Phase I: Selection of resistant Klebsiella pneumoniae strains and their screening for insertion sequences
Scientific and technical report
During this phase there was build up a collection of multidrug resistant (MDR) Klebsiella pnemoniae strains (total = 49), this resistance phenotype being established using Vitek II automated system. All of the strains were carbapenemase producers, as revealed using the Blue Carba phenotypical test.
Four selected strains were investigated using whole genome sequencing technology. The genomes were assembled using SPAdes software (http://cab.spbu.ru/software/spades/) and annotated subsequently.
For the investigation of the antibiotic resistance genetic background, the genomes were annotated using online tools hosted by Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) and CARD (The Comprehensive Antibiotic Resistance Database) (https://card.mcmaster.ca/), being revealed that all sequenced strains were resistant to beta-lactams antibiotics due to presence of carbapenemase genes blaNDM-1, blaKPC-3 and blaOXA-48, and beta-lactamases genes blaSHV-26/28/33, blaTEM-1A/1B,blaCTX-M-15, blaOXA-1/9. Aminoglycoside resistance was encountered due to the presence of genes encoding aminoglycoside modiying enzymes (acetyltransferases and/or phosphotransferases) or 16S rRNA methyltrasferases. Plasmid mediated fluoroquinolone resistance was encounterd in three strains. Tetracycline resistance was encoded by tetD gene in one strain. All strains harbored multiple plasmid specific genes (replicon type), thus being observed the plasmids associated with antibiotic resistance genes.
WGS also revealed the MLST (multilocus sequence typing) profile (respectively ST101 and ST 383 clones). The insertion sequences were annotated using IS Finder database (https://isfinder.biotoul.fr/), being observed a multitude of IS, some of them previously described being associated with antibiotic resistance.
The mgrB gene was investigated in all colistin resistant strains, being revealed that, for one strain, the above mentioned gene was disrupted by a IS903B insertion, thus not being able to regulate the bacterial lipopolysaccharides (LPS) structure and ultmately not allowing the colistin molecule to penetrate the LPS layer.
Etapa I - Rezumat
În cadrul acestei etape s-a realizat colecția de tulpini de Klebsiella pneumoniae cu fenotip de rezistență de tip MDR (multidrug resistance) (total = 49 tulpini), aspect relevat de antibiograma realizată cu ajutorul sistemului automat Vitek II. Pentru aceste tulpini a fost evidențiată producerea de carbapenemaze, prin testul fenotipic Blue Carba. Tulpinile au fost tipizate conform schemei MLST, au fost investigate privind prezența transpozonilor de tip IS (secvențe de inserție) și au fost caracterizate din punct de vedere al substratului genetic al antibiorezistenței prin tehnologia whole genome sequencing. De asemenea, pentru familia de transpozoni IS903 au fost construiți primeri specifici și s-a evidențiat implicarea acestora în rezistența la colistin, corelat cu inserția în gena mgrB.