Emergence of KPC-134, a KPC-2 variant associated with ceftazidime-avibactam resistance in a ST11 Klebsiella pneumoniae clinical strain

ABSTRACT The emergence of various new Klebsiella pneumoniae carbapenemase (KPC) variants leading to ceftazidime-avibactam treatment failure is a new challenge in current clinical anti-infection treatment. Here, we report a ceftazidime-avibactam-resistant K. pneumoniae 1072–2 clinical strain carrying a novel KPC variant, KPC-134, which differs from KPC-2 by both single mutation (D178A) and 8-amino acid insertions (asp-asp-asn-arg-ala-pro-asn-lys). The results of antimicrobial susceptibility testing showed that the isolate was resistant to meropenem (MIC = 4 mg/L), ceftazidime (MIC ≥ 32 mg/L), cefepime (MIC ≥128 mg/L), aztreonam (MIC ≥128 mg/L), and ceftazidime-avibactam (MIC ≥128 mg/L) but sensitive to imipenem (MIC = 0.5 mg/L), imepenem-relebactam (MIC = 0.5 mg/L), meropenem-vaborbactam (MIC = 2 mg/L), and aztreonam-avibactam (MIC = 4 mg/L). The plasmid containing bla KPC-134 was isolated from K. pneumoniae, and the bla KPC-134 gene was cloned into plasmid pHSG398 and transformed into an Escherichia coli DH5α to observe changes in antimicrobial resistance. The results indicated that the transformant was positive for bla KPC-134 and increased MICs of ceftazidime-avibactam, ceftazidime, cefepime, and aztreonam by 512-fold, 256-fold, 16-fold, and 4-fold, respectively, compared with the recipient. The results of third-generation sequencing showed that the bla KPC-134 gene was carried by a 133,789 bp IncFII-IncR plasmid, and many common resistance genes (including bla CTX-M-65, bla TEM-1B, bla SHV-12, rmtB, and catB4) along with the IS26, tnpR, ISkpn8, ISkpn6-like, and Tn1721 elements were identified. IMPORTANCE The emergence of various new KPC variants leading to ceftazidime-avibactam treatment failure is a new challenge for clinical anti-infection treatment. Here, we describe the characterization of a ceftazidime-avibactam-resistant blaKPC-134-positive Klebsiella pneumoniae clinical strain for the first time. K. pneumoniae bearing with KPC variant often mislead clinical anti-infection treatment because of their unique antimicrobial susceptibility profile and the tendency of conventional carbapenemase assays to give false negative results. Therefore, timely identification of KPC variants and effective anti-infective therapy are key to saving infected patients.


