开发一种基于聚合酶链反应-试纸条DNA色谱分析的工具，用于检测从赞比亚卡富埃和卡特特地区患者中分离出的产CTX-M和TEM的大肠杆菌和肺炎克雷伯菌。
Development of a PCR-dipstick DNA chromatography-based tool for the detection of CTX-M- and TEM-producing Escherichia coli and Klebsiella pneumoniae isolated from patients in Kafue and Katete districts of Zambia.
作者信息
Shawa Misheck, Hayashida Kyoko, Nao Naganori, Paudel Atmika, Kamboyi Harvey, Chambaro Herman, Mulavu Malala, Khumalo Cynthia Sipho, Nundwe Mike, Zorigt Tuvshinzaya, Nakamura Chinatsu, Qiu Yongjin, Kawai Naoko, Kasanga Maisa, Chizimu Joseph, Moonga Lavel, Ndebe Joseph, Simbotwe Manyando, Ogata Shohei, Samutela Mulemba, Nakajima Chie, Chilengi Roma, Mutengo Mable, Kajihara Masahiro, Sawa Hirofumi, Hang'ombe Bernard, Suzuki Yasuhiko, Higashi Hideaki
机构信息
Hokudai Center for Zoonosis Control in Zambia, Hokkaido University, Lusaka, 10101, Zambia.
Division of Infection and Immunity, International Institute for Zoonosis Control, Hokkaido University, N20 W10, Kita-Ku, Sapporo, 001-0020, Japan.
出版信息
BMC Infect Dis. 2025 Apr 16;25(1):541. doi: 10.1186/s12879-025-10628-9.
In Zambia, 40% of clinical Gram-negative bacteria are either Escherichia coli or Klebsiella pneumoniae, with a high third-generation cephalosporin (3GC) resistance prevalence. Therefore, 3GC resistance surveillance is a crucial indicator for guiding focused intervention policies. However, the lack of genotypic diagnostic tools limits the ability to elucidate trends, especially in peri-urban and rural areas of developing countries. This study aimed to develop a rapid, cost-effective tool for the genotypic surveillance of 3GC resistance. Here, 900 stool samples collected from patients in Kafue (peri-urban, n = 400) and Katete (rural, n = 500) districts of Zambia were used for bacterial isolation on MacConkey agar supplemented with 1 μg/ml cefotaxime. Isolated 3GC-resistant strains were characterized by sequencing the 16S rRNA gene and screening for bla and bla genes using single polymerase chain reaction (PCR). Furthermore, selected 3GC-resistant strains were subjected to whole-genome sequencing (WGS) using MiSeq/HiSeq (n = 34) and MinION (n = 1). Using the data from this and other previous studies, we developed a rapid PCR-dipstick DNA chromatography-based tool for detecting bla, bla, E. coli-specific yaiO, and K. pneumoniae-specific khe genes. The prevalence of isolated 3GC resistant strains was 15.4% (139/900), dominated by E. coli (102/139, 73.4%). On PCR, the bla gene was detected in 72.7% (101/139) of the isolates, while bla was found in 46.8% (65/139) of the strains. The developed tool displayed a high level of agreement with WGS and single PCR/Sanger sequencing, with sensitivity and specificity ≥ 95% and Kappa ≥ 0.95 for each of the four target genes. We envisage that the simplicity and adaptability of this tool will be a significant advantage for the surveillance of 3GC resistance in Zambia and elsewhere.
在赞比亚，40%的临床革兰氏阴性菌为大肠杆菌或肺炎克雷伯菌，对第三代头孢菌素（3GC）的耐药率很高。因此，3GC耐药监测是指导针对性干预政策的关键指标。然而，缺乏基因诊断工具限制了阐明趋势的能力，尤其是在发展中国家的城郊和农村地区。本研究旨在开发一种快速、经济高效的3GC耐药基因监测工具。在此，从赞比亚卡富埃（城郊，n = 400）和卡特特（农村，n = 500）地区的患者中收集了900份粪便样本，用于在添加1μg/ml头孢噻肟的麦康凯琼脂上进行细菌分离。通过对16S rRNA基因进行测序并使用单聚合酶链反应（PCR）筛选bla和bla基因，对分离出的3GC耐药菌株进行表征。此外，使用MiSeq/HiSeq（n = 34）和MinION（n = 1）对选定的3GC耐药菌株进行全基因组测序（WGS）。利用本研究及其他先前研究的数据，我们开发了一种基于PCR试纸条DNA色谱法的快速工具，用于检测bla、bla、大肠杆菌特异性yaiO和肺炎克雷伯菌特异性khe基因。分离出的3GC耐药菌株的患病率为15.4%（139/900），以大肠杆菌为主（102/139，73.4%）。在PCR检测中，72.7%（101/139）的分离株检测到bla基因，而46.8%（65/139）的菌株检测到bla基因。所开发的工具与WGS以及单PCR/Sanger测序显示出高度一致性，四个目标基因各自的灵敏度和特异性均≥95%，kappa值≥0.95。我们设想，该工具的简单性和适应性将成为赞比亚及其他地区3GC耐药监测的一项显著优势。
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