| Tigecycline is a broad-spectrum glycyclamide antibiotic against multidrug-resistant gram-positive and gram-negative bacterium.Although tigecycline has not been approved for use in animals,tigecycline-resistant genes have been found in Acinetobacter in poultry.Tet（X3）is a gene that has been discovered in recent years and can confer resistance to all tetracycline antibiotics.Poultry,as food animals,are also used with different antimicrobial agents for the prevention and treatment of diseases,and are considered a potential reservoir of bacteria that may pose a threat to human public health.In this study,a total of 615 samples were collected from poultry farms in Foshan,Zhuhai,Jiangmen and other places in Guangdong Province.The samples were collected from feces,sewage,soil and dust.The bacteria were isolated by Acinetobacter selective chromogenic medium containing 2μg/m L tegacycline,and the bacterial species were identified by 16S r RNA sequencing.The tetracycline-resistant genes tet（X3）in the isolated strains were detected by PCR.The results showed that a total of 49 Acinetobacter strains were obtained from 615 samples,including 2 Acinetobacter schindleri,3 Acinetobacter towneri,6 Acinetobacter rudis,13 Acinetobacter indicus,20 Acinetobacter variabilis,and 5unknown Acinetobacter strains,all of which were further confirmed to be positive for the tet（X3）gene.The resistance phenotypes of tet（X3）positive Acinetobacter strains were determined by agar dilution method and its conjugations were determined by micro-broth dilution method.The results showed that the drug resistance rate of all Acinetobacter to tigecycline,tetracycline,ciprofloxacin and sulfamethoxazole/trimethoprim was 100%.In addition,the drug resistance rates of florfenicol,ampicillin,fosfomycin and cefotaxime were 69.1%,54.5%,47.3%and 36.4%,respectively.Resistance rates to ceftazidine,gentamicin,amikacin and meropenem were 34.5%,32.7%,7.3%and 1.8%,respectively.But all strains were sensitive to colistin.The 49 Acinetobacter strains which were positive for tet（X3）gene were sequenced and the drug-resistant genes were analyzed.The results showed that there were 9 major classes of drug-resistant genes.The detection rate of the tetracyclines resistance gene tet（X3）was100%,followed by tet（39）with 81.6%.Next,the detection rates of macrolide resistance genes mph（E）and msr（E）were all 91.8%.The detection rate of sulfonamides resistance gene sul2 was 100%,followed by sul1 with 57.1%.Then,the detection rate of florfenicol resistance flo R was 79.6%.Among the aminoglycosides,the drug resistance gene aph（3’’）-Ib had the highest detection rate of 71.4%,followed by aph（3’）-Ia with the detection rate of 69.4%.The detection rates of beta-lactams resistance genes bla OXA-58and bla DHA-1were 34.7%and 30.6%,respectively.Then,the detection rate of trimethoprim resistance gene dfr A1 was 24.5%,and that of dfr A16 and dfr A19 was 16.3%.The detection rate of rifampicin resistance gene arr-3 was 34.7%.And the detection rate of quinolones resistance gene qnr D1 was 32.6%,and that of aac（6’）-Ib-cr was 26.5%.The conjugation experiments of all strains showed that 6 Acinetobacter strains were successfully conjugated and the remaining Acinetobacter strains failed.All the tet（X3）positive Acinetobacter and their conjugants were conducted with S1-PFGE、I-Ceu I PFGE and Southern-blot experiments.The results showed that tet（X3）genes in 30 strains were located on plasmids of different sizes ranging from 78 to 210 kb.Among them,the genes in12 strains were located on plasmids with a size of about 78 kb,7 strains on plasmids with a size of about 104 kb,3 strains on plasmids with a size of about 138 kb,and 7 strains on plasmids with a size of about 180 kb,only one strain on the plasmid with a size of 210 kb.In addition,25 strains of tet（X3）genes were located on the chromosome.The whole genome DNA were sequenced for all strains,and then the upstream and downstream gene sequences of tet（X3）were analyzed to obtain the genetic environment of tet（X3）in combine with a complete plasmid containing the tet（X3）gene.The results showed that there were 5 categories of gene environments（A,B,C,D,E）,among which 5strains belonged to category A,and the gene environment structure was IS26-tnp F-tet（X3）-par A-hp-ISCR2.There were 31 strains belonging to category B,and the gene environmental structure was IS26-Δtnp F-tet（X3）-par A-hp-ISCR2.And there were 2 strains belonging to category C,and the gene environment structure wasΔISCR2-Δtnp F-tet（X3）-par A-hp-ISCR2.There were 5 strains belonging to category D,with the genetic environment structure ofΔISCR2-Δtnp F-tet（X3）-par A-hp-ISCR2,as well as a truncation betweenΔtnp F and tet（X3）.And there are 6 strains belonging to category E,and the gene environment structure wasΔISCR2-tnp F-tet（X3）-par A-hp-ISCR2.In summary,Acinetobacter in this study has a serious resistance status to antibiotics commonly used in veterinary clinic,most of them are multi-resistant strains,and the detection rate of drug-resistant genes is high.The transmission of tet（X3）gene is related to mobile components such as plasmids and insertion sequences,and the selection pressure caused by the wide use of antibiotics is also the main driving force for the transmission of drug-resistant genes.Therefore,we should use antibiotics reasonably and appropriately in poultry to prevent them from becoming a potential reservoir of important drug-resistant genes,bringing harm to public health and human health. |
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