低氧/厌氧产品案例——肠道微生物大肠杆菌研究

文章题目：Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co‑ evolved

一氧化氮(NO)引起大肠杆菌氨基糖苷耐受性，但抗生素耐药基因携带和NO 敏感性并没有共同进化

文章出处：Archives of Microbiology (2021) 203:2541–2550 .英国肯特大学生物科学学院

工作站使用情况：InvivO₂ 300

使用气体浓度：厌氧（0% O₂）及微需氧（2% O₂)

摘要：革兰氏阴性菌的多药耐药传播是临床面临的主要挑战，需要新的方法来对抗这些微生物。一氧化氮(NO)是一种众所周知的抗菌素，是免疫系统在应对感染时产生的，许多研究表明，NO是一种具有抑菌和杀菌特性的呼吸抑制剂。然而，已知有氧呼吸复合物的丧失会降低抗生素的效力，假设有效的呼吸抑制剂NO 会引起类似的作用。事实上，目前的工作表明，暴露于NO释放体前，庆大霉素对致病性大肠杆菌的IC50值提高了10倍(即致命性大大降低)。因此，假设细菌病原体中可能出现了对NO的超敏感，这种特性可以通过使细胞在有毒水平的抗生素存在下持续存在，从而促进抗生素耐药性机制的获得。为了验证这一假设，我们利用基因组学和微生物学方法对一组大肠杆菌临床分离株进行了抗生素敏感性和NO耐受力的筛选，尽管数据并不支持抗生素耐药基因携带增加与NO耐受力之间的相关性。然而，目前的工作对未来如何测量抗生素敏感性(即±NO)具有重要意义，并强调了细菌病原体在维持对NO毒性水平的耐受力方面的进化优势。

Fig. 2 Nitric oxide releasers abrogate the lethality of gentamicin. Suspensions of 108 cells/mL of E. coli EC958 in M9 minimal media supplemented with 0.1% casamino acids were exposed to 15 mM GSNO (a) and 1 mM NOC12 (b) for 30 min, followed by incubation with diferent concentrations of gentamicin for 90 min. Serial dilutions were performed in PBS and plated on LBagar. CFU/mL values were determined after overnight incubation at 37 °C. Data were ftted to a four parameter Hill equation for calculation of IC50 values, and magnitude changes were normalised to 100% for display on a linear yaxis (insets). Values shown represent mean ± SD from two biological repeats, each comprising three technical repeatsL.

Fig. 3 Antibiotic susceptibilities of E. coli phylogroups and sequence types. a Phylogenetic relationships of 50 E. coli clinical isolates were inferred using the core genome gene sequences of all isolates (compared to E. coli MG1655) obtained by pangenome analysis with Roary. Phylogroups for each isolate were determined using in silico PCR, and each colour represents one phylogroup in the tree (inner circle). b Antibiotic resistance profles were predicted using bioinformatics approaches and were also experimentally screened for antibiotic resistance using disc susceptibility testing (Andrews 2001, 2002) (the total number of antibiotic classes that each isolate is resistant to, based on discsusceptibility, is shown in the outer ring of (a)). ‘% antibiotic resistance’ is defned as the % of isolates within a sequence type that are resistant to a particular antibiotic. ST sequence type, AMX amoxicillin, CTX cefotaxime, CAP chloramphenicol; CIP cip rofoxacin, Gen gentamicin, MEM meropenem, NIT nitrofurantoin, TMP trimethoprim, PME polymyxin E, MLS macrolides, TET tetra cycline, SUL sulfanilamides.

大肠杆菌培养条件：在37°C，常氧、微需氧(2%的氧气)或厌氧条件下培养

一氧化氮释放剂可消除庆大霉素的致命性（图2），表明一氧化氮会降低抗生素的效力；

大肠杆菌系统群和序列类型的抗生素敏感性（图3），利用Roary 全基因组分析获得的所有分离株的核心基因组基因序列，推断了50 个大肠杆菌临床分离株的系统发育关系（图3a）;使用BioInformatics 方法预测抗生素耐药性，抗生素耐药程度最高的克隆组即ST131、ST69、ST73（图 3b）。