The poultry industry has become more receptive to alternatives that allow reductions in production costs. Moreover, the industry also seeks to improve the quality of its final product, so as to meet consumer expectations. Consumers are increasingly becoming concerned about food safety and seek food that is free from antibiotics1.
As explained by Martins2, early use of probiotics is extremely important for sanitary management of poultry. Considering that these birds originate from the extremely clean and disinfected environment of an incubator, the first bacteria to come into contact with their gastrointestinal tract will tend to colonize the region more rapidly. This creates a competitive environment that is difficult for other bacteria to colonize. Salmonella is among the early colonizers.
Thus, the use of probiotics has steadily increased. Another reason for this increase is that probiotics improve the intestinal microbiota of poultry, which then leads to avoidance of indiscriminate use of antibiotics when rearing these birds3.
The presence of Lactobacillus spp. in the microbiota of poultry has been proven to be beneficial4. In addition to decreasing colonization by pathogenic microorganisms such as Salmonella spp., Lactobacillus spp. can act in other ways in its environment. These characteristics make this microorganism a good probiotic for use in the poultry industry.
The mechanisms used by Lactobacillus spp. to inhibit other bacteria in vitro are believed to involve production of hydrogen peroxide, specific proteins known as bacteriocins and organic acids. These acids include lactic and acetic acids, which decrease pH and secrete lactate, acetate, succinate and ethanol, thereby aiding the proliferation of other beneficial bacteria. Moreover, Lactobacillus spp. adheres to the intestinal mucosa and limits multiplication of pathogenic bacteria such as Escherichia coli, Salmonella spp. and Campylobacter spp.5.
According to Miyamoto et al.6, the Lactobacillus species found in the intestinal microbiota of poultry are Lactobacillus acidophilus, Lactobacillus salivarius and Lactobacillus fermentum. Other species are rarely observed.
On the other hand, Salmonella spp. is an undesirable microorganism for the intestinal microbiota of poultry. Not only it is harmful to the birds health, but also it is considered to be a public health risk and can trigger large outbreaks of food poisoning7,8.
According to Matte9, outbreaks of food poisoning related to Salmonella Heidelberg have increased considerably over recent years. Infection due to Salmonella Heidelberg has been shown to be more serious than other Salmonella infections and may lead to conditions of septicemia, myocarditis and extra-intestinal infections and even death.
Thus, these microorganisms started to be studied using methods and techniques to investigate the antagonism among them. One of the main techniques currently used is the Spot on the Lawn test, which has yielded the most significant results for research. In these studies, Lactobacillus spp. have gained the spotlight and have revealed positive results when challenged with pathogenic samples10.
Concomitant cultures are another method used to observe antagonism. In addition to defining the degree of inhibition caused by Lactobacillus spp., it can also be measured every hour, which is an additional advantage of this method11.
The objective of the present study was to correlate the results obtained using two methods for measuring antagonist activity between Lactobacillus spp. and Salmonella Heidelberg and to observe whether the correlation between the two methods tested was positive or negative.
MATERIALS AND METHODS
A total of 10 samples of Lactobacillus spp. and one sample of Salmonella Heidelberg were used. All the samples originated from birds and were taken from the bacterial collection of the Avian Pathology Laboratory of the School of Veterinary Medicine and Animal Science (FMVZ), located in the Municipality of Botucatu, São Paulo.
The first day of the technique consisted of enriching the samples of Lactobacillus spp. in DeMan-Rogosa-Sharpe (MRS)* agar in an incubator at 38°C for 18 h. After this period, on the second day, 10 μL were individually seeded at three points in a petri dish containing MRS agar. The petri dish was also placed in the incubator at 38°C for 18 h. On the same day, the Salmonella Heidelberg sample was enriched in a brain-heart infusion (BHI) broth and was placed in the incubator at 38°C for 18 h.
On the third day, 200 μL of Salmonella Heidelberg were transferred to a new tube containing BHI broth with 0.65% agar-agar at 40°C. This content was immediately placed on a dish that had been pre-cultured with Lactobacillus spp. After the agar had solidified, the dish was placed in an incubator at 38°C for 12 h. The inhibition halos were then measured.
The second part of the technique involved simultaneous mixtures of the two microorganisms (Salmonella Heidelberg and Lactobacillus spp.), in equal proportions of the culture mediums (BHI and MRS, respectively). These mixtures were kept in an incubator at 38°C for 9 h.
The samples were then removed from the incubator and serial decimal dilutions were performed to count colony forming unit (CFU) every hour. This technique consists of transferring the mixture of bacteria to a phosphate-buffered saline solution at proportions of 1:10. The other dilutions were obtained with serial decimal dilutions, with subsequent plating of all dilutions in brilliant green agar (BGA). This same process was repeated every hour over a period of 9 h using the mixture of microorganisms.
After the plating process, the plates were stored in an incubator for a period of 12 h at 37°C. This enabled counting of the Salmonella Heidelberg present, which was expressed in CFU mL1 of culture medium.
The antagonistic action of Lactobacillus spp. towards Salmonella Heidelberg in the Spot on the Lawn method is shown in Table 1.
All samples of Lactobacillus spp. submitted to the Spot on the Lawn test, showed inhibition against the indicator microorganism (Salmonella Heidelberg), but with different potentials of inhibition. As for example, sample 1 and 2 had the minimum and the maximum potential of inhibition against Salmonella Heidelberg, respectively.
Table 2 lists the colony-forming unit vales obtained by concomitantly culturing Lactobacillus spp. and Salmonella Heidelberg over a 9 h period of mixture, starting from bringing these two microorganisms together.
Table 2 shows the amount of colony forming units of Salmonella Heidelberg after concomitant culturing with the different strains of Lactobacillus spp. for 9 h.
