Carlos Gusils, Marina Bujazha and Silvia González

Carlos Gusils. Researcher, National Research Council of Argentina and Centro de Referencia para Lactobacilos (CONICET-CERELA). Instructor of Virology and Microbiology, National University of Tucumán (UNT).

Marina Bujazha. Researcher, Research Council of Tucumán National University (CIUNT), Tucumán, Argentina.

Silvia González. Researcher, CONICET-CERELA. Associate Professor of Public Health, UNT. Address: Centro de Referencias para Lactobacilos (CERELA-CONICET), Chacabuco 145, 4000, S.M. de Tucumán, Argentina. e-mail: sgonzal@cerela.org.ar

During inhibitory activity screening of 100 strains of lactic acid bacteria isolated from the gastrointestinal tract of pigs only six, four identified as Enterococcus faecium and two as Lactobacillus acidophilus, showed inhibition against enteric indicator strains: Salmonella enteritidis, S. cholera suis, S. typhimurium and Yersinia enterocolitica. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. Furthermore, these two L. acidophilus showed agglutination with treated yeast. The agglutination of one strain was inhibited by maltose, this suggests the presence of a lectin-like structure in their cell walls, which could be responsible for its adhesion ability. The selected strains were resistant to pH 3.0 and bile salts. These strains fulfil the conditions of probiotic bacteria and could be selected for elaborating pig probiotic feed, in order to prevent infectious diseases.

De 100 cepas de bacterias lácticas aisladas del tracto gastrointestinal de cerdos, solamente seis, cuatro identificadas como Enterococcus faecium y dos como Lactobacillus acidophilus, mostraron acción antagonista frente a bacterias patógenas entéricas: Salmonella enteritidis, S. cholera suis, S. typhimurium y Yersinia enterocolitica. La capacidad inhibitoria no fue afectada por el agregado de catalasa o por neutralización. Dos cepas de L. acidophilus presentaron capacidad de aglutinar con levaduras tratadas. Solamente, la aglutinación de una de ellas fue inhibida por el agregado de maltosa, esto sugiere la presencia de una estructura en su pared celular responsable de la capacidad de adhesión. Las cepas seleccionadas fueron resistentes a pH bajos (3,0) y presencia de sales biliares. Estas cepas podrían ser seleccionadas para el diseño de un alimento probiótico para cerdos con el objeto de prevenir enfermedades infecciosas.

De 100 cepas de bactérias lácticas isoladas do tracto gastrointestinal dos cerdos, somente seis, quatro identificadas como Enterococcus faecium e dois como Lactobacillus acidophilus, mostraram ação antagonista frente a bactérias patógenas entéricas: Salmonella enteritidis, S. cholera suis, S. typhimurium e Yersinia enterocolitica. A capacidade inibitória não foi afetada pelo agregado de catalasa ou por neutralização. Duas cepas de L. acidophilus apresentaram capacidade de aglutinar com levaduras tratadas. Somente, a aglutinação de uma delas foi inibida pelo agregado de maltosa, isto sugere a presença de uma estrutura em sua parede celular responsável da capacidade de adesão. As cepas selecionadas foram resistentes a pH baixos (3,0) e presença de sais biliares. Estas cepas poderiam ser selecionadas para o desenho de um alimento probiótico para cerdos com o objetivo de prevenir doenças infecciosas.

Received: 02/28/2002. Accepted: 06/12/2002

Infectious diarrhea of neonatal animals is one of the most common and economically devastating conditions encountered in the animal agriculture industry (Muralidhara et al., 1977). Traditionally, lactic acid was added (1%) to the drinking water for 10 days after weaning to avoid pathogen multiplication.

Probiotic foods can be administered to humans or animals in order to prevent infectious diseases, to strengthen the barrier function of the gut microflora and/or for a non-specific enhancement of the immune system (Sögaard and Suhr-Jessen, 1990). In the specific case of swine livestock they should be administered immediately before the weaning, at the beginning of the breeding stage, which represents the second unit in the program of intensive breeding. Probiotic foods can help pigs develop local immunity in the intestine and lactobacilli, generally included in this type of foods, ferment carbohydrates with lactic acid production.

The composition of the intestinal microflora in healthy animals remains steady, but if the stability is broken, pathogenic microorganisms, such as Yersinia or Salmonella can colonize the intestinal tract, leading to serious infections (Garriga et al., 1998).

According to Fuller (1989) a probiotic is a feed with live microorganisms, which beneficially affects the host animal by improving its intestinal microbial balance. Most probiotics contain single or multiple strains of lactic acid bacteria (LAB), which are considered as GRAS (generally regarded as safe) microorganisms.

Probiotic strains can be selected due to their condition of normal intestinal inhabitants of the host and several beneficial properties such as being active antimicrobial agents against pathogens (hydrogen peroxide, bacteriocins, and some organic acid such as lactic, acetic and propionic acids), the hydrophobic nature of the bacterial surface, stimulation of the immune system, presence of substances with adherence capacity to the epithelium (polysaccharides, lectins), coaggregation ability, and pH and bile salts resistance, among others.

