Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk

The Veterinary Journal 185 (2010) 193–198

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Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk Rocío Martín a, Mónica Olivares b, Manuela Pérez a, Jordi Xaus b, Celina Torre c, Leonides Fernández a, Juan M. Rodríguez a,* a

Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain Department of Nutrition and Health, Puleva Biotech, 18004 Granada, Spain c Department of Research and Development, Affinity Petcare, 08174 Sant Cugat del Valles, Spain b

a r t i c l e

i n f o

Article history: Accepted 18 April 2009

Keywords: Lactobacillus Canine Bitch Milk Probiotics Lactation

a b s t r a c t Canine milk protects puppies against infectious diseases through a variety of mechanisms. In this study, the presence of potentially probiotic lactic acid bacteria grown on MRS-Cys agar plates from milk of nine bitches was investigated. The Gram positive catalase negative bacilli identified in this study belonged to four Lactobacillus species (Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus murinus, Lactobacillus animalis), as well as one isolate that was identified as Weissella viridescens. Random amplified polymorphic DNA profiling revealed 28 different genetic profiles among the lactobacilli isolates. Their probiotic potential was evaluated through different assays, including survival in conditions that resemble those existing in the gastrointestinal tract, production of antimicrobial compounds, adherence to intestinal mucin, degradation of mucin and pattern of antibiotic sensitivity. Some strains showed potential for future applications as canine probiotics. Ó 2009 Elsevier Ltd. All rights reserved.

Introduction Lactation is a critical period in canine breeding and precocious weaning is usually associated with high mortality and morbidity. Canine milk not only fulfils all the nutritional requirements for rapidly-growing puppies, but also protects them against infectious diseases. The protective effect of canine milk is due to the combined action of a variety of protective factors present in colostrum and milk, such as immunoglobulins, immunocompetent cells, antimicrobial fatty acids, polyamines, fucoylated oligosaccharides, lysozyme and lactoferrin. Microbiological studies focussed on canine milk are scarce and have been restricted to the identification of potential pathogenic bacteria in clinical perinatal infections, including lactational mastitis in bitches and septicaemia in neonatal puppies (Jung et al., 2002; Schäfer-Somi et al., 2003; Ververidis et al., 2007). In contrast, human breast milk has been recently shown to be a source of probiotic lactic acid bacteria to the infant gut (Heikkilä and Saris, 2003; Martín et al., 2003, 2007a, 2009; Olivares et al., 2006). Probiotics are living micro-organisms which, upon ingestion in certain numbers, exert health affects beyond inherent basic nutrition (Guarner and Schaafsma, 1998). Probiotic bacteria selected for

* Corresponding author. Tel.: +34 91 3943747; fax: +34 91 3943743. E-mail address: [email protected] (J.M. Rodríguez).

1090-0233/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2009.04.014

practical applications must retain the characteristics for which they were originally selected (Salminen et al., 1996). These include growth and survival during production and commercialisation, as well as during transit through the stomach and small intestine. Consequently, it is necessary to test the stability of these characteristics during manufacture, storage and/or ingestion to ensure that they are retained in different types of feeds and in the host (Lee and Salminen, 1995). Therefore, the initial screening and selection of probiotic strains must include testing of the following important criteria: identification at the species level, carbohydrate utilisation patterns, acid and bile tolerance, adhesion to mucin or intestinalderived epithelial cells, production of antimicrobial substances, antibiotic resistance patterns and ability to influence metabolic activities in the host (e.g. b-galactosidase activity or production of vitamins) (Tuomola et al., 2001). The objective of the present study was to determine whether canine milk can also be a source of lactic acid bacteria to the puppy gut and to evaluate the probiotic potential of such isolates. Materials and methods Isolation and enumeration of lactobacilli from canine milk The protocol (B-16/07) was approved by the Ethical Committee on Animal Experimentation of Universidad Complutense de Madrid, Spain. The nine bitches from which the milk samples were obtained belonged to the following breeds: Boston Terrier, Cocker Spaniel, Dalmatian, Golden Retriever (two bitches), Spanish Mastiff, Belgian Shepherd and Yorkshire Terrier (two

