Characterization of Lactobacillus acidophilus Isolated from Piglets and Chicken

Lactic acid bacteria were isolated from piglets and chicken and characterized. Lactic acid bacteria showing resistance to low pH and bile, adhesion to intestinal epithelium cells, and the inhibition of Escherichia coli and Salmonella spp. were identified as Lactobacillus acidophilus. L. acidophilus PF01 survived for 2 h in MRS broth adjusted to pH 2. L. acidophilus CF07 was less resistant than L. acidophilus PF01 to pH 2, but survived at pH 2.5 for 2 h. Both of isolates were able to grow in MRS broth containing 0.3% (w/v) bile, with L. acidophilus CF07 being more tolerant to bile than L. acidophilus PF01. L. acidophilus PF01 and CF07 adhered specifically to the duodenal and jejunal epithelium cells of piglet, and the cecal and duodenal epithelium cells of chicken, respectively. Both of isolates did not adhere to the epithelium cells of the various animal intestines from which they were isolated. When L. acidophilus was cultured with E. coli and Salmonella spp. in MRS broth, MRS broth containing 2% skim milk powder or modified tryptic soy broth at 37°C, L. acidophilus PF01 and CF07 inhibited the growths of E. coli K88 and K99, and S. enteritidis and S. typhimurium, respectively. Both of isolates were found to possess the essential characteristics of probiotic lactic acid bacteria for piglet and chicken. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 12 :1790-1797)


INTRODUCTION
and 42°C, respectively, for 18 h before experimental use.

Identification of lactic acid bacterial isolates
Lactic acid bacteria growing well in MRS broth adjusted to pH 4.0 as well as containing 0.2% (w/v) porcine bile extract (Sigma Chemical co., St. Louis, MO) or 0.2% (w/v) oxgall (Difco) were selected and used for further study. Identification of lactic acid bacterial isolates was based upon the species description in the Bergey's Manual (Kandler and Weiss, 1986). Utilization of carbohydrates by lactic acid bacteria was assayed using API kit (API 50 CHL, bioMėrieux, France), and the results were analyzed using the APILAB Plus software (Ver. 3.3.3; bioMėrieux, France).

Characteristics of lactic acid bacteria
Each overnight culture (10 4 CFU/ml) of lactic acid bacteria in MRS broth was inoculated into new MRS broth, incubated at 32, 37, 42, and 47°C, and plate-counted on MRS agar. Growth of lactic acid bacteria at various temperatures was analyzed using IFR MicroFit software (Ver. 1.0; UK Ministry of Agriculture, Fisheries and Foods) based on the model of Baranyi and Roberts (1994).
Survival of lactic acid bacteria isolated from piglets in MRS broth adjusted to pH 2 with 1 N HCl at 37°C was studied. Culture samples were taken every 20 min up to 2 h of incubation to count surviving cells. The culture samples were diluted serially in 0.1% (w/v) peptone solution, and 0.1 ml aliquots of appropriate dilutions were spread on MRS agar, which were then incubated at 37°C for 48 h. Survival of chicken isolates in MRS broth adjusted between pH 2 to 4 with 1 N HCl was determined. The surviving cells were counted after grown on MRS agar at 42°C, as described for lactic acid bacteria from piglets.
Lactic acid bacteria from piglets were inoculated into MRS broth containing 0.1, 0.3, and 0.5% (w/v) bile extracts (Sigma), and incubated at 37°C for 24 h. The surviving cells after bile challenge were tested for bile tolerance as described above for the acid tolerance. Bile tolerance of lactic acid bacteria from chicken was tested in MRS broth containing 0.3, 0.4, and 0.5% (w/v) oxgall (Difco) at 42°C using the same procedures as those for piglets. To estimate the growth inhibition of lactic acid bacterial isolates by bile, the inhibition coefficient (C-Inh) was calculated using the modified method of Gopal et al. (1996). Log 10 numbers of viable cells of lactic acid bacterial isolates were calculated at the end of the exponential growth phase, and C-Inh was calculated according to the following formula.
C-Inh= (A-B):A where, A is Log 10 number of viable cells without bile, and B is Log 10 number of viable cells with bile

