Comparison of the in vitro activities and resistance mechanisms against imipenem–relebactam and ceftazidime–avibactam in clinical KPC-producing Klebsiella pneumoniae isolated in China

Yingyi Guo; Likang Yao; Jiong Wang; Yan Zhang; Chuyue Zhuo; Yijing Wang; Xu Yang; Jiahui Li; Nanhao He; Jiakang Chen; Yexin Lin; Shunian Xiao; Zhiwei Lin; Chao Zhuo

doi:10.1007/s15010-025-02474-3

. 2025 Feb 15;53(4):1469–1481. doi: 10.1007/s15010-025-02474-3

Comparison of the in vitro activities and resistance mechanisms against imipenem–relebactam and ceftazidime–avibactam in clinical KPC-producing Klebsiella pneumoniae isolated in China

Yingyi Guo ^1,^2,^#, Likang Yao ^3,^#, Jiong Wang ^4,^5,^#, Yan Zhang ^2,^#, Chuyue Zhuo ², Yijing Wang ², Xu Yang ², Jiahui Li ², Nanhao He ², Jiakang Chen ², Yexin Lin ², Shunian Xiao ², Zhiwei Lin ^6,^✉, Chao Zhuo ^2,^✉

PMCID: PMC12316821 PMID: 39954208

Abstract

Background

Ceftazidime–avibactam (CAZ–AVI) and imipenem–relebactam (IMI–REL) are both antibiotics with promising prospects for treating Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) infections. However, differences in the in vitro activities and resistance mechanisms to CAZ–AVI and IMI–REL in clinical KPC-Kps have not been described.

Methods

In this study, KPC-Kp isolates from hospitalized patients in China were collected and subjected to antimicrobial susceptibility testing of IMI–REL and CAZ–AVI using the broth microdilution method. Whole-genome sequencing (WGS) and functional validation of mutations were performed on resistant strains, and RT-qPCR was used to determine the expression levels of bla_KPC.

Results

The results showed that 21 (2.7%) of 782 clinical KPC-Kp strains were CAZ–AVI-resistant, 6 (0.8%) of 782 strains were IMI–REL-resistant, and 5 strains among them were resistant to both CAZ–AVI and IMI–REL. Strains resistant to both CAZ–AVI and IMI–REL can be effectively inhibited by tigecycline and polymyxin B. WGS and complementation experiments showed that KPC mutations are linked to high-level resistance to CAZ–AVI; while OmpK36 mutations may be the vital mechanism of IMI–REL resistance, confers resistance to CAZ–AVI simultaneously. Furthermore, RT-qPCR indicated that elevated bla_KPC expression may play an important role in both CAZ–AVI and IMI–REL resistance.

Conclusions

In summary, this study suggested that IMI–REL may have superior inhibitory effects in vitro on KPC-Kps than CAZ–AVI, and described the differences in resistance mechanisms between the two antibiotics.

Supplementary Information

The online version contains supplementary material available at 10.1007/s15010-025-02474-3.

Keywords: Klebsiella pneumoniae, Ceftazidime–avibactam, Imipenem–relebactam, Resistance

Introduction

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health, and its resistance rate has increased dramatically over time, from 3% in 2005 [1] to 22% in 2020 [2]. Furthermore, the mortality rate of patients infected with CRKP in China is alarmingly high (44.82%) [3]. Production of Klebsiella pneumoniae carbapenemases (KPCs) remains the most prevalent mechanism underlying carbapenem resistance in CRKP [4]. KPC-producing Klebsiella pneumoniae (KPC-Kp) exhibits resistance to commonly used antimicrobials; therefore, treatment options are limited for this bacterium, which poses a serious threat to public health. However, new β-lactam/β-lactamase inhibitor combinations have recently emerged, such as ceftazidime–avibactam (CAZ–AVI) and imipenem–relebactam (IMI–REL), providing new hope for the treatment of KPC-Kp infections.

KPC-Kp is highly susceptible to both CAZ–AVI and IMI–REL. Previous in vitro experiments showed that AVI can restore the activity of CAZ in more than 80% of CAZ-non-susceptible Klebsiella pneumoniae and all KPC-Kp [2, 5]. Similarly, REL can restore IMI activity in more than 93% of imipenem–resistant Klebsiella pneumoniae and all KPC-Kp [6]. CAZ–AVI is a inhibitor combination of cephalosporin/β-lactamase [2]. Avibactam (AVI) can activate class A, class C, and some class D carbapenemases. CAZ–AVI represents one of the last lines of defense against KPC-Kp that exhibits resistance to almost all available antibiotics [7]. Relebactam (REL) is a novel bicyclic diazabicyclooctane β-lactamase inhibitor with activity against dual class A and class C β-lactamases [8]. In 2019, the Food and Drug Administration (FDA) approved the use of IMI–REL for the treatment of complicated urinary tract and intra-abdominal infections [9].