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bitches). All bitches fulfilled the following criteria: (1) healthy bitches without present or past underlying conditions; (2) normal pregnancy; (3) no use of antibiotics and/or probiotics during the previous 2 months; and (4) absence of any kind of perinatal problems in both puppies and bitches. Nipples and mammary areolae were cleaned with soap and sterile water, soaked in chlorhexidine (Cristalmina, Salvat) for 5 min, then dried with a sterile cloth. Milk samples (days 7–12 after parturition) were then collected into a sterile tube by manual expression using sterile gloves. The first drops (500 lL) were discarded. The samples were kept on ice until delivery to the laboratory and processed within the first 2 h after collection. Peptone water dilutions of the milk samples were plated in triplicate onto Man, Rogosa and Sharpe (MRS, Oxoid; a medium for the isolation of lactic acid bacteria) agar plates, which were aerobically incubated at 37 °C for 24 h. In parallel, the same samples were also cultured on MRS supplemented with L-cysteine (0.5 g/L) (MRSCys) agar plates, which were incubated anaerobically (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in a anaerobic workstation (MINI-MACS, DW Scientific) at 37 °C for 48 h. In parallel, to evaluate potential faecal contamination, the samples were also cultured on Violet Red Bile Agar (VRBA; Difco; a selective medium for the isolation of enterobacteria) agar plates, which were aerobically incubated at 37 °C for 24 h. In both growth media, the lower limit of detection was 50 colony-forming units (CFU)/mL (1.69 log10 CFU/mL).

Determination of antimicrobial activity An overlay method (Magnusson and Schnürer, 2001) was used to determine the ability of the strains to inhibit the growth of various species of bacteria, moulds and yeasts. It was performed using MRS agar plates on which the lactobacilli were inoculated as approximately 2 cm long lines and incubated at 37 °C for 48 h in anaerobic jars (Oxoid). The plates were then overlaid with the indicator microorganisms in 10 mL of soft (0.7%) BHI (Oxoid; a general-purpose medium suitable for the cultivation of non-fastidious bacteria) at an approximate concentration of 104 CFU. The following bacteria, from our own culture collection, were employed as indicator organisms: Lactococcus lactis MG1614, Lactobacillus sakei NCFB2714, Enterococcus faecium P21, Enterococcus faecalis TAB28, Pediococcus acidilactici 347, Listeria monocytogenes ScottA, L. monocytogenes Ohio, Listeria seeligeri RdC, Staphylococcus aureus CECT 5191, Staphylococcus epidermidis CECT 231, Escherichia coli CECT 4076 (O157:H7), E. coli RJM1, E. coli RJM2, E. coli K12 CECT 433, Salmonella choleraesuis CECT4155, Klebsiella pneumoniae CECT 142, Proteus vulgaris CECT 484 and Klebsiella oxytoca CECT 860T. The plates overlaid with bacterial indicators were incubated at 32 or 37 °C (depending on the optimal temperature) for 48 h. Finally, the plates were examined for clear zones of inhibition (>2 mm) around the lactobacilli streaks. All experiments assaying inhibitory activity were performed in triplicate.

Identification of bacterial isolates from canine milk Production of bacteriocins Initially, a total of 1660 colonies, including at least one representative of each morphological type, were selected from the agar plates. Subsequently, the colonies were inoculated in to MRS broth tubes, which were incubated aerobically to exclude those with fastidious incubation requirements and, therefore, not suitable for practical applications. The isolates (n = 490) that showed good growth (>108 CFU/mL in MRS broth after 24 h) were examined by phase-contrast microscopy to determine cell morphology and Gram staining reaction and tested for oxidase and catalase activities. The isolates were identified by 16S rRNA sequencing. For each strain, a single bacterial colony was used as a template for PCR amplification of a segment of a 16S rRNA gene variable region. The two primers used were those described by Kullen et al. (2000), namely pbl16 (50 -AGAGTTTGATCCTGGCTCAG-30 ) and mlb16 (50 GGCTGCTGGCACGTAGTTAG-30 ). The PCR conditions were as follows: 96 °C for 30 s, 48 °C for 30 s and 72 °C for 45 s (40 cycles), then a final extension at 72 °C for 4 min. The amplified fragment was purified and sequenced using the above primers. Sequencing reactions were prepared using the PRISM ABI BigDye Ready Reaction Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase according to the manufacturer’s instructions and were run on an ABI 377A automated sequencer (Applied Biosystems). The resulting sequences were used to search sequences deposited in the EMBL database using the BLAST algorithm. The identity of the amplified strain was determined on the basis of the highest scores (>98%). RAPD genotyping of lactobacilli isolated from canine milk Once the isolates were identified, those belonging to the genus Lactobacillus were genotyped by randomly amplified polymorphic DNA (RAPD)-PCR analysis. The RAPD technique involves the PCR amplification of random segments of genomic DNA with a single primer of arbitrary nucleotide sequence and allows the differentiation between genetically distinct individuals belonging to the same bacterial species. For this purpose, genomic DNA was isolated from 10 mL of overnight MRS cultures using the DNeasy tissue kit (Qiagen), following the protocol recommended by the supplier for isolation of genomic DNA from Gram positive bacteria. The DNA was used in subsequent PCR amplifications that were performed using the Readyto-Go RAPD Analysis kit (GE Healthcare). Then, 5 lL of the PCR mixtures were analysed on 1.2% (w/v) agarose (Sigma) gels that were run for approximately 2 h at 80 V and the DNA was visualised and analysed in a gel documentation system (Gel Doc 2000, Bio-Rad), using the Diversity Database software package (Bio-Rad).

