Cultivation and sequencing-free protocol for Serratia marcescens detection and typing

Alessandro Alvaro; Aurora Piazza; Stella Papaleo; Matteo Perini; Ajay Ratan Pasala; Simona Panelli; Tiago Nardi; Riccardo Nodari; Lodovico Sterzi; Cristina Pagani; Cristina Merla; Daniele Castelli; Emanuela Olivieri; Silvia Bracco; Maria Laura Ferrando; Francesca Saluzzo; Sara Giordana Rimoldi; Marta Corbella; Annalisa Cavallero; Paola Prati; Claudio Farina; Daniela Maria Cirillo; Gianvincenzo Zuccotti; Francesco Comandatore

doi:10.1016/j.isci.2024.109402

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing

iScience. 2024 Mar 1;27(4):109402. doi: 10.1016/j.isci.2024.109402. eCollection 2024 Apr 19.

Authors

Alessandro Alvaro^{1

2}, Aurora Piazza³, Stella Papaleo¹, Matteo Perini^{1

4}, Ajay Ratan Pasala^{1

5

6}, Simona Panelli¹, Tiago Nardi^{1

7}, Riccardo Nodari¹, Lodovico Sterzi¹, Cristina Pagani⁸, Cristina Merla⁹, Daniele Castelli¹⁰, Emanuela Olivieri¹¹, Silvia Bracco¹², Maria Laura Ferrando¹³, Francesca Saluzzo¹³, Sara Giordana Rimoldi⁸, Marta Corbella⁹, Annalisa Cavallero¹⁰, Paola Prati¹¹, Claudio Farina¹², Daniela Maria Cirillo¹³, Gianvincenzo Zuccotti^{1

14}, Francesco Comandatore¹

Affiliations

¹ Department of Biomedical and Clinical Sciences, Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", University of Milan, 20157 Milan, Italy.
² Department of Biosciences and Pediatric Clinical Research Center "Romeo Ed Enrica Invernizzi", University of Milan, 20133 Milan, Italy.
³ Unit of Microbiology and Clinical Microbiology, Department of Clinical-Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia 27100, Italy.
⁴ Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY 10032, USA.
⁵ Biochemistry, Microbiology and Immunology Department, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
⁶ Centre for Innovation, Canadian Blood Services, Ottawa, ON K1G 4J5, Canada.
⁷ Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy.
⁸ Laboratorio di Microbiologia Clinica, Virologia e Diagnostica delle Bioemergenze, ASST Fatebenefratelli Sacco, 20157 Milan, Italy.
⁹ Department of Microbiology & Virology, Fondazione IRCCS Policlinico San Matteo, Viale Camillo Golgi 19, 27100 Pavia, Italy.
¹⁰ Microbiology Unit, Fondazione IRCCS San Gerardo, 20900 Monza, Italy.
¹¹ Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), 27100 Pavia, Italy.
¹² Laboratory of Microbiology and Virology, Azienda Socio-Sanitaria Territoriale (ASST) Papa Giovanni XXIII, 24127 Bergamo, Italy.
¹³ Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
¹⁴ Department of Paediatrics, Children's Hospital "V. Buzzi", 20154 Milano, Italy.

Abstract

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Keywords: Biological sciences; Biological sciences research methodologies; Environmental Biotechnology; Microbiology.