Perspective One Health Genetic Surveillance Of Bla_CTX-M Positive Klebsiella Pneumoniae In Yangzhou

Antibiotic resistance（AMR）is a complex global issue,and it is now regarded as an important One Health concern.The interrelated fields of One Health contribute to the emergence,evolution,and spread of antibiotic-resistant microorganisms at local and global scales,which is a significant risk factor for global health.factor in global health.Given the ongoing presence and spread of antimicrobial resistance and its interdependence of humans,animals,and the environment,adopting the One Health strategy to address AMR is meaningful.Klebsiella pneumoniae is an important opportunistic pathogen that cause human and animal infections and is also recognized as a carrier of resistance genes between different ecological niches.Extended-spectrum β-lactamases（ESBLs）producing K.pneumoniae has been recognized as a crucial priority pathogen by the World Health Organization.CTX-M type βlactamases are currently the most prevalent ESBLs that can be transmitted between different sources of K.pneumoniae through mobile elements such as plasmids and insertion sequences,posing a threat to public health.This study aimed to explore the epidemiological distribution of bla_CTX-M-positive K.pneumoniae on a One Health basis in humans,animals and the environments,and analyzed the characteristics and spread correlation of bla_CTX-M-positive K pneumoniae from different sources using whole-genome sequencing.In this study,2470 samples were collected from different sources in Yangzhou City,and 332 cefotaxime-resistant K.pneumoniae isolates were obtained,including 103 isolates from human sources,112 isolates from animal sources and 117 isolates from environmental sources.The minimum inhibitory concentration（MIC）of 16 antibiotics against the isolates was determined by agar dilution or broth microdilution methods.The results showed that all isolates were highly resistant to tetracycline,cefazolin,and compound sulfamethoxazole,with resistance rates of 93.67%,91.27%,and 85.54%,respectively.The resistance rates of ciprofloxacin,chloramphenicol,and nalidixic acid were also above 50.00%.The resistance rates of meropenem,tigecycline,and polymyxin were relatively low,with rates of 25.60%,18.98%,and 3.31%,respectively.Isolates from different sources showed significant differences in resistance rates to certain antibiotics,especially human isolates which showed significantly higher resistance rates to meropenem,tigecycline and amikacin compared to other sources.Animal isolates showed significantly higher resistance rates to tetracycline and gentamicin compared to other sources.In addition,cefotaxime-resistant K.pneumoniae exhibited severe multidrug resistance with all isolates being multi-drug resistant,and 18.07%of isolates were resistant to 11 antibiotics.Whole-genome sequencing of 332 isolates revealed that all isolates carried the bla_CTX-M gene,mainly divided into seven different variants.The bla_CTX-M-24 was only present in environmental isolates,while the other six variants were detected in isolates from humans,animals and environmental sources.In addition to the bla_CTX-M gene,the isolates carried other antibiotic resistance genes,such as aminoglycosides（aac（3）-Ⅱd,aac（6’）-Ⅰb-cr）,quinolones（qnrS1）,tetracyclines（tet（A））,sulfonamides（sul1,sul2）,and etc.Notably,the carbapenem resistance genes blaKPC-2,blaNDM-1 and blaNDM-5 were detected in human and environmental isolates,while tigecycline resistance genes tmexC1-tmexD1-toprJ1 were also detected in human,animal and environmental source isolates.Therefore,there were shared resistance genes among isolates from humans,animals and environments.The diversity of ST types of bla_CTX-M-positive K.pneumoniae was extensive,and the same ST type isolates were present in different sources.ST11,ST15,ST45,and ST290 were present in isolates from humans,animals,and environmental sources.The core genome phylogenetic tree based on SNPs showed that the isolates different sources have clone relationships.Additionally,all isolates carried multiple plasmid types,with IncF plasmids（311/332,93.67%）being the most common and the main bla_CTX-M group.To further explore the transmission of bla_CTX-M,some strains were subjected to the third-generation sequencing,which revealed that the bla_CTX-M-14 gene was mainly located on IncFIB-IncFII plasmids and also existed in chromosome.BLAST analysis revealed highly similar plasmids in isolates from human,animals and the environments.For example,plasmid p22HJ094₁ from an environmental source carrying bla_CTX-M-14 had 96%coverage and 99.99%nucleic acid identity with plasmid pSBH247₁ from a human source carrying the same gene.Regardless of whether it was plasmid-carried or chromosomal,they shared a core conserved genetic structure of ISEcp1-bla_CTX-M-14-IS903B.The bla_CTX-M-15 gene was also mainly predominantly located on IncFIB-IncFII plasmids,and also existed in chromosomal locations.Genetic environment analysis revealed the presence of a 2959 bp core genetic structure of ISEcp1-bla_CTX-M-15-Δorf477 in all isolates.The core genetic structures were all located in the larger multi-drug resistance region,which also includes multiple antibiotic resistance genes such as aac（6’）-Ib-cr,strA/B,blaOXA-1,sul1 and tet（A）,as well as multiple different intact or truncated insertion sequences and transposons（such as IS26,IS5075,IS6100,IS3000,ΔTnAs1,and etc.）.These resistance genes and insertion sequences formed common resistance gene cassette arrays.The acquiring multiple resistance modules confirmed the important role of insertion sequences in resistance gene propagation and plasmid evolution,thus facilitating the co-transfer of resistance genes.In summary,bla_CTX-M-positive K.pneumoniae are widely distributed in humans,animals and the environments in Yangzhou City and are associated with severe multi-drug resistance.The shared resistance genes,limited clonal relationships,similar multi-drug resistance plasmids and core genetic structures presented in isolated from different sources indicate an associated transmission risk.Despite the limited evidence,the potential risk prompts us to strengthen multidisciplinary research and surveillance on antibiotic resistance.This information can serve as the basis for implementing monitoring programs and targeting potential key control points for the sources of transmission.