Lactic acid bacteria in silage – growth, antibacterial activity and antibiotic resistance

Ensiling is a method that has been known for hundreds of years and been used in Sweden since 18 century. Ensiling is a better way to preserve forage than hay making because the method is not as rain sensitive. Lactic Acid Bacteria (LAB) are the organisms responsible for the preservation; they ferment Water Soluble Carbohydrates (WSC) under anaerobic conditions and produce lactic acid, which lowers the pH. These conditions inhibit growth of other microorganisms. To control the ensiling process, improve quality and to inhibit non-wanted microorganisms, LAB can be used as additives in silage making. The aim of this study was to follow two strains of LAB, Lactobacillus plantarum M14 and L. coryniformis Si3, in silage during 90 days to see how they grow, and also, after 90 days see if they have improved the quality of the silage. Both strains were labelled with selectable antibiotic resistance markers, and used to inoculate grass-dominated crop in mini silos. The growth of the labelled strains was followed on agar plates containing antibiotics and with PCR. After 60 days, a contamination of both labelled strains was discovered in the control silo. Further investigation showed that the contamination had been present since the packing of the silos, which calls for a revision of the methods used to inoculation and packing. Following the growth of the labelled strains showed differences between them but both strains were able to outgrow the epiphytic flora and probably dominate the fermentation. When using LAB as feed additives or starter culture in silage, antibacterial properties in addition to antifungal properties are desirable. Four strains of LAB were tested against one strain of Clostridium butyricum and three strains of C. turybutyricum with the agar well method. All of the four strains tested were able to inhibit two or more clostridial strains. Antibiotic resistance can be transferred from non-pathogenic bacteria like LAB to pathogenic bacteria and thereby cause problems in the human and veterinary medicine. It is important that LAB strains that are going to be used as feed additives do not carry resistance genes that can be transferred. The occurrence of antibiotic resistance among LAB was determined with three different methods and culture media in this study. The epiphytic flora of grass was screened against six antibiotics. LAB from the culture collection of Department of Microbiology, SLU were screened against antibiotics in different concentrations and finally four strains of LAB were tested with VetMICTM plates to determine their MIC value of 16 antibiotics. There are differences between the methods used but it can be seen that almost all strains tested seems to be resistant to or have high MIC values for tetracycline often exceeding the breakpoint. Similarly, the MIC values for chloramphenicol were close to the breakpoint. Tetracycline and streptomycin seems to be the two antibiotics most affected by method and media. It is very important to find a method and media suitable for testing antibiotic resistance among LAB in a way that can be repeated and also reliable in laboratories all over the world.