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					Annals of Microbiology, 53 (3), 299-313 (2003)




   Isolation of bifidobacteria from food and human faeces
   and rapid identification by Fourier transform infrared
                         spectroscopy

                      A. MAYER1*, H. SEILER2, S. SCHERER2
1 SzentIstván University, Faculty of Food Sciences, Department of Brewing and Distilling,
      Budapest, Hungary; 2Institut für Mikrobiologie, Forschungszentrum für Milch
           und Lebensmittel Weihenstephan, Technische Universität München,
                 Weihenstephaner Berg 3, D-85354 Freising, Germany




Abstract – Bifidobacteria are suggested to include a number of probiotic species which are
added to dairy products. A total of 67 Bifidobacterium strains were isolated from food stuff
and human faeces and were identified by fermentation tests and, in many cases, by 16S
rDNA sequencing. In food products, only B. animalis was found. B. longum was present in
starter cultures, but not in dairy products. B. bifidum, B. pseudocatenulatum, B.
longum/infantis, B. breve, and B. dentium were isolated from faeces of infants and adults.
The identification of the species of this genus by physiological methods is time consuming
(seven days, starting from purified colonies) while molecular identification methods are
rapid, but quite expensive. Therefore, we tested Fourier transform infrared (FT-IR) spec-
troscopy, an emerging physicochemical identification method, which correctly identified 62
out of the 67 isolates within two days. With respect to the overall cellular composition,
which is the basis of FT-IR spectroscopy, the type strains of B. bifidum, B. breve and B.
infantis appear to be rather untypical and did not cluster near the isolates of these species.
The discrimination between B. infantis and B. longum as well as between B. lactis and B.
animalis was difficult with all methods applied.

Key words: Bifidobacterium, probiotic bacteria, dairy products, identification of bacteria,
FT-IR spectroscopy.



                                   INTRODUCTION

Since it is known that bifidobacteria play an important role in human health (Bal-
longue, 1998; Orrhage and Nord, 2000; Rolfe, 2000; Kaur et al., 2002) an increas-
ing number of probiotic products containing bifidobacteria are introduced to the
food market (Biavati et al., 2000; Heller, 2001; Stanton et al., 2001). However,
among the 31 Bifidobacterium species which originate from diverse sources, at
least those derived from humans are expected to have beneficial effects (Charteris
et al., 1998) and probiotic bacteria sold to the customers should be identified


* Corresponding author. Phone/fax: +36-1-372-6214; E-mail: mayer@omega.kee.hu


Ann. Microbiol., 53 (3), 299-313 (2003)                                                  299
unequivocally. Many food producers appear to have difficulties to correctly label
the species they distribute (Mattarelli et al., 2002; Reuter et al., 2002), and there-
fore may welcome a rapid, simple, low-cost and reliable technique for identifica-
tion of bifidobacteria at the species level (Bonaparte, 1997). Several methods have
been used to identify bifidobacteria: Fermentation and enzymatic tests (Scardovi et
al., 1979; Gavini et al., 1991; Crociani et al., 1994; Orban and Patterson, 2000),
rDNA restriction patterns (Mangin et al., 1994), amplified ribosomal DNA restric-
tion analysis (Ventura et al., 2001a), denaturing gradient gel analysis of amplified
16S rDNA (Satokari et al., 2001), oligonucleotide probes (Yamamoto et al., 1992;
Langendijk et al., 1995; Kaufmann et al., 1997), recA (Kullen et al., 1997) and 16S
rDNA targeted PCR (Matsuki et al., 1999; Brigidi et al., 2000; Ventura et al.,
2001b). However, only a selection of bifidobacterial species have been investigat-
ed in most of these individual studies and, in general, molecular methods for the
comprehensive identification of Bifidobacterium species are still lacking (Tannock,
1999). In addition, most of these methods do not fulfil the above-mentioned
requirements: They are laborious, time consuming and, in general, they are still dif-
ficult to be performed on a routine basis in laboratories of the food industry.
     Identification of microorganisms by Fourier transform infrared spectroscopy
(FT-IR) was originally suggested by Naumann and co-workers (Helm et al., 1991;
Neumann et al., 1994). FT-IR spectra of bacteria are fingerprint–like patterns that
are highly reproducible and species-specific. The spectra represent the infrared
absorbance pattern of the total composition of cellular components such as pro-
teins, membranes, cell wall and nucleic acids. Using this method, unknown
microorganisms can be identified very easily and quickly once an extensive spec-
tral reference library is available. For identification, the infrared spectrum of an
unknown isolate is compared with all spectra present in the reference library and is
matched to the library strain whose spectrum is most similar. However, spectral
reference libraries of sufficient complexity have only been published for the genus
Listeria (Holt et al., 1995), food-borne yeasts (Kümmerle et al., 1998) and coryne-
form bacteria (Oberreuter et al., 2001). A limited number of isolates of the Bacillus
cereus group (Beattie et al., 1998) and the genus Lactobacillus (Curk et al., 1994)
have also been investigated. Here we report on the isolation and FT-IR identifica-
tion of bifidobacteria originating from food stuff and human faeces.


