Phenotypic and Molecular Characterization of an Enterobacter ludwigii Clinical Isolate Carrying a Plasmid-Mediated blaIMI-6 Gene

ABSTRACT We report a plasmid-encoded IMI-6 carbapenemase in a clinical isolate of Enterobacter ludwigii from Spain. The isolate belongs to ST641 and was susceptible to expanded-spectrum cephalosporins and resistant to carbapenems. The modified carbapenem inactivation method (mCIM) test was positive, but β-Carba was negative. Whole-genome sequencing identified the blaIMI-6 gene located in a conjugative IncFIIY plasmid and associated with the LysR-like regulator imiR. Both genes were bracketed by an ISEclI-like insertion sequence and a putatively defective ISEc36 insertion sequence. IMPORTANCE IMI carbapenemases confer an unusual resistance pattern of susceptibility to broad-spectrum cephalosporins and piperacillin-tazobactam but decreased susceptibility to carbapenems, which may make them difficult to detect in routine practice. Commercially available molecular methods for the detection of carbapenemases in clinical laboratories do not usually include blaIMI genes, which could contribute to the hidden dissemination of bacteria producing these enzymes. Techniques should be implemented to detect minor carbapenemases that are not very frequent in our environment and control their dissemination.

IMPORTANCE IMI carbapenemases confer an unusual resistance pattern of susceptibility to broad-spectrum cephalosporins and piperacillin-tazobactam but decreased susceptibility to carbapenems, which may make them difficult to detect in routine practice. Commercially available molecular methods for the detection of carbapenemases in clinical laboratories do not usually include bla IMI genes, which could contribute to the hidden dissemination of bacteria producing these enzymes. Techniques should be implemented to detect minor carbapenemases that are not very frequent in our environment and control their dissemination.
In October 2021, a carbapenem-resistant organism (HURS_212964) was isolated from a rectal swab culture during a surveillance study for CRE in a 77-year-old patient with a Burkitt lymphoma. The patient was admitted to the hematology unit of our hospital for a cycle of programmed polychemotherapy according to the Burkimab scheme (18), which he received without any infections or other complications. He was discharged after 5 days of hospitalization. The isolate was initially identified by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) (MALDI Biotyper A system; Bruker Daltonics, Madrid, Spain) as Enterobacter cloacae complex.
Whole-genome sequencing (WGS) was performed using Illumina technology. DNA pairedend libraries were generated using the Nextera XT DNA sample preparation kit (Illumina Inc., San Diego, CA, USA). Libraries were sequenced using the Illumina NextSeq500 sequencer system with 2-by 150-bp paired-end reads. Comparison of Enterobacter genomes using average nucleotide identity showed that isolate HURS_212964 belonged to the Enterobacter ludwigii species with a score of 98.9%. The isolate belonged to the sequence type ST641 (6). The presence of the bla IMI-6 gene in association with the regulator imiR was detected on the only identified plasmid belonging to the IncFIIY group. Both genes were found to be flanked by the IS3-family insertion sequences ISEc36-like and ISEcl1-like located downstream and upstream of bla IMI-6 and imi-6R, respectively. The ISEc36 sequence presented a point mutation leading to a premature stop codon, and the ISEcl1 transposase showed a deletion of two amino acids in the OrfB (His293 and Ser294). Sequenced reads were mapped against the previously described bla IMI-6 -carrying plasmid pIMI-6 from the Enterobacter cloacae isolate N14-0444 (accession number KX78618) leading to a ca.-160-kb plasmid named pHURS_212964 that shares high nucleotide sequence identity (.99%) with pIMI-6 and pRJ46C also carrying the bla IMI-6 gene identified from a Raoultella ornithinolytica isolate (accession number KT225520) (3). Shared sequences included the umuC-umuD genes responsible for mutagenesis regulation, parA-parB genes for partition, traM to traX genes for conjugative transfer, and a type VI secretion system (T6SS) region, which is involved in bacterial virulence (see Fig. S1 in the supplemental material). WGS also showed the presence of the chromosomal bla ACT-12 gene with two amino acid substitutions (Ala214Ser and Gln217Pro).
Plasmid transferability of bla IMI-6 was assessed by conjugation experiments using Escherichia coli J53 Rif-R as the recipient strain. Transconjugants were selected on MH agar plates containing 100 mg/mL of rifampicin and 100 mg/mL of ampicillin. PCR amplification followed by Sanger sequencing was used to confirm plasmid transfer. E. coli transconjugants carrying bla IMI-6 confirmed the carbapenem resistance pattern, thus demonstrating that the bla IMI-6 gene was responsible for carbapenem resistance in the parental Enterobacter ludwigii isolate (Table 1).
IMI carbapenemases confer the unusual resistance pattern of susceptibility to broadspectrum cephalosporins and piperacillin-tazobactam but decreased susceptibility to carbapenems, which may make them difficult to detect in routine practice. IMI-6 was not detected by the b-Carba test, and previous studies also indicate that IMI-6 enzymes are not detected by the Carba-NP test (3). In addition, commercially available molecular methods for the detection of carbapenemases in clinical laboratories do not usually include bla IMI genes, which could contribute to the hidden dissemination of bacteria producing these enzymes.
Techniques should be implemented to detect minor carbapenemases that are not very frequent in our environment and control their dissemination.
Accession number(s). Sequence data have been deposited in NCBI under accession number JAPHQK000000000.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.

ACKNOWLEDGMENTS
We are grateful to Martha E. Gaustad for revising the English. This work was partially supported by The Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC).