WORLDCOM

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The Project #WORLDCOM

Start: 1 January 2020
Duration: 2.5 Years
Domain: Antimicrobial Resistance
Keywords: AMR, Zoonoses, On-site, Diagnostics, Surveillance
Contact: Terry Smith (NUIG)

WORLDCOM: Development of new tools for real-time detection of zoonotic bacteria and antimicrobial resistance in veterinary, human and environmental sources

The WORLDCOM project aimed to develop diagnostic tools linked with mobile referencing technology for the detection of antimicrobial resistance (AMR) in zoonotic bacterial pathogens. Resulting technologies would enable rapid on-site AMR and pathogen detection from animal and human populations, and the environment, facilitating early investigation of emerging resistances and the development of machine-learning algorithms for AMR detection and prediction.

Extended-spectrum beta-lactamase (ESBL) and carbapenemase enzymes produced by Enterobacteriaceae confer resistance to beta-lactam antimicrobials presenting a major public health concern. A genomic approach was used to analyse publicly available sequences of relevant pathogens (Escherichia coli, Klebsiella spp., Salmonella spp. and Acinetobacter spp.) for circulating antibiotic resistance genes (ESBL, carbapenemase and mobile colistin resistance genes). Based on this information, targeted de novo sequencing and phenotypic characterisation of bacterial pathogens from human, animal and environmental origin was performed among partners, resulting in the generation of an in-house database (with a total of 685 isolates) for use in assay development. Biomarker genes associated with highly prevalent ESBLs (CTX-M), carbapenemases (OXA, KPC and NDM) and colistin resistance (MCR-1) were selected as AMR diagnostic targets. Loop-mediated isothermal amplification (using LEC-LAMP and LAMP) was selected as the in vitro amplification technology for detection of these targets due to its speed, specificity and robustness, as well as its ease of application in the field, including on-farm and / or in remote settings.

Additionally, OXA-48, OXA-23, KPC, VIM, MCR-1 and MCR-9 AMR biomarkers were targeted using real-time singleplex LAMP assays. The assays were developed, optimised and validated against a wide range of Gram-positive and Gram-negative bacterial isolates to confirm sensitivity and specificity using both fluorescent and colorimetric detection. The assays were further developed and implemented to successfully detect AMR biomarkers from environmental water and pig faecal samples within less than an hour. Additionally, a smartphone app has been developed to provide automated detection for LAMP diagnostic tests. The smartphone app automatically analyses captured images of colorimetric LAMP assays to facilitate field testing.

Closely-related CTX-M biomarkers, preferentially associated with animal (CTX-M-1) and human (CTX-M-15) infection, were targeted with an internally controlled multiplex loop-primer endonuclease cleavage (LEC)-LAMP assay enabling differential point-mutation detection. Assay validation using (i) isolates from animal, human or environmental sources and also (ii) DNA isolated directly from animal and environmental samples demonstrated rapid detection (10-20 min), complete analytical specificity, and single-digit genome copy sensitivity. Development of a portable workstation incorporating the LEC-LAMP assay with a rapid DNA extraction protocol and validation for on-site application was completed with a research article relating to this work accepted for publication with Microbiology Spectrum. This is the first report of portable nucleic acid diagnostics applied to differential detection of CTX-M-1 and CTX-M-15, providing novel transferable diagnostic technology for improved epidemiological surveillance. Additionally, a duplex LEC-LAMP assay for the differential detection of closely related OXA variants, OXA-48 and OXA-181/232, was also developed and validated.

Key Project Outputs and Outcomes

  • Generation of an in-house reference database of sequences from currently circulating AMR pathogenic strains (with a total of 685 isolates) for use in assay development.
  • Application of isothermal amplification technologies (including LAMP and LEC-LAMP) led to the development of AMR detection assays of biomarkers from environmental water and pig faecal samples for use in a laboratory setting and in the field. This diagnostics technology is detailed in the research article in Microbial Biotechnology (shown below).
  • Development of a custom-designed smartphone application for linking to relevant reference laboratories. This platform will facilitate early warning of potential zoonotic threats and enable follow up epidemiological investigations to be conducted.
  • Development and validation of a portable nucleic acid diagnostics workstation (combined LEC-LAMP assay and rapid DNA extraction protocol) for on-site application. This diagnostics tool is detailed in the research article in Microbiology Spectrum (shown below).

Hassan, M. M., van Vliet, A. H. M., Higgins, O., Burke, L. P., Chueiri, A., O’Connor, L., Morris, D., Smith, T. J., & La Ragione, R. M. (2023). Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples. Microbial biotechnology, 10.1111/1751-7915.14227. Advance online publication. DOI: https://doi.org/10.1111/1751-7915.14227

Higgins, O., Chueiri, A., O’Connor, L., Lahiff, S., Burke, L. P., Morris, D.,  Pfeifer, N. M., González Santamarina, B., Berens, C., Menge, C., Caniça, M., Manageiro, V., Kisand, V., Hassan, M. M., Gardner, B., van Vliet, A. H. M., La Ragione, R. M., Gonzalez-Zorn, B. & Smith, T. J. (2022). Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification. Microbiology Spectrum. e03316-22. Advance online publication. DOI: https://doi.org/10.1128/spectrum.03316-22

WORLDCOM has succeeded in developing a novel combination of modified isothermal amplification technology, NALF devices capable of being used on-site and mobile technology to provide an innovative, early warning tool-kit for prediction of antimicrobial resistance in various environments.

