Isolation of bacteria from oral mucosa and colostrum

The experimental animals consisted of five lactating calves (30 d of age) and five multiparous adult cows in lactation, all Friesian Holstein from College of Postgraduates (Colegio de Postgraduados, Campus Montecillo) installations. Colostrum samples were taken from five newly-calved Holstein cows on the private Xalapango ranch. Both sites are located in the Texcoco Valley, in the State of Mexico, Mexico (18°21’ and 20°17’ N; 98°36’ and 100°36’ W)⁹.

Sampling

Oral mucosa: Duplicate exudate samples were taken of the oral mucosa from each lactating calf, by rubbing the mucosa for 3 sec with a swab (3MTMSwab-sampler) prior to the morning feeding. Each swab was placed inside a sterile tube with 10 ml buffered peptone water (RS96010BPW).

Colostrum and milk: Before sampling, the cows’ nipples were cleaned, disinfected and pulled down, and 5 ml of colostrum and 10 ml of milk collected per group of cows. Samples were deposited in sterile vials, kept at 4 °C and immediately transferred to the laboratory for analysis following standardized procedures¹⁰.

Sample processing

The samples were pre-enriched (to favor LAB growth) in liquid culture medium¹¹. Duplicate samples (1 ml) of the oral mucosa bacterial suspension were taken and placed in tubes containing 5 ml de Man/Rogosa/Sharp (MRS) broth. The inoculated tubes were divided into two random groups: one under aerobic conditions and the other under anaerobic conditions, both were incubated at 37 °C for 18 h. In the colostrum and milk samples, 200 μl were taken in duplicate and deposited in tubes containing 5 ml MRS broth, and kept in a desiccator under an anaerobic environment (induced by a burning candle) for 18 h at 37 °C. Samples were then taken from the tubes with an inoculation loop and sown in Petri dishes containing MRS agar¹², and incubated at 37 °C for 48 h under anaerobic and aerobic conditions. A strain of Lactobacillus casei ATCC was used as a positive control and one of E. coli O42 as a negative control (both donated by the Universidad Autónoma de Querétaro).

Bacteria selection

During the sowing process the cultures were seriated according to sample duplication in the solid MRS medium. The result was a total of 54 colonies with LAB characteristics based on colony size, shape, surface, elevation, edge and color¹³. Strain characterization was done by the Gram stain test, cell morphology, spore staining and the catalase test¹². Indole production and motility tests were done in SIM (hydrogen sulfide, indole, motility) culture medium; gelatin hydrolysis and nitrate reduction tests were also done¹⁴. A second selection of the isolated colonies was made based on the best scores and ideal coccobacilli and bacilli morphology. A total of 27 colonies were identified which were cultured in 5 ml MRS broth for 18 h for later evaluation as probiotic bacteria. Duplicate 800 µl samples were taken from each bacterial suspension and transferred to Eppendorf tubes containing 800 µl sterile 50% glycerol as a cryoprotectant. These were stored at -20 ºC for 3 h and subsequently at -80 ºC indefinitely.

Resistance and survival of selected strains under gastrointestinal conditions

Biochemical identification and collection of strains with probiotic potential

Colonies with LAB characteristics were identified using the APICHL system (BioMerieux SA, France). In this procedure the colonies were reactivated in 5 ml MRS broth under anaerobic conditions at 37 ºC for 18 H, adding 50CH diluent (supplied with the gallery: API50CH) following manufacturer instructions (see Figure 1 for summary of procedure). The prepared suspension was added to 50 microtubes in the gallery, and the domes of these microtubes filled with sterile mineral oil to generate anaerobic conditions. The inoculated galleries (one per colony) were kept at 37 ºC for 48 h to establish each colony’s biochemical profile. Results interpretation was done based on color change in the API50CHL medium of each microtube: blue is negative, and yellow and black indicate positive values (plate safety sheet). Results were analyzed with the Apiweb^® computer system.

Figure 1: Probiotic bacteria isolation and selection procedure 

Colony isolation and growth

The colonies cultured in the anaerobic environment exhibited better growth than those cultured under aerobic conditions. Based on morphology, the selected colonies were Gram positive coccobacilli and bacilli, with no spores, catalase negative, no motility, no indole or gelatinase production and nitrate reduction negative.

The colonies from the oral mucosa samples had an average size of 2 to 4 mm in diameter with homogeneous morphological characteristics; circular shape, convex elevation, complete edge, smooth surface and white color without pigments. Of the milk samples only 20 % supported bacterial colony growth, which had an average size of 2.5 mm diameter. Those colonies isolated from the colostrum were beige in color and varied in size from 1 to 5 mm diameter. Probiotic bacteria have generally been isolated from the oral, vaginal, and intestinal mucosa of healthy calves and from milk samples^11,20. Lactobacilli colonies isolated from the oral mucosa and milk have the capacity to adapt and survive¹³. This is due to the presence of the hemin group, which allows them to activate the respiratory chain with oxygen as the electron recipient²¹. The different LAB genera share morphological, metabolic and physiological characteristics such as shape, elevation, edge, color and biochemical reactions¹³. For the purpose of probiotic strain selection, their cell morphology and biochemical tests have been reported as basic^5,22, although it is recommended that selection be complemented with molecular studies²³.

API biochemical strain identification

Colony identification based on carbohydrate fermentation profile (API50CHL-BioMerieux) produced a 96 to 99 % effectiveness interval (Table 1). Identified colonies from the oral mucosa included six Lactobacillus, five Leuconostoc and one Pediococcus, while those from the colostrum included Leuconostoc and Lactobacillus.

