Hydrolytic activity of KPC-producing Klebsiella pneumoniae clinical isolates.
Hydrolytic activity of KPC-producing Klebsiella pneumoniae clinical isolates.
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DOI：
10.1080/1120009x.2021.2004363
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发表时间：
2022-09
期刊：
影响因子：
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通讯作者：
Shields RK
中科院分区：
文献类型：
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作者：
Tam VH;Hudson CS;Merlau PR;Shields RK
耐碳青霉烯类肠杆菌是一种紧迫的抗生素耐药性威胁[1]。在美国，肠杆菌科细菌对碳青霉烯类抗生素的耐药性主要是由于丝氨酸型碳青霉烯酶(如KPC)的产生。表达KPC的临床分离株可能对广泛的抗菌剂产生耐药性，限制了有效的治疗选择。值得注意的是，这些分离株的水解力与感染部位的细菌密度(例如脓胸与不复杂的尿路感染)、酶变体(例如KPC-2与KPC-3)、其他β-内酰胺酶的存在及其转录水平以及底物稳定性有关。新的β-内酰胺酶抑制剂(如阿维巴坦、瓦波巴坦、瑞巴坦)已被用于恢复β-内酰胺类药物的活性，当它们联合使用时[2]。这些β-内酰胺酶抑制剂有望减少合伙β-内酰胺类药物的酶降解，并保持其治疗效果。从理论上讲，β-内酰胺酶抑制剂所需的有效浓度应该与目标菌株的水解力相匹配(或超过)。只要有可能，微生物实验室的分子检测旨在检测目标β-内酰胺酶基因的存在，而不是定量地检测功能能力。因此，常规检测可能不能很好地指导β-内酰胺酶抑制剂的剂量。我们检测了几个KPC阳性分离物的水解活性，以提供对这一方法的实用性的见解。对6株耐亚胺培南的肺炎克雷伯菌进行了检测。编码KPC的基因(S)和其他β-内酰胺酶(S)的存在已被聚合酶链式反应和DNA测序证实[3，4]。用傅里叶变换红外光谱(FTIR)(Bruker，Billerica，MA)对分离株的亲缘关系进行了评估。用分光光度法检测粗细胞裂解物(归一化为总蛋白含量的40μg)的酶活性。头孢他啶(50μM)在35℃、260 nm、20分钟的磷酸盐缓冲液(pH7.4)中的吸光度跟踪降解。肺炎克雷伯菌ATCC 13883(美国典型培养物收藏，马纳萨斯，弗吉尼亚州)作为阴性对照。为了进行比较，采用改良的肉汤稀释法测定头孢他啶的敏感度，同时增加阿维巴坦的浓度(直至mg/L)。在24小时观察到的浓度反应(即最低抑菌浓度曲线)由乙状结肠抑制最大效应(Emax)模型[5]表征。持续的分离物特征如表1所示。来自同一机构的三个分离物(KP1438、KP1491和KP1587)被认为是相关的(数据未显示)。与人们普遍认为的相反，多个β-内酰胺酶基因的存在并不会导致更高的水解力。似乎并不是所有的基因都能最大程度地表达，因此它们对总的水解性没有额外的贡献。对头孢他啶/阿维巴坦敏感的所有菌株的裂解物(MIC4/4 mg/L)对头孢他啶具有相似的水解性。然而，对头孢他啶/阿维巴坦耐药的分离株降解率较高(KP1491，附录1)。在该分离株中，需要更高的阿维巴坦浓度才能将头孢他啶的MIC恢复到敏感范围(数据未显示)。这些见解是重要的，并将被用来开发一种定量方法来指导最佳的β-内酰胺酶抑制剂剂量。尽管有不同的基因编码不同的β-内酰胺酶，但头孢他啶/阿维巴坦和…似乎合理地预测了各种kpc阳性分离株的水解性。
Carbapenem-resistant Enterobacterales is an urgent antibiotic resistance threat [1]. In the US, carbapenem resistance in Enterobacterales is primarily due to the production of serine-based carbapenemase (eg KPC). Clinical isolates expressing KPC could be resistant to a broad range of antimicrobial agents, limiting effective treatment options. Notably, the hydrolytic activity of these isolates varies as a function of bacterial density at the site of infection (eg empyema vs. uncomplicated urinary tract infection), enzyme variants (eg KPC-2 vs. KPC-3), presence of other beta-lactamases and their transcriptional levels, as well as substrate stability. Novel beta-lactamase inhibitors (eg avibactam, vaborbactam, relebactam) have been used to restore the activity of beta-lactam agents, when they are used in combination [2]. These beta-lactamase inhibitors are expected to reduce enzymatic degradation of the partnering beta-lactam agents and preserve their therapeutic effect. Theoretically, the effective concentration needed for a beta-lactamase inhibitor should match (or exceed) the hydrolytic activity of the target isolates. Where available, molecular testing in microbiology laboratories aims to detect the presence of target beta-lactamase genes, but not the functional capacity quantitatively. Accordingly, routine testing may not be very informative to guide dosing of beta-lactamase inhibitors. We examined the hydrolytic activity of several KPC-positive isolates to provide insights on the utility of this approach. Six clinical isolates of Klebsiella pneumoniae resistant to imipenem were examined. The presence of gene (s) encoding KPC and other beta-lactamase (s) were confirmed previously by PCR and DNA sequencing [3, 4]. The relatedness of the isolates was assessed in triplicate using Fourier-transform infrared spectroscopy (FTIR)(Bruker, Billerica, MA). Enzymatic activity of crude cell lysate (normalized to 40μg of total protein content) was assessed using a spectrophotometric assay. Ceftazidime (50μM) degradation in phosphate buffered saline (pH ¼ 7.4) was tracked by absorbance at 260nm over 20 minutes at 35 C. K. pneumoniae ATCC 13883 (American Type Culture Collection, Manassas, VA) was used as a negative control. For comparison, ceftazidime susceptibility (MIC) was determined by a modified broth dilution method, in the presence of escalating avibactam concentration (up to 64 mg/L). The concentrationresponse (ie MIC profiling) observed at 24h was characterized by the sigmoid inhibitory maximum effect (Emax) model [5].Pertinent isolate characteristics are shown in Table 1. Three isolates (KP1438, KP1491 and KP1587) from the same institution were deemed to be related (data not shown). In contrast to common belief, the presence of multiple beta-lactamase genes did not result in a higher hydrolytic activity. It appeared that not all genes were expressed to the maximal extent and thus they did not contribute additively to the overall hydrolytic activity. Lysates from all isolates susceptible to ceftazidime/avibactam (MIC 4/4mg/L) were found to have a similar hydrolytic activity against ceftazidime. However, a higher degradation rate was noted for an isolate resistant to ceftazidime/avibactam (KP 1491, Appendix 1). In this isolate, a higher avibactam concentration was needed to restore ceftazidime MIC to the susceptible range (data not shown). These insights are important and will be used to develop a quantitative method guiding optimal beta-lactamase inhibitor dosing. Despite harboring diverse genes encoding for different beta-lactamases, the hydrolytic activity of various KPC-positive isolates appeared to be reasonably predicted by ceftazidime/avibactam …