                         MATERIALS AND METHODS

Bacterial strains. The following DMSZ (Braunschweig, Germany) type strains of
the genus Bifidobacterium were used in this study: adolescentis, angulatum, ani-
malis, asteroides, bifidum, boum, breve, catenulatum, choerinum, coryneforme,
cuniculi, denticolens, dentium, gallicum, infantis, inopinatum, lactis, longum, mag-
num, merycicum, minimum, pseudocatenulatum, pseudolongum subsp. gobosum,
pseudolongum subsp. pseudolongum, pullorum, ruminantium, saeculare, subtile,
suis, thermophilum; besides Bifidobacterium asteroides DSM 20431 and Gard-
nerella vaginalis. Among the above mentioned species two species, Bifidobacteri-
um denticolens and Bifidobacterium inopinatum have been described as different
genera: Parascardovia denticolens and Scardovia inopinata respectively (Jian and
Dong, 2002; Modesto et al., 2003). The selective media NPNL agar (Teraguchi et


300                                                                    MAYER et al.
al., 1978) and DP agar (Bonaparte, 1997) were used for strain isolation from food
and faeces. The Bifidobacterium-like colonies were picked, purified and identified
at the genus level by the F6PPK test (Scardovi, 1986). Cell morphology was rou-
tinely observed with a phase-contrast microscope. Ten strains isolated and identi-
fied by Bonaparte (1997) were provided by Dr. Wandig, Institut für Fleischhy-
giene, Berlin, Germany.

Fermentation tests. A microtiter plate similar to the API 50CH test kit (Bio-
Mérieux) containing 53 substrates was produced (acid from glucose, galactose,
sorbose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rham-
nose, sucrose, maltose, trehalose, α-methylglucoside, cellobiose, salicin, arbutin,
melibiose, lactose, raffinose, melezitose, gycerol, erythritol, adonitol, xylitol, L-
arabitol, sorbitol, D-mannitol, dulcitol, m-inositol, D-lyxose, D-turanose, N-acetyl-
glucosamine, D-arabitol, gluconate, 2-ketogluconate, amygdalin, α-methylxylo-
side, α-methylmannoside, fructose, mannose, gentibiose, inulin, starch, glycogen,
dextrin, esculine, porcine gastric mucin, gum guar, amylopectin, D-glucuronate, L-
fucose, arabinogalactan, and argininedihydrolase). Cells grown for 48 h in “Rein-
forced Clostridial Medium” (RCM) agar medium (Oxoid) were suspended in mod-
ified RCM medium (yeast extract 3 g, meat extract 10 g, peptone 10 g, cystein-HCl
0.5 g, NaCl 5 g, Na-acetate 3 g, agar 0.1 g, distilled water ad 1000 ml; transfer 12
ml in a tube and autoclave at 121 °C for 15 min). 25 µl aliquots of the C-source
solutions were transferred into the wells of the microtiter plate and 150 µl aliquots
the bacterial suspensions were added. The concentration of the easy soluble C-
sources was 0.8 %, of complex C-sources 0.5%. Bromcresol purple (30 mg/l) was
used as an indicator. Growth monitored by colour changes was recorded after one
week of incubation at 37 °C in anaerobic jars and evaluated using the software
BIFMTP (H. Seiler and R. Braatz, this laboratory, unpublished). This computer
program compares the results of all fermentation characteristics of the Bifidobac-
terium species given in the literature (e.g. API) and of all Bifidobacterium type
strains and of the Bonaparte strains tested in our laboratory. Altogether, 65 refer-
ence patterns could be defined.

