Identification And Functional Analisis Of The Drug Resistance Genes Of Riemerella Anatipestifer M949₀011 And M949₀459

Riemerella anatipestifer?RA?,a member of the Flavobacteriaceae family of the rRNA superfamily V infects young ducks and geese,causing a severe form of Riemerellosis containing fibrinous pericarditis,perihepatitis,and meningitis.Riemerellosis is one of the most detrimental and lethal enteric diseases and is a major animal welfare and economic problem for the poultry industry.Because repeated infectious episodes are possible,eradication is difficult in duck flocks.Despite advances in novel vaccines,to date Riemerellosis has mainly been controlled by chemotherapy.Quinolones,tetracyclines,and cephalosporins are widely used for controlling R.anatipestifer infection in the avian breeding industry,and consequently have led to the emergence of antibiotic-resistant strains.However,in published work,few studies focus on the mechanism of R.anatipestifer antibiotic resistance.In the present work,two drug resistance genes from the R.anatipestifer strain were found and analyzed.Using the method of PCR,open reading frame?ORF?of the M949₀011 gene was amplified and sequenced.The length of M949 0011 ORF is 624 bp,encodes a putative polypeptide of 207 amino acids with a calculated molecular mass 22.89 kDa.BLAST and FASTA algorithms analyses showed that M949 0011 gene was 100%identity with RA-CH-1 and 26%identity with RA-CH-2 and RA-GD?accession no.CP002562?.Blast analyses showed that the M949₀011 gene is an outer membrane protein beta-barrel domain consisting gene.Determination of the median lethal dose?LDso?using 14-day-old Cherry Valley ducks showed that the virulence of the M949 0011 mutant strain was more than 100 times attenuated,compared to that of the wild-type strain.Further studies showed that the bacterial adherence and invasion abilities of the M949 0011 mutant strain were decreased,as compared with the WT strain,indicating that the M949 0011 gene played roles on the bacterial infection.We also examined the LPS pattern of the strain.Using the SDS-PAGE followed by silver staining,no difference was found in the LPS pattern for the M949₀011 mutant strain,compared with that of the WT strain,suggesting that M949₀011 gene has no effect on LPS biosynthesis.Furthermore,Live/dead BacLight Bacterial Viability staining showed that the mutant strain displayed an increase in the abundance of biofilms after incubation for 24 h,especially,after assign the imipenem.Finally,the hydrophobic properties of the mutant were compared with wild-type through assay of the hydrophobicity and TEM.M949₀011 mutant strain had a significantly higher cell surface hydrophobicity of HPBI?hydrophobicity index?than wild-type.In our previous study,we constructed the random Tn4351 transposon insertion library.In present work,we obtained one mutant strain with increased tigecycline resistance by scereerning the library using the MIC assay.The mutated gene was identified as M949₀459,and the complete sequence was analyzed.The M949₀459 gene encodes 386 amino acids with a calculated molecular mass 42.53 kDa.By blastn analysis,we found eight high homology protein of M949₀459 in published R.anatipestifer genome sequences,including RAYM₀8235?RA-YM?,AS87₀9615?Yb2?,RIA₀365?YA-GD?,RIA₀369?YA-GD?,B739₀030?RA-CH-1?,B739₀035?RA-CH-1?,G148₁777?RA-CH-2?and G148-1767?RA-CH-2?.With the Alignment tool of M-Coffee,we alignment the upstream sequences of these nine protein.It seems that they can dive into two groups:one is the CH3,RA-YM,YA-GD;the other group is CH-1,Yb2,CH-2.We measured the uptake of tigecycline by E.coli BL21?DE3?which contained M949₀459 on a pET-28a?+?background.The results showed that M949₀459 conferred tigecycline resistance to E.coli BL21,but did not affect susceptibility to other antibiotics including kanamycin,ceftriaxone,norfloxacin,and imipenem.Also,by qRT-PCR,we quantitatively examined the expression of M949₀459 following treatment with different doses of tigecycline.We observed a non-linear effect of the dose of tigecycline on the expression of the M949₀459 gene.The results showed that the mRNA expression of M949 0459 was elevated under treatment with 4 mg/L tigecycline,but decreased with treatment with between 4 and 10 mg/L tigecycline.