А probiotic based on the Escherichia coli ZP strain. I. Efficiency assessment of the conjugative transfer of the colicin E7 activity gene into avian pathogenic E. coli strains in vitro and in vivo

The protection of farm animals against infectious diseases is a priority in veterinary medicine. The wide spread of pathogenic and opportunistic bacteria resistant to antibiotics on poultry farms requires the development of modern methods to maintain the health of birds in industrial production. A promising direction to improve measures to prevent and limit the spread of pathogens resistant to antimicrobial agents is the use of targeted bacterial drugs - probiotics. Veterinary probiotics based on genetically modified microorganisms can be used for the treatment and prophylaxis of infectious diseases in farm animals. We demonstrated the antagonistic effect of the genetically modified Escherichia coli ŽP strain against agents of avian colibacillosis, the avian pathogenic Escherichia coli (APEC) in both in vitro and in vivo models. The colE7 gene (colicin E7 synthesis gene) carried on a conjugative plasmid was efficiently transferred by conjugation in conditions of planktonic and biofilm growth in vitro. The E. coli ŽP strain was shown to actively colonize the intestine of rats and quails and to contribute to beneficial effects of the microbiota. Our aim was to evaluate the competitive ability of the E. coli ŽP strain as well as the efficiency of killing APEC based on the conjugative transfer of the colE7 gene, encoding a DNasae, in vitro and in vivo. We used the ColE7-mediated kill-anti-kill system based on the Nissle 1917 probiotic strain, the genetically modified E. coli ŽP strain (killer donor) carrying the colE7 gene on the conjugative plasmid pOX38a, as well as the immE7 gene (colicin E7 immunity gene) on the chromosome. As control, the E. coli N4i strain without colE7 gene on the conjugative plasmid pOX38 (control donor) was used. Six APEC strains with resistance to ampicillin isolated from organs of broilers with colibacillosis were used as recipients in performed conjugation assays. The phylogenetic group of used APEC strains was detected with quadruplex PCR. The conjugation transfer was conducted in Luria-Bertani (LB) medium in immunological polystyrene 96-walls flat bottom plates in plankton and in biofilm culture. The experiments with rats (Wistar line) and Manchurian quail ( Coturnix coturnix ) were conducted in the vivarium of the Wagner Perm State Medical University. The competitive ability of E. coli ŽP strain was confirmed in co-cultivation assays with APEC strains, including bacteriocin producers, in various models. Conjugative colE7 gene transfer to APEC was detected in vitro in plankton and in biofilm: in experiments with the control donor strain E. coli N4i the conjugation frequency varied from 10 -6 to10-2. In vivo, it was shown that E. coli ŽP strain was able to effectively colonize the rat (line Wistar) and Manchurian quail ( Coturnix coturnix ) intestine and persist there at least for a month. Introduced E. coli ŽP cells increased the total amount of commensal Escherichia in the intestine of the animals and reduced the growth of the pathogenic microbes without affecting the lactic acid bacteria. The transfer ability of the conjugative plasmid pOX38 in the intestinal tract of both animal species was demonstrated. The conjugation in the intestinal tract occurred with a high frequency, on average 10-2. No transconjugants were detected in both in vitro and in vivo experiments with the E. coli ŽP strain harboring the conjugative plasmid pOX38a; the recipient cells that received the colE7 gene via the conjugative transfer expressed it and were lysed due to the colicin DNase activity. This was reflected in the number of APEC recipient cells, it was namely reduced in the assays with the ŽP strain. The results obtained indicated that the E. coli ŽP strain was able to colonize the intestine of animals and had antibacterial activity against enteropathogens due to the conjugative mechanism of colicin gene transfer. As the ŽP strain was also effective on APEC cells that were resistant and tolerant to bacteriocins, it has the potential to become the basis for a highly effective new generation of probiotics.