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, bla_RSA1 and bla_RSA2, were phylogenetically close to clinically important ESBLs like bla_GES, bla_BEL and bla_L2, 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.

进化为环境细菌提供了许多对抗生素化合物具有抗性的基因。在人为选择压力下，据信其中某些基因会随着时间的推移通过水平基因转移而被募集到病原体中。在印度，从散装毒品生产中排放的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长期，空前的抗生素选择压力环境。因此，可以推测先前未知的抗性基因已经在这里进化和/或被促进了。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此类河流沉积物。对大肠杆菌中的不同抗生素具有抗性的DNA片段使用Sanger和PacBio RSII平台进行测序。我们重新捕获了以前通过相同样品的开放式shot弹枪宏基因组测序确定的大多数已知抗生素抗性基因。此外，鉴定了七个候选抗性基因（六个β-内酰胺酶和一个阿米卡星抗性基因）。两种A类β-内酰胺酶bla _RSA1和bla _RSA2在系统发育上接近于具有临床意义的重要ESBL，例如bla _GES，bla _BEL和bla _L2，并进一步对其底物光谱进行了表征。的blaRSA1蛋白质，编码为整合基因盒，有效地水解青霉素，第一代头孢菌素和头孢霉素，而blaRSA2是可诱导的A类β-内酰胺酶，能够水解碳代青霉烯尽管具有有限的效率，类似于从L2β-内酰胺酶的嗜嗜麦芽。所有检测到的新基因都与质粒动员蛋白，整合素和/或其他抗性基因相关，表明存在迁移的潜力。这项研究提供了对由异常强大和长期的抗生素选择压力形成的抵抗组的洞察力。对外部环境中动员的抗性因子的了解增加，可能使我们为将来在诊所中可能遇到的抗性挑战做好更好的准备。