IMPORTANCE
The emergence of various new KPC variants leading to ceftazidime-avi bactam treatment failure is a new challenge for clinical anti-infection treatment.Here, we describe the characterization of a ceftazidime-avibactam-resistant blaKPC-134-positive Klebsiella pneumoniae clinical strain for the first time.K. pneumoniae bearing with KPC variant often mislead clinical anti-infection treatment because of their unique antimicro bial susceptibility profile and the tendency of conventional carbapenemase assays to give false negative results.Therefore, timely identification of KPC variants and effective anti-infective therapy are key to saving infected patients.KEYWORDS Klebsiella pneumoniae, ceftazidime-avibactam, KPC-134 I n recent years, the widespread epidemic dissemination of carbapenem-resistant Klebsiella pneumoniae in the clinic has presented a major challenge for clinical anti-infective treatment (1,2).According to the results of the China Antimicrobial Surveillance Network (www.chinets.com),the resistance rate of K. pneumoniae to imipenem or meropenem is rapidly increasing (from 3% in 2005 to more than 26% in 2022) (3).Available findings suggest that the production of carbapenemases, particularly class A KPC-type serine carbapenemases, is the predominant resistance mechanism for carbapenem resistance in K. pneumoniae (4).In vitro activities have shown that ceftazidime-avibactam has high antibacterial activity against KPC-type carbapene mase-producing K. pneumoniae and is the first-line drug for the clinical treatment of infections caused by such resistant bacteria (5)(6)(7).
However, with the widespread use of ceftazidime-avibactam in clinical practice, KPC-type carbapenemase-producing K. pneumoniae has mutated to adapt to the strong selective pressure of antibacterial drugs, and various new subtypes of KPC based on KPC-2 or KPC-3 mutations have emerged, mediating the resistance of K. pneumoniae to ceftazidime-avibactam and leading to treatment failure, which is currently a new challenge in clinical anti-infection treatment (8).The results of available studies show that the emergence of new subtypes of KPC genes has been an explosive trend in the last 3 years.To date, more than 180 bla KPC subtypes have been reported in the world, according to the NCBI database (9).In this study, we describe the first characterization of bla KPC-134 , a novel bla KPC variant that confers resistance to ceftazidime-avibactam and restores susceptibility to imipenem.
The isolate K. pneumoniae 1072-2 was collected in a sputum sample from a 17-yearold female patient admitted to Sichuan Provincial People's Hospital in 2022.The patient was admitted to the local hospital on 11 May 2022 with a recurrent fever for 7 d (maximum temperature of 40°C) and was diagnosed with pulmonary infection and peritonitis septicemia on admission.Sputum culture showed carbapenem-resistant K. pneumoniae 1072-1 (which is sensitive to ceftazidime-avibactam), and the anti-infective regimen included meropenem (1.0 g every 8 h for 19 d), tigecycline (50 mg every 12 h for 2 d), cefoperazone-sulbactam (1.5 g every 6 h for 11 d), caspofungin (50 mg every 24 h for 4 d), and ceftazidime-avibactam (2.5 g every 8 h for 14 d), but the treatment was not effective (Table 1).To seek further treatment, the patient was transferred to Sichuan Provincial People's Hospital, and the anti-infective regimen was changed to imipenem (1 g every 8 h), amphotericin B (5 mg every 24 h), and polymyxin B (500,000 units q12h).The patient's infection symptoms continued to progress, carbapenem-resistant K. pneumoniae continued to be cultured in sputum and ascites, and the patient eventually died from treatment failure.One ceftazidime-avibactam-resistant K. pneumoniae 1072-2 was isolated from sputum (Table 1) (10).
PCR and DNA sequencing of the full-length bla KPC gene revealed a novel bla KPC variant, designated bla KPC-134 .Nucleotide alignment of different bla KPC variants showed that bla KPC-134 differs from bla KPC-2 by both single mutation (D178A) and 8-amino acid insertions (asp-asp-asn-arg-ala-pro-asn-lys).The plasmid containing bla KPC-134 was isolated from K. pneumoniae.In order to verify the function of the bla KPC-134 gene, we cloned the bla KPC-134 gene into the plasmid pHSG398 and transformed the recombined plasmid into E. coli DH5a to observe the change in antimicrobial resistance (Table 1).The transformant was positive for bla KPC-134 and increased MICs of ceftazidime-avibac tam, ceftazidime, cefepime, and aztreonam by 512-fold, 256-fold, 16-fold, and 4-fold, respectively, compared with the empty vector-carrying strain (E.coli-pHSG398); however, there was essentially no change in MIC for imipenem and meropenem (Table 1).
