Role of volatile fatty acids in competitive exclusion of Salmonella enteritidis

Abstract

In the early seventies, it had been discovered that the addition of an undefined mature microflora to 1-day-old broiler chickens inhibited Salmonella in the caeca of these broilers. This phenomenon is known as competitive exclusion. Thereafter, scientists have focused on developing defined competitive exclusion mixtures of caecal bacteria. One problem in developing defined mixtures is that the mechanism behind competitive exclusion is unknown. The aim of this thesis is to study the suggested role of volatile fatty acids in competitive exclusion of Salmonella enteritidis in the caeca of broiler chickens. In addition, this thesis aims to develop a defined competitive exclusion culture with a low number of bacterial species. Results from a first study showed that volatile fatty acids are responsible for the reduction of members of the family of Enterobacteriaceae in the caeca of broiler chickens during growth. Since S. enteritidis is a member of this family, these results might indicate that volatile fatty acids could be responsible for the inhibition of S. enteritidis as well. This was further studied in a sequencing fed-batch reactor which mimicked caecal ecophysiology. It was shown that S. enteritidis could be washed out from the reactor when concentrations of volatile fatty acids (representative for a maturated microflora), were applied to a transient state culture of S. enteritidis. In contrast, inhibition of S. enteritidis was less pronounced when concentrations of volatile fatty acids (representative for a caecal microflora of young broilers), were applied. In addition, it was observed that a rapid increase of volatile fatty acids in the reactor, as measured in the caeca of broiler chickens treated with competitive exclusion mixtures, caused washout of S. enteritidis as well. On basis of these results it was decided to develop a defined mixture of two caecal bacterial species, Lactobacillus crispatus and Clostridium lactatifermentans, which are capable of producing volatile fatty acids from lactose. The effect of both bacteria on S. enteritidis was studied in the same sequencing fed-batch reactor. Results showed that both bacteria were capable of inhibiting S. enteritidis, especially when both bacteria were established in the reactor before S. enteritidis. Furthermore, it was shown that inhibition occurred at caecal pH (5.8) but not at pH 7.0. This indicates that the undissociated concentrations of acetate and propionate, produced by a coculture of L. crispatus and C. lactatifermentans, are responsible for the reduction of S. enteritidis. Finally, it was studied whether L. crispatus, C. lactatifermentans and dietary lactose can increase concentrations of volatile fatty acids in the caeca of broiler chickens. It was shown that caecal concentrations of lactate were increased in treated broiler chickens but concentrations of acetate, propionate and butyrate were not. Since L. crispatus produces lactate and C. lactatifermentans acetate and propionate, it is concluded that C. lactatifermentans had not become established in the caeca of treated broilers under these experimental conditions. It is concluded from this thesis that volatile fatty acids play a pivotal role in the reduction of S. enteritidis from the caeca of broiler chickens. Furthermore, the developed competitive exclusion culture of L. crispatus and C. lactatifermentans is capable of inhibiting growth of S. enteritidis. In order to obtain reduction in broiler chickens, colonisation of the caeca by L. crispatus and C. lactatifermentans is an absolute prerequisite.