The lactobacilli were grown in MRS broth at 37 °C until the early stationary phase (A620 1.0). The cultures were centrifuged at 12,000 g for 10 min at 4 °C and the supernatants were neutralised to 6.2 with 1 M NaOH, heated at 100 °C for 5 min and filter-sterilised through 0.22 lm pore size filters (Millipore). The bacteriocinogenic activities of the cell-free supernatants were determined by an agar well diffusion assay. Aliquots (100 lL) of the supernatants were placed in wells (7 mm diameter) cut in cooled BHI agar plates previously seeded (105 CFU/mL) with the indicator strains. The plates were kept at 4 °C for 2 h, then incubated under optimal conditions for growth of the indicator. The microorganisms employed as indicators of bacteriocinogenic activity were the Gram positive bacteria used for determination of the antimicrobial spectrum: L. lactis MG1614, L. sakei NCFB2714, E. faecium P21, E. faecalis TAB28, P. acidilactici 347, L. monocytogenes ScottA, L. monocytogenes Ohio, L. seeligeri RdC, S. aureus CECT 5191 and S. epidermidis CECT 231. Production of reuterin by the strains of Lactobacillus reuteri The strains of L. reuteri were cultured in MRS broth at 37 °C for 16 h in anaerobic conditions. Cells were harvested by centrifugation at 4000 g for 5 min, resuspended (1:1 V/V) in a 250 mM glycerol solution and incubated for 3 h at 37 °C in anaerobic conditions. The cells were removed by centrifugation and the presence of reuterin in the cell-free supernatant was detected by the colorimetric method of Smiley and Sobolov (1962). This method is based on the colorimetric determination of acrolein formed from the 3-hydroxypropionaldehyde (3-HPA) produced from glycerol by glycerol dehydratase. L. reuteri CECT 925 and Lactobacillus coryniformis CECT 5711 (Martín et al., 2005b), two reuterin-producing strains, were used as a positive controls. In parallel, since glycerol dehydratase is essential for reuterin production, PCR was used to amplify a 279 base pair fragment of the gene encoding the 60-kDa glycerol dehydratase subunit. The primers (GD1 and GD2) and PCR conditions were selected according to the criteria of Claisse and Lonvaud-Funel (2001). GD1 and GD2 are degenerate primers deduced from a domain of the protein subunit that is conserved in Gram positive and Gram negative bacteria. L. reuteri CECT 925 and L. coryniformis CECT 5711 were used as a positive control. Production of riboflavin by the strains of L. reuteri

Survival after exposition to different conditions The concentrations of viable cells of the strains after their exposure to different conditions that can be of importance for probiotic applications were tested in MRS broth. The following growth conditions were assayed under aerobiosis: 20, 25, 32 and 42 °C (pH 6.2), 37 °C (pH 4.5), presence of canine bile salts (5%), freeze-drying and storage at 20 °C for 15 days and at 80 °C for 30 days in the presence of 15% (v/v) glycerol; in addition, growth was also tested under anaerobiosis at 37 °C. All assays were performed in quadruplicate and the values were expressed as the mean ± standard deviation (SD). MRS cultures incubated at 37 °C under aerobiosis were used as controls.

