# Molecular Microbial Analysis ofLactobacillus Strains Isolated from the Gut of Calves for Potential Probiotic Use

Authors: Lorena P. Soto; Laureano S. Frizzo; Ezequiel Bertozzi; Elizabeth Avataneo; Gabriel J. Sequeira; Marcelo R. Rosmini
Journal: Veterinary Medicine International
 (2010)

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## Abstract

The intestinal microbiota has an influence on the growth and health status of the hosts. This is of particular interest in animals reared using intensive farming practices. Hence, it is necessary to know more about complexity of the beneficial intestinal microbiota. The use of molecular methods has revolutionized microbial identification by improving its quality and effectiveness. The specific aim of the study was to analyze predominant species ofLactobacillus in intestinal microbial ecosystem of young calves. Forty-two lactic acid bacteria (LAB) isolated from intestinal tract of young calves were characterized by: Amplified Ribosomal DNA Restriction Analysis (ARDRA), by using Hae III, Msp I, and Hinf I restriction enzymes, and 16S rDNA gene sequencing. ARDRA screening revealed nine unique patterns among 42 isolates, with the same pattern for 29 of the isolates. Gene fragments of 16S rDNA of 19 strains representing different patterns were sequenced to confirm the identification of these species. These results confirmed that ARDRA is a good tool for identification and discrimination of bacterial species isolated from complex ecosystem and between closely related groups. This paper provides information about the LAB species predominant in intestinal tract of young calves that could provide beneficial effects when administered as probiotic.


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## Body

## 1. Introduction

The natural microbiota of the gastrointestinal tract has an influence on the biochemistry, immunology, physiology, and nonspecific host's resistance against infectious diseases [1]. Therefore, the role of the intestinal microbiota is of vital importance in the nutritional status of the host, and particularly in farm animals that are reared in intensive systems [2]. Because of this it is necessary to determine the complexity of the intestinal flora and recognize the different microorganisms that compose it. This is particularly relevant in the probiotic therapy field where it is necessary to distinguish between probiotics and autochthonous microbiota [3].Lactobacilli are part of the normal human gastrointestinal microbiota and may also be found in other mammalian species [4–7] and birds [8]. It has been reported that some Lactobacillus  species have probiotic properties and that they are “live micro-organisms which when administered in adequate amounts confer a health benefit on the host” [9].The first step in the probiotic production is the isolation and identification of the normal components of the gut microbiota, because one of the desirable characteristics of strains used as probiotics is that they should be autochthonous to the ecosystem of which they will be part once ingested [2]. Then, we must assess the probiotic and technological properties of the strains [4] in order to select the best examples that will form the probiotic inoculum. The inocula can be either monostrain or multistrain [10]. The latter is more effective because it can use the complementary and synergistic effects of each microorganism [11].To analyse and rapidly identify bacteria from microbial communities, classical physiological and biochemical tests are not adequate because the bacterial populations involved often have similar nutritional requirements and grow under similar environmental conditions. Currently, there is a wide variety of molecular strategies, such as PCR with specific primers, DGGE, RAPD, PFGE, FISH, RFLP, and PCR-ARDRA, among others [12], which are available to determine the species diversity of Lactobacillus  [13].The comparison of sequences of the 16S rDNA gene is a very reliable method for sorting and identifying bacterial species [14]. Because these genes are highly conserved and are present in large numbers of copies within each bacterial cell, their use as a molecular target has increased in the recent years [15].The ARDRA technique is a highly discriminatory method, simple and quick to identify Gram positive nonspore bacteria. Many authors have shown that this method is suitable for the discrimination of different species ofLactobacillus  [8, 16, 17]. In addition, many LAB used as starters or probiotics have been identified with the ARDRA methodology [18].The aim of this study was to analyse the predominant species ofLactobacillus  that constitute the intestinal microbial ecosystem of young calves, by means of isolating and identifying strains through the application of the ARDRA technique and 16S rDNA gene sequencing, as a prior step to the design of a probiotic inoculum for cattle.