With this test, it was clear that the different strains of Lactobacillus spp. act in different ways, as demonstrated by strain 3, which obtained the lowest count, strains 2, 4, 5 and 10 with intermediate counts and strains 1, 6, 7, 8 and 9 with the highest Salmonella Heidelberg count number.
It is suggested in this case that the inhibition may have occurred mainly by the production of lactic acids, which decrease the pH of the medium and make the environment not ideal for the development of Salmonella Heidelberg. However, there are several other factors that could have influenced this result, which may increase or decrease the inhibitory capacity of Lactobacillus spp.
Use of probiotics in the poultry industry is not recent, given that several authors have already reported their use since the beginning of this century. The main outcomes from their use are improvements of economic and zootechnical indexes among the animals, thereby increasing weight gain and improving food conversion7,6.
Several studies have proven that Lactobacillus spp. are potential inhibitors of Salmonella spp. and other microorganisms that are considered pathogenic to the gastrointestinal tract of poultry13,14,4.
Pereira and Gomez1 reported that the strain Lactobacillus acidophilus was able to inhibit strains of Escherichia coli and
Staphylococcus aureus, in vitro.
||Antagonistic effect of Lactobacillus spp. against Salmonella Heidelberg through the Spot on the Lawn method
|*Measured in centimeters from the inhibition halo obtained from the Spot on the Lawn test|
Colony-forming units from concomitant culturing of Lactobacillus spp. and Salmonella Heidelberg over a 9 h period of mixture
*Measured in centimeters from the inhibition halo obtained from the Spot on the Lawn test
They found that the peak inhibitory activity of L. acidophilus was reached after 72 h of incubation at 37°C in a MRS broth under aerobiotic conditions. Moreover, they explained that these results were attained probably due to low pH values and to the action of lactic acid on the strains studied.
In turn, Poppi et al.15 demonstrated the important role of acids produced by samples of Lactobacillus, particularly lactic acid, in the antagonistic effect of pathogenic samples. They observed that addition of sodium bicarbonate (acid neutralizer) hindered the inhibitory effect of some strains of Lactobacillus. However, not all samples in their study had the same result. Some strains presented inhibition even with addition of sodium bicarbonate, thus demonstrating that this effect is caused by a combination of various factors and products generated by Lactobacillus.
Regarding the method used, Shanthya et al.16 and Cadirci and Citak10 reported that the best method for evaluating antagonism between samples is the Spot on the Lawn method. However, Soomro et al.11 disagreed with this and stated that the best technique was in fact the paper disc method, which is considered easy to apply. Nevertheless, all authors have agreed that the inhibition that occurs with the Spot on the Lawn method is largely due to the metabolites produced, such as lactic acid, acetic acid and bacteriocins16,11.
Miyamoto et al.6 suggested that Lactobacillus could present a protective effect for poultry against colonization by Salmonella Enteritidis, considering the inhibition halos obtained through the Spot on the Lawn technique. Similarly to what was obtained in the present study, halos were formed around the samples, measuring between 0.2 and 1.6 cm.
There are no data in the literature regarding use of the concomitant culturing technique to evaluate and quantify antagonism between samples. However, the samples that showed formation of large halos in the Spot on the Lawn method did not present the best performance in the concomitant culturing and vice versa.
According to Borowsky17, in the specific case of Salmonella spp., quantification is not done routinely, which makes studying these microorganisms more difficult. Moreover, the results vary greatly according to the methods used for analyses.
This situation was observed in comparing sample numbers 2 and 3. While sample 2 presented the largest halo by means of the Spot on the Lawn technique, inhibition in this same sample using the CC technique was only completed after 7 h of contact. On the other hand, sample 3 presented an intermediate halo and was able to inhibit Salmonella Heidelberg within the first hour of contact.
The results obtained may have varied because the amount of Lactobacillus spp. inoculated in each sample was not assessed. Moreover, the pH of the environment, the concentration of the inoculum and the culture medium itself are all considered to be variables.
The medium used in the present study for culturing of Lactobacillus spp. differed from what was used by Lopes12 in their study, different culturing mediums were compared and it was demonstrated that the one with greatest production of lactic acid had been enriched with tomato extract and had been agitated. Moreover, in comparison with the medium CSN 12**, the standard medium***, can be considered to be the second best medium for production of large amounts of lactic acid from Lactobacillus spp.
Differences among the results were expected, since different strains of Lactobacillus were used. This demonstrates that some strains are more resistant towards inhibiting Salmonella Heidelberg than others.
However, more studies are still needed, particularly regarding the inhibition promoted by Lactobacillus spp. in vivo. The tables demonstrated that samples with higher inhibition capacity through the Spot on the Lawn technique did not present high inhibition capacity when cultured together in the same medium. Thus, this result shows that there was neither a positive nor a negative correlation between the tests.
It is concluded that all Lactobacillus spp. inhibit Salmonella Heidelberg, it was not possible to correlate the data obtained, since each Lactobacillus spp. presented a halo measurement that did not correspond to the quantity of Salmonella Heidelberg actually inhibited, due to other factors involved.
*MRS medium composed of dextrose, peptonate, yeast extract, beef extract, ammonium citrate, sodium acetate, sorbitan complex, magnesium sulfate, manganese sulfate, disodium phosphate and water at pH 6.512
**CSN 12 medium composed of peptonate, yeast extract, beef extract, ammonium citrate and clarified sugar cane broth at pH 6.212.
***Standard medium composed of sucrose, yeast extract, magnesium sulfate (0.2 g L1), manganese sulfate (0.01 g L1), sodium chloride (0.01 g L1), ferrous sulfate
(0.01 g L1) (Sol A), dibasic potassium phosphate (Sol B), calcium chloride (Sol C) and water at pH 6.212