A set of 48 Lactobacillus isolates recovered from the faecal swabs of different dog breeds in Andhra Pradesh were genotyped by (GTG)5 repetitive element PCR (rep-PCR). The genomic fingerprinting allowed us to differentiate five major clusters among the 48 canine faecal Lactobacillus isolates viz. cluster I (8 isolates), cluster II (6 isolates), cluster III (14 isolates), cluster IV and V (10 isolates each). The discriminatory power of rep-PCR for Lactobacillus species was found to be 0.997. Rep-PCR cluster analysis data was compared with the previous results of Lactobacillus group-specific PCR. Perfect matches occurred between these data, as evidenced by the correspondence of dendrogram clustering patterns with that of group-specific PCR patterns. This was the first report on application of rep-PCR for the genotyping of lactobacilli of dog faecal origin in India.
Lactobacillus species constitute the diverse group of microbes to be used as probiotics in dogs and are known to improve the health of dogs (Baillon et al., 2004). Probiotic characteristics of lactobacilli are linked to host specificity (Dogi and Perdigon, 2006). As most commercial canine probiotic products are of bacterial strains of human origin, probiotic strains with greater specificity for dogs are of need (Strompfova et al., 2006). Repetitive element PCR (rep-PCR) uses oligonucleotide primers complementary to the polytrinucleotide (GTG)5 sequences dispersed throughout the genome of bacterial species (Versalovic et al., 1994). It been widely applied as a fast and reliable high-throughput genotyping system for the typing of Lactobacillus strains of diverse origin (Gevers et al., 2001 and Lee et al., 2012). PCR amplification of the DNA sequences flanked between these repetitive elements was known to generate typical DNA fingerprints of lactobacilli (De Vuyst et al., 2008). To our knowledge, very few studies have actually applied rep-PCR for the molecular typing of lactobacilli commonly identified in the gastrointestinal tract of dog (Pallin, 2012 and Tang et al., 2012). The present study aimed at the (GTG)5-rep-PCR based genotyping of 48 Lactobacillus isolates, recovered from different dog breeds in Andhra Pradesh, India.
Materials and Methods
The reference strains Lactobacillus plantarum (MTCC 9496), L. delbrueckii sub-species lactis(MTCC 911), L. acidophilus (MTCC 10307) and L. rhamnosus (MTCC 1408) obtained from Microbial Type Culture Collection and Gene Bank, Chandigarh (India) were used as positive controls.
A total of 48 Lactobacillus isolates recovered from the faecal swabs of different dog breeds viz. Pomeranian (12), Labrador (15) and mongrel/indigenous dogs (21) were used in the present study. Cultural isolation was done using MRS (de Man, Rogosa and Sharpe) medium and the isolates were identified based on colony morphology (pale white/yellow), Gram’s staining (Gram positive rods), motility (non-motile), catalase (negative), oxidase (negative), IMVC (-VV-), sugar fermentation (positive for glucose, maltose, sucrose, lactose and negative for mannitol) and milk coagulation (positive) tests (Sneath and Holt, 2001). Whole cell DNA was extracted by boiling and snap chilling method (Rao, 2009). The isolates were confirmed at genus level as Lactobacillus by PCR targeting 16S rRNA gene (Garcia- Mazcorro et al., 2011). Further, all the 48 isolates were previously subjected to Lactobacillus group-specific multiplex PCR targeting 16S-23S intergenic spacer region (ISR) and its flanking 23S rRNA gene (Pena et al., 2004), where 23 isolates were found to be positive for Group IV (L. plantarum group), 6 isolates for Group I (L. delbrueckii group), 4 isolates for Group II (L. acidophilus group) and 15 isolates were not assigned into any of the group.
