Abstract：
OBJECTIVE: Although cefiderocol (FDC) is not prescribed in China, FDC-resistant pandrug-resistant hypervirulent Klebsiella pneumoniae (PDR-hvKp) is emerging. In this study, we performed FDC susceptibility testing of clinical Kp isolates to explore the prevalence of FDC-resistant isolates and the mechanism of FDC-resistance.
METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing.
RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage.
CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.
METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing.
RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage.
CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.
摘要：
目的：虽然中国没有使用头孢地洛（FDC），FDC抗性的pandrug抗性高毒力肺炎克雷伯菌（PDR-hvKp）正在出现。在这项研究中,我们对临床Kp分离株进行了FDC药敏试验,以探讨FDC耐药株的患病率和FDC耐药机制.
方法：我们回顾性选择了151株耐碳青霉烯类Kp分离株评估FDC敏感性。来自两名患者的7个携带blaSHV-12的分离株被纳入全基因组测序。抗菌素耐药性，毒力,blaSHV-12表达式，并检查了不同媒介的健身成本。通过qPCR和长读数测序进一步研究blaSHV-12的扩增。
结果：151个分离株显示FDC的MIC50/MIC90（1/4mg/L）较低。7个分离株为ST11PDR-hvKp，和两个表示的FDC电阻(MIC=32mg/L)。两个FDC抗性分离株的IncR/IncFII质粒携带6和15个blaSHV-12拷贝，而四个FDC敏感分离株携带一个拷贝，一个携带三个拷贝。这些blaSHV-12基因连接在一起，并位于IS26侧翼的同一7.3kb片段内，这有助于增加表达和FDC抗性，而无需适应成本。BlaSHV-12和FDC电阻的扩增可由FDC体外诱导，并在连续传代过程中逆转。
结论：blaSHV-12的扩增和随之而来的动态宿主内异抗性是抗生素合理应用的重要关注点。长读数测序可能是快速准确检测抗性基因扩增的一种优越方法。
方法：我们回顾性选择了151株耐碳青霉烯类Kp分离株评估FDC敏感性。来自两名患者的7个携带blaSHV-12的分离株被纳入全基因组测序。抗菌素耐药性，毒力,blaSHV-12表达式，并检查了不同媒介的健身成本。通过qPCR和长读数测序进一步研究blaSHV-12的扩增。
结果：151个分离株显示FDC的MIC50/MIC90（1/4mg/L）较低。7个分离株为ST11PDR-hvKp，和两个表示的FDC电阻(MIC=32mg/L)。两个FDC抗性分离株的IncR/IncFII质粒携带6和15个blaSHV-12拷贝，而四个FDC敏感分离株携带一个拷贝，一个携带三个拷贝。这些blaSHV-12基因连接在一起，并位于IS26侧翼的同一7.3kb片段内，这有助于增加表达和FDC抗性，而无需适应成本。BlaSHV-12和FDC电阻的扩增可由FDC体外诱导，并在连续传代过程中逆转。
结论：blaSHV-12的扩增和随之而来的动态宿主内异抗性是抗生素合理应用的重要关注点。长读数测序可能是快速准确检测抗性基因扩增的一种优越方法。