Surprisingly, several colonies of L. reuteri were yellow instead of white. Therefore, we investigated their ability to produce riboflavin (vitamin B2; a compound that is typically associated with such colour) when they were grown in MRS both. For this purpose, we followed the method described by Zafra-Gómez et al. (2006), which is based on a high performance liquid chromatography (HPLC) technique. The chromatographic separation was carried out in a 2695 Alliance chromatograph (Waters) equipped with a 2475 fluorescence detector and using a C18 Spherisorb ODS-2 column (Waters) set at 40 °C. The fluorescence detection was set at 400/ 520 nm (excitation/emission) for riboflavin determination. Millennium 4.0 software was used for data treatment.

Profiles of carbohydrate fermentation and enzymatic activities Adherence to mucin Carbohydrate fermentation patterns were obtained with API Rapid CH fermentation strips (BioMérieux) in Lactobacillus Identification Medium (CHL broth, API 50 CHL; BioMérieux) as specified by the manufacturer. Enzymatic activities were tested by using the APIzym system (BioMérieux), following the instructions provided by the manufacturer.

The adhesion of bacteria to mucin was determined according to the method described by Cohen and Laux (1995), with some modifications. One hundred microlitres of a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Hanks salt solution (HH) of canine mucin (1 mg/mL), obtained as described by

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R. Martín et al. / The Veterinary Journal 185 (2010) 193–198 Rinkinen et al. (2003), were immobilised in polystyrene microtitre plates (Maxisorp; Nunc) by overnight incubation at 4 °C. The wells were washed twice with 250 lL HH. In parallel, bacteria were grown overnight at 37 °C in MRS broth and the bacterial pellets from 1 mL fractions were obtained by centrifugation and washed with HH. Then, 10 lL of 10 mM carboxifluorescein (Sigma) were added to the pellets and the bacterial suspensions were incubated for 20 min at 37 °C. The bacterial cells were washed three times with HH, then resuspended in 1 mL HH. A suspension of 50 lL of the fluorescent-labelled bacteria (5  107 CFU) was added to each well. After incubation for 1 h at 37 °C, the plates were washed twice with 250 lL of HH to remove unattached cells, then incubated for 1 h at 60 °C with 50 lL 1% sodium dodecyl sulfate (SDS)–NaOH (0.1 mol/L) to release and lyse bound microorganisms. Fluorescence was measured in a fluorescence microplate reader (Tecan). Adhesion was assessed as the percentage of the fluorescence retained in the wells after the washing steps when compared to that present in the labelled bacterial aliquots originally added to the wells. The assays were performed in duplicate and the values were expressed as the mean ± SD.

Table 1 Enumeration and isolation of bacterial isolates grown on MRS agar plates inoculated with bitch milk. Bitch (n = 9)

MRS count (mean CFU/mL ± SD)

Initial isolatesa (n = 1660)

Identified isolatesb (n = 490)

Boston terrier Cocker spaniel Dalmatian Golden retriever A Golden retriever B Spanish mastiff Belgian shepherd Yorkshire terrier A Yorkshire terrier B

132 ± 6 148 ± 5 609 ± 18 451 ± 12 471 ± 16 507 ± 19 466 ± 14 419 ± 10 180 ± 8

64 72 298 222 233 248 229 207 87

26 56 45 48 48 72 72 70 53

(A: 26) (A: 25, B: 30, C: 1) (A: 27, B: 18) (A: 28, B: 19, C: 1) (A: 2, B: 18, C: 24) (A: 72) (B: 69, C: 3) (B: 70) (A: 43, B: 10)

