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1. Synthetic antimicrobial peptides as enhancers of the bacteriolytic action of staphylococcal phage endolysins

Author

Ana Gouveia, Daniela Pinto, Helena Veiga, Wilson Antunes, Mariana G. Pinho, and Carlos São-José

Subjects
Medicine, Science
Abstract

Abstract Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth, Staphylococcus aureus becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different S. aureus strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of S. aureus to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed.

Published
2022
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2. A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Author

Catarina Amaral, Wilson Antunes, Elin Moe, Américo G. Duarte, Luís M. P. Lima, Cristiana Santos, Inês L. Gomes, Gonçalo S. Afonso, Ricardo Vieira, Helena Sofia S. Teles, Marisa S. Reis, Manuel A. Ramalho da Silva, Ana Margarida Henriques, Miguel Fevereiro, M. Rita Ventura, Mónica Serrano, and Catarina Pimentel

Subjects
Medicine, Science
Abstract

Abstract Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

Published
2021
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3. A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Author

Helena Sofia S. Teles, Américo G. Duarte, Ana Margarida Henriques, Wilson Antunes, Cristiana C. Santos, Catarina Pimentel, Catarina Amaral, Manuel A. Ramalho da Silva, Mónica Serrano, Elin Moe, Ricardo Vieira, Luís M. P. Lima, Marisa S. Reis, Gonçalo S. Afonso, M. Rita Ventura, Inês Gomes, Miguel Fevereiro, and Instituto de Tecnologia Química e Biológica António Xavier (ITQB)

Subjects
Test strategy, Saliva, Molecular biology, Science, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Sensitivity and Specificity, Article, Colorimetry (chemical method), COVID-19 Testing, SDG 3 - Good Health and Well-being, Humans, General, Pandemics, Multidisciplinary, Chromatography, Portugal, SARS-CoV-2, Chemistry, Infectious-disease diagnostics, COVID-19, RNA, Nucleic acid amplification technique, Molecular Diagnostic Techniques, Medicine, RNA, Viral, Colorimetry, RNA extraction, Nucleic Acid Amplification Techniques, Viral load
Abstract

Funding Information: We thank the members of the COVID-19 task force of ITQB NOVA and Mariana Pinho for helpful discussions and suggestions. In particular, we thank Claudio M. Soares and Adriano O. Henriques for their unconditional support and invaluable help in making this work possible. The authors acknowledge the assistance of Paula Chi-cau, ITQB NOVA, in the development of the saliva test and are grateful to Chuck Farah, São Paulo University, for the pGTf2 plasmid. This work was supported by (i) Project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020—“Programa Operacional Competitividade e Internacionalização” (POCI), (ii) the European Union’s Horizon 2020 research and innovation programme under grant agreement No 810856 and (iii) “Fundação para a Ciência e a Tecnologia” (FCT) through programme IF (IF/00124/2015) to C.P, (iv) through the project “DETECT: Development of an Easy, fast-Track and Economical Colorimetric Test for autonomous national diagnosis of COVID-19” Ref 433_613549914 (20/7/153), attributed to C.P., under the scope of the 2nd edition of the programme RESEARCH4COVID19 and partially supported by (v) the project “STOP-COVID—Strategies to prevent COVID-19 by early detection of asymptomatic carriers at increased risk: epidemiological studies and validation of a rapid in-house diagnostic test”, Ref 072559, funded by FEDER—“Fundo Europeu de Desenvolvimento Regional” from “Programa Opera-cional Regional Lisboa”. Publisher Copyright: © 2021, The Author(s). Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH. publishersversion published

Published
2021

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