Complex carbohydrate tests. Fermentation of the complex carbohydrates ara-
binogalactan, porcine gastric mucin, gum guar and amylopectin in addition to L-
fucose and D-glucuronate are considered to be the “key for differentiation of Bifi-
dobacterium species of human origin” (Crociani et al., 1994). Although 12 bifi-
dobacterial species have been isolated from humans (Satokari et al., 2001), a data-
base is only available for B. infantis, breve, pseudocatenulatum, longum, bifidum,
dentium, adolescentis, catenulatum isolated from humans and that has been used
for our isolates belonging to these species.

16S rDNA sequence analysis. The strains were lysed and the amplification of the
almost complete 16S rDNA molecule was performed according to Von Stetten et
al. (1998). After PCR amplification, the DNA was purified using the QIAquick
PCR Purification Kit (Qiagen) according to the instruction of the manufacturer, fol-
lowed by a PEG precipitation of the purified product according to Facius et al.
(1999). After purification, the samples were subjected to a cycle-sequencing PCR
(Facius et al., 1999) using the ThermoSequenase fluorescent labelled primer cycle


Ann. Microbiol., 53 (3), 299-313 (2003)                                          301
sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) and the uni-
versal primer 5´f IRD800 [5´-AGAGTTTGATCCTGGCTC A-3´, Escherichia coli
numbering 8-26]. 11% (v/v) DMSO and 7% (v/v) formamide were added to facil-
itate the cycle sequencing PCR of these high G+C Gram-positive bacteria. DNA
sequencing was performed on a LI-COR sequencer (MWG Biotech), typically
yielding sequence length of approximately 800 to 1000 bases per run. Identifica-
tion of the strains was carried out by comparison of the partial sequences with
sequences in the Genbank database (BLAST).

FT-IR spectroscopy. RCM agar (OXOID CM151) was used to grow lawns of
bifidobacteria by incubating the plates at 37 °C for 48 h in anaerobic jars. An oxy-
gen-free atmosphere was created by using Anaerocult A (Merck). Sample prepara-
tion and measurement of FT-IR spectra was performed according to Kümmerle et
al. (1998) using an IFS-28B FT-IR spectrometer (Bruker). FT-IR parameters were
adjusted as follows: wavelengths windows, 1350-1200 cm-1, 900-700 cm-1 and
3030-2830 cm-1; relative weights and reproducibility levels were 1 and 30, respec-
tively (Kümmerle et al., 1998; Oberreuter et al., 2001). The FT-IR spectrum of
each isolate was compared with the reference library containing FT-IR spectra of
strains identified by 16S rDNA sequences.


                         RESULTS AND DISCUSSION

FT-IR spectra of the type strains
The FT-IR spectra of 32 reference strains (30 type strains of Bifidobacterium, one
type strain of Gardnerella, one non-type Bifidobacterium strain) available were
compiled in an initial spectral reference library. From at least 3 independent cul-
tures spectra were recorded and the mean FT-IR spectra were calculated. Similari-
ties were found between the species B. animalis and B. lactis (FT-IR distance value
ca 0.3), B. bifidum and B. choerinum (0.5), B. catenulatum, B. dentium and B.
pseudolongum subsp. pseudolongum (0.5) as well as between B. minimum and B.
thermophilum (0.5). All other species were separated by spectral distances greater
than 0.5. This type strain based reference spectral library was used for an initial
characterization of the strains isolated from food and faeces.

