Whole-genome sequence analysis of clinically isolated carbapenem resistant Escherichia coli from Iran.

BACKGROUND: The emergence of carbapenem-resistant Enterobacterales (CRE) continues to threaten public health due to limited therapeutic options. In the current study the incidence of carbapenem resistance among the 104 clinical isolates of Escherichia coli and the genomic features of carbapenem resistant isolates were investigated. METHODS: The susceptibility to imipenem, tigecycline and colistin was tested by broth dilution method. Susceptibility to other classes of antimicrobials was examined by disk diffusion test. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR. Molecular characteristics of carbapenem resistant isolates were further investigated by whole-genome sequencing (WGS) using Illumina and Nanopore platforms. RESULTS: Four isolates (3.8%) revealed imipenem MIC of ≥32 mg/L and positive results for modified carbapenem inactivation method and categorized as carbapenem resistant E. coli (CREC). Colistin, nitrofurantoin, fosfomycin, and tigecycline were the most active agents against all isolates (total susceptibility rate of 99, 99, 96 and 95.2% respectively) with the last three compounds being found as the most active antimicrobials for carbapenem resistant isolates (susceptibility rate of 100%). According to Multilocus Sequence Type (MLST) analysis the 4 CREC isolates belonged to ST167 (n = 2), ST361 (n = 1) and ST648 (n = 1). NDM was detected in all CREC isolates (NDM-1 (n = 1) and NMD-5 (n = 3)) among which one isolate co-harbored NDM-5 and OXA-181 carbapenemases. WGS further detected blaCTX-M-15, blaCMY-145, blaCMY-42 and blaTEM-1 (with different frequencies) among CREC isolates. Co-occurrence of NDM-type carbapenemase and 16S rRNA methyltransferase RmtB and RmtC was found in two isolates belonging to ST167 and ST648. A colistin-carbapenem resistant isolate which was mcr-negative, revealed various amino acid substitutions in PmrB, PmrD and PhoPQ proteins. CONCLUSION: About 1.9% of E. coli isolates studied here were resistant to imipenem, colistin and/or amikacin which raises the concern about the outbreaks of difficult-to-treat infection by these emerging superbugs in the future.

Advantages of long- and short-reads sequencing for the hybrid investigation of the Mycobacterium tuberculosis genome.

Introduction: In the fight to limit the global spread of antibiotic resistance, computational challenges associated with sequencing technology can impact the accuracy of downstream analysis, including drug resistance identification, transmission, and genome resolution. About 10% of Mycobacterium tuberculosis (MTB) genome is constituted by the PE/PPE family, a GC-rich repetitive genome region. Although sequencing using short read technology is widely used, it is well recognized its limit in the PE/PPE regions due to the unambiguously mapping process onto the reference genome. The aim of this study was to compare the performances of short-reads (SRS), long-reads (LRS) and hybrid-reads (HYBR) based analysis over different common investigative tasks: genome coverage estimation, variant calling and cluster analysis, drug resistance detection and de novo assembly. Methods: For the study 13 model MTB clinical isolates were sequenced with both SRS and LRS. HYBR were produced correcting the long reads with the short reads. The fastq from the three approaches were then processed using a customized version of MTBseq for genome coverage estimation and variant calling and using two different assemblers for de novo assembly evaluation. Results: Estimation of genome coverage performances showed lower 8X breadth coverage for SRS respect to LRS and HYBR: considering the PE/PPE genes, SRS showed low results for the PE_PGRS family, while obtained acceptable coverage in PE and PPE genes; LRS and HYBR reached optimal coverages in PE/PPE genes. For variant calling HYBR showed the highest resolution, detecting the highest percentage of uniquely identified mutations compared to LRS and SRS. All three approaches agreed on the identification of two major clusters, with HYBR identifying an higher number of SNPs between the two clusters. Comparing the quality of the assemblies, HYBR and LRS obtained better results than SRS. Discussion: In conclusion, depending on the aim of the investigation, both SRS and LRS present complementary advantages and limitations implying that for a full resolution of MTB genomes, where all the mentioned analyses and both technologies are needed, the use of the HYBR approach represents a valid option and a well-rounded strategy.