Identification And Potential Risk Assesment Of β-Lactamase-Coding Genes Identified In Primary Forest Soils In Yunnan

The β-lactam class antibiotics are one of the most widely used antibiotics classes in clinics;however,more and more pathogenic bacteria have exhibited resistance to βlactam class antibiotics,which threaten public health seriously.Production of β-lactamase is the main mechanism of the clinically resistant pathogens to resist against the β-lactam class antibiotics.Acquisition of β-lactamase encoding genes from the environmental microbes via horizontal gene transfer(HGT)is an important way to obtain the β-lactam resistance ability in the clinical pathogens.Therefore,identification and monitoring of the β-lactamase encoding genes in the environmental microbiomes are critical to controlling the spread of β-lactam antibiotics resistance-associated genes as well as the pathogen managements.The pristine soil microbiomes are found to serve as an important reservoir of antibiotic resistance genes,which probably harbor a variety of novel uncharacterized β-lactamase encoding genes.Therefore,it is extremely important to identify and monitor the diversity and abundance of β-lactamase encoding genes in pristine soil environments.A total of 792 β-lactamase encoding genes were identified in 8 soil samples collected from Yuanjiang pristine forest National Nature Reserve Park located in Yunnan Province,via whole-genome metagenomic analyses.The majority(87%)of the identified 792 βlactamase encoding genes were demonstrated to be novel uncharacterized β-lactamase encoding genes as revealed by phylogenetic and conserved motif analysis,with only 13%of the 792 genes exhibiting high sequence similarity(identity>50%)to known βlactamase coding genes.Furthermore,a novel conserved motif,SSN,was identified for the first time in class A β-lactamase.A total of 9 representative β-lactamase encoding genes were selected for functional determination,including 2 genes that were closely related to known clinic-originating antibiotic resistance genes,3 potential novel β-lactamase encoding genes that exhibited relatively low sequence similarity to clinic-originating antibiotic resistance genes,1 gens with novel conserved motif SSN,and 3 genes with relatively high abundance.The 9 selected genes were cloned to expression vectors,transferred to Escherichia coli,and the resistance improvement to the β-lactam class antibiotics of the bacterial hosts carrying the imported genes compared with that carrying empety vector were then determined.The results showed that these representative β-lactamase encoding genes could efficiently hydrolyze ampicillin,a representative penicillin-class antibiotics,and improved the minimum inhibitory concentration(MIC)of the E.coli host strains 8-32 times higher than the strain carrying the empty vector.All of the nine genes also exhibited certain hydrolytic ability to the third-generation antibiotics cephalosporin and carbapenems.In addition,our results demonstrated that the β-lactamase containing the novel motif SSN exhibited higher hydrolysis ability to the tested β-lactam class antibiotics compared with the classic motif SDN,which was possibly because SSN could improve the stability of the enzyme compared with SDN,.Overall,we identified a large number of novel β-lactamase encoding genes in a pristine soil environment and assessed the potential risk of these genes,which increased our understanding of the diversity of β-lactamase encoding genes and laid foundation for the development of novel antibiotics and β-lactamase inhibitors.