■氨基糖苷修饰酶(AME)在细菌对氨基糖苷类抗微生物剂的耐药性中起着至关重要的作用。随着测序技术的发展,更多的细菌基因组已经被测序,这有助于发现越来越多的新型抗性机制。
■通过16SrRNA基因同源性和平均核苷酸同一性(ANI)分析鉴定细菌种类。通过琼脂稀释法测定每种抗微生物剂的最小抑制浓度(MIC)。用pColdI载体在大肠杆菌BL21中表达蛋白质,并检测酶动力学参数。通过Illumina和PacBio测序平台获得细菌的全基因组序列。系统发育树的重建,鉴定保守的功能残基,和基因背景分析使用相应的生物信息学技术进行。
■一种新的氨基糖苷抗性基因,指定aph(3')-即,赋予对核糖霉素的抗性,卡那霉素,西索米星和巴龙霉素,在动物细菌柠檬酸杆菌DW61的染色体中鉴定出,该细菌表现出多药耐药表型。APH(3')-Ie与功能特征酶APH(3')-Ia的氨基酸同一性最高,为74.90%。酶动力学分析表明,它对四种氨基糖苷底物具有磷酸化活性,表现出最高的亲和力(Km,4.22±0.88µM)和最高的催化效率[kcat/Km,(32.27±8.14)×104]为利他霉素。与其他APH(3')蛋白相似,APH(3')-Ie包含APH家族的所有保守功能位点。aph(3')-Ie同源基因存在于不同来源的吉氏梭菌分离物中,包括一些临床意义。
■在这项工作中,一种新的染色体氨基糖苷抗性基因,指定aph(3')-即,赋予对氨基糖苷类抗微生物剂的耐药性,在兔分离株C.gilleniiDW61中鉴定。新的抗性机制的阐明将有助于有效治疗由携带这种抗性基因的病原体引起的感染。
UNASSIGNED: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms.
UNASSIGNED: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques.
UNASSIGNED: A novel aminoglycoside resistance gene, designated aph(3\')-Ie, which confers resistance to ribostamycin,
kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3\')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3\')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (K m, 4.22 ± 0.88 µM) and the highest catalytic efficiency [k cat/K m, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3\') proteins, APH(3\')-Ie contained all the conserved functional sites of the APH family. The aph(3\')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance.
UNASSIGNED: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3\')-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.