Long-Read Whole Genome Sequencing Elucidates the Mechanisms of Amikacin Resistance in Multidrug-Resistant Klebsiella pneumoniae Isolates Obtained from COVID-19 Patients

Abstract

Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacterium, which represents a global challenge to public health as one of the major causes of healthcare-associated infections worldwide. In the recent decade, the World Health Organization (WHO) noticed a critically increasing rate of carbapenem-resistant K. pneumoniae occurrence in hospitals. The situation with extended-spectrum beta-lactamase (ESBL) producing bacteria further worsened during the COVID-19 pandemic, due to an increasing number of patients in intensive care units (ICU) and extensive, while often inappropriate, use of antibiotics including carbapenems. In order to elucidate the ways and mechanisms of antibiotic resistance spreading within the K. pneumoniae population, whole genome sequencing (WGS) seems to be a promising approach, and long-read sequencing is especially useful for the investigation of mobile genetic elements carrying antibiotic resistance genes, such as plasmids. We have performed short- and long read sequencing of three carbapenem-resistant K. pneumoniae isolates obtained from COVID-19 patients in a dedicated ICU of a multipurpose medical center, which belonged to the same clone according to cgMLST analysis, in order to understand the differences in their resistance profiles. We have revealed the presence of a small plasmid carrying aph(3′)-VIa gene providing resistance to amikacin in one of these isolates, which corresponded perfectly to its phenotypic resistance profile. We believe that the results obtained will facilitate further elucidating of antibiotic resistance mechanisms for this important pathogen, and highlight the need for continuous genomic epidemiology surveillance of clinical K. pneumoniae isolates.

International Clones of High Risk of Acinetobacter Baumannii—Definitions, History, Properties and Perspectives

“…Isolate typing is crucial for conducting epidemiological surveillance and investigating the spread of particular clones of interest, including ICs. The rapid development and dramatic cost reduction of WGS for bacteria in recent years have made genomic epidemiology a promising approach for pathogen population studies and outbreak investigation [ 20 , 120 , 121 , 122 , 123 ].…”

Whole-Genome Sequencing Revealed the Fusion Plasmids Capable of Transmission and Acquisition of Both Antimicrobial Resistance and Hypervirulence Determinants in Multidrug-Resistant Klebsiella pneumoniae Isolates

“…CriePt491 was the only isolate not having conjugative plasmids and carrying a large fraction of its AMR genes on the chromosome, and these facts could be explained by its possession of a complete CRISPR-Cas type I-E system. The situation of chromosomal AMR gene location is not typical for K. pneumoniae since this species usually carries most of its acquired resistance genes on the plasmids [ 2 , 25 , 57 ], which was observed in other isolates from the current study. At the same time, all other isolates included the AcrIIA7 Cas9 inhibitor protein gene, which could be the reason why they did not have CRISPR-Cas, although AcrIIA7 has been supposed to target II-A systems [ 58 ].…”

“…High-molecular-weight plasmids belong to different incompatibility groups and usually carry genetic determinants of virulence and antibiotic resistance [ 23 ]. In recent years, whole-genome sequencing (WGS), especially long-read sequencing, has become a powerful tool for the determination of plasmid structures, as well as antimicrobial resistance (AMR) and virulence gene locations [ 17 , 24 , 25 ].…”