| Tigecycline is a new generation of tetracycline derivatives,and its unique chemical structure allows the drug to expand its antimicrobial spectrum and is known as the last line of clinical defense.Although tigecycline is not currently used in veterinary practice,tigecycline-resistant pathogens have been reported to have been isolated clinically in animals and found to be widely prevalent in animals.Current studies of tigecycline-resistant bacteria have focused on swine and poultry,but there are not many reports of tigecycline-resistant bacteria in ruminants.Escherichia coli（E.coli）,which can cause severe infections in humans and animals,has also emerged in recent years as a major reservoir of resistance genes and is an important cause of treatment failure for clinical infections in humans and animals.There is still a lack of sufficient data and information to elucidate the emergence mechanism,epidemiological trends and hazards of tigecycline-resistant E.coli of animal origin.Therefore,in this study,fecal samples of animal origin（swine,cattle and sheep）were collected to isolate tigecycline-resistant Escherichia coli from several regions including Tibet,Gansu,Ningxia and Shaanxi and so on.The resistance analysis and whole genome sequencing techniques were used to compare the resistance phenotypes,multi-locus sequence typing,phylogenetic relationships,resistance and virulence genes,plasmid types and transfer ability of the strains screened,in order to discover the characteristics,prevalence and resistance mechanisms of tigecycline-resistant E.coli strains of animal origin in different regions.The following results were obtained in this study:（1）A total of 77 tigecycline-resistant E.coli strains were isolated from all 916 collected fecal samples.Among them,the isolation rate of tigecycline-resistant E.coli in samples of porcine origin was 13.51%（47/348）,the isolation rate of tigecycline-resistant E.coli in samples of bovine origin was 0%（0/145）,and the isolation rate of tigecycline-resistant E.coli in sheep origin was 7.09%（30/423）.The isolation rates in each region were 17.11%in Shaanxi,16.00%in Shanxi,10.00%in Gansu,7.14%in Sichuan and 7.05%in Ningxia,and no tigecycline-resistant E.coli were isolated in Tibet,Xinjiang and Qinghai.The results of antibiotic susceptibility tests showed that all E.coli were multi-resistant bacteria,with severe resistance to antimicrobials such as doxycycline（100.00%）,florfenicol（100.00%）,ampicillin（97.73%）,amoxicillin/clavulanic acid（93.18%）,and sulfisoxazole（93.18%）;followed by gentamicin（65.91%）,enrofloxacin（54.55%）,ceftiofur sodium（47.73%）,and ceftazidime（38.64%）;All E.coli strains showed sensitivity to meropenem and polymyxin B.E.coli isolated from swine showed more extensive resistance and higher tigecycline MICs than E.coli of sheep origin.（2）Forty-four of these tigecycline-resistant E.coli strains were subjected to second-generation whole-genome sequencing,and analyses of MLST,resistance and virulence genes,serotypes,plasmid types,and gene sequences were performed.Twenty-four known ST types with diverse genotypes were identified,with the largest number of ST206（n=6）,followed by ST10,ST195,and ST1308.And the remaining ST types were found in individual strains with a more dispersed distribution.CC10 was the main prevalent clonal complex,found in several regions.The resistance gene profile showed that all E.coli strains carried multiple resistance genes,and the resistance phenotypes were basically consistent with the resistance genes carried.E.coli strains isolated from swine carried tet（X4）gene,while all E.coli isolated sheep carried tet（A）gene.Virulence gene statistics revealed that the E.coli in this study carried nearly 50virulence genes,and the hlyE and terC genes were carried at 100%.The serotypes of the strains in this study showed a regional distribution,three E.coli strains showing mixed serotypes.IncHI2 and IncHI2A were the most predominant replicon types in this study and were distributed in both sources of E.coli.Gene sequence analysis revealed that the tet（X4）gene has a more stable core structure,with rdm C-tet（X4）-isvsa3-IV secretion protein-flo R-reg being the most prevalent.Analysis of 35 strains positive for tet（A）gene showed that tet（A）mutants were identified in 77.14%of E.coli（sheep 12/12,swine 15/23）.Amino acid sequence analysis identified three different tet（A）variants,with type 1 tet（A）mutants being the most prevalent and a swine-derived E.coli strain carrying a tet（A）-V mutant,and a new tet（A）mutant（New-tet（A））was also identified to distinguish it from the existing tet（A）mutant.Δaph（3’’）-I-aph（6）-Id"-Relaxase-tet R-tet（A）-Permease is the core structure of the genetic environment in which tet（A）is located in this study.（3）All E.coli were initially identified by pulsed-field gel electrophoresis（S1-PFGE）as carrying at least one plasmid.The plasmid transfer ability was verified by splice transfer assay using the isolate as the donor and E.coli 26R 793 as the recipient.The in vitro splicing results showed that 57.14%（44/77）of E.coli strains were successfully spliced,and the splicing frequency ranged from 10-2 to 10-7.However,none of the 10 isolates could be successfully transfer from donors to the recipient.The S1-PFEG showed that the number of transconjugants carrying plasmids was significantly reduced compared with that of the donor strain.Drug sensitivity tests showed that spliceosomes acquired resistance to tigecycline,in addition to some other antibiotics.In summary,this study analyzed the resistance phenotypes,molecular characteristics,plasmid typing and plasmids’transfer ability of tigecycline-resistant E.coli of animal origin isolated from several provinces in China,and found that E.coli multi-drug resistance is very serious,and tigecycline resistance in swine-derived E.coli is mainly mediated by tet（X4）gene,while tet（A）variants are found in both sources of E.coli.The distribution of tet（A）variants in both sources of E.coli is a major factor contributing to tigecycline resistance in E.coli strains of sheep origin,and the coexistence of tet（A）variants and tet（X4）further complicates the mechanisms of bacterial resistance in livestock farming environments.Furthermore,the widespread presence and evolution of the tet（A）variant is a potential driver for the acquisition of tigecycline resistance in pathogenic bacteria and should not be ignored.Therefore,we should strengthen the investigation and monitoring of tigecycline-resistant bacteria from multiple sources nationwide,conduct more extensive and in-depth studies on the causes and mechanisms of their emergence,pay attention to the horizontal transmission of tigecycline-resistant genes carried by plasmids in fauna bacteria,and strictly manage drug administration and conduct antibiotic tier management to slow down the emergence of tigecycline resistance. |
PDF Full Text Request