Streptococcus alactolyticus was the dominating culturable lactic acid
bacterium species in canine jejunum and feces of four fistulated dogs
Minna L. Rinkinen∗, Joanna M.K. Koort†, Arthur C. Ouwehand‡, Elias
Westermarck∗, K. Johanna Björkroth†
∗Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, P.O.
Box 57, FIN–00014 University of Helsinki, Finland
†Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine,
P.O. Box 57, FIN–00014 University of Helsinki, Finland
‡Department of Biochemistry and Food Chemistry, University of Turku,
FIN-20014 Turku, Finland
Minna Rinkinen, Department of Clinical Veterinary Sciences, Faculty of Veterinary
Medicine, P.O. Box 57, FIN-00014 University of Helsinki, Finland
Tel: +358-50-549 78 38
Fax: +358-9-191 49 670
23 E-mail: Minna.Rinkinen@helsinki.fi
Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was
cultured and enumerated using MRS agar. LAB levels ranging from 1.4×106 to
1.5×107 CFU ml-1 were obtained in jejunal chyme. In the fecal samples 7.0×107 and
2.0×108 CFU g-1 were detected. Thirty randomly selected isolates growing in the
highest sample dilutions were identified to species level using numerical analysis of
16 and 23 S rDNA RFLP patterns (ribotyping) and 16S rDNA sequence analysis.
According to these results, Streptococcus alactolyticus was the dominant culturable
LAB species in both faeces and jejunal chyme. In addition, Lactobacillus murinus and
Lactobacillus reuteri were detected.
Keywords: Culturable canine intestinal lactic acid bacteria, Streptococcus
alactolyticus, jejunal chyme
LAB are gram-positive, aerotolerant, catalase negative rods or cocci producing
lactic acid as their main fermentation product. They form a heterogenous group of
bacteria, the genera of Enterococcus, Lactobacillus, Lactococcus, Leuconostoc,
Pediococcus, Streptococcus and Weissella being the best known. Most LAB are non-
pathogenic and they are associated with a wide variety of sources, such as plant
material and various foods . They also form a substantial part of the intestinal
microbiota, and are believed to have a major effect on host’s well-being .
The knowledge of the canine intestinal LAB is scarce. Only few studies have
previously addressed the canine intestinal microbiome [3-7]. Most of these studies
date back to times when novel molecular techniques were not available and LAB were
not identified to species level. Also the classification and nomenclature of LAB has
been subjected to various changes during recent years.
In order to obtain knowledge of the culturable LAB species in canine intestinal
microbiome, we enumerated and identified jejunal and fecal LAB associated with four
permanently fistulated beagles. Culturing was done using anaerobic incubation and
MRS agar and the predominating LAB species were identified to the species level
using molecular methods.
Materials and methods
The dogs used in the study originated from the experimental animal colony of
Helsinki University. They all had permanent jejunum nipple valve fistulas operated
into the proximal jejunum, 60 cm distally from pylorus. The operations had been
performed one to three years before this study took place according the method
described by Wilsson-Rahmberg and Jonsson . The fistulas did not cause any
clinical discomfort or gastrointestinal symptoms to the dogs. The dogs had been used
only for sampling of jejunal chyme and were not medicated. At the time of this study,
the dogs were from three to six years of age. They were fed canned commercial
balanced dog food, the main ingredients of which were cereal, meat, animal
derivatives, oils and fats, vegetable protein extract and vegetable derivatives. The
composition was as follows: raw protein 9 %, raw fat 6 %, raw fiber 0.4 %, calcium
0.3 % and phosphorus 0.25 %; moisture 80 %. The study had been approved by the
Helsinki University ethics committee.
For the microbiological analyses, a sample of approximately 8 ml of jejunal
chyme was collected from 4 permanently fistulated, healthy castrated male beagles 2
hours postprandial. Fecal samples were collected manually from rectum of two dogs.
All samples were immediately submitted to the laboratory for microbiological
Samples were homogenized in 0.1% peptone water using a Stomacher blender.
