[Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genes by PCR]

This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline [100%], chloramphenicol [79%] and florfenicol [72%]. The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples.

[Gene disruption in Salmonella typhimurim by modified Lamda Red disruption system]

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the lamda Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT [FLP recognition target] sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the lamda Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species

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

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

Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium

Among all common techniques in site directed mutagenesis, Lambda Red recombinase system has been widely used to knock out chromosomal genes in bacteria. In this method, there is always the risk of DNA Linear digestion by host's restriction enzymes that leads to the low frequency of recombination. To overcome this, we constructed a recombinant vector to disrupt phoP gene in Salmonella typhimurium. The SOEing PCR method and restriction enzymes were used to construct the vector. The resulting plasmid, pTAAZ92, contains a Kanamycin cassette with two long homologous arms flanking of the phoP gene. After electrotransformation of the pTAAZ92 into the Salmonella typhimurium, the phoP gene is replaced by the Kanamycin cassette through homologous recombination. According to the high homology of the phoP gene in many of Salmonella species the pTAAZ92 can be used to disrupt the phoP gene in most of these species

Distribution of virulence associated genes in isolated Escherichia coli from avian colibacillosis

Colibacillosis is one of the most prevalent diseases in the world that causes multimillion-dollar annual losses. In order to evaluate molecular epidemiology of some virulence associated factors in Escherichia coli, isolated from poultry, the presence of iut A, iss, hlyF, omp T, iro N, afa, sfa [S] and pap G [II] were investigated by multiplex PCR assay. Two hundred thirty four Escherichia coli isolated from avian colibacillosis [APEC] and fifty four fecal E. coli isolates from the feces of apparently healthy birds [AFEC] were investigated for presence of some virulence associated genes by two panel of multiplex PCR. Statistical analysis was performed using x[2] test, the p-value was

[aroA gene sequencing of native Escherichia coli O78: k80 isolated from avian colibacillosis in Iran]

aroA is an important gene which produces aromatic amino acids essential for bacterial life. The sequence of this gene was shown to be different in bacteria, so the purpose of this study was sequencing of aroA gene in native E. coli O78: K80 to determine the putative differences between this strain and other E. coli O78: K80 strains in gene bank. PCR was used to amplify aroA gene in native E. coli O78: K80. The amplified 1.206 kb PCR product was extracted from Agaros gel, ligated in pTZ57R and sequenced. Blast analysis showed that aroA sequence in native E. coli is different from already submitted homolog gene in Gene Bank. Two amino acids were shown to be different from those already found in other E. coli strains. This different amino acids are Isoleucine and Glycine instead of 39[th] Threonine and 240[th] Glutamic acid, respectively. Knowing these differences are important for doing molecular techniques, designing primers and future studies