title: Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.
abstract: Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n= 6) and grassland (n= 4) soil intoEscherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated fromProteobacteria, two (PEDO-1 and ESP-2) fromBacteroidetesand one (GRD33-1) fromGemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed inE. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Dereje D
authors.initials: DD
authors.lastname: Gudeta
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Valeria
authors.initials: V
authors.lastname: Bortolaia
authors.affiliation: Department of Medical Biotechnology, University of Siena Siena, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pollini
authors.affiliation: Department of Medical Biotechnology, University of Siena Siena, Italy.
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Docquier
authors.affiliation: Department of Medical Biotechnology, University of SienaSiena, Italy; Department of Experimental and Clinical Medicine, University of FlorenceFlorence, Italy; Clinical Microbiology and Virology Unit, Florence Careggi University HospitalFlorence, Italy; Don Carlo Gnocchi FoundationFlorence, Italy.
authors.forename: Gian M
authors.initials: GM
authors.lastname: Rossolini
authors.affiliation: School of Life Sciences, University of Warwick Coventry, UK.
authors.forename: Gregory C A
authors.initials: GC
authors.lastname: Amos
authors.affiliation: School of Life Sciences, University of Warwick Coventry, UK.
authors.forename: Elizabeth M H
authors.initials: EM
authors.lastname: Wellington
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of CopenhagenFrederiksberg, Denmark; Department of Biomedical Sciences, Ross University School of Veterinary MedicineSt. Kitts, West Indies.
authors.forename: Luca
authors.initials: L
authors.lastname: Guardabassi
keywords: antibiotic resistance; carbapenems; functional metagenomics; metallo-ß-lactamases; soil