Selection and characterization of lactic acid bacteria with improved antimicrobial activity to poultry diarrhea inducing-pathogen by genome shuffling

Abstract

Various kinds of antibiotics have been added to the feed as additives to promote growth of livestock, increase productivity and protect from some kinds of pathogens. But the abuse of antibiotics to livestock caused the appearance of superbacteria that have strong resistance to most kinds of antibiotics. Superbacteria are hardly eradicated by antibiotics and they can impair the microflora of the intestinal environment. For this reason, from June. 2011, The usage of antibiotics was totally banned on livestock. And the substitutes for antibiotics are needed. Phage therapy, various kinds of natural fermentation products, exogenous enzymes and probiotics were mentioned as expected substitutes for antibiotics. Among others, probiotics have many other functions except for functions of substitutes for antibiotics. These include competitive exclusion efficacy, so probiotic microorganism can inhibit colonization of pathogens on the intestinal receptor, and induction of secretion of antimicrobial substance such as bacteriocin, organic acid, hydrogen peroxide and fatty acids etc. They can also increase the secretion of lysozyme, immunoglobulin and mucin from the intestinal epithelium, and modulate the host immune system. It can also reduce the cholesterol level in blood and provide increased intestinal barrier function. Thus, probiotics with improved antimicrobial activity may not also act as good substitutes of antibiotics but also be good substances that can provide health benefits to host. Therefore, the goal of our study is to breed probiotics with improved antimicrobial activity by using genome shuffling method and to use the selected genome shuffling mutants as a substitutes for antibiotics. Genome shuffling is a method which induces homologous recombination among genes of mutated protoplasts. Ultraviolet irradiation or other chemical mutation method usually induces point mutation (mutations in nucleotide-level). But genome shuffling induces mutation in gene locus level. So we can expect more remarkable phenotypic changes of genome shuffling mutants than those of UV mutants. Genome shuffling can induce direct evolution of cells and we can select the mutants with interested phenotypic change more easily. In experiment 1, we drew up anti-pathogenic ability standard index which is a table that represents original antimicrobial activity of wild-type lactic acid bacteria for major chicken diarrhea causing pathogens by conducting pathogen double-agar layer activity test. Based on anti-pathogenic ability standard index, a pair of Pediococcus acidilactia and Salmonella Gallinarum was chosen as a combination of strain breeding. In experiment 2, we selected UV mutants with improved antimicrobial activity by repeated co-cultivating UV mutants pool with Salmonella Gallinarum and by conducting pathogen double-agar layer activity test. After 8days of co-cultivation, the live UV mutants pool was selected as mutants with improved viability against pathogens. And then repeated this step for 3 rounds. And with the final pool of UV mutants with improved viability, conducted pathogen-double agar layer activity test. 5 UV mutants (M3, M2', M4', M5', M6') which revealed increased CC ratio were finally selected as mutants with improved antimicrobial activity. To quantitatively measure the antimicrobial activity in culture supernatants of UV mutants, critical-dillution micro-method was conducted by using finally selected 5 UV mutants. Selected UV mutants showed lower optical density values than those of wild-type at the same dilution magnificiation. So supernatants of culture broth of selected UV mutants showed a little bit higher antimicrobial capacity than those of wild-type. And to quantitatively measure the antimicrobial activity of UV mutants, we conducted pathogen-LAB co-cultivation assay by using finally selected 5 UV mutants. Selected UV mutants showed lower viable cell counts of pathogens than those of wild-type after 8.5hours of co-cultivation with pathogens. Selected UV mutants were able to inhibit growth of pathogens approximately 7% more than wild-type Pediococcus acidilactia. In experiment 3, we selected genome shuffling mutants with improved antimicrobial activity by repeated co-cultivating genome shuffling mutants pool with pathogens and by conducting Pathogen double-agar layer activity test. UV mutants M3, M2', M4', M5', M6' were used as the starting population for genome shuffling strategy. After 8days of co-cultivation, the live Genome shuffling mutants pool was selected as mutants with improved viability against Salmonella Gallinarum. And then repeated this step for 3 rounds. With the pool of Genome shuffling mutants with improved viability, we conducted pathogen-double agar layer activity test. And then repeated this step for 3 rounds. And with the pool of genome shuffling mutants with improved viability, we conducted pathogen-double agar layer activity test. 4 Genome shuffling mutants (GS1, GS2, GS3, GS4) which revealed increased CC ratios than those of UV mutants were finally selected as mutants with improved antimicrobial activity. To quantitatively measure the antimicrobial activity in culture supernatants of genome shuffling mutants. critical-dillution micro-method was conducted by using finally selected 4 genome shuffling mutants. Selected genome shuffling mutants showed lower optical density values than those of wild-type at the same dilution magnificiation. So supernatants of culture broth of selected genome shuffling mutants showed a little bit higher antimcirobial capacity than those of wild-type. And to quantitatively measure the antimicrobial activity of genome shuffling mutants, we conducted pathogen-LAB co-cultivation assay by using finally selected 4 genome shuffling mutants. Selected genome shuffling mutants showed lower viable cell counts of pathogens than those of wild-type after 8.5hours of co-cultivation with pathogens. Selected genome shuffling mutants were able to inhibit growth of pathogens approximately 11% more than wild-type Pediococcus acidilactia. In experiment 4, we characterized antimicrobial activity for finally selected mutants. By conducting bacteriocin activity assay, we identified that bacteriocin induction amounts of culture supernatants of Salmonella Gallinarum-GS1 mutant co-cultured broth were much higher than those of GS1 mutant solely cultured broth. Also, Bacteriocin induction amounts of culture supernatants of Salmonella Gallinarum-GS1 mutant co-cultured broth were much higher than those of Salmonella Gallinarum-wild type LAB co-cultured broth. By conducting pH effect assay, we identified that induction amounts of acidic molecule of culture supernatants of Salmonella Gallinarum-GS1 mutant co-cultured broth were higher than those of GS1 mutant solely cultured broth. Also, Induction amounts of acidic molecule of culture supernatants of Salmonella Gallinarum-GS1 mutant co-cultured broth were higher than those of Salmonella Gallinarum-wild type LAB co-cultured broth. We also identified that there was almost no induction of hydrogen peroxide by conducting hydrogen peroxide activity assay In its final analysis, Pediococcus acidilactia showed no antimicrobial activity by production of hydrogen peroxide. And lastly, we drew up growth curve and pH curve of wild-type, UV mutants and genome shuffling mutants. by pH curve, we also confirmed that genome shuffling mutants represented slightly low pH value than that of wild-type. And when co-cultivated with pathogens, induction amounts of acidic molecule were a little bit higher than those of solely cultured Pediococcus acidilactia. In conclusion, GS1 was finally selected as a genome shuffling mutants with improved antimicrobial activity against Salmonella Gallinarum which is known as a pathogen causing poultry diarrhea and sometimes leads to death. GS1 represented 11% higher antimicrobial capacity than that of wild-type. We expect that the GS1 could have a great potential to be a specialized probiotic having preventive effect to the infectious disease such as fowl typhoid.