## 2. Materials and Methods

### 2.1. Bacterial Isolation

Isolates were taken from the mucosa of cecum and jejunum of six young calves reared in intensive conditions. For this, a selectiveLactobacillus  Anaerobic MRS broth with Vancomycin and Bromocresol green (LAMVAB, 7) was used. Forty-one colonies were multiplied in MRS broth for 24 hours at 37°C. For preservation, the cultures were frozen at -80°C with the addition of glycerol 25% v/v.


### 2.2. DNA Isolation

An aliquot of 2 mL of each 24 hours culture was centrifuged at 14000 g (for 5 minutes). The sediment was frozen at-20°C for 24 hours to facilitate the breaking of the cells. The DNA was extracted according to Marmur [19] modified by Kurzak et al. [20] and then resuspended in 50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). An aliquot of 5 μL of this template DNA was added directly to the PCR tube. The amount of DNA obtained was quantified by measuring it in an UV spectrum (260 nm) and its integrity was visualised by agarose gel electrophoresis to 0.7% w/v, by staining with ethidium bromide and visualising under UV light.


### 2.3. 16S rDNA Amplification

The 16S rDNA gene was amplified by PCR with a thermal cycler (MJ Research). DNA fragments of approximately 1.5 kpb were amplified using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). Each PCR tube (50 μL) contained a reaction mix of 10 μL 5X PCR buffer for Taq  polymerase (Promega), 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate (Promega), 0.4 μM of each primer and 2 U of Taq  Polymerase (Promega) and 5 μL of template DNA. The termocycle programme was as follows: 94°C for 5 minutes; 30 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute; and a final extension step at 72°C for 7 minute. After cycling, the PCR products were visualised by electrophoresis on a 1% w/v agarose gel (40 minute, 75 V), by staining with ethidium bromide (0.5 μg/mL) and visualising under UV light (DyNA Light UV Transilluminator, LabNet, UV light source wavelength 302 nm).


### 2.4. ARDRA

In order to achieve complete digestion, restriction mixes (20μL of final volume) were carried out for 4 hours at 37°C. Each reaction tube contained 2 μL of 10X incubation buffer, 0.2 μL of bovine serum albumin, 6 U of the respective restriction enzyme, 2.5 μL of bidistilled water and 15 μL of PCR product. Three restriction enzymes were used: Hae  III, Msp  I and Hinf  I (Promega). The resulting digestion products were visualised under UV-light (LabNet Transilluminator, UV light source wavelength 302 nm), after agarose gel electrophoresis 3% w/v (90 minutes, 75 V) by staining with ethidium bromide (0.5 μg/mL). Restriction patterns identical to the sequenced strains led to the identification of the corresponding species [17].


### 2.5. In Silico Study

For this study, Nebcutter software testing protocols (http://tools.neb.com/NEBcutter2/index.php) were used. The theoretical restriction profiles of the 16S rDNA sequence of each species, which had a high percentage of identity in the alignment of the BLAST algorithm, were compared with profiles of the isolates in this study. Besides, theoretical restriction profiles of the 16S rDNA gene sequences were obtained from other species of Lactobacillus  and Enterococcus  to determine the power of the ARDRA technique to discriminate from other species.


### 2.6. Sequencing

The PCR products of 19 representative strains of each restriction group were purified with the Wizard PCR SV Gel & PCR Clean-Up System kit (Promega) and sequenced. The sequences were compared with the sequences deposited in the GenBank database using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/; 1).


### 2.7. Nucleotide Sequence Accession Numbers

The sequences were deposited in the GenBank database using the web-based data submission tool, BankIt (http://www.ncbi.nlm.nih.gov/BankIt, 1).


## 2.1. Bacterial Isolation

Isolates were taken from the mucosa of cecum and jejunum of six young calves reared in intensive conditions. For this, a selectiveLactobacillus  Anaerobic MRS broth with Vancomycin and Bromocresol green (LAMVAB, 7) was used. Forty-one colonies were multiplied in MRS broth for 24 hours at 37°C. For preservation, the cultures were frozen at -80°C with the addition of glycerol 25% v/v.