Strain viability based on resistance to acid pH

Analysis of colony population resistance to different pH levels (Table 1), found growth at the control pH (6.5) to average 9.07 log₁₀ CFU/ml. At pH 4.0 growth decreased (P<0.001) to 5.09 log₁₀ CFU/ml. The two colonies from the milk samples did not grow at pH 4 and were not included in the final strains. Resistance to acid pH is relevant because to reach the action site and remain viable probiotic bacteria must withstand acid pH and the presence of BS in the duodenum²⁴. Several authors have developed methodologies to evaluate probiotic strain resistance under gastrointestinal conditions^7,25. The present results coincide with a study of L. plantarum and L. acidophilus in which these strains grew and remained viable at pH 5.0, but became inactive at pH 4.0 and 3.0¹⁶. Strain sensitivity may be related to the acid tolerant response or acquired resistance. For example, in a study comparing a control of L. casei cells grown at pH 6.0 to acid adapted cells at pH 4.5 for 10 and 20 min, viability decreased up to 4.0 log₁₀ CFU/ml at 10 min adaptation, and from 0.7 to 2.4 log₁₀ CFU/ml at 20 min²⁶. Cell adaptation to an acid environment caused changes in membrane lipid composition, with a dramatic increase in saturated and unsaturated fatty acids, as well as malolactic fermentation and intracellular histidine accumulation. The ability of probiotic bacteria to survive the stomach’s acid environment varies by strain^27,28,29, which would explain the differences in resistance between LAB strains observed here at pH 4.0. Lactobacilli commonly grow better at pH 4.0 than at pH 3.0³⁰, and at pH 3.0 only four of 200 known LAB strains survive³¹.

Viability under bile salts exposure

When exposed to high BS concentrations, the LAB tested in the present study continued to grow at concentrations as high as 1.5 g. Lactic acid bacteria resistance to and growth under exposure to BS has been tested at concentrations from 0.1 to 4.0 %^32,33; this is an important parameter for microorganisms in commercial products^34,35, but one rarely tested. In another study³⁶, L. plantarum resistance was exposed to four concentrations of porcine BS (0.01, 0.05, 0.10 and 0.15 g), and strain growth monitored for 24 h via OD measurements. The highest growth rate was observed at the lowest BS concentration, and, at the final density and 0.10 g BS, this strain’s growth rate was three times lower than in the control. This is higher growth inhibition at lower BS concentrations than observed in the present study: final colony OD was only 2.5 times lower at 0.3 g BS than in the control treatment. There are reports of resistance to BS at concentrations from 0.3 to 1% BS in LAB (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and probiotic bacteria (L. acidophilus, L. casei, L. rhamnosus and Bifidobacterium)^37,38. In these studies, S. thermophilus was the most sensitive LAB strain (growth inhibition at 0.5 g BS), L. lactis was the most resistant LAB (growth inhibition at 1 g BS), and all the probiotic strains exhibited resistance to 1.5 g BS. Resistance to BS exposure may differ between Lactobacillus species based on their ability to colonize and rapidly stabilize, as has been tested in the intestine of heifers⁷. For example, in an in vivo study in which L. acidophilus was administered to heifers, the total lactobacilli count in the jejunum increased from 13 to 39 %, but strains of L. plantarum and Lactococcus acidilactici exhibited better growth at pH 4.0 and 0.3 g BS⁵. In the present results, the LAB were more tolerant of BS exposure than of acid pH levels (4.0). However, their relatively good resistance to prolonged exposure to acid pH and very good resistance to high BS concentrations are effective indicators of their survival and colonization capacity during intestinal transit^28,39.

Resistance to BS was also quantified by comparing strain average OD at two BS concentrations (0.3 and 1.5 g) (Table 2). Average OD in all the evaluated strains increased 3.1 times (P<0.05) after 24 h incubation at 0.3 g SB, compared to the initial reading, but when exposed to 1.5 g BS for 24 h, it increased 2.7 times. Optical density (OD) dropped significantly (P<0.023) as BS concentration increased, but it still increased (P<0.0001) from 1 to 24 h.

Evaluations of the benefits of probiotic bacteria in heifers have been contradictory, perhaps due to a lack of diversity in probiotics for specific geographical regions and sale of unviable strains. This makes it difficult to find experimental sequences utilizing the same strain⁴⁰. The marketing of probiotic products for heifers in Latin America is limited and dubious. Most suppliers offer only L. acidophilus strain KA1-A 8 (3 trillion CFU/dose) and L. casei (3 billion CFU/dose), without specifying their use in heifers. Others promote products containing Lactobacillus rhamnosus and Bifidobacterium lactis (1 x 10¹¹ CFU/ dose or 50 units for direct application in 1,000 L milk). But they do not specify strain origin, leaving open the possibility that they have been isolated from other animal species or food, making them suboptimum options for use in heifers. For example, when human probiotic strains are administered to livestock the microorganisms cannot resist gastrointestinal conditions or colonize the intestine due to interspecies differences in physiology and food^41,42. This is why the strains isolated in the present study were taken from the species in which they are intended for use and in the region they are to be applied; their probiotic potential can therefore be stated to be for Holstein cattle in the study area. Currently, the most widely used probiotic bacteria genera in livestock are Lactobacillus, Enterococcus, Bifidobacterium, Lactococcus and Leuconostoc⁴³; Lactobacillus plantarum, L. acidophilus, L. casei, L. salivarius and Lactococcus lactis^5,44,45; and L. fermentum VC3B-08, W. hellinica V1V-30 and L. farciminis B4F-06²⁰.