According to the whole-genome sequencing (WGS) analysis for the strain, many resistance genes had been identified, including the β-lactamase genes bla KPC-134 , bla CTX-M-65 , bla TEM-1B , and bla SHV-12 , the aminoglycoside resistance genes rmtB, and the phenicol resistance gene catB4.Quinolone-related resistance gene mutations of GyrA (GyrA-83I and GyrA-87G) and ParC (ParC-80I) were found in K. pneumoniae 1072-2.According to the multilocus sequence typing result, strain K. pneumoniae 1072-2 belonged to ST11.The bla KPC-134 gene was carried by a 133,789 bp plasmid, which belonged to the IncFII-IncR type.The genetic structure of bla KPC-134 in pKPC-134 is identical to pKP36-KPC-2 (NZ_CP082761) (Fig. 1), carrying an IS26-based composite transposon, which is an 11.282 kb region including bla KPC-2 and one flanking IS26 element; the complete genetic structure was IS26-tnpR-ISKpn8-bla KPC-134 -ISKpn6-like-Tn1721 (Fig. 2).The emergence of various new KPC variants leading to ceftazidime-avibactam treatment failure is a new challenge for clinical anti-infection treatment (11).K. pneumo niae bearing with KPC-variant often mislead clinical anti-infection treatment because of their unique antimicrobial susceptibility profile and the tendency of conventional carbapenemase assays to give false negative results (12).Therefore, timely identification of KPC variants and effective anti-infective therapy are key to saving infected patients.The results of existing studies reported that the emergence of new subtypes of KPC in K. pneumoniae is overwhelmingly associated with the use of ceftazidime-avibactam (13,14).Mutation derived from the bla KPC-2 or bla KPC-3 gene is the most reported resistance mechanism of K. pneumoniae to ceftazidime-avibactam, including amino acid deletions, mutations, insertions, and tandem repeats (15,16).Compared with KPC-2, KPC-134 has both single mutation (D178A) and 8-amino acid insertions (asp-asp-asn-arg-ala-pro-asnlys).It is worth noting that mutations within the Ω-loop that embrace the active site of KPC have been proven to enhance ceftazidime affinity and restrict avibactam binding (17)(18)(19).Interestingly, while mediating bacterial resistance to ceftazidime-avibactam, the new subtype of KPC tended to reduce the hydrolytic ability of the KPC enzyme to carbapenems, showing susceptibility to carbapenems, especially imipenem.
As reported, two major genetic structures, the Tn4401 transposon and the Tn3-Tn4401 transposon chimera, are mostly associated with KPC-2 (20,21) and are consid ered the original genetic structure mediating bla KPC gene acquisition worldwide (20,22).In China, the genetic environment of the bla KPC gene was distinct, with the Tn1721bla KPC -IS26 transposon chimera being the most common (23).The full genetic structure of bla KPC-134 in our study was IS26, tnpR, ISKpn8, bla KPC-134 , ISKpn6-like element, and Tn1721 transposase.It is known that the insertion sequence IS26 can form a composite transposon, which can be excised from the plasmid to form a translocation unit (TU) (24).As mentioned above, the unique enzymatic hydrolysis characteristics and antimicrobial susceptibility profile of the new subtype of KPC raise a challenge for routine carbapene mase detection assays (12).KPC variants usually show false-negative results (12).The misleading detection in the clinical laboratory may lead to incorrect clinical decisionmaking and treatment failure.Therefore, the development of effective genetic testing methods for KPC variants and the enhancement of ceftazidime-avibactam susceptibility testing to better screen for KPC variants are ongoing efforts.In addition, studies have shown that meropenem-vaborbactam (25), for example, is considered a salvage therapy for the infection caused by ceftazidime-avibactam-resistant KPC variant-producing K. pneumoniae, but the new antimicrobial agent is not available in all countries or regions, especially in low-resource countries or regions.Therefore, for countries or regions lacking new antimicrobial agents, there is an urgent need to actively search for other alternative therapeutic options for the treatment of infections caused by KPC variant-producing strains, such as ceftazidime-avibactam in combination with imipenem (26).

FIG 1
FIG 1Alignments of plasmids.Comparison of the plasmids pKPC-134 and pKP36-KPC-2 using Proksee.A BLAST search for the sequence in GenBank showed that the sequence of pKP36-KPC-2-plasmid was very similar (99.95% coverage and 100% identity) to that of pKPC-134 (133,789 bp, GenBank accession no.OP293349.1),a plasmid of K. pneumoniae isolated from Sichuan, China.

FIG 2
FIG 2The genetic environment surrounding bla KPC-134 .Genes, genetic elements, and other traits are color coded according to functional classification.

TABLE 1
Susceptibility of K. pneumoniae clinical isolate, transformant, and recipient to antimicrobial agents a