Construction Of Chp Gene Isogenic Mutants Of Avian Pathogenic Escherichia Coli Strain E058 With Their Pathogenicity Evaluation And Prevalence Of Antimicrobial Resistance Of APEC Isolates Originated From 1993 To 2006

Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including septicaemia, granuloma (Hjarre's disease), air sac disease, chronic respiratory disease (CRD), avian cellulitis, swollen head syndrome, peritonitis, salpingitis, osteomyelitis/synovitis, panophthalmitis, and omphalitis/yolk sac infection. Colibacillosis in mammals is most often a primary enteric disease, whereas colibacillosis in poultry is typically a secondary localized or systemic disease occurring when host defence has been impaired or overwhelmed. Collectively, infections caused by Escherichia coli (E. coli) are responsible for significant economic losses to the poultry industry in many parts of the world. The pioneer serological surveys indicate that the majority of avian septicaemic E. coli strains belong to a limited number of O serogroups (O1, O2, and O78). Analysis of the involvement of these potential virulence factors using experimental models of infection just began during the past few years. Nevertheless, the actual roles of the known virulence factors are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which avian pathogenic E. coli cause infection are largely unknown.It has been reported that APEC has many virulence factors. Researchers are working hard on making clear the virulence factors, meanwhile, they pay attention to isolation and identification of the new virulence factors. Because many isolated strains don't have the virulence factors ever known, according to the pathogenic experiment of the APEC. The above-mentioned information indicates that the isolated strains have other virulence factors. It is significant to acquire new virulence factors of the APEC.1. Construction of chp genes isogenic mutants of APEC E058 and evaluation of their pathogenicity in chickensIn our previous study,some unique DNA fragments associated with APEC E058 are obtained using suppression subtractive hybridization (SSH) in which the genome of avian highly pathogenic E. coli strain E058 serves as a tester and the genome of avian low pathogenic E. coli strain E526 serves as a driver. Among these unique DNA fragments, aes-31 is selected for future study since it is found to be up-regulated in APEC E058 compared to APEC E526 in chicken sera stationary cultures when analyzed by DNA microarray. In this study, we constructed the aes-31 gene isogenic mutants as follows. The 1200 bp fragment of aes-31 consists of two opening reading frames (ORF),the 753-bp gene of chp1 encoding a conserved hypothetical protein in E. coli K-12 and the 441-bp gene of chp2, encoding another conserved hypothetical protein in E. coli K-12 characterized by blast searching in GenBank. Chp1 and chp2 genes were generated by PCR and cloned into pBSK vector respectively. Zeocin resistant gene and kanamycin resistant gene were then cloned into the target gene chp1 and chp2, respectively, the resultant vectors containing the recombinant genes were named pBSK-chp1-Zeo and pBSK-chp2-Kan respectively. Mutant derivatives of strain E058 were generated by allelic replacement with electroporation technique and named E058 (Δchp1),E058 (Δchp2) and E058 (Δchp1Δchp2).The growth curve showed that the mutant strains are slowly grown than the parental strain APEC E058 in logarithmic phase when they grew in LB broth. The 50% lethal dose (LD50) of E058, E058 (Δchp1), E058 (Δchp2) and E058 (Δchp1Δchp2) in commercial 1-day-old chickens challenged experimently via intratrachea were 104.5 CFU , 104.8 CFU , 105.1 CFU和105.4 CFU , respectively. In the chicken challenge model, the mutants were observed to have higher clearence rate of challenged strain than that of the parental strain, especially the double mutant strain E058 (Δchp1Δchp2). These results showed that the unique DNA fragment aes-31 contributes to the the virulence and persistance of APEC E058 in chickens.2. Prevalence of antimicrobial resistance of APEC isolates originated from 1993 to 2006In this study, 203 APEC isolates originated from 1993 to 2006 were characterized for antimicrobial susceptibilities. 203 APEC isolates include 58 collected during 1993-1995, 42 obtained during 1996-1999, 39 isolated during 2000-2003 and 64 recoverd during 2004-2006. Among the 21 antimicrobial agents tested, resistance of 203 APEC isolates to spectinomycin, tetracycline was the most frequent , and the resistance rate of all tested isolates for each of the two agents was 98% (199/203). None of the 203 isolates have antimicrobial resistance to polymycin B. High resistance rates were also observed in sulfamethoxazole (84%) , trimethoprim-sulfamethoxazole (78%), streptomycin (76%) and ampicillin (74%) . The results showed that a trend of increasing and broadening their antimicrobial resistance in tested isolates was observed, the nearer the isolates were isolated, the stronger and wider antimicrobial resistance were presented. It was interesting that all tested isolates collected during 2002-2003 experienced the strongest and widest antimicrobial resistance among the 14 years, especially for quinolones and sulfanilamide group agents. Our findings also showed that all isolates were multi-resistant.