a

Degradation of mucin The potential of the strains to degrade gastric mucin in vitro was evaluated following the plate procedure developed by Zhou et al. (2001). Canine mucin and agarose type I-A (Sigma) were added to a minimal anaerobic culture medium without glucose (Ruseler-van Embeden et al., 1995) at concentrations of 0.5% and 1.5% (W/V), respectively. The plates were incubated at 37 °C anaerobically for 72 h and subsequently stained with 0.1% amido black in 3.5 M acetic acid for 30 min. Then, they were washed with 1.2 M acetic acid until the mucin lysis zone around the colony of positive control culture (faecal flora) appeared. These assays were performed in triplicate. Sensitivity to antibiotics Minimum inhibitory concentrations (MICs) of 12 antimicrobial agents were determined by microdilution using the newly developed and standardised ‘lactic acid bacteria susceptibility test medium’ (LSM) broth, consisting of a mixture of Iso-Sensitest (IST; Oxoid) broth (90%) and MRS broth (10%) adjusted to pH 6.7 as previously described (Klare et al., 2005). Nine of the antimicrobials tested (ampicillin gentamicin, streptomycin, quinupristin/dalfopristin, erythromycin, clindamycin, oxytetracycline, chloramphenicol, kanamycin) were those for which the panel on additives and products or substances used in animal feed (FEEDAP) of the European Food Safety Authority (EFSA) has established microbiological breakpoints (cut-off values) that enable the distinction between strains of lactobacilli harbouring acquired antimicrobial resistances and susceptible strains (EFSA, 2008). MICs of three additional antimicrobials agents for which tentative cut-off values for lactobacilli have been suggested (Klare et al., 2007) were also determined. The antimicrobials were tested in the concentration ranges (mg/L) given in parentheses: penicillin G (0.032-64), ampicillin (0.032-64), gentamicin (1-2048), streptomycin (2-4096), quinupristin/dalfopristin (tested as 30:70 ratio: 0.032-64), erythromycin (0.016-32), clindamycin (0.032-32), oxytetracycline (0.063-128), fusidic acid (0.063-128), linezolid (0.016-32), chloramphenicol (0.125-256) and kanamycin (1-256).

Results

Fifty percent of the colonies observed in one of the MRS plates. Initial isolates that showed a good growth in MRS broth incubated in aerobiosis; A: Gram positive, catalase negative and oxidase negative rods; B: Gram positive, catalase negative and oxidase negative cocci and C: Gram positive, catalase positive and oxidase negative bacteria. b

Streptococcus. Catalase positive and oxidase negative cocci were identified as Staphylococcus spp. (Table 2). RAPD profiling revealed the existence of 28 different profiles among the Lactobacillus spp. isolates (Table 3). Subsequently, one representative of each Lactobacillus profile (from now, ‘strain’) was selected for further characterisation. Table 2 Identification of the selected isolates. Bitch

Species

Number of isolates

Boston terrier (n = 26)

L. L. L. L.

16 8 1 1

Cocker spaniel (n = 56)

L. murinus L. animalis L. fermentum E. faecium E. faecalis Strept. salivarius Staph. epidermidis

14 8 3 17 11 2 1

Dalmatian (n = 45)

L. murinus L. animalis L. reuteria L. reuterib E. faecalis Strept. bovis

5 7 8 7 13 5

Golden retriever A (n = 48)

L. murinus L. animalis L. reuteri E. faecalis Strept. bovis Staph. simulans

24 3 1 10 9 1

Golden retriever B (n = 48)

L. murinus E. faecium Staph. simulans Yeasts

2 18 24 4

Mastiff (n = 72)

L. reuteri

72

Belgian shepherd (n = 72)

Strept. bovis Staph. pseudointermedius

69 3

Yorkshire terrier A (n = 70)

E. faecalis

70

Yorkshire terrier B (n = 53)

L. fermentum L. reuteria L. reuterib W. viridescens E. faecium

27 1 9 6 10

Isolation and identification of lactobacilli from canine milk Colonies were obtained from all the milk samples analysed, both in MRS and MRS-Cys plates, with MRS counts ranging from 132 to 609 CFU/mL in different bitches (Table 1). No growth was observed on VRBA agar plates. Initially, 50% of the colonies growing on one of the MRS plates inoculated with each sample were subcultured, including at least one representative of each morphology (Table 1). All these colonies were inoculated in MRS broth and incubated at 37 °C under aerobiosis. Under such conditions, 490 isolates showed good growth (>108 CFU/mL after 24 h) and were selected for further screening. Most isolates were Gram positive, catalase negative and oxidase negative rods or cocci, while only a few of them were Gram positive, catalase positive and oxidase negative cocci. Identification of the isolates by 16S rDNA PCR sequencing revealed that the Gram-positive bacilli belonged to the Genus Lactobacillus, along with one isolate that was classified as Weissella spp. (Table 2). Coccoid isolates that showed catalase negative and oxidase negative reactions belonged to the genera Enterococcus or

murinus animalis reuteri johnsonii

L.: Lactobacillus; E.: Enterococcus; Strept.: Streptococcus; Staph.: Staphylococcus and W.: Weissella. a White colonies. b Yellow colonies.

