A large-scale surveillance revealed that KPC variants mediated ceftazidime-avibactam resistance in clinically isolated Klebsiella pneumoniae

ABSTRACT To monitor the resistance rate and gain a deeper understanding of the resistance mechanisms, we conducted over a 2-year surveillance focusing on the Klebsiella pneumoniae associated with the clinical usage of ceftazidime-avibactam (CZA) in a teaching hospital. A total of 4,641 K. pneumoniae isolates were screened to identify the CZA resistance through antimicrobial susceptibility testing. Comprehensive analyses, including homology analysis, conjugation experiments, clone assays, and whole genome sequencing, were furtherly performed on the CZA-resistant strains. In total, four CZA-resistant K. pneumoniae (CZA-R-Kp) strains were separated from four patients, in which three of them received CZA treatment during the hospitalization, accounting for a 4% (3/75) resistance development rate of K. pneumoniae under CZA stress. All CZA-R-Kp isolates were found to possess variants of blaKPC-2. The identified mutations included blaKPC-33, blaKPC-86, and a novel variant designated as blaKPC-129, all of which were located in the Ω loop of the KPC enzyme. These mutations were found to impact the amino acid sequence and spatial structure of the enzyme’s active center, consequently affecting KPC carbapenemase activity. This study underscores the importance of active surveillance to monitor the emergence of resistance to CZA, highlighting the need for ongoing research to develop effective strategies for combating antimicrobial resistance. Understanding the mechanisms behind resistance is crucial in maintaining the efficacy of CZA, a vital tool in the battle against multidrug-resistant infections. IMPORTANCE As an effective drug for the treatment of carbapenem-resistant Klebsiella pneumoniae, ceftazidime-avibactam (CZA) began to develop resistance in recent years and showed an increasing trend. In order to effectively monitor the resistance rate of CZA and understand its resistance mechanism, we monitored K. pneumoniae for more than 2 years to find CZA-resistant strains. Through comprehensive analysis of the selected CZA-resistant strains, it was found that all the CZA-resistant strains had mutation, which could affect the activity of KPC carbapenemase. This study highlights the importance of proactive surveillance to monitor the emergence of CZA resistance, which highlights the need for ongoing research to develop effective strategies to combat antimicrobial resistance. Understanding the mechanisms behind resistance is critical to maintaining the effectiveness of CZA, an important tool in the fight against multidrug-resistant infections.

carbapenem-resistant K. pneumoniae (CRKP) has become widespread (1), yet the increasing drug resistance is a concern for clinicians.Previously, the epidemics of CRKP in China and the USA during 2015 to 2019 were analyzed, revealing an average prevalence of CRKP at 10.9% (ranging from 0.6% to 32.8%) and 4.7% (ranging from 0% to 30%), respectively (2).Currently, there are few drug options for the treatment of CRKP in clinical practice, with CZA emerging as one of the few effective choices (3).Notably, a large proportion of CRKP prevalent in China is KPC producing, making CZA the most suitable treatment for infection of KPC resistance.However, reports of CZA resistance have emerged.Multiple research studies have identified KPC variants, such as KPC-33, KPC-86, KPC-87, and KPC-88 (4), with new mutation variants intermittently reported.The focus of these studies on the mechanism of CZA resistance revolves around KPC variants, which alter the Ω loop structure of KPC carbapenemase, enhancing affinity to ceftazi dime while weakening affinity to avibactam, thereby mediating bacterial resistance to CZA (5).
Several previous studies have reported the development of CZA resistance during clinical practice, but the frequency of resistance emergence after CZA treatment remains unclear.Hence, we conducted a long-term study involving prospective clinical monitor ing of CZA-resistant K. pneumoniae, aiming to explore the mechanism of drug resistance and the rate of resistance development during clinical practice.The results indicate a low incidence of CZA-resistant K. pneumoniae, with only four positives for CZA resistance.All four resistant strains exhibited Ω loop point mutations on bla KPC .Furthermore, a novel variant, designated as bla KPC-129 , was identified in one of our isolates.

