Construction of an iss deleted mutant strain from a native avian pathogenic Escherichia coli O78: K80 and in vitro serum resistance evaluation of mutant

ABSTRACT

Colibacillosis, caused by different serotypes of avian pathogenic Escherichia coli [APEC], is one of the important diseases in poultry industry. The isolate O78 is the most prevalent serotype of APEC in Iran. One of the APEC virulence factors, increased serum survival [iss] gene, is related to serum resistance. The usual form of colibacillosis in avian is extraintestinal, and serum resistance is applied one way by APEC to reach internal organs; hence, it appears that the control of colibacillosis in poultry regarding the deletion of iss and the construction of a serum sensitive APEC strain is beneficial. Additionally, the knowledge about APEC serum resistance could be extended using mutant strains. The present study was an attempt to generate an iss mutant strain from native APEC-O78 strain |c|1378 and to study the level of serum resistance of native APEC-O78 strain c1378 in comparison with its mutant [APEC-O78 strain c1378|D|iss]. The lambda red recombinase system was utilized to delete iss gene in native APEC-O78 strain c1378. This strain was first transformed with the plasmid pkD46 to introduce the lambda red recombinase system and then the PCR product with sequence homology to the iss gene and a kanamycin resistance marker was transformed into the APEC-O78 strain c1378. Serum sensitivity of mutant and wild type strain was investigated by microtiter test. The generation of mutant was successful and the iss was replaced with kanamycin resistance cassette. Also, it was observed that the mutant was sensitive to serum. However, serum sensitivity of iss deleted mutant was not statistically different from its parents. Application of lambda red recombination could be a simple and useful technique for production of a precisely defined gene deletion. Also, there may be some genes that compensate the activity of iss gene