for the bulk of the Butyrivibrio strains were 14:0, 15:1 and 16:0 (Table II). This grouping was in good accordance with rumen

2001

IDENTIFICATION OF Prevotella AND Butyrivibrio spp. BY FATTY ACID METHYL ESTER PROFILES

59

Butyrivibrio groups based on 16S r D N A s e q u e n c e comparisons and R F L P analysis (Kope~n2~ 2000; Marin~ek Logar 2000). Table 11. Characteristic ratios between major and unique FAs for 4 major rumen Butyriwbrto groups

Ratios between major FAsa Group

1/F 2/B 3/D 4/E

No. of strains

8 6 10 8

Unique FAs 14:0

15:1

16:0

1 1 1 1

0.83 1.73 0.86 0.89

1.60 2.81 3.11 5.17

14:0 (2OH) 17:0 (3OH)

aCalculated for the major FAs from the mean percentages of the total FAs.

F A M E s p r o f i l i n g was s u c c e s s f u l l y used for the identification o f 4 rumen species o f Prevotella. Butyrivibrios can be reclassified in the near future (acquired data o f F A composition o f different groups o f butyrivibrios can be used for the identification o f n e w species). The F A data h a v e been used for the creation o f our own two databases for the automatic identification o f Prevotella and Butyrivibrio isolates by the M I S system. F A M E s profiling contributes to establising t a x o n o m i c relationships a m o n g Butyrivibrio isolates; it could be useful also for checking the purity o f cultures in culture collections and for assessment o f composition o f microbial communities. Authors gratefully acknowledge the support of the Ministry of Education, Youth and Sports of the Czech Republic and Slovenian Ministry of Science (project KONTAKT 1999-2000) and the Grant Agency of the Czech Republic (project no. 524/99/0602). REFERENCES

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