Surveillance
Since the approval of CZA in China in 2019, we conducted surveillance for CZA resistance among clinically isolated Klebsiella pneumoniae in a 2,400-bed teaching hospital from 1 January 2020 to 31 May 2022, aiming to explore potential resistance with or without CZA treatment.After excluding patient duplication, a total of 4,641 K. pneumoniae isolates were collected from 1,845 patients, including 75 patients who received CZA treatment during their hospitalization.Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Mérieux, France) was utilized for species identification.Clinical information from all enrolled patients was obtained through electronic medical records.

Pulsed-field gel electrophoresis
Homology analysis of paired isolates, comprising one CZA-resistant isolate matched with one CZA-susceptible isolate selected from the same patient, was performed using pulsed-field gel electrophoresis (PFGE) after digestion with the XbaI restriction enzyme, as previously described (8).

Clone of the novel bla KPC variant
The novel bla KPC-2 variant was amplified and cloned to verify the CZA-resistant phenotype.Genomic DNA extraction was carried out using the QIAamp DNA Mini Kit (Qiagen, New York, USA) following the manufacturer's instructions.First, PCR amplification was performed using specific clonal primers (Table S1).Then, we used the TA clone method to insert PCR-amplified gene fragments with single 3′-A overhangs on each end into a plasmid vector containing a complementary 5′-T overhang.After confirmation by sequencing, the fragment of the bla KPC variant was then cloned into the pCR2.1 vector using ClonExpress technology (Vazyme, Nanjing, China) following digestion by HindIII and BamHI restriction enzymes.The resulting ligation product was transformed into E. coli DH5α competent cells using the heat shock method.Mueller Hinton (MH) plates containing 50 mg/L kanamycin were used for the selection of E. coli DH5α positive for pCR2.1-blaKPC .Colonies growing on the selection plate were confirmed by PCR.The pCR2.1 vector was introduced into E. coli DH5α as a control.AST was then conducted to determine the MICs of both E. coli DH5α (pCR2.1-blaKPC ) and E. coli DH5α (pCR2.1).

Horizontal transferability of bla KPC plasmid
S1-PFGE and southern blotting were used to confirm the location of the bla KPC gene, as previously reported (9,10).Conjugation assays were performed using filter mating, with bla KPC -positive isolates serving as donors and E. coli J53 (azide resistant) as recipients on a filter membrane resting on MH agar without antibiotics.After 18 hours of incubation, the mixed cultures were plated onto MH agar containing ampicillin (100 mg/L) and azide (200 mg/L).Transconjugants were selected after 48 hours of incubation.Plasmids were also extracted from the CZA-resistant K. pneumoniae isolates and electro-transformed into E. coli DH5α competent cells, followed by selection on plates containing 100 µg/mL ampicillin.Species identification, AST, and PCR confirmation were performed on both conjugants and electro-transformants upon acquisition (11).

Surveillance of CZA resistance
During over 2 years of CZA resistance surveillance, a total of 4,641 K. pneumoniae isolates were collected from 1,845 non-duplicate patients, among which 1,667 isolates from 491 patients were CRKP.Consequently, four CZA-resistant K. pneumoniae isolates from distinct patients were obtained, with three of them collected from patients who received CZA treatment during their hospitalization, accounting for a resistance development rate of 4.0% (3/75) of K. pneumoniae under CZA stress.Notably, the remaining isolate was obtained from a patient who had not undergone CZA therapy, although other β-lactam antibiotics were administered during the patient's hospitalization.

