摘要

进化为环境细菌提供了许多对抗生素化合物产生抗性的基因。在人为选择压力下，据信这些基因中的一些通过水平基因转移随时间推移进入病原体。印度原料药生产中氟喹诺酮类药物和其他药物污染的河流沉积物构成了一个前所未有的，长期抗生素选择压力的环境。因此，以前未知的抗性基因已经在这里进化和/或被提升，这是合理的。为了寻找新的抗性基因，我们因此通过功能性宏基因组学方法分析了这样的河流沉积物。使用Sanger和PacBio RSII平台对提供对大肠杆菌中不同抗生素的抗性的DNA片段进行测序。我们重新获得了大多数已知的抗生素抗性基因，这些基因以前通过相同样品的开放式枪支宏基因组测序确定。另外，鉴定了7个新的抗性基因候选物（6个β-内酰胺酶和1个阿米卡星抗性基因）。两种A型β-内酰胺酶，blaRSA1和blaRSA2在系统发育上与临床上重要的ESBLs如blaGES，blaBEL和blaL2接近，并进一步表征其底物谱。编码为整合子基因盒的blaRSA1蛋白有效地水解青霉素，第一代头孢菌素和头孢噻肟，而blaRSA2是诱导型A类β-内酰胺酶，能够水解碳代青霉烯尽管效率有限，类似于来自Stenotrophomonas的L2β-内酰胺酶麦芽。所有检测到的新基因都与质粒动员蛋白，整合子和/或其他抗性基因相关，这表明具有迁移潜力。这项研究提供了一个由特别强大和长期抗生素选择压力形成的抵抗素的洞察力。加强对外部环境中动员的抵抗因素的了解可能使我们能够更好地准备我们未来在诊所可能面临的阻力挑战。

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future.

https://www.sciencedirect.com/science/article/pii/S0160412017319967