Identification of SNP barcode for classify carbapenem-resistant enterobacteriaceae (CRE) and non-CRE using Nanopore sequencing

Abstract

Background: The continuous increase of carbapenem-resistant Enterobacteriaceae (CRE) prevalence has been a serious problem for public health worldwide. In the previous study, 25% of 3864 Klebsiella pneumoniae isolates were carbapenem-resistant. One of the most important risks of bacterial drug resistance is the improper use of antibiotics. The routine antimicrobial-susceptibility test requires overnight and has a turnaround time of approximately 2–3 days which delays the proper antibiotics. This study aims to develop a technique to classify CRE and non-CRE by SNP barcode. Method: Forty Klebsiella pneumoniae (KP) clinical isolates were collected from 2020–2021 at King Chulalongkorn Memorial Hospital. Twenty samples were carbapenem-resistant Klebsiella pneumoniae (CRKP), and 20 samples were not carbapenem-resistant (non-CRKP). Each isolate was sub-cultured at 37°C overnight for DNA extraction, library preparation, and Nanopore sequencing. Then, we assembled the bacterial whole genomes and analyzed their chromosomes by bioinformatic tools, including Snippy and OrthoFinder. Result: The phylogenetic tree generated by the Snippy tool can classify 40 KP clinical isolates into 3 clades. Most of non-CRKPs are in clades 1 and 2, while the CRKPs are mainly in clade 3. However, none of the SNPs is unique and useful in differentiation of CRKP from non-CRKP. Thus, SNP barcode cannot be defined from this study. In contrast, OrthoFinder, which compares amino acid sequences between orthologous genes, can clearly distinguish KPs into 4 clades, 1–2 for CRKPs and 3–4 for non-CRKPs. Two of twenty CRKPs are members of clades 3–4, while only 1 non-CRKP is in clade 1–2. Conclusion: Different tools generate quite different phylogenetic trees. SNPs from KP chromosomal DNA are not very different between CRKP and non-CRKP. In fact, plasmids and other mobile genetic elements, harboring drug-resistant genes, are frequently found in KPs. These can be horizontal transferred and may weaken the association between SNPs in bacterial chromosome and the carbapenem-resistance in KPs.