Carbapenem-Susceptible Klebsiella pneumoniae and Escherichia coli Isolates Carrying a Truncated KPC Carbapenemase: a Challenge for Rapid Molecular Diagnostics

LETTER

Carbapenems are the preferred therapeutic option to treat infections caused by multidrug-resistant Gram-negative bacteria. Rapid molecular diagnostics (RMDs; e.g., BioFire FilmArray or Luminex Verigene BC-GP and BC-GN) facilitate the identification of pathogens from positive blood cultures and offer information to predict phenotype based on their ability to detect certain antibiotic resistance genes. Phenotypic techniques, such as the modified carbapenem inactivation method (mCIM) or Rapidec Carba NP, also detect carbapenemase-producing bacteria.

This communication reports the first identification of a nonfunctional bla_KPC gene in Escherichia coli and Klebsiella pneumoniae. Both organisms were isolated from the blood culture of a 27-year-old African-American man with a past medical history of childhood brain injury, paraplegia, and seizure disorder. The patient, a resident of the Detroit area, was brought to the emergency room a day after his gastrostomy-jejunostomy tube was replaced and presented with severe dehydration, tachycardia, and coffee ground emesis. He developed fever after admission; a set of blood cultures were drawn, and he was started on vancomycin and ceftriaxone empirically. The anaerobic bottle was flagged positive the next day and showed Gram-positive cocci and Gram-negative bacilli, identified as Enterococcus faecalis and E. coli by Verigene BC-GP and BC-GN molecular assays. Assay results also reported the detection of bla_KPC. Vancomycin and ceftriaxone were then discontinued and meropenem-vaborbactam and ampicillin were initiated. Subculture revealed E. coli, E. faecalis, K. pneumoniae, and coagulase-negative Staphylococcus. Phenotypic antibiotic susceptibility testing (AST) of the isolated pathogens was performed using the Phoenix 100 system. Both E. coli and K. pneumoniae were susceptible to carbapenems and piperacillin-tazobactam (Table 1), so meropenem-vaborbactam was switched to piperacillin-tazobactam and ampicillin was discontinued. Subsequent blood cultures obtained the day after admission were negative, antibiotics were stopped 6 days after, and the patient was discharged.

TABLE 1.

Antibiotic susceptibility of E. coli and K. pneumoniae using Phoenix panel NMIC-300^a

Drug	E. coli		K. pneumoniae
	MIC(s) (μg/ml)	Interpretation	MIC(s) (μg/ml)	Interpretation
Ampicillin-sulbactam	16, 8	I	16, 8	S
Ampicillin	>16	R	>16	R
Aztreonam	≤1	S	≤1	S
Cefazolin	4	I	2	S
Cefepime	≥0.5	S	≥0.5	S
Cefoxitin	16	I	≤4	S
Ceftazidime	≤0.5	S	≤0.5	S
Ceftriaxone	≤0.5	S	≤0.5	S
Ertapenem	≤0.125	S	≤0.125	S
Gentamicin	≤1	S	1	S
Imipenem	≤0.25	S	≤0.25	S
Meropenem	≤0.125	S	≤0.125	S
Piperacillin-tazobactam	8, 4	S	4, 4	S
Tobramycin	1	S	≤0.5	S
Trimethoprim-sulfamethoxazole	≤0.5, 9.5	S	≤0.5, 9.5	S

^aI, intermediate; R, resistant; S, susceptible.

A discrepancy between the carbapenem-susceptible results and the detection of bla_KPC was further explored. Both the E. coli and K. pneumoniae isolates were retested on the Verigene system and by a standard KPC PCR analysis (1, 2); the bla_KPC gene was detected in both isolates. AST was repeated, yielding the same results (carbapenem susceptibility), and the lack of phenotypic carbapenemase expression in both isolates was confirmed by mCIM testing. The newly added Appendix H in CLSI document M100 (edition 2019) outlines the suggested resolution and reporting for discordant phenotype-to-genotype results (3).

PCR amplicons of the bla_KPC gene and upstream region were sequenced. The translated bla_KPC amino acid sequences were 100% identical in both bacteria and almost a perfect match to KPC-2 (NCBI reference sequence NG_049253.1). However, there was a C-to-T mismatch that changed the glutamine at amino acid position 85 to a stop codon (CAG to TAG), resulting in a truncated, nonfunctional carbapenemase enzyme. KPC-2 amino acid numbering was assigned as in the crystal structure of Protein Data Bank accession number 2OV5. The bla_KPC upstream sequence was 100% identical to the sequence of Tn4401a.

This is the first report of a truncated and inactive bla_KPC gene. As RMD platforms are used for routine genotypic testing, this type of mutation will be detected more frequently, hence the need for confirmatory phenotypic testing. Perhaps this mutation arose and conferred better fitness to the bacteria when they were not under selective pressure to produce the KPC β-lactamase (4, 5). Our findings suggest that the detection of genes conferring antibiotic resistance by molecular methods should be confirmed by CLSI-approved phenotypic testing, as the results obtained support antibiotic stewardship decisions.