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Table 3 Groups of Lactobacillus isolates after RAPD genotyping and selection of a strain from each genotype. Species

RAPD group

Type strain

Origin (bitch)

L. fermentum

A B C

PNA1 PNC3 PND4

Yorkshire terrier B Yorkshire terrier B Yorkshire terrier B

L. reuteri (white colonies)

D E F G H

PND1 PDD2 PYD7 R13 M9

Yorkshire terrier B Dalmatian Boston terrier Mastiff Mastiff

L. reuteri (yellow colonies)

I J K L M N

PDA3 PDH3 PDD1 PNF4 MA25 R18

Dalmatian Dalmatian Dalmatian Yorkshire terrier B Mastiff Mastiff

L. murinus

O P Q R S T V W

PKB1 PKD3 PDH1 PDF4 PYH2 PYE7 PGAC7 PGBA1

Cocker spaniel Cocker spaniel Dalmatian Dalmatian Boston terrier Boston terrier Golden retriever A Golden retriever B

L. animalis

X Y Z AA BB CC

PKG5 PKH9 PDA1 PYA1 PYB8 PGAH8

Cocker spaniel Cocker spaniel Dalmatian Boston terrier Boston terrier Golden retriever A

the widest antimicrobial spectrum and the strongest antimicrobial activity, which was particularly effective against the Listeria, Staphylococcus and Salmonella spp. and E. coli strains used as indicator organisms. In contrast, those strains belonging to the species L. murinus and L. animalis showed narrower inhibition zones against all the indicators tested in this study. Subsequently, all the strains were screened for production of bacteriocins and/or reuterin. None of them showed bacteriocin activity against the spectrum of indicator organisms used and none of them produced reuterin. Other properties All the strains of L. reuteri with yellow colonies produced riboflavin when they grew in MRS broth; the concentration ranged from 3.54 (L. reuteri PDH3) to 10.09 mg/L (L. reuteri PNF4). In contrast, riboflavin could not be detected in the MRS cultures of the strains of L. reuteri which had typical white colonies. The strains tested showed a variable ability to adhere to mucin but L. reuteri showed the highest adherence ability. None of the strains were able to degrade gastric mucin in vitro. Antibiotic susceptibility profiles showed that the strains were sensitive to all antibiotics tested. MIC values demonstrated that the strains were sensitive to b-lactam antibiotics such as penicillin G and ampicillin, to aminoglycosides such as gentamicin, to macrolides such as erythromycin and to other broad-spectrum antibiotics such as chloramphenicol or tetracycline.

Discussion Survival after exposition to different conditions All the strains grew in MRS broth at 20, 25, 30 and 42 °C, at pH 4.5, under anaerobiosis and in the presence of canine bile (5%). They were also able to grow after conventional freeze-drying and after storage at 20 and 80 °C for 15 and 30 days, respectively. Under such conditions, the bacterial concentrations after incubation were P85% of that found in the control cultures (MRS broth, 37 °C, aerobiosis, initial pH 6.2). Profiles of fermentation and enzymatic activities All the strains fermented the following carbohydrates: ribose, galactose, D-glucose, D-fructose (with the exceptions of strains PDA3 and PNF4), maltose, lactose, melibiose, saccharose and D-raffinose. In contrast, none of them were able to ferment the following carbohydrates: erythritol, L-xylose, adonitol, L-sorbose, rhamnose, glycogen, xylitol, dulcitol, inositol D-turanose, D-lyxose, a-methyl-D-mannoside, inulin, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. All strains displayed the following enzymatic activities: acid phosphatase, leucine and cystine arylamidases (except strain PNA1), a-galactosidase (except strain PYA1), b-galactosidase and naphthol-AS-BI-phosphohydrolase. All the strains of Lactobacillus fermentum and L. reuteri displayed a-glucosidase activity, whereas this enzyme was not detected among the isolates of Lactobacillus murinus and Lactobacillus animalis. None of the strains showed b-glucuronidase activity. Antimicrobial activity The strains showed a clear inhibitory antimicrobial activity (inhibition zone >2 mm around the streak) against all indicator organisms used in this study, with the exception of L. animalis PKG5. The strains of L. fermentum and L. reuteri displayed both

