Molecular Characterization And Evolution Of SHV-type Beta-lactamases In Gram-negative Bacilli

BackgroundInfections caused by Gram-negative bacteria are on the rise in clinic due to antimicrobial abuse and irrational use,especially the emergence of drug-resistant strains and even multidrug-resistant strains has increased the difficulty for clinical anti-infective treatment.As an important method of antimicrobial therapy of Gram-negative bacilli,?-lactam antibiotics are widely used.However,the emergence of drug-resistance strains,especially extended-spectrum?-lactamase-producing strains,poses greater challenges in preventing and controlling the epidemic of drug-resistant strains.SHV-type?-lactamases are one of the representative types of earlier?-lactamases.Majority of bla_SHV genes are located on plasmids,a few on chromosomes.Since the first SHV-type ESBL has been reported in Germany in 1983,the detection rate of SHV ESBLs has increased gradually,which has become the highest detection rate of extended-spectrum?-lactamases in Gram-negative bacilli in some areas.SHV-type?-lactamases have been widely spread in Europe,South America,Asia and Africa,particularly in Asia.Globally,there are more than 180 bla_SHV variants that have been published worldwide and have emerged in clinically related pathogens causing infections in health care.Although SHV-type?-lactamases have not been disseminated explosively as CTX-M ESBLs,they have been gradually detected in other clinical hosts besides Klebsiella pneumonia and Escherichia coli.Allelic variability in SHV-type?-lactamases is increasing and appears in different clinical environments.At present,researches about molecular evolution of?-lactamases in Gram-negative bacilli have become hotspots.The application of bioinformatics provides a practical tool for the study of the evolutionary mechanism of drug-resistant genes,and genome sequencing technology provides us with a large amount of available data on microbial genomes.Thus,the evolutionary study of SHV-type?-lactamases can be analyzed by sequence alignment,phylogenetic tree reconstruction,detection of potential positive selection sites and protein structure analysis.These studies are of great importance to understand the prevalence of resistance genes,explore the evolutionary origin and kinetics of resistance genes and to analyze the potential evolutionary direction of SHV-type?-lactamases.The distribution characteristics of SHV-type?-lactamases have regional variations.The epidemic of SHV-type?-lactamases varies in different countries,even in distinct areas of the same country.When SHV-type?-lactamases in different regions were not well connected,the evolutionary path results based only on regional source data according to bioinformatics were not accurate enough.The molecular evolutionary pathway combined with drug resistance history and clinical data can restore the clinical microenvironment changes under antibiotic selection pressure as much as possible,and improve the reliability of results.Thus,this study explored the clinical drug resistance characteristics and epidemic distribution of SHV beta-lactamases and constructed the molecular evolutionary pathway of SHV beta-lactamases to understand the emergence and development of resistance genes under antimicrobial action.It has a great significance for clinical treatment and the rational use of antimicrobial agents.Methods and materials1.Collection of bacteria isolates.A total of 374 non-duplicate gram-negative bacteria?resistant to the 3rd,4th-generation cephalosporins?were collected from inpatients at the First Affiliated Hospital of Zhengzhou University between July 2016and December 2016.All strains were performed by a VITEK 2 System.2.Antimicrobial susceptibility testing and ESBL screening.Antimicrobial susceptibility test was performed by the VITEK 2 System.The results of drug sensitivity testing and ESBL phenotype confirming were interpreted according to CLSI 2015 guidelines.3.PCR amplification of antibiotic resistance genes.The bla_SHV gene and other antibiotic resistance genes were detected by PCR and agarose gel electrophoresis,and bla_SHVHV genotypes were identified by DNA sequencing.4.Strains homology of bla_SHV-positive isolates.Genetic similarity of bla_SHV-positive strains was detected by ERIC-PCR.5.Evolutionary analysis of SHV?-lactamases.The molecular evolutionary trajectory of SHV?-lactamases was analyzed by sequence alignment and phylogenetic tree reconstruction.6.Protein structure analysis of SHV?-lactamases.The structure and function of amino acid sites in SHV?-lactamases were analyzed by positive selection site detection and protein structure construction.Results1.Detection of bla_SHV-positive strains.A total of 67 bla_SHV-positive strains were detected in 374 Gram-negative bacilli,including 61 Klebsiella pneumoniae,2 Escherichia coli,1 Enterobacter cloacae,1Klebsiella ozaenae,1 Citrobacter freundii,and 1 strain of non-fermentative bacteria,Acinetobacter baumannii.2.Genotype and distribution of resistance genes.All bla_SHV-producing isolates were identified for 12 genotypes,comprising SHV-1,-2,-2a,-11,-12,-27,-31,-33,-41,-61,-71 and-190.Most strains carried along with TEM?n=39?,CTX-M-1 group?n=11?,CTX-M-9 group?n=25?,OXA-1group?n=4?,KPC?n=16?and NDM?n=7?.3.Genetic similarity by ERIC-PCR.61 strains of Klebsiella pneumoniae showed 56 different DNA genotypes,in which 8 strains presented three related clones.Additionally,two Escherichia coli isolates and other isolates with heterogeneous ERIC profiles were clonally unrelated with each other.4.Evolutionary pathway of SHV?-lactamases.The evolutionary pathways of SHV-11?SHV-2a/SHV-31?SHV-12 were found.Amino acid positions 238 and 240 were detected positive selection sites,both of which are important in hydrolytic activity of SHV?-lactamases.Conclusions1.SHV?-lactamases were mainly found in Enterobacteriaceae.In addition,they were found not only in Klebsiella ozaenae and Citrobacter freundii,but also in Acinetobacter baumannii for the first time.It is suggested that the host strains of SHV?-lactamases have a broadening trend.2.SHV-12 is the most epidemic ESBL-type.And this is the first report of SHV-61and SHV-190 in Henan Province.Most bla_SHV-positive strains harbored more than two resistance genes.3.SHV-1 and SHV-11 are the main evolutionary nodes,and there are diversified evolutionary pathways of SHV-11?SHV-2a/SHV-31?SHV-12 in this region.Continuity monitoring of genotypes should be strengthened to prevent and control the further transmission and evolution of drug resistance genes.