Characterization of KPC-82, a KPC-2 Variant Conferring Resistance to Ceftazidime-Avibactam in a Carbapenem-Nonsusceptible Clinical Isolate of Citrobacter koseri

ABSTRACT KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/β-lactamase inhibitor combinations.

C arbapenem-resistant Enterobacteriaceae (CRE) are a significant threat to modern medicine. In particular, isolates producing carbapenem-hydrolyzing b-lactamase enzymes (carbapenemases) are increasingly prevalent and a cause for further concern given their ability to spread, the severity of infections, and the lack of effective therapeutics (1). Though colistin and tigecycline have been used as first-line treatment, newer antimicrobials with better safety profiles and potent activity against CRE are increasingly being employed as preferable therapeutic options (2).
Among them, ceftazidime-avibactam (CZA) is a b-lactam/b-lactamase inhibitor combination recently introduced into clinical practice (2). It has proven active against serine b-lactamases, including Klebsiella pneumoniae carbapenemases (KPC), which otherwise confer resistance to most b-lactams and b-lactam/b-lactamase inhibitor combinations (1). Despite limited clinical use worldwide, acquired resistance has been reported in multiple independent occurrences and by several mechanisms in both patients with or without a history of CZA therapy (3)(4)(5)(6)(7)(8)(9)(10). Most frequently, resistance is caused by KPC variants exhibiting amino acid substitutions, insertions, or deletions in one of 4 loops (loop Leu 102 to Ser 106 , X-loop Arg 164 to Asp 179 , or loops Cys 238 to Thr 243 and Ala 267 to Ser 275 ) (11). At the time of writing (April 2021), 82 bla KPC alleles have been deposited in GenBank, including 20 conferring CZA resistance. In this report, we use genomic and molecular genetic approaches to characterize KPC-82, a KPC-2 variant that confers CZA resistance.
Citrobacter koseri MRSN 755319 was cultured from the blood of a patient in a U.S. hospital in 2020. The patient had been hospitalized for several months after suffering a gunshot wound to the abdomen. During this time, the patient had frequent infections caused by multidrug-resistant (MDR) bacteria, including a recurrent respiratory infection due to a carbapenem-susceptible Klebsiella aerogenes (days 159, 197, and 231) as well as a bloodstream infection caused by a carbapenem-resistant (CR), bla KPC-2 -carrying Serratia marcescens (MRSN 696556, day 109), that ultimately resolved after ;4 weeks of treatment with CZA ( Fig. 1A). Two and a half months after CZA was discontinued, the patient developed another infection, and blood cultures yielded C. koseri (MRSN 755319, day 250). The isolate was carbapenem resistant (Table 1), and the bla KPC gene was detected using the Cepheid Xpert Carba-R assay. On day 252, the patient was prescribed tigecycline and CZA, which was substituted on day 260 with meropenem-vaborbactam (MVB) following extended antibiotic susceptibility testing (AST) that indicated the isolate was nonsusceptible to CZA (MIC, 128 mg/ml) but susceptible to MVB (MIC, 0.125 mg/ml).
Genome analysis revealed that CR and CZA-susceptible (Table 1) S. marcescens MRSN 696556 carried the bla KPC-2 allele. In contrast, CR and CZA-nonsusceptible C. koseri MRSN 755319 carried a mutated bla KPC-2 allele (hereby named bla KPC-82 ; GenBank accession no. MW485086) and no other acquired b-lactamase. The mutated allele was identical to bla KPC-2 with the exception of a 6-nucleotide (ACAGCG) tandem To investigate whether the two-amino-acid insertion identified within KPC-82 was responsible for the phenotypic resistance to CZA, the bla KPC-82 gene was cloned into vector pBCSK (Stratagene, La Jolla, CA) and expressed in E. coli TOP10. AST showed that KPC-82 conferred resistance to all b-lactams, including ceftazidime, as well as high-level resistance to CZA (Table 1). Importantly, and similar to KPC-50 (3), E. coli expressing bla KPC-82 remained susceptible to the carbapenems (ertapenem, imipenem, and meropenem).
Further investigations into the genetic context of bla KPC-82 in MRSN 755319 revealed that it was carried by an ;10-kb Tn4401-like transposon that inserted into the chromosome and disrupted the gene coding for the outer membrane protein PhoE (Fig. 1C and D). Porin loss, such as OprD in P. aeruginosa (14) and OmpK36 in K. pneumoniae (15), has been widely implicated in b-lactam and carbapenem resistance in other bacterial species. Notably, PhoE downregulation has been hypothesized as a possible reason for carbapenem resistance in K. pneumoniae (16), suggesting that its inactivation in MRSN 755319 could cause the otherwise unexplained low-level carbapenem resistance (Table 1).
Interestingly, in S. marcescens MRSN 696556 from the same patient, the bla KPC-2 allele was also carried by a nearly identical Tn4401 (only 2 synonymous mutations in istB in addition to the TR insertion in bla KPC ). However, unlike MRSN 755319 but similar to previous reports (17), this transposon was not chromosomally located and was instead carried by an ;76-kb IncN plasmid named pBWC01 (Fig. 1C and D). The backbone of pBWC01 was absent in MRSN 755319, but both S. marcescens and C. koseri isolates carried an identical ;4-kb Col440i-type plasmid (Fig. 1C). Similar Col440i cryptic plasmids have been identified in a variety of Enterobacteriaceae and have been documented to coconjugate with a larger IncN KPC-carrying plasmid (including across genus, in vitro) (18). Altogether, and despite missing intermediate isolates, a hypothesis for the emergence of bla KPC-82 would be that (i) both plasmids cotransferred from Serratia to Citrobacter within the host, and (ii) Tn4401 inserted into the chromosome of Citrobacter while the remaining of pBWC01 was lost. In this proposed chain of events, whether bla KPC-82 evolved from bla KPC-2 in Serratia, as a result of CZA exposure, or once acquired by Citrobacter MRSN 755319 still remains unresolved. In summary, a novel KPC-type enzyme conferring resistance to CZA was identified from a multidrug-resistant C. koseri. Similar to other KPC mutants conferring resistance to CZA, KPC-82 showed decreased carbapenemase activity and remained susceptible to carbapenem/b-lactamase inhibitor combinations, including meropenem-vaborbactam, which successfully cleared the infection in this patient.