Transition of the intestinal microbiota of cats with age

The transition of intestinal microbiota with age has been well described in humans. However, the age-related changes in intestinal microbiota of cats have not been well studied. In the present study, we investigated the composition of intestinal microbiota of cats in 5 different age groups (pre-weanling, weanling, young, aged, senile) with a culture-based method. For lactobacilli and bifidobacteria, we also quantified with molecular-based method, real-time PCR. The results suggested that the composition of the feline intestinal microbiota changes with age, while the changes were different from those of humans and dogs. Bifidobacteria which are predominant in human intestine or lactobacilli which are predominant in dog intestine, did not appear to be important in cat intestines. Enterococci, instead, seem to be major lactic acid producing bacteria in cats. We also identified lactobacilli and bifidobacteria at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age.


Introduction
The cat is one of the most popular companion animals around the world and is playing important roles as a member of the family. Recent advances of veterinary practices have dramatically prolonged the life span of cats, making disorders associated with aging in the aged cat population more serious problem.
It is now well accepted that the intestinal microbiota has great impacts on health and disease of the host, and the impacts are both in beneficial and harmful ways. Therefore, maintaining the intestinal microbiota in good condition is important for good health of the host. It has been reported that the composition of the intestinal microbiota is influenced by various factors. Among the many factors that influence the composition of the intestinal microbiota, age is one of the most critical [1]. PLOS  It has been reported that the composition of the human intestinal microbiota changes with age, and "aging of the intestinal microbiota" is thought to be somehow related to the health of the host [1]. For example, it was revealed that bifidobacteria, which are also often used as probiotics, decrease during the transition from middle age to old age, while the numbers of Clostridium perfringens, lactobacilli, enterococci and Enterobacteriaceae increase [1]. Recently, the authors have reported the transition of intestinal microbiota of dogs with age [2]. However, although the compositions of the intestinal microbiota of various animal species have been studied [1,3,4], transition of the intestinal microbiota of cats with age has not been well studied.
In this study, we analyzed the composition of the intestinal microbiota in cats of different age groups to elucidate the age-dependent transitions.

Animals
In this study, we collected fresh fecal samples from 5 different age groups of cats, and each group contained 10 animals (Table 1). Animals in pre-weanling and weanling groups were 12 to 13 days old and 7 to 8 weeks old respectively. Young cats were 2 to 3 years old, and aged cats were 10 to 14 years old. Pre-weanling, weanling, young and aged cats were mixed breed bred and maintained at Kitayama Labes Co., Ltd (Nagano, Japan). Pre-weanling cats were breast-fed and housed in group. Weanling, young and aged cats were housed individually and fed CF6 diet (Oriental Yeast Co., Ltd, Tokyo, Japan). On the other hands, senile cats were 16 to 19 years old mixed breed except for one American shorthair. Two of them were maintained individually at Kitayama Labes Co., Ltd and fed CF6 diet. Eight senile cats were kept in owner household and fed commercial diet (JP-style dietetics kidney keep (Nisshin Petfood) for 1, Neko Genki series (Unicharm) for 2, Gin no spoon series (Unicharm) for 2, KalKan (Mars) for 2, Royal Canin pH control (Royal Canin) for 1). Owners provided written informed consent before collection of the samples. All cats were apparently healthy and had no symptom of illness except for a senile cat with urinary calculus and a senile cat with renal disease.

Collection of fecal samples
Fresh fecal samples were collected immediately after defecation avoiding contamination from the environment and kept under anaerobic conditions with AnaeroPack 1 Kenki (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan). In the case of the pre-weanling group, kittens were provoked to defecate. Samples were refrigerated and transported to Laboratory of Veterinary Public Health, the University of Tokyo, the next day. Samples from the senile cats were aseptically collected immediately after defecation at their veterinary clinic and transferred to the laboratory in the same way.

