Mechanisms Of Novel Metallo-?-lactamases Variants In Carbapenem-resistant Organisms From Livestock And Poultry

The discovery and application of antibiotics brought benefit to human and animal health,which promoted the development of modem medicine.However,the plentiful and extensive use of the antibiotic has also led to the bacterial resistance,especially the emergence of super-resistant bacteria by producing MBLs(Metallo-?-lactamase)Enterobacteriaceae.It is a serious threat to the healthy development of livestock and poultry husbandry and the safety of public health.Carbapenems are the few antibiotics to effectively treat clinical infection caused by multi-drug resistant gram-negative.Currently,the MBLs(NDM and VIM)-producing bacteria,with resistance to carbapenems by efficiently hydrolyzing carbapenem,have spread in the clinic.The variants of MBLs are evolving with enhanced hydrolytic activity,the importance of which can't be emphasized too much by medical clinic.Notably,carbapenems are unproved for use in livestock and poultry husbandry in our country,but the MBLs-producing bacteria has been identified and spread in livestock and poultry.The documented studies can only show that NDM and VIM are the most prevalent and typical MBLs.But there is no relevant research to clarify the characteristics of MBLs-producing bacteria prevalence and character evolutionary in livestock breeding environment.This study is aims to explore the prevalence of NDM and VIM in carbapenem-resistant pathogens of livestock and poultry in China,and the resistance mechanisms of novel variants.In this study,1,160 samples were collected by Shandong and Guangdong province from 2015 to 2017,including 818 chicken origin samples and 342 swine origin samples.433 blaNDM positive bacteria,including 7 isolates from swine and 426 isolates from chicken,and 4 blaVIM positive bacteria were identified.The major host strain of blaNDM from chicken origin were E.coli(n=344,80.8%)and Klebsiella pneumoniae(n=79,18.5%);and that in swine origin were E.coli(n=4,57.1%)and Acinetobacter baumannii(n=3,42.9%).Sequence alignment showed that 364 isolates were successfully assigned into NDM subtype:The NDM-5(n=207,55.8%)was the main NDM subtype,followed by NDM-9(n=94,25.3%)and NDM-1(n=63,17.0%).The blaVIM sequencing results showed only one can be classified into blaVIM-2 in 4 blaVIM sequences.It is noteworthy that blaNDM in 7 E.coli strains and blaVIM in 3 Pseudomonas putida isolates were unclassified,indicating that novel variants of MBLs were possibly generated in 10 isolates carbapenem-resistant bacteria.Two novel NDM-1 variants,NDM-17 and NDM-20,were identified and named in 6 E coli isolates and 1 E coli isolates,respectively;which were firstly described in livestock and poultry.Compared to NDM-1,NDM-17 and NDM-20 have two same amino acid substitutions V88L and M154L,also possessed by NDM-5,and still hold two amino acid substitutions firstly described E170K and R270H,respectively.The bblaNDM-17 can confer bacteria increased resistance to carbapenems than blaNDM-1 and blaNDM-5,while the blaNDM-20 can mediate the same carbapenems resistance to blaNDM-5 but higher than blaNDM-1.The enzyme kinetic confirmed that NDM-17 possessed significantly increased substrate hydrolysis efficiency compared with NDM-1 and enhanced affinity relative to NDM-5,suggesting that NDM-17 possesses better enzymatic activity.NDM-20 increase the catalytic activity toward penicillins and cephalosporins,but depressed carbapenemase activity,indicating that NDM-20 go up the specific enzyme activity,further imply that NDM are evolving to increase enzyme activity or hydrolytic activity to specific class ?-lactams.The melting test results showed that the stability of NDM-17 and NDM-20 are significantly higher than that of NDM-1,which can be attributed to M154L amino acid substitutions.The location analysis showed blaNDM-17 and blaNDM-20 were harbored in the transferrable IncX3-type plasmid(46,161 bp).A novel VIM variant,VIM-48,was identified in 3 untyped blaVIM gene.VIM-48 presented successive 11 amino acids(aa)alteration at the C-terminal compared with VIM-2.It is a new model of evolutionary for VIM that VIM-48 evolutionary by 11 aa alteration differed from traditional single aa substitution in other variants.To study the role of successive 11 amino acids on VIM-48,a deletion isolate VIM-D(A)with 11 amino acids deletion at C-terminal and VIM-2 was constructed as the reference isolates.Functional analysis demonstrated that VIM-48 could mediate high resistance level to carbapenems compared with VIM-2 and VIM-D(A).VIM-48 showed increased hydrolytic activity against carbapenems by enhanced affinity relative to VIM-2,while VIM-D(?)had significantly decreased catalytic efficiency,implying C-terminal amino acids was crucial to enzyme activity.Thermostability test results confirmed the stability of VIM-48 was high relative to VIM-2,while that of VIM-D(?)significantly was the lower than that of VIM-48,demonstrated that C-terminal successive 11 amino acids(aa)alteration increase the thermostability of VIM-48.All this founding suggests that VIM-48 maybe a dominant with increased enzyme activity and stability,and indication of VIM evolution shifts from amino acid point substitutions to multisite or continuous fragment insertions.In summary,three MBLs variants,NDM-17,NDM-20 and VIM-48,were identified on basis of investigating the prevalence of NDM and VIM in carbapenem-resistant pathogens from livestock and poultry,in some area of China;and their characteristics of biological activities were illuminated,suggesting that Metallo-beta-lactamases may be evolving to increase biological activity and stability.It will inevitably increase the difficulty in the prevention and control of carbapenem-resistant bacteria and the development of new drugs.Meanwhile,our study showed the evolution of MBLs I not only in medical clinical pathogens,but also in pathogenic bacteria from livestock and poultry.Once such strains are widespread in livestock and poultry,it will be a risk of transmission by food chain and will inevitably bring great difficulties to clinical treatment.The research and understanding of novel MBLs variants will lay a theoretical foundation and provide data for the prevention and control of carbapenemase-resistant bacteria and risk assessment of public health safety.