Identification of bifidobacteria from foodstuff
A total of 27 own isolates and 9 strains isolated by Bonaparte (1997) were screened
by FT-IR spectroscopy (Fig. 1). Only the type strains of B. animalis, B. lactis and
B. longum showed similarities to the food isolates. Strain B77 clustered with the
type strain of B. longum at a high similarity level (cluster 1). Two isolates of B.
longum from the starter culture of the suppliers Hansen and SKW formed cluster 2.
Many strains of B. animalis formed the large cluster 3 in which 4 subclusters can
be identified. Strain B55 formed cluster 4 which is well separated from all other
isolates. A number of strains from each cluster, indicated in Fig. 1, were identified
by their 16S rDNA sequences. This sequence information together with the relative
similarity of the FT-IR spectra allowed assignment of all isolates to a Bifidobac-
terium species.
     To further characterize the isolates, 53 fermentation tests were applied (Table


302                                                                   MAYER et al.
FIG. 1 – Dendrogram of the FT-IR spectra of 36 bifidobacterial isolates from foodstuff
         and four type strains. The dendrogram was constructed using an unweighted pair
         group method algorithm, correlation of FT-IR spectra was done with normalized
         to reproducibility level. Frequency ranges with weights and reprolevels were as
         follows: 1350-1200 cm-1 / 1/ 30; 900-700 / 1/ 30; 3030-2830 / 1/ 30. Numbers
         indicate arrangements into clusters and subclusters.



1) which resulted in most isolates being identified as B. animalis. Two isolates
which originated from the same culture supplier were identified as B. lactis. A
very high similarity was found between these two species, which were separated by
a maximum of three fermentation reactions only. Using complex carbohydrate fer-
mentation tests (Crociani et al., 1994), the species B. animalis and B. lactis are not
yet characterized. Their similarity at the 16S rDNS sequence level is more than
99% (Meile et al., 1997; Ventura et al., 2001b) and the two species are also
extremely similar when HSP60 heat shock gene sequences are compared (Jian et
al., 2001). Subcluster 3a of Figure 1 includes the type strains of B. lactis and B.


Ann. Microbiol., 53 (3), 299-313 (2003)                                             303
               TABLE 1 – Identification of 36 bifidobacterial strains isolated from from foodstuff from Germany, Hungary and Poland




304
               Strain         Origin                                                                Identification method

                                                          16S rDNA analysis          FT-IR spectroscopy            53 fermentation tests   Complex carbohydrate tests
                                                                                     Cluster no. / Species

               Ga1435         Starter culture             B. infantis/longumb         2 / not identifiable             B. longum                   B. longum
               G1452          Yoghurt                     B. animalis/lactisb          3d / B. animalis                B. animalis                      -
               G1453          Yoghurt                               -                  3d / B. animalis                     -                           -
               G1454          Yoghurt                      B. animalis/lactis          3b / B. animalis                B. animalis                      -
               G1455          Yoghurt                               -                  3b / B. animalis                     -                           -
               G1456          Yoghurt                               -                  3b / B. animalis                     -                           -
               G1458          Yoghurt                      B. animalis/lactis          3d / B. animalis                B. animalis                      -
               G1459          Yoghurt                               -                  3d / B. animalis                     -                           -
               G1461          Yoghurt                               -                  3d / B. animalis                     -                           -
               G1462          Yoghurt                               -                  3d / B. animalis                     -                           -
               G1463          Yoghurt                      B. lactis/animalis          3d / B. animalis                B. animalis                      -
               G1546          Starter culture              B. lactis/animalis          3c / B. animalis                B. animalis                      -
               G1547          Starter culture             B. infantis/longum          2 / not identifiable             ~B. longum                  B. longum
               G1548          Starter culture                       -                  3d / B. animalis                 B. lactis                       -
               G1549          Starter culture                       -                  3d / B. animalis                 B. lactis                       -
               G1550          Starter culture              B. lactis/animalis          3d / B. animalis                B. animalis                      -
               G1552          Infant formula               B. lactis/animalis          3c / B. animalis                B. animalis                      -
               G1553          Quark                                 -                  3c / B. animalis                     -                           -
               G1554          Yoghurt mild                          -                  3c / B. animalis                     -                           -
               G1555          Starter culture                       -                  3c / B. animalis                     -                           -
               G1747          Yoghurt drink                B. lactis/animalis          3c / B. animalis                     -                           -