Patients
The four patients from whom CZA-resistant K. pneumoniae isolates were collected suffered from severe infectious diseases (Table 1).Among them, three patients are male and one is female, aged between 28 and 69 years, all of whom had a medical history of intensive care unit (ICU) admission.For the three patients who received CZA treatment, multiple antimicrobial agents were administered prior to the isolation of CZA-resistant strains.The duration of CZA treatment ranged from 8 to 33 days.A series of K. pneumoniae strains were isolated from each of these four patients, and we selected one of the CZA-susceptible strains identified in each patient before the isolation of the CZA-resistant K. pneumoniae for subsequent homolog comparison.The paired strains, which were susceptible to CZA before the CZA-R-KP isolated, were selected in our study and designated as XYJ-CZA-S/-R, GYH-CZA-S/-R, ZZX-CZA-S/-R, and WHY-CZA-S/-R, respectively (Table 2).

AST results
The AST results confirmed that all paired isolates were pan-drug resistant, display ing resistance to β-lactams, carbapenems, fluoroquinolones, aminoglycosides, and tetracyclines.Additionally, the CZA-resistant isolates exhibited varying degrees of resistance to cefepime, amikacin, trimethoprim/sulfamethoxazole, and tobramycin.Notably, apart from CZA, the resistance pattern was generally consistent for each paired strain, although some differences were observed, particularly in susceptibility to carbapenems.All four CZA-resistant strains showed reversed susceptibility to carbape nems, such as meropenem with MIC 2 or <0.125 µg/mL compared with CZA-susceptible strains.The phenomenon of regaining carbapenem susceptibility due to the acquisition of CZA resistance was consistent with previous reports (13).Furthermore, all paired strains demonstrated sensitivity to tigecycline, colistin, meropenem-vaborbactam, and imipenem-relebactam (Table 2).The MICs of CZA for the four CZA-resistant strains were greater than 16 µg/mL, indicating a high level of resistance to this antibiotic combination (Table 2).

Homology analysis
Each paired strains exhibited the same PFGE pattern, with fewer than three different bands observed, indicating that the two isolates in each pair originated from the same clone (Fig. S1A).Genome analysis of each paired strains also revealed the same ST type, with three pairs identified as ST11 and the remaining pairs identified as ST716, and the latter one is rarely reported for CZA resistance dissemination.The SNP analysis of each paired strains demonstrated the most detailed genome variants.Three out of four pairs of strains exhibited extremely few SNP differences, all fewer than 10 SNP differences,    except for the pair ZZX-CZA-S/-R.However, the differences between each pair of strains did not occur in the known antibiotic resistance genes, except for the bla KPC-2 gene.

bla KPC mutations contribute to CZA resistance
We conducted a comparison of changes in bla KPC genes between CZA-susceptible and CZA-resistant isolates from the same patient, revealing the contribution of KPC mutations to CZA resistance.Using NG-Test CARBA 5, all CZA-susceptible isolates tested positive for the carbapenem phenotype, while the corresponding CZA-resistant isolates tested negative (Fig. S2).The PCR and sequencing results showed that all four CZA-sus ceptible isolates harbored the bla KPC-2 gene, whereas the paired CZA-resistant isolates were positive for bla KPC-33 (two isolates), bla KPC-86 , and a novel bla KPC allele designated as bla KPC-129 in this study, respectively (Table 2).According to the alignment results of the amino acid sequences of KPC carbapenemase, XYJ-CZA-R and WHY-CZA-R exhibited a point mutation compared with the KPC-2 protein, resulting in a change from D to Y at site 179 of the Ω loop.ZZX-CZA-R exhibited a D to G point mutation at site 179 and expressed a KPC-86 phenotype.Furthermore, GYH-CZA-R exhibited a novel N to H mutation at site 170 of the Ω loop, named KPC-129.KPC-129 represents the first report of this mutation phenotype in clinically CZA-resistant KPC strains (Fig. S3).

bla KPC plasmids and their locations
We employed S1-PFGE to investigate the presence of plasmids in each of the isola ted strains carrying bla KPC resistance determinants.Southern blotting experiments confirmed that bla KPC genes among the four paired strains were indeed located on plasmids, with sizes ranging from 54.7 kb to 216.9 kb (Fig. S2B).In the strains XYJ-R and WHY-R, the bla KPC-33 gene was located on a plasmid of approximately 78.2 kb.
The bla KPC-129 gene was detected in the GYH-R strain, associated with a plasmid of approximately 104.5 kb, while the bla KPC-86 gene was identified in the ZZX-R strain, linked to a plasmid of approximately 138.9 kb (Fig. S2B).

