Table_2_Overexpression of Efflux Pumps, Mutations in the Pumps’ Regulators, Chromosomal Mutations, and AAC(6′)-Ib-cr Are Associated With Fluoroquinolone Resistance in Diverse Sequence Types of Neonatal Septicaemic Acinetobacter baumannii: A 7-Year Single Center Study.doc

This study investigates susceptibility toward three fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), multiple fluoroquinolone-resistance mechanisms, and epidemiological relationship of neonatal septicaemic Acinetobacter baumannii. Previous studies on fluoroquinolone resistance in A. baumannii focused primarily on ciprofloxacin susceptibility and assessed a particular mechanism of resistance; a more holistic approach was taken here. Epidemiological relationship was evaluated by Multi Locus Sequence Typing. Minimum Inhibitory Concentrations of fluoroquinolones was determined with and without efflux pump inhibitors. Overexpression of efflux pumps, resistance-nodulation-cell-division (RND)-type, and multidrug and toxic compound extrusion (MATE)-type efflux pumps were evaluated by reverse transcriptase-qPCR. Mutations within regulatory proteins (AdeRS, AdeN, and AdeL) of RND-pumps were examined. Chromosomal mutations, presence of qnr and aac(6′)-Ib-cr were investigated. A. baumannii were highly diverse as 24 sequence-types with seven novel STs (ST-1440/ST-1441/ST-1481/ST-1482/ST-1483/ST-1484/ST-1486) were identified among 47 A. baumannii. High resistance to ciprofloxacin (96%), levofloxacin (92%), and particularly moxifloxacin (90%) was observed, with multiple mechanisms being active. Resistance to 4th generation fluoroquinolone (moxifloxacin) in neonatal isolates is worrisome. Mutations within GyrA (S83L) and ParC (S80L) were detected in more than 90% of fluoroquinolone-resistant A. baumannii (FQRAB) spread across 10 different clonal complexes (CC1/CC2/CC10/CC25/CC32/CC126/CC149/CC216/CC218/CC513). Efflux-based FQ resistance was found in 65% of FQRAB with ≥2 different active pumps in 38% of strains. Overexpression of adeB was highest (2.2−34-folds) followed by adeJ, adeG, and abeM. Amino acid changes in the regulators (AdeRS/AdeN/AdeL) either as single or multiple substitutions substantiated the overexpression of the pumps. Diverse mutations within AdeRS were detected among different CCs whereas mutations within AdeN linked to CC10 and CC32. Chromosomal mutations and active efflux pumps were detected simultaneously among 64% of FQRAB. Presence of aac(6′)-Ib-cr was also high (74% of FQRAB) but qnrS were absent. As most FQRABs had chromosomal mutations, this was considered predominant, however, isolates where pumps were also active had higher MIC values, establishing the critical role of the efflux pumps. The high variability of FQ susceptibility among FQRAB, possessing the same set of mutations in gyrA, parC, and efflux pump regulators, was also noted. This reveals the complexity of interpreting the interplay of multiple resistance mechanisms in A. baumannii.

本研究针对新生儿败血症源性鲍曼不动杆菌（Acinetobacter baumannii），探究其对3种氟喹诺酮类（fluoroquinolones）——环丙沙星（ciprofloxacin）、左氧氟沙星（levofloxacin）、莫西沙星（moxifloxacin）的敏感性、多重氟喹诺酮耐药机制及流行病学关联。既往针对鲍曼不动杆菌氟喹诺酮耐药性的研究多聚焦于环丙沙星敏感性，并仅评估单一耐药机制；本研究则采用更为全面的研究策略。流行病学关联通过多位点序列分型（Multi Locus Sequence Typing）进行评估。本研究测定了添加与不添加外排泵抑制剂（efflux pump inhibitors）时的氟喹诺酮类最低抑菌浓度（Minimum Inhibitory Concentrations）；通过逆转录定量PCR（reverse transcriptase-qPCR）检测了外排泵的过表达情况，包括耐药结节分化（RND）型以及多药耐药及毒素外排（MATE）型外排泵；同时检测了RND型外排泵的调控蛋白（AdeRS、AdeN及AdeL）编码基因的突变情况，并分析了染色体突变、qnr基因与aac(6′)-Ib-cr基因的携带情况。本次纳入的47株鲍曼不动杆菌共分为24种序列型（sequence-types），其中包含7种新型序列型（ST-1440/ST-1441/ST-1481/ST-1482/ST-1483/ST-1484/ST-1486），显示出较高的菌株多样性。研究观察到菌株对环丙沙星（96%）、左氧氟沙星（92%），尤其是莫西沙星（90%）均表现出较高的耐药性，且存在多种活性耐药机制。新生儿分离株对第四代氟喹诺酮类（莫西沙星）的耐药性值得警惕。在超过90%的氟喹诺酮耐药鲍曼不动杆菌（fluoroquinolone-resistant A. baumannii, FQRAB）中检测到GyrA（S83L）与ParC（S80L）位点的突变，这些菌株分属于10种不同的克隆群（CC1/CC2/CC10/CC25/CC32/CC126/CC149/CC216/CC218/CC513）。65%的FQRAB存在基于外排泵的氟喹诺酮耐药性，其中38%的菌株可同时激活≥2种不同的外排泵。adeB基因的过表达幅度最高（2.2~34倍），其次为adeJ、adeG与abeM。调控蛋白（AdeRS/AdeN/AdeL）的氨基酸单突变或多重突变均证实了外排泵的过表达。不同克隆群中均检测到AdeRS编码基因的多样突变，而AdeN编码基因的突变则与CC10及CC32克隆群相关。64%的FQRAB同时携带染色体突变与活性外排泵。aac(6′)-Ib-cr基因的携带率也较高（占FQRAB的74%），但未检测到qnrS基因。由于多数FQRAB均携带染色体突变，该机制被认为是主要的耐药途径；但同时激活外排泵的菌株具有更高的最低抑菌浓度值，证实了外排泵在耐药过程中的关键作用。研究还观察到，尽管部分FQRAB在gyrA、parC及外排泵调控蛋白编码基因上携带相同的突变位点，但其氟喹诺酮类敏感性仍存在较高异质性。这一结果揭示了鲍曼不动杆菌多重耐药机制之间相互作用的复杂性。