                                                                                                                                                            (followed)




MAYER et al.
                                          TABLE 1 – Identification of 36 bifidobacterial strains isolated from from foodstuff from Germany, Hungary and Poland (continued)

                                          Strain            Origin                                                                 Identification method

                                                                                       16S rDNA analysis            FT-IR spectroscopy             53 fermentation tests   Complex carbohydrate tests
                                                                                                                    Cluster no. / Species

                                          G1748             Yoghurt                              -                     3c / B. animalis                      -                          -
                                          G1749             Yoghurt mild                         -                     3c / B. animalis                      -                          -
                                          G1750             Yoghurt                              -                     3c / B. animalis                      -                          -
                                          G1751             Yoghurt                              -                     3c / B. animalis                      -                          -
                                          G1752             Yoghurt                              -                     3c / B. animalis                      -                          -
                                          G1753             Yoghurt drink               B. lactis/animalis             3c / B. animalis                 B. animalis                     -




Ann. Microbiol., 53 (3), 299-313 (2003)
                                          Bc11              Starter culture                      -                     3a / B. animalis                 B. animalis                     -
                                          B15               Dairy industry                       -                     3a / B. animalis                 B. animalis                     -
                                          B16               Cultured milk                        -                     3d / B. animalis                 B. animalis                     -
                                          B17               Cultured milk                        -                     3d / B. animalis                 B. animalis                     -
                                          B22               Cultured milk                        -                     3b / B. animalis                 B. animalis                     -
                                          B23               Starter culture                      -                     3d / B. animalis                 B. animalis                     -
                                          B39               Starter culture                      -                     3a / B. animalis                 B. animalis                     -
                                          B55               Starter culture             B. lactis/animalis             4 / ~B. animalis                 B. animalis                     -
                                          B77               Starter culture                      -                      1 / B. longum                   B. longum                  B. longum

                                            a own   isolates.
                                            b infantis/longum    and animalis/lactis: both species had very similar homology levels. The species with priority is named first.
                                            c see Bonaparte (1997).
                                            d at all strains of this type an unspecificated “butyrate-producing bacterium T1” was named first.
                                            ~, low similarity with the corresponding reference strain.