Hydrolytic activity of the bla KPC gene of the CZA-R K. pneumoniae isolate
Several attempts of filter mating experiments to transfer CZA-resistant plasmids to E. coli J53 recipients failed, indicating that the plasmids in these strains were incapable of being transferred by conjugation.Since the CZA resistances mediated by KPC-86 and KPC-129 carbapenemases were not reported previously, E. coli DH5α transformants containing pKPC-GYH-CZA-R and pKPC-ZZX-CZA-R were furtherly constructed.Both plasmids were found to mediate increases in the MICs of β-lactams, including ampicil lin, ceftazidime, ampicillin-sulbactam, piperacillin-tazobactam, ceftazidime-avibactam, aztreonam, gentamicin, and amikacin.As a novel carbapenemase, KPC-129 was also shown to confer resistance to both ceftazidime and CZA, as revealed by TA clone experiments (Table 2).

Genomic analysis of bla KPC plasmids
WGS was performed to analyze the genomes of the plasmids isolated from the four paired K. pneumoniae isolates.As depicted in Fig. 1, the presence of identical genetic organization regions suggested a conservative structure among the four CZA-resist ant isolates compared with their paired CZA-sensitive isolates.In the XYJ-CZA-R and WHY-CZA-R strains, the resistance gene bla TEM-1B was located downstream of ISKpn27.Additionally, the WHY-R strain exhibited the presence of a qnrS1 resistance gene downstream of ISKpn19.The ZZX-CZA-R and GYH-CZA-R isolates showed resistance genes, including bla TEM-1 , rmtB, and bla SHV-12 , flanked by IS26, which differed from the presence of bla KPC-57 and bla KPC-79 in relative proportions.Furthermore, all four CZA-resistant strains were found to exhibit mercury resistance genes of merT and merC.These results collectively suggest that mutations in plasmid resistance genes may induce amino acid substitutions, which could subsequently influence the formation and function of the Ω loop of KPC variants, thereby affecting the hydrolyzation function of KPC enzymes to CZA.