## 2.2. DNA Isolation

An aliquot of 2 mL of each 24 hours culture was centrifuged at 14000 g (for 5 minutes). The sediment was frozen at-20°C for 24 hours to facilitate the breaking of the cells. The DNA was extracted according to Marmur [19] modified by Kurzak et al. [20] and then resuspended in 50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). An aliquot of 5 μL of this template DNA was added directly to the PCR tube. The amount of DNA obtained was quantified by measuring it in an UV spectrum (260 nm) and its integrity was visualised by agarose gel electrophoresis to 0.7% w/v, by staining with ethidium bromide and visualising under UV light.


## 2.3. 16S rDNA Amplification

The 16S rDNA gene was amplified by PCR with a thermal cycler (MJ Research). DNA fragments of approximately 1.5 kpb were amplified using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). Each PCR tube (50 μL) contained a reaction mix of 10 μL 5X PCR buffer for Taq  polymerase (Promega), 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate (Promega), 0.4 μM of each primer and 2 U of Taq  Polymerase (Promega) and 5 μL of template DNA. The termocycle programme was as follows: 94°C for 5 minutes; 30 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute; and a final extension step at 72°C for 7 minute. After cycling, the PCR products were visualised by electrophoresis on a 1% w/v agarose gel (40 minute, 75 V), by staining with ethidium bromide (0.5 μg/mL) and visualising under UV light (DyNA Light UV Transilluminator, LabNet, UV light source wavelength 302 nm).


## 2.4. ARDRA

In order to achieve complete digestion, restriction mixes (20μL of final volume) were carried out for 4 hours at 37°C. Each reaction tube contained 2 μL of 10X incubation buffer, 0.2 μL of bovine serum albumin, 6 U of the respective restriction enzyme, 2.5 μL of bidistilled water and 15 μL of PCR product. Three restriction enzymes were used: Hae  III, Msp  I and Hinf  I (Promega). The resulting digestion products were visualised under UV-light (LabNet Transilluminator, UV light source wavelength 302 nm), after agarose gel electrophoresis 3% w/v (90 minutes, 75 V) by staining with ethidium bromide (0.5 μg/mL). Restriction patterns identical to the sequenced strains led to the identification of the corresponding species [17].


## 2.5. In Silico Study

For this study, Nebcutter software testing protocols (http://tools.neb.com/NEBcutter2/index.php) were used. The theoretical restriction profiles of the 16S rDNA sequence of each species, which had a high percentage of identity in the alignment of the BLAST algorithm, were compared with profiles of the isolates in this study. Besides, theoretical restriction profiles of the 16S rDNA gene sequences were obtained from other species of Lactobacillus  and Enterococcus  to determine the power of the ARDRA technique to discriminate from other species.


## 2.6. Sequencing

The PCR products of 19 representative strains of each restriction group were purified with the Wizard PCR SV Gel & PCR Clean-Up System kit (Promega) and sequenced. The sequences were compared with the sequences deposited in the GenBank database using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/; 1).


## 2.7. Nucleotide Sequence Accession Numbers

The sequences were deposited in the GenBank database using the web-based data submission tool, BankIt (http://www.ncbi.nlm.nih.gov/BankIt, 1).