Rep-PCR genomic fingerprinting of a set of 48 Lactobacillus isolates and four reference strains of Lactobacillus species was done using single oligonucleotide primer (GTG)5 (5’-GTG GTG GTG GTG GTG-3’)(Gevers et al., 2001). PCR reactions were optimized in 25 μl volume reaction mixture containing 3.0 µl of DNA template (50 ng/μl); 2.5 μl of Taq buffer [10x]; 0.5 μl of dNTP mix [10mM]; 1.5 μl of MgCl2 [25mM]; (GTG)5 primer (10 pmol/μl) – 2.0 μl; 1 μl of Taq DNA polymerase (1 U/μl) and 14.5 μl of nuclease free water, under the following standardized cycling conditions – initial denaturation at 95ºC for 5 min, 30 cycles of denaturation at 94ºC for 30 sec, annealing at 40ºC for 1 min, elongation at 65ºC for 8 min, final elongation at 65ºC for 16 min. The PCR products were subjected to 1.5% agarose gel electrophoresis (Sambrook and Russell, 2001). Rep-PCR DNA fingerprints were photographed and analyzed using BIO-RAD Gel Documentation system (USA) and the supplied image lab software. The position of bands was compared using 100 bp and 1 kb DNA ladder as an external reference. Genotyping of the strains was performed using binary coding based on presence (1) or absence (0) of a particular band in the given isolate. The binary data was analyzed using dollop programme of phylip version 3.6 software and dendrograms were constructed. The similarity of band patterns was calculated using the Pearson’s correlation coefficient. Clusters were considered at a 70% similarity cut-off (Dos Anjos Borges et al., 2003). The numerical index of discrimination was calculated using the formula of Simpson’s index of diversity, D = 1-1/N (N-1) ∑ nj (nj-1), where D corresponds to the discriminatory power, N corresponds to the total number of strains, nj corresponds to the number of strains belonging to the jth type (Hunter and Gaston, 1988).
Results and Discussion
Rep-PCR typing generated 1-13 fragments per isolate, with 46 rep-PCR patterns obtained out of 48 Lactobacillus isolates analyzed. Three Lactobacillus isolates (L4, L39 and L40) recovered from three different Labrador dogs were found to have identical rep-PCR pattern. Cluster analysis of rep-PCR profiles revealed that the canine faecal Lactobacillus isolates were clustered with those of the four reference strains and were found to be dispersed over five major clusters (I to V) (Fig. 1). Cluster I encompassed isolates that were previously found positive for L. delbrueckii group (L6, L46, L38, L23, L34, L17), clustered with the reference strain L. delbrueckii (MTCC 911). Cluster II encompassed isolates that were previously found positive for L. acidophilus group (L8, L28, L24, L22), clustered with the reference strain L. acidophilus (MTCC 10307). Cluster III encompassed isolates that were previously found positive for L. plantarum group (L39, L4, L40, L16, L9, L45, L2, L42, L25, L19, L20, L21, L11, L44), clustered with the reference strains L. plantarum (MTCC 9496) and L. rhamnosus (MTCC 1408). Cluster IV also encompassed isolates that were previously found positive for L. plantarum group (L13, L37, L7, L14, L18, L33, L36, L27, L5). The remaining 15 isolates (which were previously found unassigned to any of the group) were dispersed over four clusters, i.e. cluster V (L43, L1, L30, L41, L15, L32, L29, L10, L31, L12), cluster IV (L26), cluster II (L48, L47) and cluster I (L35, L3). Comparing the results obtained by rep-PCR clustering (Fig. 1) with that of group-specific PCR results, in almost all cases lactobacilli clustered as per the grouping patterns of Lactobacillusgroup-specific PCR, with their respective reference strain, indicating the usefulness of this technique for identification or typing of Lactobacillus strains.
Fig 1: Dendogram cluster analysis of rep-PCR DNA fingerprints. L1-L48 Lactobacillus isolates, LP: L.plantarum(MTCC 9496),LD: L. delbrueckii sub species lactis(MTCC 911), LA: L. acidophilus (MTCC 10307) and LR: L. rhamnosus (MTCC 1408).
The rep-PCR has been widely used for the differentiation of Lactobacillus strains in various studies on poultry (Lee et al., 2003) and dogs (Pallin, 2012 and Tang et al., 2012). Pallin (2012) differentiated 19 genotypes among 32 Lactobacillus isolates recovered from gastrointestinal tract of dogs. Differentiation of 58 Lactobacillus isolates by rep-PCR was reported in a study on lactobacilli recovered from the beagle dogs (Tang et al., 2012). In the present study, rep-PCR discriminated 46 genotypes among 48 Lactobacillus isolates, with discriminatory power of 0.997, as calculated by Simpson’s index of diversity. In a study on discrimination of probiotic lactobacilli of poultry origin, discriminatory power of 0.889 for rep-PCR was reported (Lee et al.,2012). In the present study, rep-PCR was found to be highly desirable genotyping tool, since the discriminatory powers above 0.90 are considered highly significant (Hunter and Gaston, 1988).
In summary, (GTG)5-rep-PCR cluster analysis of the present study was in line with the grouping patterns of Lactobacillus group-specific multiplex PCR. Collectively, the present research and these past studies highlight the utility of rep-PCR genotyping tool for the rapid and reliable discrimination of canine lactobacilli.
Financial support as postgraduate fellowship and other contingency support for the work were provided by Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India.