Species identification of isolates
The DNA of isolated bacteria was extracted using a Simple Prep 1 DNA Extraction Kit (Takara Bio Inc., Kusatsu, Shiga), according to the manufacturer's protocol. The 16S rRNA gene was amplified from the DNA extracts using a Bacterial 16S rDNA PCR Kit (Takara Bio), and the PCR products were purified with NucleoSpin 1 Gel and PCR Clean-up (Macherey-Nagel GmbH & Co. KG., Düren, Germany), according to the manufacturer's instructions. The sequences of the purified products were analyzed by FASMAC Co., Ltd. sequencing service (Kanagawa, Japan). The sequences obtained were compared with those available in nucleic acid databases using the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/) [7] and species were identified as the top hit species with <98% similarity using the EzTaxon-e database.

Quantification of Lactobacillus group and Bifidobacterium by real-time PCR
Bacterial DNA was extracted directly from fecal samples using QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's protocol. The real-time PCRs were carried out by using MyiQ real-time PCR system (Bio-Rad, Tokyo, Japan). The reaction mixture (20 μl) contained 10 μl of the iQ SYBER Green Supermix (Bio-Rad), 1 μl of fecal DNA, and 400 μmol/l of each primer. For quantification of Lactobacillus group, the primers 5'-AGCAGTAGGGAATCTTCCA-3' (forward) and 5'-CACCGCTACACATGGAG-3' (reverse) were used [8]. For quantification of Bifidobacterium, the primers g-Bifid-F (5'-CTCCTGGAAACGGGTGG -3') and g-Bifid-R (5'-GGTGTTCTTCCCGATATCTACA -3') were used [9]. The thermal program consisted of initial denaturation at 95˚C for 3 min followed by 40 cycles of at 94˚C for 10 s, primer annealing at optimum temperature for 30 s and at 72˚C for 40 s. The annealing temperatures for Lactobacillus group and Bifidobacterium were 58˚C and 55˚C, respectively. Fluorescent products were detected at the last step of each cycle. Melting curve analysis of the product was performed after completion of the amplifications to determine the specificity of the PCR. A plasmid containing a partial sequence of the 16S rRNA gene identical to the targeted bacteria was constructed in our laboratory and used as a standard DNA for the real-time PCR.

Statistical analysis
Bacterial numbers and gene copy numbers of Lactobacillus group and Bifidobacterium were compared among 5 groups by Tukey's t-test. Detection frequencies were compared by Fisher's exact tests. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). [10] Results The composition of the intestinal microbiota of cats in different age groups The compositions of the fecal microbiota in different age groups are shown in Table 3. Enterococci were detected in almost all animals, while the mean number of enterococci was significantly lower in the senile group than that in the pre-weanling group. On the other hand, bifidobacteria were detected in only 1 out of 10 aged and 3 out of 10 senile cats. Lactobacilli were also detected only in 3 out of 10 pre-weanling, 4 out of 10 young, 1 out of 10 aged and 4 Table 3. Fecal microbiota of the different age groups of cats.

Bacterial groups
10.5 ± 0.3 a (10) 9.6 ± 0.2 b (10) 9.5 ± 0.5 (10) 9.4 ± 0.4 Megasphaera (0) a (0) b 6.9 ± 1.5 (6) a,b,c (0) c 5.9 ± 0.9 out of 10 senile cats. Although clostridia were detected in only 1 out of 10 aged cats, this bacterial group was detected in many of the cats of four other age groups. The number of Enterobacteriaceae was significantly higher in the pre-weanling kittens compared with four other age groups. Compared with the pre-weanling, young and senile cats, the number of Bacteroidaceae was significantly higher in the weanling cats. The number of eubacteria was significantly higher in the pre-weanling kittens compared with the weanling and young cats. Megasphaera and staphylococci were detected only in the young and senile cats. The detection rate of Megasphaera in the young cats was higher compared with three other age groups, while those of staphylococci in the young and senile cats were also higher compared with the three other groups. The total bacterial count became the highest in the weanling cats.