## 3. Results

### 3.1. Identification of LAB Isolates by ARDRA

Lactic acid bacteria isolated from calves’ intestinal tract samples yielded nine unique ARDRA patterns among the 42 isolates tested (Figure1). One ARDRA pattern clearly dominated the samples, accounting for 29 of the 42 colonies tested. The other ARDRA patterns from the isolated bacteria were present at a low frequency (Table 2). Most of the ARDRA patterns derived from lactobacilli. Although the isolation medium was specific for Lactobacillus  spp., two of the patterns found belonged to Enterococcus  spp.Agarose gel with different groups of restriction. Line 1 and 11, MW ladder (100 bp);  line 2, ARDRA group 1 (L. plantarum); line 3, ARDRA group 2 (P. acidilactici); line 4, ARDRA group 3 (W. paramesenteroides); line 5, ARDRA group 4 (L. salivarius); line 6, ARDRA group 5 (L. ruminis); line 7, ARDRA group 6 (L. curvatus); line 8, ARDRA group 7 (L. farciminis); line 9, ARDRA group 8 (L. mucosae); line 10, ARDRA group 9 (E. hirae). Restriction fragments obtained with each enzyme: (a)  Hae  III, (b) Msp  I, (c)  Hinf  I.
(a)(b)(c)The restriction of the amplified fragment of the 16S rDNA gene withHae  III generated six different profiles. Lactobacillus plantarum, Weissella paramesenteroides, L. salivarius, L. ruminis  and  L. mucosae  presented specific profiles for each of these species. Instead, Pediococcus acidilactici, L. farciminis, L. curvatus  and E. hirae  showed restriction fragments different from the species listed above but not distinguishable among them (Figure 1(a)).The enzymeMsp  I also showed six different restriction profiles. Species that showed characteristic profiles were: L. salivarius, L. curvatus, L. mucosae  and E. hirae. It was not possible to differentiate between  P. acidilactici  and W. paramesenteroides  and between L. plantarum, L. ruminis  andL. farciminis  (Figure 1(b)).Hinf  I produced seven restriction profiles, five of which were typical of L. ruminis, L. curvatus, L. farciminis, L. mucosae  and E. hirae. The restriction profiles produced by the species L. plantarum  and P. acidilactici,  and by W. paramesenteroides  and L. salivarius were not able to distinguish between them (Figure 1(c)).The restriction profile of each isolate and its association with the concerned species are  detailed in Table1.Table 1
List of bacterial isolated in this study and their closest affiliation according to the 16S rDNA sequencing (1500 pb) or by belonging to the same ARDRA group.
Lactobacilli isolatesARDRA groupCalfSpecieIdentity valueAccession numberDSPV 320T91Enterococcus hirae98%FJ751777DSPV 321T81Lactobacillus mucosae99%FJ751778DSPV 322T41Lactobacillus salivarius99%FJ751779DSPV 323T41Lactobacillus salivariusDSPV 324T41Lactobacillus salivarius99%FJ751780DSPV 325T41Lactobacillus salivarius95%FJ751781DSPV 326T42Lactobacillus salivariusDSPV 327T42Lactobacillus salivarius99%FJ751782DSPV 328T42Lactobacillus salivariusDSPV 329T42Lactobacillus salivarius99%FJ751783DSPV 330T42Lactobacillus salivariusDSPV 331T42Lactobacillus salivariusDSPV 332T42Lactobacillus salivariusDSPV 333T43Lactobacillus salivarius99%FJ751784DSPV 334T43Lactobacillus salivariusDSPV 335T43Lactobacillus salivariusDSPV 336T43Lactobacillus salivariusDSPV 337T43Lactobacillus salivariusDSPV 338T43Lactobacillus salivariusDSPV 339T43Lactobacillus salivariusDSPV 340T44Lactobacillus salivarius99%FJ751785DSPV 341T44Lactobacillus salivariusDSPV 342T44Lactobacillus salivariusDSPV 343T54Lactobacillus ruminis99%FJ751786DSPV 344T45Lactobacillus salivarius99%FJ751787DSPV 345T45Lactobacillus salivariusDSPV 346T95Enterococcus hirae98%FJ751788DSPV 347T15Lactobacillus plantarumDSPV 348T25Pediococcus acidilactici99%FJ751789DSPV 349T35Weissella paramesenteroides90%FJ751790DSPV 350T25Pediococcus acidilacticiDSPV 351T35Weissella paramesenteroidesDSPV 352T65Lactobacillus curvatus99%FJ751791DSPV 353T75Lactobacillus farciminis94%FJ751792DSPV 354T15Lactobacillus plantarum99%FJ751793DSPV 355T46Lactobacillus salivarius99%FJ751794DSPV 356T46Lactobacillus salivariusDSPV 357T46Lactobacillus salivariusDSPV 358T26Pediococcus acidilactici99%FJ751795DSPV 359T46Lactobacillus salivariusDSPV 360T46Lactobacillus salivariusDSPV 361T46Lactobacillus salivariusTable 2
Number of isolates for each ARDRA group; frequency of occurrence of each species and portion of the intestine in which the isolates were obtained.
ARDRA group(a)Related speciesIsolates(b)Frequency(c)Portion of intestine4L. salivarius28/426/6Cecum2P. acidilactici3/422/6Cecum/jejunum6L. curvatus1/421/6Cecum1L. plantarum2/421/6Jejunum7L. farciminis1/421/6Jejunum9E. hirae2/422/6Cecum3W. paramesenteroides2/421/6Jejunum5L. ruminis1/421/6Cecum8L. mucosae1/421/6Cecum(a)The numbers correspond to the ARDRA groups of the agarose gel electrophoresis (Figure 1). (b)Isolates: number of isolates for each group/total isolates. (c)Frequency: number of calves in which each species was isolated/total number of calves studied.