Serial 10-fold dilutions of the homogenized samples were made from 10-2 to 10-8 in
0.1% peptone water. LAB were enumerated on MRS agar (Oxoid, Basingstoke,
England) inoculated using the spread plate technique. All plates were incubated in an
2 atmosphere (Anaerogen, Oxoid, 9-13% CO 2 according to the
manufacturer) at 30°C for 3 to 4 days. Five colonies from each sample were picked
randomly from the plates showing growth of less than 100 colonies. Depending on the
sample, these dilutions were 106 × or 107 × of the original sample. Isolates were
cultured to purity using MRS agar/broth for species identification. Gram staining and
catalase testing were performed before the molecular analysis.
Two ml of cultures grown overnight at 30°C in MRS broth were used for
DNA isolation. DNA was isolated by guanidium thiocyanate method by Pitcher and
others  as modified by Björkroth and Korkeala  by the combined lysozyme and
mutanolysin (Sigma) treatment. HindIII and EcoRI enzymes were used for restriction
endonuclease treatment of 4 μg of DNA as specified by the manufacturer (New
England Biolabs), and Restriction Endonuclease Analysis (REA) was performed as
described previously . Southern blotting was done using a vacuum device
(Vacugene, Pharmacia), and the rDNA probe for ribotyping  was labelled by
reverse transcription (AMV-RT, Promega and Dig Labelling Kit, Roche Molecular
Biochemicals) as previously described . Membranes were hybridized at +58 ºC
overnight, and the detection of the digoxigenin label was performed as recommended
by the manufacturer.
For pattern analysis, the membranes were scanned with a Hewlett-Packard
(Boise, Idaho, USA) Scan-Jet 4c/T scanner. The EcoRI and HindIII ribopatterns were
compared with the corresponding patterns in the previously established LAB database
at the Department of Food and Environmental Hygiene. Ribopatterns were analyzed
using the BioNumerics 3.0 software package (Applied Maths, Sint-Martens-Latem,
Belgium). The similarity between all pairs was expressed by Dice coefficient
correlation, and UPGMA clustering was used for the construction of the dendrogram.
Based on the use of internal controls position tolerance of 1.5% was allowed for the
bands. For the dendrogram combining the information from EcoRI and HindIII
ribopatterns, equal weight was given to both banding pattern types.
Chromosomal DNA for use in PCR was isolated as for ribotyping. The nearly
complete (at least over 1400 bases sequenced) 16S rRNA gene was amplified by PCR
with a universal primer pair, 5’-CTGGCTCAGGAYGAACGCTG-3’ as the forward
primer, corresponding to positions 19-38 in Escherichia coli 16S numbering, and 5’-
AAGGAGGTGATCCAGCCGCA-3’ as the reverse primer, complementary to
positions 1541-1522. Sequencing of the purified (QIAquick PCR Purification Kit,
Qiagen) PCR product was performed by Sanger’s dideoxynucleotide chain
termination method as two long and two shorter reactions. Samples were run in a
Global IR32 using LiCor sequencing device with e-Seq 1.1 software (LiCor) according
to the manufacturer’s recommendation. Overlapping complementary sequences were
joined by the Align IR 1.2 program (LiCor). Nucleotide sequence data were analyzed
with version 32.0 of the BioNumerics software package (Applied Maths).
Phylogenetic analysis of the 16S rDNA sequence of strains was performed by using
the Bionumerics 3.0 software package (Applied Maths). Calculation of the level of
similarity and construction of a phylogenetic tree was based on the neighbour-joining
method. Bootstrap probability values were calculated to branching points resampling
LAB levels ranging from 1.4×106 to 1.5×107 CFU ml-1 were obtained in the
jejunal chyme. In the two fecal samples, 7.0×107 and 2.0×108 CFU g-1 were detected.
All isolates were gram positive and catalase negative. Twenty of them possessed
coccal morphology while 10 were rod shaped.
Three LAB species, S. alactolyticus, L. murinus and L. reuteri were detected
by the means of the RFLP database and 16 S rDNA sequencing. Fig. 1 a and b show
the dendrograms generated by EcoRI and HindIII restriction enzymes, respectively.
Fig. 1c was made by combining the information from both restriction enzyme
analyses together. All types of analyses resulted in species-specific clusters showing
pattern similarity values ranging from 46.2 to 100%. In the distance matrix tree based
on the 16S sequences (Fig. 2), strains were located in 3 branches corresponding well
to the species-specific clusters obtained by ribotyping.