Species identification of lactobacilli and bifidobacteria
Since bifidobacteria and/or lactobacilli in the intestinal microbiota are thought to play beneficial roles in maintaining host well-being and often used as probiotics, isolated strains of bifidobacteria and lactobacilli were subjected to nucleotide sequencing of the 16S rRNA genes to delineate them at the species level (Table 4). Among the 4 strains of bifidobacteria, one strain from aged cat was identified as Bifidobacterium longum subsp. longum, while one B. longum subsp. suis and two B. pseudocatenulatum strains were isolated from senile cats.
In pre-weanling and young groups, only Lactobacillus reuteri was detected, while this species was not detected from aged and senile cats. Various species other than L. reuteri, L. johnsonii, L. animalis, L. fermentum and L. mucosae, were isolated from the senile group (Table 4).

The numbers of gene copies of Lactobacillus group and Bifidobacterium
The numbers of 16S rRNA gene copies of Lactobacillus group and Bifidobacterium were measured by real-time PCR as an indicator of cell number. With PCR method, Lactobacillus group was detected from all samples. There was no significant difference in detection rate and number of gene copies of Lactobacillus group among age groups. On the other hand, Bifidobacterium were detected from 35 out of 50 samples and the number of gene copies of Bifidobacterium in senile cats was significantly higher than that in young and aged cats (Table 5).

