The Contribution of Antibiotic Resistance Mechanisms in Clinical <i>Burkholderia cepacia</i> Complex Isolates: An Emphasis on Efflux Pump Activity

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.鉴于目前临床洋葱伯克霍尔德菌复合物（Burkholderia cepacia complex）分离株中各类抗生素耐药机制的贡献信息尚不充分，本研究针对66株临床洋葱伯克霍尔德菌复合物分离株，系统探究了其抗生素耐药机制，涵盖整合子分析、喹诺酮耐药决定区突变鉴定、外排泵活性检测以及外排泵调控基因序列分析等内容。菌株通过recA限制性片段长度多态性（recA-RFLP）与基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）完成鉴定，经recA-RFLP共区分出4个基因组型，其中类鼻疽伯克霍尔德菌（基因组型III）为优势基因组型，占比达90.1%。绝大多数分离株（60/66，90.9%）可经MALDI-TOF分析得到准确鉴定。脉冲场凝胶电泳（PFGE）分析揭示的克隆相关性结果显示，共存在30个脉冲型，其中2个主要脉冲型分别占分离株总数的22.7%与18.2%。共有17株（25.8%）分离株携带包含多种耐药基因组合的1类整合子（class 1 integron）。在6株左氧氟沙星耐药分离株中，5株在促旋酶A亚基基因（gyrA）中存在单碱基替换，另有3株表现出外排泵活性。在42株对至少1种抗菌药物呈现耐药的分离株中，94.4%的头孢他啶耐药分离株（17/18）与72.7%的氯霉素耐药分离株（16/22）均检测出外排泵活性。对外排泵RNA水平的定量检测与序列分析结果显示，RND型3型外排泵（RND-3 efflux pump）的过表达源于该外排泵调控基因的特定突变。综上，外排泵的高表达在洋葱伯克霍尔德菌复合物分离株中普遍存在。RND-3外排泵调控基因的突变是引发外排泵活性的主要原因，进而导致临床洋葱伯克霍尔德菌复合物分离株产生抗生素耐药性。