Table 1 shows the LAB species distribution within the 30 randomly selected
isolates identified to the species level. Within a species, identical ribopatterns were
obtained from the isolates by both enzymes used. Fig 1. shows the representative
patterns of all different types obtained. S. alactolyticus was found to be the dominant
LAB species isolated from both faeces and jejunal chyme. L. murinus was associated
with 3 of the dogs while 2 dogs were found to carry L. reuteri (Table 1).
S. alactolyticus was found to be the dominating culturable LAB species in the
jejunal and faecal samples associated with the dogs in the present study. It was found
in all the dogs and in every sample. In addition to S. alactolyticus, strains belonging to
species L. reuteri and L. murinus were detected to a lesser extent (Table 1).
To our knowledge, this is the first report on the composition of the most
prevalent culturable LAB species in the canine jejunal chyme and faeces. S.
alactolyticus was described by Farrow and others , they isolated it from the
intestines of pigs and the faeces of chicken. This organism has also been documented
to reside in the pigeon intestines, although only as a minor part of the microbiota .
Ureolytic Streptococcus intestinalis was reported to be the predominant member of
the pig colonic microbiota . Later work by Vandamme and co-workers 
revealed that S. intestinalis is a junior subjective synonym of S. alactolyticus and
therefore pigeons must also be considered as a host of S. alactolyticus.
In a recent study , the faecal microbiota of four Labrador retrievers was
examined, and S. bovis and L. murinus were found to be the most prevalent culturable
LAB species. In this study, there was variation in the occurrence of LAB species
between the different samples. This was not clearly evident in our work. However, it
has been documented that the canine intestinal microbiota may change in time , so
the finding could reflect natural variation. The composition of intestinal bacterial flora
is known to be host species specific and dependent on dietary and environmental
factors . This may also explain the differences in LAB strains between the present
study and the work published by Greetham and co-workers . In addition, their
study dealt only with the faecal microbiota whereas we identified the most prevalent
culturable small intestinal LAB, too. However, the dogs we studied live in a colony of
experimental animals. They have very few contacts with dogs outside the colony and
their lives do not fully resemble the life of a domestic pet. On the other hand, the
possibilities to examine the small intestinal microbiota in healthy, non-medicated pet
dogs are practically nonexistent.
LAB are reported to have several beneficial effects on host’s well being. They
may suppress the growth of intestinal pathogens by the means of competitive
exclusion [18, 19], and they have been documented to enhance the immune functions
in humans and mice [20, 21]. It is noteworthy that with the exception of L. reuteri,
none of the LAB strains detected in this study are used in commercial probiotic
Human gut microbiome has already been studied using various culture-
independent methods whereas in association with canine intestinal microbiome these
studies are only on their way. Therefore, our results form a basis for the future either
culture-dependent or independent studies dealing with canine intestinal microbiota.
We conclude that knowledge of the dominant culturable LAB in the dog is necessary
for further studies on the canine intestinal microbial ecology.
We would like to thank Ms. Henna Niinivirta for the excellent technical
assistance. Financial support from the Academy of Finland (project 100479) for the
identification of the LAB strains is gratefully acknowledged.
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Table 1. Species division (number of isolates) within the LAB 30 isolates cultured
pure from jejunal chyme or feces of 4 castrated male dogs with permanent jejunum
nipple valve fistulas. Species were identified by the means of a RFLP database and
16S rDNA sequencing.
Dog 1 Dog 2 Dog 3 Dog 4
LAB species jejunal
4 5 3 3 2 3
1 2 2 1
Fig. 1. (a), (b) and (c) present numerical analysis of 16 and 23S RFLP patterns
(ribotypes) generated by EcoRI, HindIII and an analysis combining the information of
both restriction enzymes, respectively. Clusters show representative patterns of all
different types obtained. Numerical analyses of the patterns are presented as
dendrograms, left side of the EcoRI and HindIII banding patterns possesses high
molecular weights, < 23 kbp, and right side >1000 bp.
Scales show percentual similarities of the patterns.
Fig. 2. Phylogenetic tree based on similarity values of almost entire 16S rDNA
sequences (at least 1400 bp). Bootstrap probability values from 1000 trees resampled
are given at the branch points. C. jejuni was used to root the tree.
Scale shows 5% difference.
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