| Riemerella anatipestifer is an imortant pahtogen which can cause infection with high morbidity and mortality in waterfowl worldwide, leading ecomomic losses. Use of antimicrobials can cause rapid resistance in R. anatipestifer, resulting in reduced therapeutic efficacy even failure. Currently studies on the molecular mechanism and dissemination of antimicrobial resistance of R. anatipestifer are rare.Brain and liver tissue samples were collected from sick?dead? ducks or geese suffering from typical symptoms from nine cities of Guangdong province, during the period of February 2013 and July 2015. The presumptive colonies with typical morphology were confirmed by automated microbial identification system, and were further identified by PCR with specific primers. 115 R. anatipestifer strains were identified, among which 54 were from duck and 61 from goose. The MICs of 32 antimicrobial agents were determined by the agar dilution method. Susceptibility testing showed the resistance rates observed were quite high. A high incidence of multidrug resistance in 103?89.6%? R. anatipestifer isolates were observed.Resistance genes and integrons were detected by PCR and then confirmed by sequencing in R. anatipestifer isolates.Twenty-five resistance genes were identified in five classes of antimicrobial agents. To our knowledge, this is the first report of aph?3'?-?, aph?3'?-?, aph?4'?-?a, three kinds of aminoglycoside phosphotransferase genes and aminoglycoside adenylyl transferase gene aad B in R. anatipestifer isolates. This is also the first report of the erm B, erm C and sul3 genes in R. anatipestifer isolates, which mediate resistance to erythromycin and sulfonamide antimicrobial agents. Nine R. anatipestifer isolates harbored class 1 integrons, including 6 isolates carrying 978 bp aad A2 gene cassette and 3 isolates carrying 2287 bp aac?6'?-?-cat B3-aad A1 gene cassettes.The PFGE experiment were carried out by Sma I-digested chromosomal DNA. In total, sixteen different Sma I-PFGE types?with similarity percentage of ?90%? were detected among 58 available PFGE profiles in R. anatipestifer isolates, including 11 clusters and 5 single types. The result suggested that there was a wide clonal spread in R. anatipestifer isolated from Guangdong province of China. Clonal spread exists in strains isolated from different species of host animals?ducks and geese? and same or different regions.To confirm whether or not antibiotic resistance in R. Anatipestifer are biofilm-related, biofilm formation assay was determined quantitatively in 96-well flat-bottomed microtitre plates by crystal violet staining. The result showed that 40?34.8%? strong-biofilm-producer isolates were resistant to 12-23 antimicrobial agents tested, 25?21.7%? moderate-biofilm-producer and 30?26.1%? weak-biofilm-producer strains can be resistant to 7-12 kinds of antimicrobial agents. 20?17.4%?strains, non biofilm producer, were resistant to less than 6 antimicrobial agents. To a certain extent, biofilm formation ability in R. Anatipestifer was parallel to the multi-drug resistance profiles.In addition, the flo R gene was located on both the chromosome and the plasmid, when performing S1-PFGE and hybridization. A novel plasmid carrying flo R gene different from previous study was identified, whose size was more than 23 kb. Conjugation experiment suggested that the novel plasmids can be transferred to Escherichia coli C600 with transfer frequency from 10-6 to 10-9, however the stability of this plasmid was poor. |
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