305
                                            -, not tested.
animalis which are quite similar when their FT-IR spectra are considered. Sub-
cluster 3d contains also B. animalis and B. lactis isolates which are extremely sim-
ilar. We were not able to distinguish between B. animalis and B. lactis and the pre-
viously proposed rejection of the species B. lactis by Cai et al. (2000) is in accor-
dance with FT-IR spectroscopical data. On the other hand, Ventura et al. (2001b)
differentiated B. lactis from all other bifidobacteria by a pair of 16S rDNA primers.
This study was based on three B. lactis and four B. animalis isolates. The taxono-
my of some very closely related bifidobacterial species, concerning their species or
subspecies status, including B. lactis and B. animalis, is currently under critical
evaluation by the International Committee on Systematic Bacteriology.
      Three isolates from starter cultures were identified as B. longum, but these
showed high dissimilarities to the type strain pattern (6-10 out of 53 fermentation
reactions). It was difficult to unambiguously separate B. longum and B. infantis.
Strains of these species were identified with highest priority as B. longum by the
physiological methods and as B. infantis by the rDNA sequencing method (Table 1,
compare also Table 2). The products of starter culture suppliers are declared to con-
tain B. bifidum, B. longum and B. infantis. Preparations of two suppliers were
investigated, but only B. animalis and B. longum were found. Bonaparte (1997)
isolated strains of B. animalis and B. longum from food. Surprisingly, from all
foodstuff samples collected in retail markets in Germany, Hungary and Poland,
only B. animalis was isolated. It is not possible to conclude whether this species
predominantly is used to produce probiotic dairy products in these countries, or
whether we have by chance only investigated foodstuff containing this species, or
whether other species do not survive in fermented milk products. It is, however,
noticeable that in an earlier study of 6 fermented milk samples in contrast to the
label information only B. animalis was found (Biavati et al., 1992) and that
Yaeshima et al. (1996) isolated bifidobacteria from 15 dairy products, 11 of which
were B. animalis, which was also in contrast to the product label information. In a
recent review, Reuter et al. (2002) noted that false declarations of bifidobacteria
may occur frequently.

Identification of bifidobacteria from human faeces
The FT-IR spectra of 29 isolates were compared to those of 32 reference type
strains. Similarities to type strains were not always obvious and the spectral vari-
ability of the isolates from faeces was much higher (up to a spectral distance of 5,
data not shown). Therefore, we decided to identify all isolates by their 16S rDNA
sequence (Table 2). Most of the infant isolates were B. bifidum, followed by B.
infantis/longum, B. breve, and B. dentium. Strains G1436 and G1443, also isolated
from infants, were identified as B. pseudocatenulatum. All of the adult’s isolates
were B. infantis/longum except one, which was B. dentium. Evaluation of six com-
plex carbohydrate fermentations confirmed many of the identifications resulting
from 16S rDNA sequences. Only the strain B. pseudocatenulatum G1443 was iden-
tified as B. catenulatum (Table 2).
     The characterization of 28 bifidobacterial isolates from faeces with 53 fermen-
tation tests revealed strains of the species B. longum (11 isolates), B. bifidum (8 iso-
lates), B. dentium (3 isolates), B. breve (4 isolates), B. animalis (1 isolate) and B.
angulatum (1 isolate). Thus, most of the 16S rDNA identifications could be con-
firmed. Most strains of B. longum showed sufficient similarities to the patterns of


306                                                                      MAYER et al.
                                          TABLE 2 – Identification of 31 bifidobacterial strains isolated from human faeces

                                          Strain           Origin                                                          Identification method

                                                                                16S rDNA analysis           FT-IR spectroscopy            53 fermentation tests   Complex carbohydrate tests
                                                                                                            Cluster no. / Species

                                          G1436            Infant                B. p´catenulatum          7a / B. longum/infantis           ≈B. angulatum            B. p´catenulatum
                                          G1437            Infant                    B. dentium                 4 / B. dentium                ~B. dentium                B. dentium
                                          G1438            Infant               B. infantis/longumd        5b / B. longum/infantis             B. longum                  B. longum
                                          G1439            Infant                    B. bifidum                 1a / B. bifidum               ~B. bifidum                B. bifidum
                                          G1440            Infant                    B. bifidum                1b / B. bifidum                 B. bifidum                B. bifidum
                                          G1441            Infant                    B. dentium                4 / B. dentium                 ~B. dentium                B. dentium