### 3.2. In Silico Study

The size of the fragments obtained by the theoretical restriction of the sequences obtained from GenBank that had a higher percentage of identity with the isolations coincided with the restriction fragments obtained in the in vitro study.On the other hand, some nonisolated species that belonged to the same genus or phylogenetic group as that of the isolates were distinguishable with thein silico  study, in most cases by restriction with the Hinf  I enzyme (Table 3).Table 3
In silico  study.
Phylogenetic groupIsolated speciesRelated species(a)Enzymes(b)Enterococcus  groupE. hiraeE. faeciumHinf  IE. faecalisHinf  IE. lactisHinf  IE. sanguinicolaHinf IE. thailandicusHinf  ILeuconostoc   groupW. paramesenteroidesLeuconostoc paramesenteroidesHae  IIIW. confusaHinf  IW. minorHinf  IW. viridensesHinf  IL. casei-Pediococcus   groupL. mucosaeL. fermentumHinf  I and Msp  IL. reuteriHinf  I and Msp  IL.  salivariusL. maliMsp  IP. acidilacticiP. pentosaceusHinf  IL. curvatusL. caseiHinf  IL. sakeiHinf  IP. parvolusHinf  I(a)Species related with the isolates using the BLAST algorithm, and that differ in the restriction of the 16S rDNA gene profiles. (b)Enzymes for differentiating species isolated from related species.


### 3.3. Identification by Sequencing of the 16S rRNA Gene

Nineteen representative clones of the ARDRA profiles observed were selected for sequencing.The sequences of the gene fragments obtained from the 16S rDNA were aligned with those from GenBank using the BLAST algorithm. Table1 shows the percentage of identity of the isolated strains in relation to those found in the database and the access number to GenBank for each of the sequences obtained.We found that the isolates DSPV 322T, 324T, 325T, 327T, 329T, 333T, 340T, 344T, and 355T represented the ARDRA patterns that were observed most frequently (29 times) among the 42 isolates tested, and that their 16S sequence was most closely related toLactobacillus salivarius.This species was found in all the calves studied. The other species were represented by one, two or three isolates and were found in one or two calves depending on the case. On the other hand, with the exception ofP. acidilactici,  which was found in the jejunum and in the cecum, the species isolated in the large intestine were different from those isolated in the small intestine (Table 2).