Some microorganisms are able to bind to epithelial cells of the gastrointestinal tract through lectins present in their surface structures. Lectins are carbohydrate-binding proteins or glycoproteins from non-immune origin which agglutinate cells with receptors such as yeast and red blood cells (Slifkin and Doyle, 1990).

An important factor controlling in vivo adhesion and colonization is the animal species specificity of microorganisms, which indicates that bacterial strains isolated from the indigenous microflora of one animal species will not necessarily colonize the same site in another animal species.

Morata de Ambrosini et al., (1999) determined that Lactobacillus casei from human origin showed higher adhesion ability to ileal epithelial cells than L. casei from dairy origin (50% and 8% respectively). Furthermore, the pH range with highest adhesion capacity was between 6 and 7.5, which is within the normal pH range of the intestinal environment, and adhesion was only observed at 37ºC. On the other hand, in previous experiments we found higher adhesion values after incubation at 42ºC than at 37 or 30ºC, but in that case the study was performed on poultry, where the normal body temperature is 42ºC. This observation can be deemed host-specific, as lactobacillus strains and tissue fragments were obtained from chickens (Gusils et al., 1999).

The present study was designed to isolate, characterize and further select beneficial lactobacillus strains for the elaboration of a swine probiotic feed.

Lactic Acid Bacteria (LAB) were isolated from pig faeces at the Technological Ecophysiology Laboratory of CERELA, Tucumán, Argentina. Serial dilutions of faeces were plated (de Man et al., 1960) on Man, Rogosa, and Sharpe (MRS) agar (Merck), Streptococcus Selective Medium (Merck). Incubation was at 37ºC in a microaerobic atmosphere. The strains were identified with API 50 CH (Biomeriec) and other complementary tests according to the criteria of Bergey’s Manual of Determinative Bacteriology, 9th Edition. Strains of Salmonella and Yersinia were provided by the Enterobacteria Service and the Service of Special Bacteriology Department of the Instituto Nacional de Enfermedades Infecciosas (INEI) Dr. Carlos Malbrán, Buenos Aires, Argentina. Saccharomyces cerevisae was provided by the Planta Piloto de Procesamientos Industriales Microbiológicos (PROIMI), Tucumán, Argentina; and Mycobacterium sp. was provided by the Instituto de Microbiología Dr. Luis Verna, Universidad Nacional de Tucumán, Argentina

All LAB strains were kept at -20°C in a LAPTg broth (Raibaud et al., 1961) with 30% glycerol (v/v). Lactobacilli were activated and grown in a LAPTg medium.

The producer strain was grown in 1 liter of MRS broth at 30ºC for 18-20h, without pH control. The culture was heated to 70ºC for 30min to inactivate proteases and to kill cells, and the pH was adjusted to 6.5 with 4 M NaOH. Cells were collected by centrifugation (15,000g, 15 min), the pellet washed twice in 5mM sodium phosphate buffer (pH 6.5), resuspended in 10ml of 100mM NaCl at pH 2.0 (adjusted with 5% phosphoric acid), and mixed with a magnetic stirrer (29,000g, 20min at 4ºC), and the supernatant stored at -20ºC (Yang et al., 1992).

Bacterial cells were collected by centrifugation (15,000g for 10min) and suspended to one tenth of the original volume in phosphate buffered saline (PBS), pH 7.4. Agglutination was monitored visually on microscopic slides by mixing 10µl of the sample with 5µl PBS, and 10µl of a suspension of treated Saccharomyces cerevisiae (108 cells·ml-1 PBS). The yeast cells were prepared by preincubation in PBS with glutaraldehyde (1mg·ml-1) for 1h at 25ºC, washed twice with PBS, incubated for 30min at 25ºC with 10mg/ml glycine and washed as above. The treated yeast cells were stored at 4ºC as a suspension in PBS (0.1g·ml-1) containing 0.02% (w/v) sodium azide (Eshdat et al., 1978).

The ability of different sugars to inhibit agglutination was tested by mixing 10µl of suspension in PBS with 5µl of the sugar solution (fucose, galactose, glucose, lactose, mannose, N-ac. galactosamine, N-ac. glucosamine, sialic acid, sucrose were individually added at 1; 0.5; 0.2; 0.1 and 0.05 M), prior to the addition of 10µl of the yeast suspension.

Bacterial cells were collected by centrifugation (15,000g, 10min) and suspended to one tenth of the original volume in (PBS), pH 7.1. Hemagglutination was carried out at room temperature with a 96-well microtiter plate using PBS as a diluent. 50µl of a 2-fold diluted sample was mixed with 10µl of 2% pig erythrocyte suspension (SIGMA) in PBS. The strength of agglutination was read with the naked eye after 1h of incubation.

