| Duck Salmonella disease,also known as duck paratyphoid,spreads quickly and has high incidence and mortality rates.In recent years,with the continuous expansion of Chinese duck farming scale,the infection rate of Salmonella in ducks has been constantly rising,resulting in serious economic losses.As animal-source Salmonella has a wide host range,in the process of preventing and controlling duck Salmonella disease,the irrational use of antibiotics has led to increasing antibiotic resistance.Therefore,understanding the evolution of duck Salmonella resistance patterns is beneficial for in-depth research into resistance mechanisms,and provides scientific support for the prevention and treatment of Salmonella disease in duck farms.This study investigated the antibiotic resistance of 110 duck-derived Salmonella with 7serotypes isolated from 5 regions in Shandong Province and surrounding areas,China.This research conducted detection of drug sensitivity phenotypes,resistance genes,plasmid incompatibility groups,and class I integron,as well as multiple-locus sequence typing（MLST）analysis,further analyzed the plasmid-mediated aac（6’）-Ib-cr cooperative resistance and the whole genome sequencing of multidrug-resistant strain SD33.This study has grasped the drug resistance patterns and epidemic trends of duck-derived Salmonella with different origins and serotypes,analyzed the mobile elements and modes of transmission for drug resistance dissemination,and has scientific guidance significance for the study of drug resistance mechanisms,gene transmission,and clinical medication of duck-derived Salmonella.The results of this study are as follows:1.The distribution pattern of antimicrobial resistance phenotypes and genotypesAmong the 15 antibiotics tested,110 Salmonella strains had the highest resistance rate to erythromycin（98.18%）,followed by streptomycin（84.54%）and tetracycline（48.18%）.In contrast,they were most sensitive to ofloxacin and amikacin（87.27%）.71.82%（79/110）of all strains showed multidrug resistance,and 17 Salmonella（15.45%）showed resistance to 10 or more drugs,including 2 strains of 15-drug resistant bacteria.A total of 47 resistance patterns were found in this study,and the rate of multidrug resistance in S.Newlands,S.Enteritidis,S.Kottbus and S.Typhimurium was between 62.50%and 88.89%.The resistance rate of strains isolated from Heze to 10 antibiotics exceeded 60%,and the severity of resistance was much higher than that of strains from other regions and sources.A total of 20 resistance genes were detected,and 12 of them were found,with ere A（34.55%）,tet A（33.64%）,bla TEM（32.73%）,and aph（3’）-Ia（32.73%）having a high prevalence rate.S.Kottbus carried all detected genes.Except for quinolones,the concordance rate of resistance phenotypes and genotypes forβ-lactams,aminoglycosides,tetracyclines,and macrolides was over 70%.2.Transmission characteristics of drug resistance genes in duck-derived Salmonella110 Salmonella strains were divided into 7 serotypes and 13 ST types.The ST types and the serotypes had a significant correlation in Salmonella.As the dominant serotypes,S.Kottbus and S.Typhimurium had multiple ST types.Molecular phylogenetic analyses revealed close evolutionary relationships between ST19 and ST1544,ST808 and ST1690 from duck embryo isolates.A total of 12 resistance genes were detected in all strains,and 9 of them were mainly distributed in ST19,ST1546,and ST808 strains.ST19 Salmonella was distributed in duck embryos isolated from Tai’an,Liaocheng,Jining,and Guantao,carrying 10 antibiotic resistance genes.The detection rate of bla TEMand tet A exceeded 40%.ST1546（duck embryo,duck body,and environment isolates from Tai’an and Liaocheng）and ST808（duck embryo isolates from Tai’an）also carried antibiotic resistance genes.Among them,ST1546Salmonella mainly carried aph（3’）-Ia,while ST808 strains carried aac（6’）-Ib-cr,aad A1,aac（3）-IV,ere A,and qnr D.The above results showed that duck-derived Salmonella with ST types as the main type were important carriers of antibiotic resistance genes.Bla TEMand tet A were widely prevalent in duck embryo isolates from different regions,while aph（3’）-Ia exhibited clonal transmission among Salmonella isolated from different breeding stages in the same region.In addition,ST321 was distributed in the duck body and environmental isolates from Heze and Liaocheng,and carried aph（3’）-Ia and tet A with a 100%detection rate,showing resistance to aminoglycosides and tetracyclines,respectively.This indicated that non-mainstream ST types were more conducive to the development of bacterial resistance to high levels and multidrug resistance at the genetic level,which further increases the difficulty of preventing and controlling duck Salmonella disease.3.Relationship between plasmid incompatibility groups,class I integrons,and antibiotic resistance6 plasmid incompatibility groups were identified in 47 of all strains,with the highest detection rate（26.36%）being Inc FIIs.One strain carried three plasmid incompatibility groups（Inc HI2+Inc FIB+Inc Frep B）.The positive rates of plasmid incompatibility groups in S.Typhimurium,S.Enteritidis,and S.Newlands were much higher than those in other serotypes.86 Salmonella strains（78.18%）carried class I integrons.The carriage rates of various antibiotic resistance genes in class I integrons-positive strains were much higher than those in class I integrons-negative strains,and the detection rate of class I integrons in multidrug-resistant strains（87.34%）was significantly higher than that in non-multidrug-resistant strains（54.84%）.68.09%of the 47 plasmid incompatibility groups-carrying Salmonella strains carried class I integrons,indicating that class I integrons and plasmids were key mobile elements for the formation and transmission of resistance in duck-origined Salmonella.4.Synergistic resistance mechanism mediated by aac（6’）-Ib-crThe transfer conjugation experiment of plasmids showed that both aac（6’）-Ib-cr and other resistance genes could be spread through plasmid-mediated horizontal transfer,which was an important pathway for bacteria to acquire antibiotic resistance.When aac（6’）-Ib-cr existed alone,the MIC of ciprofloxacin in Escherichia coli J53 increased 4-fold,which was significantly higher than those that other types of resistance genes existed independently.Among all double-plasmid strains,the change in MIC of ciprofloxacin for J53-aac（6’）-Ib-cr+aad A1 was the most significant,which was 32 times that of Escherichia coli J53 and 8 times that of J53-aac（6’）-Ib-cr.At the same time,the coexistence rate of aac（6’）-Ib-cr and aad A1 was the highest（65.5%,19/29）,indicating that when these two genes existed in the same strain,they had a synergistic effect in conducive the development of bacteria towards high-level resistance,leading to the formation of multidrug-resistant strains.It may be a relatively common reason for the co-spreading phenomenon of aac（6’）-Ib-cr and aad A1 in duck-derived Salmonella in present study,posing a potential threat to the prevention and control of resistance.5.Whole-genome sequencing analysisThe whole-genome sequencing analysis of SD33,a representative multi-drug resistance strain,showed that 78 antibiotic resistance genes,23 antibiotic target genes,and two antibiotic synthesis-related genes were detected in chromosome.Among them,we found efflux pump systems（acr AB-tol C,acr D,acr EF,emr AB,mac AB,mdf A,mdt K,and mdt ABC）related to Salmonella resistance,genes（omp C and omp F）encoding outer membrane proteins,and mutations in the gyr A gene’s fluoroquinolone resistance-determining region（QRDR）with aspartic acid being replaced by glycine at position 87.In addition,8 prophages were found to carry the efflux pump mdt K,outer membrane protein omp S1,multidrug transporter emr E,as well as rsp L,tuf A,and fus A interfere with protein synthesis.The above results indicated that drug resistance mechanisms were mainly located on chromosomes. |