## 3.1. Identification of LAB Isolates by ARDRA

Lactic acid bacteria isolated from calves’ intestinal tract samples yielded nine unique ARDRA patterns among the 42 isolates tested (Figure1). One ARDRA pattern clearly dominated the samples, accounting for 29 of the 42 colonies tested. The other ARDRA patterns from the isolated bacteria were present at a low frequency (Table 2). Most of the ARDRA patterns derived from lactobacilli. Although the isolation medium was specific for Lactobacillus  spp., two of the patterns found belonged to Enterococcus  spp.Agarose gel with different groups of restriction. Line 1 and 11, MW ladder (100 bp);  line 2, ARDRA group 1 (L. plantarum); line 3, ARDRA group 2 (P. acidilactici); line 4, ARDRA group 3 (W. paramesenteroides); line 5, ARDRA group 4 (L. salivarius); line 6, ARDRA group 5 (L. ruminis); line 7, ARDRA group 6 (L. curvatus); line 8, ARDRA group 7 (L. farciminis); line 9, ARDRA group 8 (L. mucosae); line 10, ARDRA group 9 (E. hirae). Restriction fragments obtained with each enzyme: (a)  Hae  III, (b) Msp  I, (c)  Hinf  I.
(a)(b)(c)The restriction of the amplified fragment of the 16S rDNA gene withHae  III generated six different profiles. Lactobacillus plantarum, Weissella paramesenteroides, L. salivarius, L. ruminis  and  L. mucosae  presented specific profiles for each of these species. Instead, Pediococcus acidilactici, L. farciminis, L. curvatus  and E. hirae  showed restriction fragments different from the species listed above but not distinguishable among them (Figure 1(a)).The enzymeMsp  I also showed six different restriction profiles. Species that showed characteristic profiles were: L. salivarius, L. curvatus, L. mucosae  and E. hirae. It was not possible to differentiate between  P. acidilactici  and W. paramesenteroides  and between L. plantarum, L. ruminis  andL. farciminis  (Figure 1(b)).Hinf  I produced seven restriction profiles, five of which were typical of L. ruminis, L. curvatus, L. farciminis, L. mucosae  and E. hirae. The restriction profiles produced by the species L. plantarum  and P. acidilactici,  and by W. paramesenteroides  and L. salivarius were not able to distinguish between them (Figure 1(c)).The restriction profile of each isolate and its association with the concerned species are  detailed in Table1.Table 1
List of bacterial isolated in this study and their closest affiliation according to the 16S rDNA sequencing (1500 pb) or by belonging to the same ARDRA group.
Lactobacilli isolatesARDRA groupCalfSpecieIdentity valueAccession numberDSPV 320T91Enterococcus hirae98%FJ751777DSPV 321T81Lactobacillus mucosae99%FJ751778DSPV 322T41Lactobacillus salivarius99%FJ751779DSPV 323T41Lactobacillus salivariusDSPV 324T41Lactobacillus salivarius99%FJ751780DSPV 325T41Lactobacillus salivarius95%FJ751781DSPV 326T42Lactobacillus salivariusDSPV 327T42Lactobacillus salivarius99%FJ751782DSPV 328T42Lactobacillus salivariusDSPV 329T42Lactobacillus salivarius99%FJ751783DSPV 330T42Lactobacillus salivariusDSPV 331T42Lactobacillus salivariusDSPV 332T42Lactobacillus salivariusDSPV 333T43Lactobacillus salivarius99%FJ751784DSPV 334T43Lactobacillus salivariusDSPV 335T43Lactobacillus salivariusDSPV 336T43Lactobacillus salivariusDSPV 337T43Lactobacillus salivariusDSPV 338T43Lactobacillus salivariusDSPV 339T43Lactobacillus salivariusDSPV 340T44Lactobacillus salivarius99%FJ751785DSPV 341T44Lactobacillus salivariusDSPV 342T44Lactobacillus salivariusDSPV 343T54Lactobacillus ruminis99%FJ751786DSPV 344T45Lactobacillus salivarius99%FJ751787DSPV 345T45Lactobacillus salivariusDSPV 346T95Enterococcus hirae98%FJ751788DSPV 347T15Lactobacillus plantarumDSPV 348T25Pediococcus acidilactici99%FJ751789DSPV 349T35Weissella paramesenteroides90%FJ751790DSPV 350T25Pediococcus acidilacticiDSPV 351T35Weissella paramesenteroidesDSPV 352T65Lactobacillus curvatus99%FJ751791DSPV 353T75Lactobacillus farciminis94%FJ751792DSPV 354T15Lactobacillus plantarum99%FJ751793DSPV 355T46Lactobacillus salivarius99%FJ751794DSPV 356T46Lactobacillus salivariusDSPV 357T46Lactobacillus salivariusDSPV 358T26Pediococcus acidilactici99%FJ751795DSPV 359T46Lactobacillus salivariusDSPV 360T46Lactobacillus salivariusDSPV 361T46Lactobacillus salivariusTable 2
Number of isolates for each ARDRA group; frequency of occurrence of each species and portion of the intestine in which the isolates were obtained.
ARDRA group(a)Related speciesIsolates(b)Frequency(c)Portion of intestine4L. salivarius28/426/6Cecum2P. acidilactici3/422/6Cecum/jejunum6L. curvatus1/421/6Cecum1L. plantarum2/421/6Jejunum7L. farciminis1/421/6Jejunum9E. hirae2/422/6Cecum3W. paramesenteroides2/421/6Jejunum5L. ruminis1/421/6Cecum8L. mucosae1/421/6Cecum(a)The numbers correspond to the ARDRA groups of the agarose gel electrophoresis (Figure 1). (b)Isolates: number of isolates for each group/total isolates. (c)Frequency: number of calves in which each species was isolated/total number of calves studied.