Cell surface hydrophobicity was determined by the bacterial adherence to hydrocarbons assay (Rosenberg et al., 1980). The test bacteria were grown at 37ºC in LAPTg broth. Unless otherwise stated, bacteria were harvested (10,000g, 10min) at the early logarithmic growth phase (12-18h), washed twice and resuspended in physiological solution (PS) to an optical density (OD600) of 0.5-0.7. To test tubes containing 3ml of washed cells, 1ml of test hydrocarbon (Hexadecane, Toluene and Xylene) were added. The mixtures were blended on a vortex mixer for 90s. The tubes were left to stand for 15min for separation of the two phases and the OD of the aqueous phase was measured. Hydrophobicity was calculated from three replicates as the percentage decrease in the optical density of the original bacterial suspension due to cells partitioning into a hydrocarbon layer. Mycobacterium sp was used as positive control and Lactobacillus acidophilus CRL 730 as negative control (Morata de Ambrosini et al., 1999). The percentage of hydrophobicity was calculated using the equation

In order to examine the resistance to bile salts, lactobacilli were grown in LAPTg broth supplemented with Oxgall (Difco) 0.1% or 0.4% (w/v)(1 or 4% bile salts). The cultures were incubated at 37°C for 24h and growth was monitored by measuring the optical density at 560nm.

Resistance of the isolates to pH 3.0 was tested as follows: overnight cultures of the isolated strains were centrifuged at 5,000g for 10min. After resuspending the pellet in the same buffer of saline solution, it was diluted 1/10 in sterile physiological solution (PS) at pH 3.0. After 3h at 37ºC, the appropriate dilutions were plated in selective agar medium and incubated at 37ºC for 48h.

The aggregation test was performed according to Reniero et al., (1992). Aggregation was scored positive when clearly visible sand-like particles, formed by the aggregated cells, gravitated to the bottom of the tubes, leaving a clear supernatant fluid within 2h.

At 48h of incubation, bacteria were harvested (15,000g, 10min), supernatants were used for determination of bacteriocin yield by an agar spot test, as previously described; and cellular pellets were washed and resuspended in PS for determination of lectin production by agglutination assay, as previously described.

Experiments were carried out in triplicate. Significant differences were tested using Tukey´s test (Minitab Student R12) (Rossman and Chance, 1998).

The screening of 100 lactic acid bacteria, isolated from intestinal content and fecal swabs of 25 pigs, determined that only 10 strains presented antimicrobial activity against pathogenic indicator bacteria. Between them 9 strains were able to inhibit the growth of Yersinia enterocolitica, S. enteritidis, S. typhimurium and S. cholerae suis; only one, the remaining strain was effective only against the two first pathogens above mentioned (Table I). However, the supernatants sterilized by heat, neutralized with NaOH solution or treated with catalase or proteolytic enzymes, were unable to inhibit the growth of pathogens. From the results it can be assumed that these lactic acid bacteria do not have capacity for antimicrobial production, but some microorganisms adsorb on their external structures, like-bacteriocin substances. For this reason bacteriocin presence on cell walls of pig lactic acid bacteria was studied, but the results determined that the assayed strains did not present this kind of substance in its external layers.

The adhesiveness of two strains isolated from pig was recorded as positive concerning the aggregation test of Reiniero et al. (1992), agglutination of treated yeasts and hemagglutination assays (Table I). The addition of sucrose (0.05M) inhibited agglutination and hemagglutination from the strain named 4c; these results could be correlated to the presence of an external lectin-like structure with sucrose affinity.

In previous works we found lectin-like structures in two lactobacilli isolated from chickens; the adhesion characterization of Lactobacillus animalis indicated that a lectin-like structure has glucose and mannose as specific binding sugars. The results about adhesion related to Lactobacillus fermentum showed that sialic acid and mannose are involved in the binding (Gusils et al., 1999).

The strains with antimicrobial and/or adhesive activity were resistant to bile salts (1 and 4%) and pH 3.0 (Table I). High acidity in the stomach and high concentration of bile components in the small intestine are the first host attributes that affect strain selection. Gilliland et al., (1984) observed a great variability among Lactobacillus acidophilus strains isolated from calf intestinal contents in their ability to grow in vitro in the presence of bile salts. When a strain exhibiting low tolerance to bile and another strain exhibiting high tolerance to bile were administered orally to calves, the more resistant strain caused greater increase in the number of facultative lactobacilli than the one possessing low tolerance (Nousiainen and Setälä, 1998).

Competition assays were carried out using different double mixed cultures between potentially probiotic strains and pathogenic microorganisms in order to study benefical properties. After incubation during 24h at 37°C the different mixed cultures, lactobacillus counts and lectin-like substance production did not present significant differences (P>0.05) with respect to control cultures (data not shown). On the contrary, in the same mixed cultures, partial inhibition of pathogens was observed when mixed and control cultures were compared (Figure 1). The antipathogenic effect observed in mixed cultures could be explained as a nutritional competition.

After physicochemical assays, four strains were identified as Enterococcus faecium and two as Lactobacillus acidophillus by the fermentation pattern in the API test and another biochemical determinations (Table II).

Among 100 strain isolates, only six were selected because of their potentially probiotic (adhesion capacity, antipathogenic activity against enteric bacteria, resistance to bile salts and pH 3.0, high hydrofobicity values of cell walls, etc. Studies related to the effect of oral administration to pigs of these potentially probiotic strains are in progress.

This research was supported by CIUNT under program D26/126 and by CONICET.

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