Ann. Microbiol., 53 (3), 299-313 (2003)
                                          G1442            Infant                    B. bifidum                1b / B. bifidum                ~B. bifidum                B. bifidum
                                          G1443            Infant                B. p´catenulatum              5b / B. animalis               ~B. animalis             B. catenulatum
                                          G1444            Infant               B. infantis/longum         5b / B. longum/infantis            ~B. longum                  B. longum
                                          G1445            Adult                B. infantis/longum           6 / not identifiable              B. longum                  B. longum
                                          G1447            Adult                     B. dentium                5a / B. dentium                ~B. dentium                B. dentium
                                          G1448            Infant                    B. bifidum                1b / B. bifidum                ~B. bifidum                B. bifidum
                                          G1537            Infant                    B. bifidum                 1a / B. bifidum               ~B. bifidum                B. bifidum
                                          G1538            Infant                    B. bifidum                 1a / B. bifidum                     -                          -
                                          G1539            Infant                    B. bifidum                 1a / B. bifidum                B. bifidum                B. bifidum
                                          G1541            Adult                B. infantis/longum         7a / B. longum/infantis             B. longum                  B. longum
                                          G1542            Adult                B. infantis/longum         3a / B. longum/infantis             B. longum                  B. longum
                                          G1543            Adult                B. infantis/longum         7a / B. longum/infantis             B. longum                  B. longum
                                          G1544            Adult                B. infantis/longum         3a / B. longum/infantis             B. longum                  B. longum




307
                                                                                                                                                                                   (followed)
308
               TABLE 2 – Identification of 31 bifidobacterial strains isolated from human faeces (continued)

               Strain           Origin                                                           Identification method

                                                     16S rDNA analysis            FT-IR spectroscopy            53 fermentation tests          Complex carbohydrate tests
                                                                                  Cluster no. / Species

               G1545            Adult                B. infantis/longum          7a / B. longum/infantis                B. longum                      B. longum
               G1563            Infant                   ~B. bifidum                  1a / B. bifidum                  B. bifidum                      B. bifidum
               G1564            Infant                         -                      1a / B. bifidum                  B. bifidum                      B. bifidum
               G1565            Infant                     B. breve                    2 / B. breve                     ~B. breve                       B. breve
               G1572            Infant                     B. breve                    2 / B. breve                          -                              -
               G1573            Infant                     B. breve                    2 / B. breve                     ~B. breve                       B. breve
               G1575            Infant                     B. breve                    2 / B. breve                     ≈ B. breve                      B. breve
               G1576            Infant                     B. breve                    2 / B. breve                     ≈ B. breve                      B. breve
               G1577            Infant               B. infantis/longum          3a / B. longum/infantis               ~B. longum                      B. longum
               G1581            Infant               ~B. infantis/longum           8 / not identifiable                ~B. longum                      B. longum
               G1582            Infant               ~B. infantis/longum           8 / not identifiable                      -                              -
               B12 c            Adult                          -                      3b / B. longum                    B. longum                      B. longum
               a own isolates.
               b infantis/longum and animalis/lactis = both species had very similar homology levels. The species   with priority is named first.
               c see Bonaparte (1997).
               d of all strains of this type an unspecificated “butyrate-producing bacterium T1” was named first.
               ~, low similarity with the corresponding reference strain.
               ≈, very low similarity with the corresponding reference strain.
               -, not tested.




MAYER et al.
                                      Single colony



             Cell lysis               Cultivation on                 F6PPK
           PCR reactions               RCM agar                   enzymatic test
           Sequencing gel                2 days                      2 hours
             Data base
              Search
               1 day                                              Identification at
                                                                  the genus level


                                         FT-IR                    Fermentation
                                      spectroscopy                  reactions
                                         1 hour                      7 days


                            Identification at the species level

FIG. 2 – Comparison of identification methods for Bifidobacterium species using 16S
         rDNA sequencing, FT-IR spectroscopy and fermentation tests.