Adhesions of lactic acid bacterial cells to the intestinal epithelium cells
The adhesions of lactic acid bacterial cells to the intestinal epithelium cells of piglets and chicken were examined in vitro. Suspensions of the intestinal epithelium cells of piglets and chicken were prepared using the method of Mäyrä-Mäkinen et al. (1983). The duodenum and jejunum of piglets were cut into 2×4 cm pieces, and the cecum and duodenum of chicken into 1×4 cm pieces. The epithelium tissue samples were held in buffered phosphate saline (PBS) at 4°C for 30 min to loosen the surface mucus, and washed seven times with PBS. Surfaces of epithelium tissues were scraped off with the edge of a sterile microscope slide, and the epithelium cells were suspended in PBS. All cell suspensions were examined microscopically to verify the removal of adherent bacteria. The adhesion of the lactic acid bacterial cells was tested using the method of Fuller et al. (1978). Each overnight culture broth of L. acidophilus cell was centrifuged at 6,000 rpm for 15 min at 4°C, and the cells (10 8 CFU/ml) were suspended in PBS. The epithelium cell suspension (1 ml) was then mixed with the lactic acid bacterial cell suspension (1ml), and the tubes containing the cell mixture were rotated at 20 rpm/min at 37°C for 30 min. Adhesion of the bacterial cells to the epithelium cells was examined using light microscopy (×1,500) (AXIOPHOT; Zeiss, Germany) after Gram-staining.

Inhibitions of E. coli and Salmonella spp. by lactic acid bacteria
Inhibitions of E. coli and Salmonella spp. by lactic acid bacteria were examined using the method of Ahn et al. (1997). MRS broth containing lactic acid bacteria (10 5 CFU/ml) were incubated with the same number of E. coli or Salmonella spp. cells at 37 and 42°C for isolates from piglet and chicken, respectively. Viable cells of lactic acid bacteria, E. coli, and Salmonella spp. on MRS agar containing 0.02% (w/v) sodium azide, EMB agar (Difco), and bismuth sulfite agar (Difco), respectively, were determined as follows. Serial dilutions of the bacterial suspensions were prepared in 0.1% (w/v) peptone water. Aliquots of the dilutions were plated out on each selective medium mentioned above and were incubated at 37 or 42°C for 48 h before counting typical colonies.

Identification of lactic acid bacterial isolates
Among the lactic acid bacterial isolates from piglets and chicken, two strains able to grow well in MRS broth adjusted to pH 4.0 as well as containing 0.2% (w/v) bile extract or 0.2% (w/v) oxgall were chosen for further study. Based upon the Bergey's Manual (Kandler and Weiss, 1986) and the utilization of carbohydrates, they were identified as Lactobacillus acidophilus: L. acidophilus PF01 and CF07 from piglet and chicken. Both of L. acidophilus strains were Gram positive, homofermentative, catalasenegative, and non-sporeforming rods. Their characteristics and fermentation of carbohydrates are listed in Tables 1 and 2, respectively. Their carbohydrates fermentation gave the same patterns with the exception of mannose, salicin and trehalose: L. acidophilus PF01 fermented trehalose but did not ferment mannose and salicin, and L. acidophilus CF07 fermented mannose and salicin but not trehalose.

Growth of L. acidophilus
In order to survive and thrive in the gastrointestinal tract of the host, a short generation time is of utmost importance; otherwise the bacteria are washed off by the contractive motion of the gut (Havenaar et al., 1992). The doubling times of L. acidophilus PF01 and CF07 were estimated to be 1.71 and 1.07 (32°C), 0.52 and 0.57 (37°C), 0.44 and 0.47 (42°C), and 1.46 and 0.74 h (47°C), respectively. The two isolates generally grew slightly faster at 42°C than at 37°C. L. acidophilus PF01 isolate grew better near its optimum temperature (between 37 and 42°C), whereas L. acidophilus CF07 was able to grow well at broader range of temperatures and grew slightly better at above the temperature considered to be the optimum for L. acidophilus ( Figure 1). This difference in growth temperatures is probably due to the adaptation of the bacteria to the body temperature of the host. That is, L. acidophilus CF07 isolate from chicken grew better at the body temperature of chicken.

Acid tolerance of L. acidophilus
L. acidophilus PF01 from piglet survived for 2 h in MRS broth acidified to pH 2.0 ( Figure 2a), while L. acidophilus CF07 survived at pH 2.5 for 2 h (Figure 2b). The microorganisms in the stomach are generally inhibited by the gastric acid, which contains hydrochloric acid (Gilliland, 1979). Conway et al. (1987) claimed that microbial cultures for use as probiotics should be screened for their resistance to strong acidity. L. acidophilus is generally more resistant to low pH than other lactobacilli (Pettersson et al., 1983;Ahn et al., 1999). Ahn et al. (1999) reported that most L. acidophilus isolated from fermented milk products on Korean markets survived well (>90%) for 2 h in 12% (w/v) skim milk adjusted to pH 2.5 except L. casei ssp. rhamnosus. However, Jin et al. (1998) reported variations in the survival of the lactic acid bacteria at low pH depended on the origin of L. acidophilus strains, and those living in the cecum showed better survival rate in acidic environment than those from the ileum.