DISCUSSION
We conducted a surveillance spanning over 2 years, focusing on clinically isolated K. pneumoniae strains to monitor the CZA-R-KP strains.All four identified strains were found to possess a bla KPC-2 variant, including bla KPC-33 , bla KPC-86 , and a novel variant designated as bla KPC-129 .The observed resistance rate of K. pneumoniae was lower than the rate reported by CHINET 2023 (China Antimicrobial Surveillance Network), which reported that the CZA resistance rate for CRKP is around 11.0%.These data were based on antimicrobial susceptibility testing in vitro of 16,125 CRKP isolates collected from 73 hospitals across China (available from http://www.chinets.com/Data/AntibioticDrugFast).We attribute this lower rate of CZA resistance to differences in drug application choices resulting from single-center studies and variations in antibiotic resistance patterns within large urban populations.Although CZA has not been extensively used in clinical practice for an extended period, reports have emerged indicating the occurrence of drug resistance, particularly in organisms such as K. pneumoniae (14,15).For instance, there have been reports of a clinical isolate of K. pneumoniae demonstrating high-level resistance to CZA, attributed to the production of KPC-53 due to a Leu167Glu168 duplication in the Ω loop and subsequent loss of carbapenemase activity (16).In our study, three patients received CZA treatment, suggesting that the development of drug resistance may not necessarily correlate with the duration of drug use and therefore underscoring the importance of cautious and supervised use throughout the treatment process.We also report a case where CZA resistance occurred in a patient with no history of CZA usage but had been treated with various antibiotic drugs, particularly ceftazidime.The patient received ceftazidime treatment for 28 days before developing CZA resistance, prompting the hypothesis that the introduction of ceftazidime might have induced CZA resistance (17).Previous research has shown that CRKP could acquire resistance to CZA and tigecycline in vivo under the exposure to β-lactam antibiotics and TGC (18).
The active site within the Ω loop plays a crucial role in the catalytic activity of class A β-lactamases, located adjacent to the catalytic residue S70.The Ω loop also contains a position for a catalytic water molecule necessary for the diacylation reaction (25).Within this loop, residues LEU162-ASP179 are present, forming part of the active site of KPC-2 containing ASN170 and ASP179 (Fig. S4).However, the conversion of amino acid types, resulting in HIS170 and GLY179 in KPC-129 and KPC-86, respectively, has been found to cause ceftazidime resistance.Although similar mechanisms of bla KPC mutations have been reported, such as bla KPC-2 , bla KPC-33 , and bla KPC-86 , our research introduces a novel mutation named bla KPC-129 .KPC-129 represents a newly discovered mutation phenotype isolated from the GYH-CZA-R strain, featuring a mutation at site 170 of the Ω loop.
In previous studies, CZA-R strains demonstrated collateral sensitivity to β-lactam antibiotics.This suggests that the mutations in the KPC-2 and KPC-3 β-lactamase sequences responsible for CZA resistance can also affect susceptibility to other β-lactam antibiotics (26).In our research, a similar phenomenon was observed, where the development of CZA resistance in K. pneumoniae strains resulted in the regaining of sensitivity to several antibiotics like imipenem and meropenem.On the one hand, most clinical laboratories may not test or report ceftazidime-avibactam resistance if carbape nem is tested susceptible.The selection of this compound preparation requires caution.On the other hand, this revival of drug sensitivity may assist in selecting appropriate drugs for treating K. pneumoniae infections.From a clinical perspective, the shift in drug resistance phenotype during our treatment with CZA underscores the importance of promptly assessing changes in the in vitro susceptibility of the isolates and adjusting antibiotics as needed, particularly for patients receiving CZA.The mechanism underly ing this susceptibility change may lie in the protein structural alterations caused by carbapenemase mutations, which alter the hydrolysis profile of the enzyme toward antibiotics.When the variant exhibits enhanced hydrolytic activity toward one antibiotic, it may imply a decreased hydrolytic activity toward another antibiotic.For researchers, exploring the mechanisms could lead to a deeper understanding of the resistance mechanisms of KPC.
In summary, our study presented four clinical cases of bla KPC mutants in CZA-resistant isolates, notably including a novel mutation named KPC-129.Given the relatively high incidence of CZA usage in treating K. pneumoniae infections, caution should be exercised in the prescription and monitoring of CZA throughout the entire course of treatment.This study underscores the significance of active surveillance to monitor the emergence of resistance to CZA, emphasizing the necessity for further research to combat antimicro bial resistance.
KPC gene of CZA-R K. pneumoniae isolate detected by electro-transformation and TA clone numbers in bold mean resistance a,b ; SAM, ampicillin-sulbactam;

FIG 1
FIG 1Characteristics and alignment of plasmids containing CZA-resistant genes.Circular map of the KPC-R and comparative genomics analysis with its similar plasmids.Starting from the center: (i) GC skew (G−C/G+C), with a positive GC skew toward the inside and a negative GC skew toward the outside.(ii) GC content of pNDM4-191773 with an average of 54.03% (used as reference).(iii) pNDM4-191773 plasmid sequence (CP116494.1).(iv) Gene annotation.Red, antibiotic resistance; gray, transfer conjugation; sea-blue, insertion sequence; gray-blue, the relative KPC-S strain plasmid gene sequence.Arrowhead indicates the direction of transcription.A scale of 1,000 bp is attached to the corner.

TABLE 2
The genotypic and phenotypic characteristics of the four paired isolates and hydrolytic activity of bla