Project Assets

Hassan, M. M., van Vliet, A. H. M., Higgins, O., Burke, L. P., Chueiri, A., O’Connor, L., Morris, D., Smith, T. J., & La Ragione, R. M. (2023). Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples. Microbial biotechnology, 10.1111/1751-7915.14227. Advance online publication. DOI: https://doi.org/10.1111/1751-7915.14227

Higgins, O., Chueiri, A., O’Connor, L., Lahiff, S., Burke, L. P., Morris, D.,  Pfeifer, N. M., González Santamarina, B., Berens, C., Menge, C., Caniça, M., Manageiro, V., Kisand, V., Hassan, M. M., Gardner, B., van Vliet, A. H. M., La Ragione, R. M., Gonzalez-Zorn, B. & Smith, T. J. (2022). Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification. Microbiology Spectrum. e03316-22. Advance online publication. DOI: https://doi.org/10.1128/spectrum.03316-22

González-Santamarina, B., Weber , M., Menge C., Berens, C. (2022). Comparative Genomic Analysis of Antimicrobial-Resistant Escherichia coli from South American Camelids in Central Germany. Microorganisms. 10(9), 1697. DOI: https://doi.org/10.3390/microorganisms10091697

Manageiro, V., Salgueiro, V., Rosado, T., Bandarra, N. M., Ferreira, E., Smith, T., Dias, E., Caniça, M. (2022). Genomic Analysis of a mcr-9.1-Harbouring IncHI2-ST1 Plasmid from Enterobacter ludwigii Isolated in Fish Farming. Antibiotics. 11(9), 1232. DOI: https://doi.org/10.3390/antibiotics11091232

Thomson, N. M., Gilroy, R., Getino, M., Foster-Nyarko, E., van Vliet, A, H, M., La Ragione, R. M., & Pallen, M. J. (2022). Remarkable genomic diversity among Escherichia isolates recovered from healthy chickens. Peer J. 10, e12935. DOI: https://doi.org/10.7717/peerj.12935

Tedersoo, T., Roasto, M., Mäesaar, M., Häkkinen, L., Kisand, K., Ivanova, M., Valli, M. H., & Meremäe, K. (2022). Antibiotic Resistance in Campylobacter spp. Isolated from Broiler Chicken Meat and Human Patients in Estonia. Microorganisms. 10(5), 1067. DOI: https://doi.org/10.3390/microorganisms10051067

González-Santamarina, B., Schnee, C., Koehler, H., Weber, M., Methner, U., Seyboldt, C., Berens, C. & Menge, C. (2022). Survey on shedding of selected pathogenic, zoonotic or antimicrobial resistant bacteria by South American camelids in Central Germany. Berliner Und Münchener Tierärztliche Wochenschrift. 135, 1–16. DOI: https://doi.org/10.2376/1439-0299-2021-21

Tedersoo, T., Roasto, M., Mäesaar, M., Kisand, V., Ivanova, M., Meremäe, M. (2022). The prevalence, counts, and MLST genotypes of Campylobacter in poultry meat and genomic comparison with clinical isolates. Poultry Science. 101 (4), 101703. DOI: https://doi.org/10.1016/j.psj.2022.101703

Hassan, M. M., Grist, L. F., Poirier, A. C., & La Ragione, R. M. (2022). JMM profile: Loop-mediated isothermal amplification (LAMP): for the rapid detection of nucleic acid targets in resource-limited settings. Journal of Medical Microbiology. 71(5), 001522. DOI: https://doi.org/10.1099/jmm.0.001522

Delgado-Blas, J. F., Valenzuela Agüi, C., Marin Rodriguez, E., Serna, C., Montero, N., Saba, C., & Gonzalez-Zorn, B. (2022). Dissemination Routes of Carbapenem and Pan-Aminoglycoside Resistance Mechanisms in Hospital and Urban Wastewater Canalizations of Ghana. mSystems. 7(1), e0101921. DOI: https://doi.org/10.1128/msystems.01019-21

van Vliet, A., Thakur, S., Prada, J. M., Mehat, J. W., & La Ragione, R. M. (2022). Genomic Screening of Antimicrobial Resistance Markers in UK and US Campylobacter Isolates Highlights Stability of Resistance over an 18-Year Period. Antimicrobial Agents and Chemotherapy. 66(5), e0168721. DOI:  https://doi.org/10.1128/aac.01687-21

Delgado-Blas, JF., Ovejero, CM., David, S., Montero, N., Calero-Caceres, W., Garcillan-Barcia, MP., de la Cruz, F., Muniesa, M., Aanensen, DM., Gonzalez-Zorn, B. (2021). Population genomics and antimicrobial resistance dynamics of Escherichia coli in wastewater and river environments. Communications Biology. 4(1), 457. DOI: https://doi.org/10.1038/s42003-021-01949-x

Project

Project Events

One Health EJP Project Kick-off meeting- 13th November 2019, Berlin, Germany

WORLDCOM Kick-off meeting- 27-28th January 2020

WorldCOM First annual review meeting- September 2020

WorldCOM Second annual review meeting- September 2021

WORLDCOM Final annual review meeting- December 2022

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