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

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 ß-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.

In vivo selection of KPC-94 and KPC-95 in Klebsiella pneumoniae isolates from patients treated with ceftazidime/avibactam.

Ceftazidime/avibactam (CZA) is used to treat infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp). Resistance to CZA is commonly related to point mutations in the blaKPC gene. Here we describe the in vivo emergence of CZA resistance in clinical isolates of KPC-Kp from four patients treated with this combination therapy. Four pre-therapy and five post-therapy KPC-Kp isolates were examined. Antibiogram (microdilution and gradient strips) and whole-genome sequencing were performed. The role of KPC mutations was validated by cloning blaKPC genes into competent Escherichia coli. All KPC-Kp isolates recovered before treatment with CZA were susceptible to CZA and produced KPC-3. Five KPC-Kp isolates recovered after treatment were resistant to this combination. Three post-therapy isolates from two patients produced KPC-31 (D179Y mutation). Additionally, we identified the novel substitution LN169-170H (KPC-94) in one isolate, and the combination of two independently described mutations, D179Y and A172T (KPC-95), in another isolate. All KPC-Kp isolates belonged to sequence type 512 (ST512). All CZA-resistant isolates with blaKPC variants had restoration of carbapenem susceptibility. In conclusion, resistance to CZA was related to blaKPC mutations, including the new KPC-94 and KPC-95 alleles, which do not cause carbapenem resistance.