## 3.2. In Silico Study

The size of the fragments obtained by the theoretical restriction of the sequences obtained from GenBank that had a higher percentage of identity with the isolations coincided with the restriction fragments obtained in the in vitro study.On the other hand, some nonisolated species that belonged to the same genus or phylogenetic group as that of the isolates were distinguishable with thein silico  study, in most cases by restriction with the Hinf  I enzyme (Table 3).Table 3
In silico  study.
Phylogenetic groupIsolated speciesRelated species(a)Enzymes(b)Enterococcus  groupE. hiraeE. faeciumHinf  IE. faecalisHinf  IE. lactisHinf  IE. sanguinicolaHinf IE. thailandicusHinf  ILeuconostoc   groupW. paramesenteroidesLeuconostoc paramesenteroidesHae  IIIW. confusaHinf  IW. minorHinf  IW. viridensesHinf  IL. casei-Pediococcus   groupL. mucosaeL. fermentumHinf  I and Msp  IL. reuteriHinf  I and Msp  IL.  salivariusL. maliMsp  IP. acidilacticiP. pentosaceusHinf  IL. curvatusL. caseiHinf  IL. sakeiHinf  IP. parvolusHinf  I(a)Species related with the isolates using the BLAST algorithm, and that differ in the restriction of the 16S rDNA gene profiles. (b)Enzymes for differentiating species isolated from related species.


## 3.3. Identification by Sequencing of the 16S rRNA Gene

Nineteen representative clones of the ARDRA profiles observed were selected for sequencing.The sequences of the gene fragments obtained from the 16S rDNA were aligned with those from GenBank using the BLAST algorithm. Table1 shows the percentage of identity of the isolated strains in relation to those found in the database and the access number to GenBank for each of the sequences obtained.We found that the isolates DSPV 322T, 324T, 325T, 327T, 329T, 333T, 340T, 344T, and 355T represented the ARDRA patterns that were observed most frequently (29 times) among the 42 isolates tested, and that their 16S sequence was most closely related toLactobacillus salivarius.This species was found in all the calves studied. The other species were represented by one, two or three isolates and were found in one or two calves depending on the case. On the other hand, with the exception ofP. acidilactici,  which was found in the jejunum and in the cecum, the species isolated in the large intestine were different from those isolated in the small intestine (Table 2).