the reference strain but strains of B. breve, B. dentium and B. angulatum were
rather dissimilar to the fermentation patterns of the reference strains. Strains of B.
bifidum and B. longum exhibited varying similarities to the corresponding refer-
ence strain patterns. We conclude that identification of bifidobacteria by fermenta-
tion patterns needs to be optimized which is in accordance to the notion that the
identification of Bifidobacterium species is considered difficult due to phenotypic
and genetic heterogeneities (Leblond-Bourget et al., 1996).
     Using FT-IR spectroscopy, it was difficult to distinguish between B. longum
and B. infantis. The 16S rDNA and HSP60 sequences (Jian et al., 2001) have been
found to be very similar. All isolates were identified as B. longum by fermentation
pattern and complex carbohydrate testing. However, FT-IR spectroscopy grouped
the B. infantis/longum isolates in different clusters. Interestingly, the type strains of
B. longum and B. infantis showed high dissimilarity in their FT-IR spectra. It has
been reported previously that different spectral types can be found in a single
species (Kümmerle et al., 1998; Oberreuter et al., 2001). This phenomenon may
provide a possibility to characterize some species at the strain level using FT-IR
spectroscopy.

External validation of FT-IR identification
FT-IR spectra of all isolates and all type strains were combined in a single spectral
reference library. All strains were labeled with species names derived from the 16S
rDNA sequences and phenetic identification. The average spectrum of a strain was
excluded from the complete reference library and then its spectrum was tested
against the remaining database. In case the first hit matched with an average spec-


Ann. Microbiol., 53 (3), 299-313 (2003)                                               309
trum of a strain of the same species and the spectral distance value was below 1.0,
the result was counted as a correct identification at the species level. A misidentifi-
cation was noted if a spectrum of an isolate did not fulfill these conditions. In total,
we found that a correct identification was achieved in 62 out of 67 cases: B. ani-
malis/lactis 100% (33 isolates), B. bifidum 100% (9 isolates), B. breve 100% (5 iso-
lates), B. dentium 100 % (3 isolates), B. infantis/longum 80% (15 isolates), and B.
pseudocatenulatum 0% (2 isolates). The reason for misidentification of the latter
species is due to the fact that the isolates G1436 and G1443 have spectra more sim-
ilar to B. animalis than to the type strain of B. pseudocatenulatum. This example
clearly demonstrates that for reliable identification by FT-IR spectroscopy the dep-
osition of the spectrum of the type strain only in the reference library is insuffi-
cient. It is necessary to collect spectra of approximately ten different strains of each
species, which must originate from different sources. Only this procedure will
ensure coverage of most of the spectroscopical types of a species.

Concluding remarks
The three methods used in this study to identify bifidobacteria are compared in Fig
2. Nowadays, 16S rDNA sequencing is considered to be the gold standard for the
identification of bacteria. While it is fast and reliable, it is comparatively expen-
sive, laborious and will rarely be performed in a routine laboratory of a food com-
pany. The use of broad fermentation patterns (either API 50 CH or individually
designed microtiter plates) currently is extremely time-consuming and the results
may be questionable. While the examination of complex carbohydrates in
microtiter plates is easy to perform, only few Bifidobacterium species can be char-
acterized by this method (Crociani et al., 1994). As an alternative, FT-IR spec-
troscopy is a fast and inexpensive approach. As shown in Fig. 2, it reduces the time
needed for identification from nine to two days. Accuracy of identification at least
appears to be comparable to phenotypic methods and the identification quality can
be emended by the deposition of an increased number of spectra from different
strains (Naumann et al., 1994; Oberreuter et al., 2001). Since this biometrical
method is easy to perform, it might be a step towards the goal of biomonitoring of
microbial populations in food industry laboratories.

Achnowledgment
This project was supported by the FEI, the AiF and the German Ministry of Eco-
nomics and Technology (Project No. 11627N), as well as by an Erasmus scholarship
to A.M. Thanks are expressed to Dr. Wanding, Berlin and Dr. Meile, Zürich for pro-
viding strains and Dr. Ralf Zink, Lausanne for helpful comments on the manuscript.
The excellent technical assistance of Cornelia Fischer is greatly appreciated.


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