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<p>In Zambia, 40% of clinical Gram-negative bacteria are either <i>Escherichia coli</i> or <i>Klebsiella pneumoniae</i>, 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, <i>n</i> = 400) and Katete (rural, <i>n</i> = 500) districts of Zambia were used for bacterial isolation on MacConkey agar supplemented with 1&#xa0;μg/ml cefotaxime. Isolated 3GC-resistant strains were characterized by sequencing the 16S rRNA gene and screening for <i>bla</i><sub>CTX-M</sub> and <i>bla</i><sub>TEM</sub> genes using single polymerase chain reaction (PCR). Furthermore, selected 3GC-resistant strains were subjected to whole-genome sequencing (WGS) using MiSeq/HiSeq (<i>n</i> = 34) and MinION (<i>n</i> = 1). Using the data from this and other previous studies, we developed a rapid PCR-dipstick DNA chromatography-based tool for detecting <i>bla</i><sub>CTX-M</sub>, <i>bla</i><sub>TEM</sub>, <i>E. coli</i>-specific <i>yaiO,</i> and<i> K. pneumoniae</i>-specific <i>khe</i> genes. The prevalence of isolated 3GC resistant strains was 15.4% (139/900), dominated by <i>E. coli</i> (102/139, 73.4%). On PCR, the <i>bla</i><sub>CTX-M</sub> gene was detected in 72.7% (101/139) of the isolates, while <i>bla</i><sub>TEM</sub> 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.</p>

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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

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  • 发表日期：

    2025年4月16日

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  • Misheck Shawa,
  • Kyoko Hayashida,
  • Naganori Nao,
  • Atmika Paudel,
  • Harvey Kamboyi,
  • Herman Chambaro,
  • Malala Mulavu,
  • Cynthia Sipho Khumalo,
  • Mike Nundwe,
  • Tuvshinzaya Zorigt,
  • Chinatsu Nakamura,
  • Yongjin Qiu,
  • Naoko Kawai,
  • Maisa Kasanga,
  • Joseph Chizimu,
  • Lavel Moonga,
  • Joseph Ndebe,
  • Manyando Simbotwe,
  • Shohei Ogata,
  • Mulemba Samutela,
  • Chie Nakajima,
  • Roma Chilengi,
  • Mable Mutengo,
  • Masahiro Kajihara,
  • Hirofumi Sawa,
  • Bernard Hang’ombe,
  • Yasuhiko Suzuki,
  • Hideaki Higashi

摘要

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 blaCTX-M and blaTEM 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 blaCTX-M, blaTEM, 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 blaCTX-M gene was detected in 72.7% (101/139) of the isolates, while blaTEM 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.