Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid

Carbapenem-resistance in Enterobacter spp due to acquisition of mobile carbapenemases is of concern. An Enterobacter spp grew on ChromID CARBA medium and was positive for the mCIM carbapenemase detection assay. Susceptibility testing showed resistance to aztreonam and reduced susceptibility to imipenem. Conventional PCR using FRI primers detected a blaFRI gene. Whole genome sequencing reveled a new variant; blaFRI−12 was closest in sequence to blaFRI−5 differing by 13 amino acids and was found on a unique 110Kb IncR plasmid. Given the intrinsic nature of Enterobacter spp. to be carbapenem non-susceptible, blaFRI-types may be under reported globally. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-024-04907-7.


Introduction
A report on the global burden of bacterial antimicrobial resistance (AMR) from 2019 was published and indicated Enterobacter species were in the top 9 bacterial species associated with global deaths attributable to and associated with bacterial AMR in infections [1].In 2020, Enterobacter species were found to be the top species to produce carbapenemases in Canadian hospitals, just behind Escherichia coli [2].Enterobacter cloacae complex (ECC) are common nosocomial pathogens and often associated with multi-drug resistance including the carbapenems [3].Though carbapenem resistance in ECC may be related to overexpression of a β-lactamase or AmpCs combined with decreased outer membrane permeability, it is the production of carbapenemases PCR containing FRI specific primers (FRI-F-U1: 5

'-T A A A C T C A G C T A T T C C A G G C-3' and FRI-R-U2: 5'-A C A G G T G C C T G T T T T A T C G C C-3'
) was conducted yielding a positive result [11].Antimicrobials susceptibility testing was done using the commercial Sensititre™ panel from Thermo Scientific™.

Whole genome sequencing
Once confirmed positive by FRI primers the isolate was sent for whole genome sequencing (WGS).DNA was extracted using Qiagen DNeasy kits (Qiagen, Toronto, Canada) and sequenced on an Illumina NextSeq™ platform using Nextera XT libraries.MinION sequencing (Nanopore Technologies, Oxford, UK) was conducted using the rapid kit (SQK-RBK 004) on R9.4.1 flowcells and run on Guppy 6.3.7 using the super accurate basecalling model.De novo hybrid assemblies were done using Unicycler 0.4.7 [13].Analysis was conducted using the StarAMR pipeline (https://github.com/phac-nml/staramr) for resistance gene detection, plasmid detection and multilocus sequence typing.Analysis of the genome using FastANI (https://github.com/ParBLiSS/FastANI)was used to determine species.Data for this project was deposited on NCBI under BioProject PRJNA865257.

Results and discussion
A 91-year-old patient presenting with severe neurocognitive disorders entered the emergency department after suffering injuries from a fall during 2021.She was hospitalized for three weeks where she received a 2 day course of tazobactam.She had no known history of travel or acute infections or stay in palliative care.She was screened for CPE following a suspected outbreak of KPC on the floor where she was hospitalized.The result of her mCIM came back positive and no carbapenemase genes were found following testing for NDM, KPC, OXA-48like, IMP, VIM, GES, NMC and IMI.The patient was

(forward-G A T T T C T C A T T G T A T A C C A A C C) and FRI-12B (reverse-T G G C G G A C A T T T C A T G G C G C
), cloned into pCR21.TOPO (ThermoFisher Scientific).pCR2.TOPO contains an ampicillin resistance gene (bla TEM−1 ) and kanamycin resistance gene (aphII) moved to a residential and long-term care center and in June 2022 and tested positive for mCIM following routine screening practices.At this time PCR targets for FRI had been developed due to a separate FRI case reported in Canada [11].The CPE isolate (N22-01531) was confirmed to be FRI-positive as were all retrospective isolates from this patient (data not shown).
The bla FRI−12 gene was found on a 104 Kb IncR plasmid (pN22-01531, Fig. 1).The closest hit to a known plasmid was pKeioCLMIC23 harbouring bla FRI−5 (accession number AP028423.1)which shared 44% coverage at 95% identity.Indeed, when all other FRI plasmids were BLAST against pN22-01531only 5-17% coverage was observed.Previous reports have shown all bla FRI−type genes are harboured on plasmids containing the replicon IncFll-type [10].However, some of these (FLC-1, FRI-2, FRI-4, FRI-9 and FRI-11) are multi-replicon plasmids that also contain IncR [10].Interestingly, the IncFII-type FRI-5 plasmid (pKeioCLMIC23), which shared the closest identity to our FRI-12 plasmid did not contain the IncR replicon as observed in other FRIvariants.The alignment of FRI surrounding regions in different variants highlights a diverse arrangement of coding sequences (Supplementary Fig. 2).The FRI-12 surrounding region was most similar to FRI-5 where we observed a common 7 Kb of shared sequence representing FRI-12, FriR (a LysR transcriptional regulator), SafC and SafB (components of a saf operon for pili functionality).No additional resistance genes were found on the FRI-12 IncR plasmid.
Similar to our previous reports [11,12], we were able to successfully transform pN22-01531 into Escherichia coli TOP10 (labeled pFRI-12TF), however, conjugation experiments with Escherichia coli J53AzR were not successful.Other than with FRI-6 [11] this phenomenon is not uncommon in FRI-type plasmids.We cloned bla FRI−12 into pCR2.1TOPOand subsequently transformed this plasmid into E. coli TOP10 (pblaFRI-12) to test its antimicrobial susceptibility (Table 1).The transformant showed higher MICs than the clinical strain to aztreonam, ceftriaxone, cefoxitin, ertapenem, meropenem, imipenem, ceftolozone/tazobactam and piperacillin/tazobactam.However, the cloned bla FRI−12 gene showed higher MIC to piperacillin/tazobactam only.Further work will need to be conducted on FRI-12 plasmid copy number and gene expression to establish if they affect MIC to selected antimicrobials.
To our knowledge, this is the first published report of bla FRI−12 identified globally.This study highlights the benefits of phenotypic screening of carbapenemases in Enterobacter sp.isolates exhibiting non-susceptible MICs to carbapenems despite susceptibility to cephalosporins.

Fig. 1
Fig. 1 FRI-12 plasmid generated with Proksee.Innermost two tracks represent forward and reverse open reading frames of pN22-01531.Third track represents BLASTn of FRI-12 plasmid to its closest related

Table 1
Antimicrobial susceptibilities (mg/L) for strains tested in this report a E.coli TOP10 electrotransformed with pFRI-12 b bla FRI−12 and upstream region amplified from N22-01531 with primers FRI-12 A