Filip Božić1*, Mirjana Hadnađev2, Ljuban Blanuša3, Jovana Kabić4, Ina Gajić4, Miloš Jovićević4, Dušan Kekić4 and Anika Trudić5
1Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad, Serbia; Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia
2Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia
3Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia
4Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
5Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia; University of Novi Sad, Faculty of Medicine, Novi Sad, Serbia
flpbzc [at] gmail.com
Abstract
Carbapenemase-producing Enterobacterales represent a major therapeutic and infection-control challenge, but are most frequently associated with Klebsiella pneumoniae, whereas VIM-producing Proteus mirabilis remains an uncommon finding. In Serbia, VIM-producing P. mirabilis has only sporadically been reported, and genomic data on blaVIM allelic diversity, associated resistance determinants, and hospital-associated clonal relatedness remain limited.
This study aimed to genomically characterize five clinical P. mirabilis isolates recovered from intensive care and pulmonary units of a tertiary care hospital in Serbia, focusing on sequence type, antimicrobial resistance gene repertoire, blaVIM allelic diversity, plasmid replicon content and core-genome relatedness.
Whole-genome sequencing was performed on an Illumina NovaSeq 6000 platform using 150 bp paired-end reads. Quality-filtered reads were assembled with SPAdes and assessed with QUAST. Downstream analyses included Proteus MLST, antimicrobial resistance gene detection using ResFinder v4.1 and ABRicate-based database screening, plasmid replicon detection with PlasmidFinder, blaVIM mapping with BWA-MEM/SAMtools and Snippy-based core-genome SNP analysis for assessment of isolate relatedness.
All isolates belonged to ST135 and carried blaVIM carbapenemase genes together with multiple resistance determinants affecting beta-lactams, aminoglycosides, macrolides, sulfonamides, tetracyclines, trimethoprim and phenicols. Comparative analysis of the 801 bp blaVIM coding sequence identified distinct allelic variants: blaVIM-1 in isolate 5, blaVIM-4 in isolate 1, blaVIM-75 in isolate 4 and blaVIM-78 in isolates 2 and 3. Relative to the blaVIM-1 reference sequence, these variants differed by substitutions at positions 179 A→G and/or 613 A→C. IncQ1 replicons were detected in four isolates; however, they were located on contigs distinct from blaVIM-containing contigs, indicating that short-read assemblies did not resolve their physical linkage. Core-genome SNP analysis separated isolate 3 from a closely related cluster comprising isolates 1, 2, 4 and 5, despite isolates 2 and 3 carrying identical blaVIM-78 coding sequences.
These findings indicate that blaVIM-positive P. mirabilis isolates from the same hospital belonged to a single sequence type, yet blaVIM allelic distribution did not fully mirror core-genome relatedness. This suggests that blaVIM diversity may not be explained solely by clonal expansion of ST135 and may reflect acquisition or rearrangement of blaVIM-harbouring elements, although their precise genetic context could not be resolved using short-read sequencing alone.
Keywords: Proteus, blaVIM, carbapenemase, multidrug-resistant, WGS
Acknowledgement: This research was funded by the Science Fund of the Republic of Serbia, Grant No. 7042, Tracking antimicrobial resistance in diverse ecological niches – one health perspective – TRACE