Bile tolerance of L. acidophilus
Bile tolerances of L. acidophilus PF01 and CF07 were investigated, and the results are shown in Figure 3. Although the growths of L. acidophilus PF01 and CF07 decreased with increasing concentration of bile extract or oxgall, both isolates grew well in the presence of 0.3% (w/v) bile in MRS broth. The inhibition coefficients (C-Inh) of L. acidophilus PF01 in the presence of bile extract were 0.11 (0.1%), 0.25 (0.3%), and 0.39 (0.5%), whereas those of L. acidophilus CF07 in the presence of oxgall were 0.13 (0.3%), 0.19 (0.4%), and 0.28 (0.5%) ( Table 3). L. acidophilus CF07 was more tolerant to bile than L. acidophilus PF01. The bile tolerance of L. acidophilus is an important characteristic for the survival and growth of the bacteria in the intestinal tract (Gilliland, 1979;Gilliland et al., 1984).

Adhesion of L. acidophilus to the intestinal epithelium cells
Attachment of the lactic acid bacterial cells to the intestinal mucosa of the host is one of the main selection criteria for probiotic bacteria and is considered as a prerequisite for colonization (Ouwehand et al., 1999). L. acidophilus PF01 cells from piglet adhered heavily to the jejunal epithelium cells, whereas less to the duodenal epithelium cells of piglet (Figure 4). On the other hand, L. acidophilus CF07 cells from chicken adhered well to the cecal and duodenal epithelium cells of chicken ( Figure 5). However, L. acidophilus cells did not adhere to the intestinal epithelium cells of the various hosts from which they were isolated (Figures 4 and 5). These results confirmed the host-specific adherence of the lactobacilli tested, as reported by many other researchers (Fuller, 1975;Suegara et al., 1975;Barrow et al., 1980;Mäyrä-Mäkinen et al., 1983).

Inhibition of E. coli and Salmonella spp. by L. acidophilus
Growth retardations of E. coli and Salmonella spp. cells were evident when these pathogens were challenged by L. acidophilus cells in MRS broth. L. acidophilus PF01 cocultured with the same number of E. coli K88 or K99 cells in MRS broth reached the stationary growth phase after about 10 h of incubation, and the number of viable cells reached above 10 9 CFU/ml (Figure 6a and b). However, the growths of E. coli K88 (Figure 6a) and K99 (Figure 6b) were retarded considerably at the same growth phase,    Ahn et al. (1997) reported that the growths of E. coli O157 and S. typhimurium were considerably inhibited when L. acidophilus NCFM reached the stationary growth phase. Co-culturing with L. acidophilus CF07 resulted in the growth of S. enteritidis and S. typhimurium up to 6 h of incubation in MRS broth; however, the number of viable pathogens decreased when pH of the culture broth reached 5.73 and 5.56, respectively, and the growth of L. acidophilus CF07 reached the mid-exponential phase (Figure 7). E. coli survived better than Salmonella spp. in MRS broth when co-cultured with L. acidophilus. Since MRS broth is not a good medium for both E. coli and Salmonella, 0.2% (w/v) skim milk powder was added into the MRS broth to determine its effect on the growth of E. coli. Effect of modified TSB containing 1% (w/v) dextrose and 0.1% (w/v) yeast extract on the growth of Salmonella  spp. was determined. When L. acidophilus PF01 was cocultured with E. coli K88 or K99 in MRS broth containing 0.2% (w/v) skim milk powder, L. acidophilus PF01 grew slightly slower, and the growths of E. coli K88 and K99 were inhibited when the culture exhibited pH 4.45 ( Figure  8a and b). On the other hand, S. enteritidis and S. typhimurium outgrew L. acidophilus CF07 in modified TSB during 14 h of incubation. The growths of S. enteritidis and S. typhimurium were retarded at pH 4.62 and 5.08, respectively (Figure 9). Although some differences were observed depending on the nutritional environments, Salmonella spp. was more easily inhibited by L. acidophilus than E. coli, which is slightly more resistant to low pH than Salmonella spp.. L. acidophilus has antagonistic activities against Staphylococcus aureus, S. typhimurium, enterophothogenic E. coli, and Clostridium perfringens (Gilliland and Speck, 1977), mainly due to low pH and organic acids, particularly lactic acid (Ahn et al., 1997;Jin et al., 1996b;Hechard et al., 1990;Reinheimer et al., 1990). The organic acids produced by L. acidophilus PF01 and CF07 appeared to be the main factors for the antagonistic activities of L. acidophilus against E. coli and Salmonella spp. tested.
In conclusion, L. acidophilus PF01 and CF07 were confirmed to have most of the required characteristics, including acid and bile tolerances, specific adhesion to the host cells, and inhibition of pathogens. Furthermore, they are found in large numbers in animal feces (unpublished data), an indication that they survive and grow well in the gastrointestinal tracts of piglet and chicken.