This study showed that canine milk contains lactobacilli and may be a natural source of such microorganisms for the suckling puppy. MRS counts ranged from 1.3 to 6.1  102 CFU/mL; such bacterial concentration values are similar to those reported from hygienically-obtained fresh human milk (Perez et al., 2007). The fact that no growth was detected on VRBA plates inoculated with the same samples confirmed the hygienic collection of the milk samples. Several Lactobacillus spp. (L. reuteri, L. fermentum, L. animalis, L. murinus, L. johnsoni) were identified in this study. Lactobacilli belonging to the same or closely related species have been frequently isolated or detected in canine faeces (Beasley et al., 2006; Mentula et al., 2005; Greetham et al., 2002) and seem to be dominant in suckling puppies, even 1 day after birth (Buddington, 2003). The lactobacilli pattern of canine milk seems to be hostspecific, a finding that has also been reported for human milk (Martín et al., 2007a), canine faeces (Simpson et al., 2002; Suchodolski et al., 2005) and faeces of lactating piglets (Simpson et al., 2000). In addition, this pattern seems to be restricted to a low number of Lactobacillus spp. Similarly, the lactobacilli composition of human infant faeces and breast milk usually includes a low number of species of lactobacilli (Heikkilä and Saris, 2003; Martín et al., 2007a); examination of Lactobacillus spp. colonisation in 112 infants showed that, during the first 6 months of life, 26% of infants had no lactobacilli, 37% carried a single strain, 26% two strains and only 11% three or more strains (Ahrné et al., 2005). A total of 28 strains of Lactobacillus (a representative of each RAPD genotype) were screened for the presence of potentially probiotic properties. All of them were able to grow in a variety of conditions and grow or survive at a wide range of incubation or storage temperatures (80–42 °C), an acidic pH or presence of bile. Survival of the lactobacilli when exposed to conditions found in the gastrointestinal tract (low pH and bile) or during production and commercialisation appear to be important pre-requisites for probiotic strains (Marteau et al., 1997; Tuomola et al., 2001).

R. Martín et al. / The Veterinary Journal 185 (2010) 193–198

API 50 CH profiles for the 28 strains of lactobacilli demonstrated phenotypic diversity, even among strains belonging to the same species. This result is not unusual, since application of carbohydrate fermentation profiling to lactobacilli can lead to high intraspecies diversity. In a previous study, none of the 18 isolates of Lactobacillus crispatus tested shared the same phenotypic pattern, 20 isolates of Lactobacillus gasseri yielded 18 different patterns, 20 isolates of Lactobacillus vaginalis isolates yielded 15 different patterns and 19 isolates of Lactobacillus jensenii produced 11 different patterns (Boyd et al., 2005). In the same study, all the Lactobacillus iners isolates and three of the L. vaginalis isolates were non-reactive for all of the tests in the API 50 CH system. It has been demonstrated that the use of the current API 50 CH database for identification of commensal Lactobacillus spp. leads to a high frequency of misidentification (Nagy et al., 1991; Klein et al., 1998); therefore, its usefulness for identification of lactobacilli is very limited and the use of genomic methods, such as 16S rRNA sequencing, is preferable (Wilks et al., 2004). However, carbohydrate fermentation profiling may provide useful information to discriminate among strains of lactobacilli belonging to the same species. Lactobacilli have been long considered to constitute the primary microbiological barrier to infection by intestinal and urogenital pathogens. The production of inhibitory substances, such as lactic acid, bacteriocins or reuterin, may affect undesirable or pathogenic bacteria. Bacteriocin or reuterin production could not be detected in any of the lactobacilli tested in this study. Our results suggest that differences among the antimicrobial activities displayed by the strains tested in this work may merely be due to differences in growth and production of organic acids. Although it has been suggested that lactic acid can help to maintain a low pH in the gut (Aiba et al., 1998), its production and efficacy in vivo remain uncertain. Another important property of potential probiotic strains is their ability to adhere to intestinal or gastric mucin, since this is considered a pre-requisite for gut colonisation and may lead to the competitive exclusion of pathogenic bacteria (Martín et al., 2005a). Some of the strains tested, such as L. fermentum PNA1, L. reuteri M9 and L. murinus PKB1, showed a strong ability to adhere to mucin, reaching values comparable to other highly adhesive strains of lactobacilli (Olivares et al., 2006). In addition, six strains of L. reuteri (isolated from three different bitches) were able to synthesise riboflavin. This is a relevant observation since, up to the present, it was thought that lactobacilli were unable to produce this vitamin and, therefore, were dependent on external riboflavin for growth (Ledesma et al., 1976; Møretrø et al., 1998). The verification that probiotic strains lack acquired antimicrobial resistance properties is an important requisite when considering them to be safe for animal consumption. Antimicrobial susceptibility testing of lactobacilli can be performed by several methods, but dilution methods generally provide a more reliable indication of the intrinsic or acquired nature of a given resistance phenotype (Klare et al., 2007). All the strains of lactobacilli tested in this study did not demonstrate antimicrobial resistance when tested according to EFSA guidelines (EFSA, 2008). The species of lactobacilli isolated from canine milk in this study are among those considered as potential probiotic bacteria. L. reuteri, L. fermentum or L. animalis have already been tested as canine probiotics (Manninen et al., 2006; Biagi et al., 2007). Therefore, the milk of healthy bitches may be a source of potentially probiotic lactobacilli, with a role in protecting mothers and/or puppies against infectious diseases. Although cross-species efficacy has been demonstrated for some probiotic strains, one criterion for selection of a probiotic is host species specificity. However, most of the commercial probiotic strains for dogs are not of canine

