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
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<sub>CTX-M</sub> and bla<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 (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<sub>CTX-M</sub>, bla<sub>TEM</sub>, 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<sub>CTX-M</sub> gene was detected in 72.7% (101/139) of the isolates, while bla<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.
Authors
- Hideaki Higashi (ORCID: https://orcid.org/0000-0002-5370-7865)
- Chinatsu Nakamura
- Harvey Kamboyi (ORCID: https://orcid.org/0000-0002-3316-7611)
- Chie Nakajima (ORCID: https://orcid.org/0000-0002-9194-1062)
- Herman M. Chambaro (ORCID: https://orcid.org/0000-0003-1407-6818)
- Malala Mulavu
- Tuvshinzaya Zorigt
- Joseph Ndebe (ORCID: https://orcid.org/0000-0002-3070-8830)
- Hirofumi Sawa (ORCID: https://orcid.org/0000-0003-2569-2755)
- Masahiro Kajihara
- Mulemba Samutela
- Naganori Nao
- Shohei Ogata
- Lavel C. Moonga (ORCID: https://orcid.org/0000-0001-7814-9997)
- Joseph Yamweka Chizimu (ORCID: https://orcid.org/0000-0002-2126-5759)
- Manyando Simbotwe
- Maisa Kasanga (ORCID: https://orcid.org/0000-0003-1724-7934)
- Yongjin Qiu (ORCID: https://orcid.org/0000-0001-8621-5472)
- Roma Chilengi (ORCID: https://orcid.org/0000-0003-0221-9527)
- Mable Mutengo (ORCID: https://orcid.org/0000-0003-3973-0345)
- Atmika Paudel (ORCID: https://orcid.org/0000-0003-3478-9452)
- Cynthia Sipho Khumalo (ORCID: https://orcid.org/0000-0002-1382-6827)
- Bernard M. Hang’ombe (ORCID: https://orcid.org/0000-0002-4201-6401)
- Yasuhiko Suzuki (ORCID: https://orcid.org/0000-0002-1313-1494)
- Misheck Shawa
- Kyoko Hayashida (ORCID: https://orcid.org/0000-0001-9266-5162)
- Naoko Kawai
- Mike Nundwe (ORCID: https://orcid.org/0009-0002-4110-9312)
Institutions
- Hokkaido University of Science (JP)
- Centre for Infectious Disease Research in Zambia (ZM)
- Ministry of Health (ZM)
- Hokkaido University (JP)
- Global Virus Network (US)
- University Teaching Hospital (ZM)
- Lusaka Apex Medical University (ZM)
- Sapporo International University (JP)
- University of Zambia (ZM)
- National Institute of Infectious Diseases (JP)
- Ministry of Agriculture and Livestock (ZM)
Publication Details
- Journal
- BMC Infectious Diseases
- Published
- 2025-04-16
- DOI
- https://doi.org/10.1186/s12879-025-10628-9
- Citations
- 1
- Primary Topic
- Antibiotic Resistance in Bacteria
- Type
- article
- Field-Weighted Citation Impact
- 0.59
Funders
- Japan Agency for Medical Research and Development
- Ministry of Education, Culture, Sports, Science and Technology
- Hokkaido University
- Japan Society for the Promotion of Science