Characterization of plasmids mediating carbapenem resistance in Klebsiella pneumoniae in Pretoria, South Africa

Abstract

pneumoniae is a Gram-negative bacterium belonging to the Enterobacteriaceae family. This pathogen is implicated in community- and hospital-acquired infections, particularly in neonates, the elderly, and immunocompromised patients. Risk factors such as extended hospital stay, being immunocompromised, and excessive antibiotic use lead to disease severity and potential acquisition of resistance to antibiotics. Carbapenems are usually the treatment of choice in multidrug-resistant K. pneumoniae infections. Since the last decade, carbapenems has become less effective as K. pneumoniae stains develop mechanisms of resistance against them. Among the various mechanisms of carbapenem resistance, enzyme (carbapenemases, ESBLs, AmpCs) production is the most dominant. Carbapenemases are classified into three classes viz., class A, B and D, and have the ability to hydrolyse β-lactams antibiotics, including “last resort” carbapenems. The most commonly reported carbapenemases worldwide in K. pneumoniae strains include the blaKPC, blaNDM, blaVIM, blaIMP and blaOXA genes. In South Africa, blaOXA-48/181 and blaNDM-1 have been reported in K. pneumoniae in almost all provinces, with outbreaks being reported in other provinces within the past six years. Plasmids are shuttles that mediate the acquisition and dissemination of carbapenemases. This is because plasmids are mobile and may be transferred from one specie to another via horizontal gene transfer. Conjugative plasmids such as IncF, A/C, IncL/M, IncN and IncX plasmids have been associated with commonly reported carbapenemases worldwide. In South Africa, little is known about plasmid types associated with carbapenemase genes and their molecular characteristics. This study aimed to identify and characterise plasmids mediating carbapenem resistance in Pretoria, South Africa. Sixty K. pneumoniae clinical isolates were collected from the National Health Laboratory service (NHLS, Pretoria) in 2018. Carbapenem resistance and carbapenemase production was determined using MicroScan automated system and PCR assays, respectively. The isolates’ plasmids were characterized to determine their size, number and replicon types using gel electrophoresis, and PCR-based replicon typing techniques, respectively. Molecular characteristics of the isolates’ carbapenemases and plasmids were analysed using both PCR assays and whole-genome sequencing (WGS). All K. pneumoniae isolates were multidrug resistant. Carbapenemase production was identified in 65% (blaOXA-48-like) and 29% (blaNDM-1) of the isolates. Multi-locus typing revealed five sequence types: ST307, ST607, ST17, ST39, and ST3559. Both PCR and WGS revealed multiple plasmid replicons associated with the carbapenem-resistant K. pneumoniae (CRKP) isolates viz., IncF, A/C, IncL/M and IncX3 plasmids. WGS proved to be more useful in characterising plasmids over the PCR-based replicon typing (PBRT). The PBRT could not identify the IncX3 replicons, which were detected by WGS. Identified plasmids could be transferred from donor CRKP strains to recipient E. coli strains. Phylogenomic analysis showed that strains in this study were closely related to stains from the United States, China, Thailand, and South Korea more than other countries. These strains shared similar antimicrobial resistance mechanisms, however, they belonged to different sequence types including ST14, ST11, ST147, and ST152. This study shows an ongoing plasmid-mediated dissemination of carbapenemase genes in 2018 in Pretoria, with blaOXA-48-like and blaNDM-1 genes being the major resistance determinants. This study has also shown the important role conjugative or mobile plasmids play in the acquisition and dissemination of carbapenemase genes from one specie to another via conjugation.