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origin. In a previous study, it was shown that Lactobacillus rhamnosus GG, a human isolate, survived gastrointestinal transit in dogs, but faecal colonisation was less efficient than in humans (Weese and Anderson, 2002). The origin of the lactobacilli that colonise the neonatal gut is a subject of debate. In the past, it was suggested that they were acquired by oral contamination with maternal lactobacilli during the transit through the birth canal; however, molecular studies have shown that human Lactobacillus colonisation is not significantly related to the delivery method (vaginal delivery or Caesarean section) (Matsumiya et al., 2002; Martín et al., 2003; Ahrné et al., 2005). In contrast, breast milk constitutes a good source of lactobacilli (Heikkilä and Saris, 2003) and is responsible for the vertical mother-to-child transmission of lactobacilli and bifidobacteria inhabiting the gut (Matsumiya et al., 2002; Martín et al., 2003, 2007b, 2009). Recently, it has been shown that live commensal bacteria can spread from the maternal gut to the epithelium of the lactating mammary gland through an endogenous route involving dendritic cells and macrophages: the enteromammary pathway (Martín et al., 2004; Perez et al., 2007). Work is in progress to compare the probiotic potential of strains of lactobacilli isolated from canine milk with that of strains recovered from canine faeces or gut. Conclusions Some strains of lactobacilli isolated from canine milk have potential for future applications as canine probiotics. In particular, L. reuteri and L. fermentum exhibited high antimicrobial activities, high survival rates after exposure to different adverse physical conditions and desirable enzymatic activities (such as production of a-glucosidase). In addition, these strains did not degrade mucin and the MICs of several antibiotics were within the values recommended by the EFSA. Conflict of interest statement C. Torre is employed by Affinity Petcare. None of the other authors have a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements We are grateful to Elena Aguado (Clínica Veterinaria Sevilla la Nueva, Spain) for the collection of the milk samples. This work was funded by Affinity Petcare S.A. (Sant Cugat del Valles, Spain). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tvjl.2009.04.014. References Ahrné, S., Lönnermark, E., Wold, A.E., Aberg, N., Hesselmar, B., Saalman, R., Strannegard, I.L., Molin, G., Adlerberth, I., 2005. Lactobacilli in the intestinal microbiota of Swedish infants. Microbes and Infection 7, 1256– 1262. Aiba, Y., Suzuki, N., Kabir, A.M.A., Takagi, A., Koga, Y., 1998. Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model. American Journal of Gastroenterology 11, 2097–2111. Beasley, S.S., Manninen, T.J.K., Saris, P.-E.J., 2006. Lactic acid bacteria isolated from canine faeces. Journal of Applied Microbiology 101, 131–138. Biagi, G., Cipollini, I., Pompei, A., Zaghini, G., Matteuzzi, D., 2007. Effect of a Lactobacillus animalis strain on composition and metabolism of the intestinal microflora in adult dogs. Veterinary Microbiology 124, 160–165.

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