Table_1_Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: Predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii.XLSX

ObjectivesLittle is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period.
Materials and methodsIMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the β-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.
ResultsFifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and β-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent.
ConclusionIMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination.

研究目的
目前欧洲地区对产IMP酶的肠杆菌目细菌（IMP-producing Enterobacterales, IMP-Ent）的认知仍较为匮乏。本研究针对9年间在西班牙流行的IMP-Ent分离株，从基因组与表型层面开展系统性分析。

材料与方法
本研究纳入所有提交至本参考实验室的IMP-Ent分离株。采用微量肉汤稀释法（欧盟药敏试验委员会（EUCAST）标准）进行抗生素敏感性试验；通过碳青霉烯酶抑制剂试验、β-CARBA法、改良霍奇试验（modified Hodge test, MHT）以及改良碳青霉烯酶抑制试验（modified carbapenemase inhibition method, mCIM）检测IMP碳青霉烯酶活性。对所有收集的分离株进行高分辨率单核苷酸多态性（single-nucleotide polymorphism, SNP）分型、核心基因组多位点序列分型（core genome multilocus sequence typing, cgMLST）以及耐药组分析。

结果
本研究共检出50株IMP-Ent分离株，采集自西班牙13个省份的19家医院，具体菌种分布如下：肺炎克雷伯菌（Klebsiella pneumoniae, IMP-Kpn）24株（占比48%）、罗根坎普肠杆菌（Enterobacter roggenkampii）13株（26%）、霍马切肠杆菌（Enterobacter hormaechei）8株（16%）、产酸克雷伯菌（Klebsiella oxytoca）2株（4%）、阿氏肠杆菌（Enterobacter asburiae）1株（2%）、黏质沙雷菌（Serratia marcescens）1株（2%）以及大肠埃希菌（Escherichia coli）1株（2%）。所有分离株的改良霍奇试验与β-CARBA检测均呈阳性；其中48株（96%）改良碳青霉烯酶抑制试验结果为阳性；分别有12株（24%）与26株（52%）对二吡啶甲酸（美罗培南）与乙二胺四乙酸（EDTA，厄他培南）呈现阳性抑制效应。本研究共鉴定出5种IMP碳青霉烯酶型别：IMP-8（22株，44%）、IMP-22（17株，34%）、IMP-13（7株，14%）、IMP-28（2株，4%）以及IMP-15（2株，4%）；其中肺炎克雷伯菌以IMP-8型为主，罗根坎普肠杆菌以IMP-22型为主。IMP-28仅在产酸克雷伯菌中检出，IMP-15仅在霍马切肠杆菌中检出。肺炎克雷伯菌分离株的优势序列型别为ST405（29.2%）、ST15（25%）与ST464（20.8%）；罗根坎普肠杆菌的优势序列型别为ST96（100%）；霍马切肠杆菌的优势序列型别为ST182（62.5%）。对于IMP-Ent分离株，黏菌素与阿米卡星为活性最佳的非碳青霉烯类抗生素。

结论
尽管近年来产IMP酶的肠杆菌目细菌在西班牙已出现流行并引发医院内暴发疫情，但整体仍属少见菌株；本研究中检出率最高的为产IMP-8酶的肺炎克雷伯菌与产IMP-22酶的罗根坎普肠杆菌。部分产IMP酶菌株在乙二胺四乙酸或二吡啶甲酸抑制试验中呈现假阴性结果。为更好地理解并控制IMP-Ent的传播，持续开展有效的微生物学与分子流行病学监测至关重要。