Discussion
In this study, we analyzed the composition of the intestinal microbiota of cats in different age groups. Although we did not follow up the same cohort of animals for prolonged years, our results seem to suggest aging of the intestinal microbiota in cats. Although the population of eubacteria was lower in the pre-weanling cats compared with the weanling and young cats, Bacteroidaceae and eubacteria were predominant throughout life. Enterobacteriaceae and enterococci were predominant in the pre-weanling kittens, while the number of Enterobacteriaceae and enterococci appeared to decrease as the animals got older. Lactobacilli were detected from less than half of the animals in all age groups, and bifidobacteria were detected only from one aged and three senile cats. These results basically agree with the previous findings that the detection rate of lactobacilli was low and bifidobacteria was hardly detected from all age groups in cats [4].
The present study employed a culture-based method, which was essentially the same as those described by Mitsuoka et al. [1,5], to analyze the composition of the intestinal microbiota of cats, because we aimed to not only identify beneficial age-related changes in the composition of feline intestinal microbiota but also to isolate particular bacteria for future development of probiotics targeting cats. On the other hand, recent studies in which they employed a culture independent method, pyrosequencing, have reported that lactobacilli and bifidobacteria are abundant in cats [11]. Therefore, we further tried to quantify these two bacterial groups using molecular method, real-time PCR. Unlike culture-based method, Lactobacillus group were detected from all cats and Bifidobacterium was detected from 70% of cats by real-time PCR. The selective media used in the present study might not be perfectly suitable for lactobacilli and bifidobacteria of cat origin. Furthermore, molecular method, containing real-time PCR, is able to not only live but also dead bacterial DNA. However, the highest gene copy numbers detected were 7.0 ± 0.7 (log 10 /g wet weight of feces) in pre-weanling group for Lactobacillus group and 5.8 ± 0.8 in pre-weanling and senile group for Bifidobacterium. The present study revealed that lactobacilli and bifidobacteria are the minor components of cat intestinal microbiota.
Recent advances in the molecular methods for the studies on microbiota have made quick and more comprehensive analysis of the intestinal microbiota possible. A previous study utilizing molecular method reported that the composition of the intestinal microbiota of four healthy conventionally raised cats (ages 13-18 months) were comprised of about 68% of phyla Firmicutes group, followed by Proteobacteria (14%), Bacteroidetes (10%), Fusobacteria (5%) and Actinobacteria (4%) [12]. Another previous study with Illumina sequencing has reported that Lactobacillus and Bifidobacterium are extremely common (35% and 20% respectively) in cats at about 18 weeks of age [11]. Furthermore, the previous study with pyrosequencing reported that the most abundant phylum was Firmicutes, followed by Actinobacteria in the intestinal microbiota of twelve healthy cats (ages 0.7-6.7 years) [13]. Most of the bacteria classified in lactobacilli, enterococci, clostridia and eubacteria in this study belong to phylum Firmicutes, while most of the bacteria classified in Bacteroidaceae in this study belong to the phyla Bacteroidetes or Fusobacteria. In addition, bacteria identified as Enterobacteriaceae in this study belongs to phylum Proteobacteria, indicating that our results obtained by the culture method did not coincide with these two previous findings obtained by using molecular techniques. On the other hands, the previous study with Mean ± SD of log 10 /g feces when the organism was present (number of subjects in which the organism was detected by real-time PCR) a,b The same superscript letters in the same horizontal line indicate significant differences (P<0. metagenomics analysis reported that the composition of intestinal microbiota of five healthy cats (from 3-16 years) were comprised of~68% of phylum Bacteroides/Chlorobi group, followed by Firmicutes (~13%) and Proteobacteria (~6%) [14]. From the perspective of that phylum Bacteroides/Chlorobi group was the most dominant, this study corresponds to our results obtained by using the culture method. To elucidate the cause of these contradictions, the influence of various factors including age, breed and environment such as diet and rearing conditions should be further studied. It has been reported that the composition of the intestinal microbiota of human begins changes with age. The dominance of bifidobacteria observed in infancy is not evident in the middle-aged population, and there are slight reductions in total bacterial counts. Furthermore, bifidobacteria become completely undetectable in some individuals in old age, while the prevalence and the numbers of Clostridium perfringens, Lactobacillus, Enterobacteriaceae and Enterococcus markedly increase [1,4]. Although bifidobacteria, the most predominant bacteria in infants and one of the predominant bacteria in adults in humans [4], are thought to confer some health benefits, the finding in this study that bifidobacteria were isolated only from one aged and three senile cats suggested that bifidobacteria may not play important roles in cats in contrast to humans. Furthermore, although our previous study showed that lactobacilli may exert some health benefits in dogs similar to those anticipated for bifidobacteria in humans [2], lactobacilli were only minor constituents in cats. On the other hand, enterococci were predominant among lactic acid producing bacteria in pre-weanling cats, and the number of enterococci appeared to decrease in elder individuals, in contrast to the finding in humans that the numbers of enterococci in human gut increased with age [4]. It seems, therefore, likely that enterococci may have some health benefits in cats similar to those anticipated for bifidobacteria in human and lactobacilli in dogs. The roles played by each component bacteria in microbiota might be different depending on the species of animals in question.
Although the number of Lactobacillus strains isolated by culture-based method was limited, L. reuteri was the only species isolated from pre-weanling and young cats, while various species of Lactobacillus other than L. reuteri were isolated from elder cats. In dogs, L. johnsonii strains were mostly isolated from pre-weanling dogs, while L. animalis was isolated from all age groups except for the senile group [2], suggesting that L. johnsonii might be a specific species for infant dogs. The present study clearly indicated that the age related changes in the intestinal microbiota are different between cats and dogs, also at the level of bacterial species.
The present study showed that the intestinal microbiota of cats undergoes age-dependent changes at the levels of both bacterial groups and species, as in the case of human and dog intestinal microbiota, although the changes in senile cats should be further studied because only the senile cats were kept under different conditions. The present study also suggested that the roles played by some intestinal bacterial groups of cats might be different from those of humans and dogs. Our results suggested that Bifidobacterium and Lactobacillus species may not be as important for cat health as in the case of humans and dogs. Instead, enterococci were suggested to be one of the major lactic acid producing bacteria playing important roles to control intestinal conditions of cats. The present study indicates the importance of development of probiotics specific to cats. Considering that some major human probiotic species are predominant in healthy infants and decrease in elder individuals [15][16][17], further studies on the age related changes of enterococci at the species level should be performed. It is known that age-associated changes, such as reduced digestive function and constipation, often occur in senile cats and these symptoms may also be related to intestinal microbiota in cats [18]. It is expected that bacterial species specific to cats will play an important part in reducing these symptoms as probiotics.