Mechanism Of Erythromycin Resistance In Rimerella Anatipestifer

Riemerella anatipestifer is one of two species in the genus Riemerella within the family Flavobacteriaceae, which is one of the largest branches in the phylum Bacteroidetes. R.anatipestifer infection causes major economic losses in the duck industry through high mortality, reduced growth rate, poor feed conversion, increased condemnation, and high treatment costs. Endemic infections are restricted to commercial duck and turkey flocks,although other poultry species, such as chicken and geese, are also susceptible. Usually, the main ways to cure this disease are vaccination and using antibiotics, erythromycin has been used to successfully treat R. anatipestifer infection in some duck. Howere, erythromycin treatment failures have been noted on several occasions in past years. In recent years, the rate of erythromycin-resistant R. anatipestifer isolates is growing increase, but the mechanism of erythromycin resistance in R. anatipestifer has not been described. So this research focuses on the mechanism of erythromycin resistance in R. anatipestifer.In this research,from 1996 to 2014, a total of 79 R. anatipestifer isolates were isolated from sick ducklings and a R. anatipestife type strain ATCC11845 as research subjects, by using broth dilution method to detect the erythromycin minimum inhibitory concentration(MIC). Forty-three of 80(53.8%) tested R. anatipestifer strains showed resistance to erythromycin, while 37(46.2%) were susceptible. Furthermore, 41(73.2%) of 56 R.anatipestifer isolates obtained from 2005 to 2014 exhibited erythromycin resistance, while 2(8.3%) of 24 R.anatipestifer strains, which were isolated from sick ducklings in China between 1996 and 2004, were resistance to erythromycin. In addition, the presence of ermF or ereD in erythromycin-resistant R. anatipestifer strains were determined by PCR. The results showed that 30(70.0%) of 43 erythromycin-resistant strains encoded the ermF or ermFU gene, the MICs rang from 32 to 2048 mg/L, while the other 13 strains carrying the ereD gene exhibited an MIC of 4–16 mg/L. Moreover, 27 out of 30(90.0%) ermF+ R. anatipestifer strains carried the ermFU gene, while three other strains carried ermF.The ermF genes from different R. anatipestifer strains were cloned and sequenced, theresults showed that they shared an amino acid homology of 98.5%–100%. Genome walking was performed to clone the upstream flanking sequence from HXb2、GuiZ-1、YXb15、HGb1、NJ-4、JY-6、YZ-1、ZJb2 strains. Sequence homology analysis showed that the upstream sequence of the ermFU gene from R. anatipestifer strains shared 85.8%–88.7% homology with that of CTnDOT, so the ermFU gene may be derived from CTnDOT-like element.Howere, the upstream sequence of the ermF gene(-1 to-600)from R. anatipestifer strains shared high homology with that of Tn4351, so the ermF gene may be derived from Tn4351.Moreover, sequence analysis revealed that ermF, ermFU and ereD genes were located in the multiresistance region of the R. anatipestifer genome.To further determine whether the identified ermF, ermFU and ereD genes were responsible for erythromycin resistance in R. anatipestifer, three recombinant shuttle plasmids carrying the ermF, ermFU and ereD cassettes were introduced into an erythromycin--susceptible R. anatipestifer CH3. The results of MICs showed that all the three recombinant strains CH3(pRES-HXb2-ermFU), CH3(pRES-YZ-1-ermF) and CH3(pRES-SX-ere D) exhibited erythromycin resistance, and two ermF carrying strains CH3(pRES-HXb2-erm FU) and CH3(pRES-YZ-1-ermF) showed high resistance to clindamycin,while CH3 carrying plasmid pRES-SX-ereD did not exhibit clindamycin resistance. The relative mRNA expression of the CH3 strains which carried theermFU/ ermF/ereD cassette is3.3/ 80/ 6.7 folds more than that of HXb2/ YZ-1/ SX strains.These rusults suggested that ermF, ermFU and ereD genes were located in the multiresistance region of the R. anatipestife genome, which can cause R. anatipestife erythromycin resistance.