## 4. Discussion

The identification of microbial species through the use of phenotypic methods can sometimes be uncertain, complicated and time-consuming. The use of molecular methods has revolutionised their identification, by improving the quality and effectiveness of this identification. Some of these methodologies use either the rDNA spacer region or its target. These techniques are useful for both the identification and reliable detection of different bacterial species as well as the monitoring of the species [21]. In this way, members of a probiotic multistrain inoculum can be identified and distinguished from strains that share the same environment such as starters in foods (yogurt, cheese, etc.).The use of species-specific primers or probes is not applicable in environments where there are severalLactobacillus  species because prior knowledge of them is required. In these cases, more general molecular tools should be applied [21]. The techniques used to identify Lactobacillus species in different environments are the comparison of total or partial sequences of 16S rDNA, ARDRA patterns of 16S rDNA or the intergenic region of the 16S-23S rDNA [5, 8, 22].While the use of 16S DNA sequencing methods gives a high resolution of the diversity of microbial species in an environment, it is very time-consuming and too costly to be used for routine screening of samples. Methods for the initial analysis of faecal samples should be rapid and able to give a broad view of the microbial ecology. ARDRA has been used to compare bacterial isolates within a wide range of microbial communities. The advantages of ARDRA are that it is rapid, reproducible, relates to microbial diversity, and will be invaluable in analysing a greater number of samples together with experimental objectives such as dietary interventions [6].In the present work, ARDRA allowed us to differentiateEnterococcus hirae  from the rest of the Lactobacillus  spp. isolates. This differentiation was observed by restricting with any of the three enzymes used.TheLactobacillus  isolated belonged to two groups: the L. casei-Pediococcus  group and the Leuconostoc group. The latter includes the species Weissella  and Lactobacillus paramesenteroides, which can be differentiated from the L. casei-Pediococcus  group by the typical profile obtained with the restriction enzymes Hae III and Hinf I. This methodology also allowed the distinction between phylogenetically related species belonging to the L. casei-Pediococcus  group. These species were L. ruminis, L. salivarius, L. curvatus, P. acidilactici, L. farciminis, L. plantarum,   and L. mucosae, which have a 16S rDNA homology of 90.3 to 99% [23].The similarity between the profiles obtained by thein silico  study of the sequences of the GenBank and the isolates revealed that the strains of the same species had similar profiles. This result proved to be another tool for the identification of the species. The possibility to obtain these profiles, distinguishable between the isolates, together with the differentiation of these isolates from other related species (Table 3), shows that this technique allows the distinction of species with high homology. Such is the case of E. faecium  and E. faecalis, which were also found in the intestines of calves [7] and can be distinguished from E. hirae by the restriction enzyme Hinf I. There are species within the same phylogenetic groups, such as L. fermentum  and L. reuteri, which have higher homology than others and are most closely related to L. mucosae  [24]. Despite these similarities, in the present work the in silico study showed that the latter could be distinguished from the first two by the ARDRA methodology (Table 3). These results show that the ARDRA technique is a tool that highly discriminates between LAB species and seems to group the isolates by species and then sequence some exponents of each group. This may save both time and money when it is necessary to analyse large numbers of isolates.L. salivarius was the predominant species in the gastrointestinal tract of calves. It was found in the cecum of all individuals (Table 2) and in some animals it was the only species isolated. This species was also detected by Schneider et al. [7] in calves reared in the same geographical area. L. salivarius is an inhabitant of the gastrointestinal tract of other species such as chickens [8], pigs [25], and humans [4, 5]. Many strains that correspond to this species have been studied to evaluate their probiotic properties. Some strains isolated from infants have shown antimicrobial capacity against pathogens [26], and, in particular, L. salivarius  CTC2197 was able to prevent the colonization of Salmonella enteritidis  in chickens [27].The probiotic properties of microorganisms are characteristic of each strain. Therefore, belonging to a species is not sufficient to guarantee the possession of such properties. For a strain to be used as a probiotic, it should be considered GRAS, that is, possessing probiotic effects and technological capabilities suitable for its propagation and preservation over time. Therefore, in order to select the best specimens, in future works we aim at evaluating the probiotic properties of each isolate obtained in this study (in vitro: aggregation, coaggregation with pathogens, production of inhibitory substances, bile and pH resistance; in vivo: effect on calves performance, challenge with pathogen). The knowledge of such properties will allow the development of an inoculum for young calves to improve their performance in intensive farming systems.



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*Source: 274987-2009-09-01.xml*