Computational protocol: Clinically Relevant ESBL-Producing K. pneumoniae ST307 and E. coli ST38 in an Urban West African Rat Population

Similar protocols

Protocol publication

[…] This study was performed as part of a broader study investigating the role of rodents as potential infection source for various pathogens, in collaboration and under authorization by the Ministry of Livestock and Animal Products (Ministere de l'Elevage et des productions animales) in Conakry, Guinea.Twenty-nine rats from three different species (Rattus rattus n = 22, Rattus norvegicus n = 6, Cricetomys gambianus n = 1) were randomly captured in Conakry (Guinea, West Africa) between November and December 2015. Animals were trapped with life traps (Sherman LFA live trap; H.B. Sherman Traps, Inc., Tallahassee, FL, USA) inside and outside of households in three different, densely populated districts of Conakry (Dixinn [UTM 28P 645031 1055828], Ratoma [UTM 28P 648851 1063078], Matoto [UTM 28P 657469 1066350]; Figure ). Trapped rodents were handled using appropriate personal protective equipment and were euthanized with a lethal dose of fluorane according to animal welfare guidelines. Each animal was sampled once and rectal swabs (MASTASWAB, Mast Diagnostics Reinfeld, Germany) were shipped to the Institute of Microbiology and Epizootics, Berlin, Germany.Selection for cefotaxime-resistant and thus ESBL/AmpC-producing E. coli and K. pneumoniae ssp. pneumoniae was carried out by streaking on CHROMagar™ orientation plates (with and without 4 μg/ml cefotaxime; Mast Diagnostica, Reinfeld, Germany). Enterobacteriaceae-like isolates were species-typed by the automated VITEK®2 system (BioMérieux, Germany), which was also used to determine phenotypic antimicrobial resistance to carbapenems and other classes of antimicrobials (card GN38). In addition, isolates growing on CHROMagarTM that contained cefotaxime were confirmed as ESBL-producers using the phenotypic confirmatory test for ESBL-production (CLSI, ). Confirmed E. coli and K. pneumoniae isolates were subjected to whole-genome sequencing (WGS) and plasmid profile analysis as described previously (Schaufler et al., ). In brief, WGS was performed using MiSeq Illumina 300 bp paired-end sequencing and a coverage greater than 50 was obtained. After quality control using the NGS tool kit (Patel and Jain, ) (70% of bases with a phred score > 20), high quality filtered reads were used for a de novo assembly into contiguous sequences (contigs) using Velvet (Zerbino and Birney, ). Assembled draft genomes of the isolates were annotated using RAST (Aziz et al., ). WGS data was used for genotypic characterization including determination of the multi-locus sequence type (MLST), resistance genes (ResFinder 2.1), and plasmids (PlasmidFinder1.3); all available on the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org).The plasmid sequence (KY271406) of pKPN3-307_TypeC (Villa et al., ) originating from a clinical ST307 isolate from Italy was mapped to the whole-genome of the ST307 isolate IMT38405 using Geneious 6.6. Plasmid contents of both genomes were then compared using Blast Ring Image Generator (BRIG) (Alikhan et al., ) (Figure ).Genes related to bacterial fitness (π-fimbrial cluster, capsule 2 Enterobacter, glycogen synthesis cluster; (Villa et al., )) were analyzed accordingly. The number of SNPs in the core genome between isolates of the same ST was calculated using Harvest Suite 1.0 (Parsnp) and MEGA 6.0 (http://www.megasoftware.net/). […]

Pipeline specifications

Software tools Velvet, RAST, PlasmidFinder, Geneious, BRIG, Parsnp, MEGA
Databases ResFinder
Applications WGS analysis, Nucleotide sequence alignment, Genome data visualization
Organisms Klebsiella pneumoniae, Escherichia coli, Rattus norvegicus, Homo sapiens
Diseases Infection
Chemicals beta-Lactams