Although CAZ–AVI and IMI–REL have shown significant activity against KPC-Kp and are the representation of promising antibiotics for the treatment of KPC-Kp, reports on the emergence of CAZ–AVI and IMI–REL resistance in KPC-Kp have recently surfaced [10]. Multiple resistance mechanisms of KPC-Kp to CAZ–AVI have been identified, including mutations in β-lactamase, porins (OmpK35 and OmpK36), and PBP3, as well as the increased expression of bla_KPC and efflux pumps [11]. The mechanism of IMI–REL resistance to KPC-Kp is not fully understood [12], and reports suggested that the mechanisms underlying resistance to CAZ–AVI are distinct from those to IMI–REL; for instance, different mutation sites in KPC do not equally contribute to resistance to these two antibiotics [13]. Currently, the differences between the in vitro efficacies of these two antibiotics against KPC-Kp strains have not been clearly reported. Moreover, systematic studies on the resistance mechanism of IMI–REL are lacking, and there is inadequate evidence available to elucidate the differences in intrinsic resistance mechanisms of KPC-Kp strains towards CAZ–AVI and IMI–REL.

In this study, we aimed to elucidate the in vitro activities of CAZ–AVI and IMI–REL against clinical KPC-Kp strains, reveal the underlying resistance mechanisms, and explore the effects of different resistance mechanisms of KPC-Kp against CAZ–AVI and IMI–REL through molecular experiments. These findings provide further evidence for controlling the generation and spread of antibiotic-resistant bacteria.

Materials and methods

Bacterial collection

All Klebsiella pneumoniae strains included in this study were isolated from inpatients of three tertiary hospitals in Guangzhou, China between 2017 and 2022. Duplicate isolates from the same site in the same patient were excluded. All strains were identified as Klebsiella pneumoniae using a VITEK-2 Compact fully automatic microorganism analyzer (bioMénière, France). Common carbapenemase-encoding genes (bla_KPC, bla_NDM, bla_OXA-48, bla_IMP, and bla_VIM) were identified using PCR and Sanger sequencing, and isolates that only carry bla_KPC (without carrying any of bla_NDM, bla_OXA-48, bla_IMP, or bla_VIM) were included. All wzi genes in KPC-Kp were amplified by PCR, and capsular serotypes were classified using Abricate and BIGSdb [14, 15]. Primers used in this study are listed in Table S1.

String tests were performed to determine the hypermucoviscosity phenotype. Briefly, an isolate was considered positive when a viscous string > 5 mm in length was generated using an inoculation loop by stretching a single colony on a Columbia blood agar plate (Dijing, Guangzhou, China).

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) of CAZ, IMI, CAZ–AVI, IMI–REL, polymyxin B (PB), ampicillin (AMP), aztreonam (AZT), levofloxacin (LEV), minocycline (MIN), ciprofloxacin (CIP), gentamycin (GM), and amikacin (AMK) against KPC-Kp strains were determined using the broth microdilution method according to the guidelines of the American Clinical and Laboratory Standardization Institute (CLSI) M100 [16]. The MIC of tigecycline (TGC) were determined according to the US Food and Drug Administration standards [17]. The control strains were Klebsiella pneumoniae ATCC700603 and Escherichia coli ATCC25922. MIC₅₀ was defined as the lowest concentration at which 50% of the strains were inhibited by an antimicrobial agent, whereas MIC₉₀ was defined as the minimum concentration at which an antimicrobial agent had an inhibitory effect on 90% of the bacterial strains [18]. The breakpoints for interpretation were as follows: IMI, MIC ≥ 4 mg/L; CAZ, MIC ≥ 16 mg/L; CAZ–AVI, MIC ≥ 16 /4 mg/L; PB, MIC ≥ 4 mg/L; TGC, MIC ≥ 8 mg/L; IMI–REL, MIC ≥ 4 /4 mg/L for antibiotics resistance.

Whole-genome sequencing (WGS) and bioinformatic analysis

WGS was performed on all CAZ–AVI- and IMI–REL-resistant KPC-Kp strains. Single colonies from the agar plates were incubated in 6 ml of fresh LB broth at 37 °C for 12 h. Genomic DNA was extracted using a FastPure Bacteria DNA Isolation Mini Kit (Vazyme, Nanjing, China). WGS was performed by Novogene (Beijing, China) using an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Raw data were trimmed and assembled using Shovill, and annotation of the assembled contigs was performed using Prokka [19, 20]. Multilocus sequence typing (MLST) was performed using MLST software [21]. Antibiotic resistance genes and virulence genes were detected using the NCBI database and VFDB database in ABRicate v.1.0.1, respectively [22]. Mutations in common proteins associated with antibiotics resistance (KPC, OmpK35/36, and PBP2/3) were identified using snippy [23]. The sequences of wild-type bla_KPC (NCBI reference sequence KPHS_p200360), OmpK35 (NCBI reference sequence WP_135730820.1), OmpK36 (GenBank accession number AEW62399.1), PBP2 (encoded by mrda, NCBI reference sequence CP117227.1), and PBP3 (encoded by ftsi, NCBI reference sequence KPHS_08040) were used as reference sequences [24]. To confirm these mutations, PCR was performed to amplify the target genes and Sanger sequencing was conducted using BGI (Beijing, China).

Determination of bla_KPC expression levels

The strains were resuspended overnight in LB broth, inoculated into 6 ml of fresh LB broth, and incubated at 37 °C with shaking at 220 rpm until they reached the logarithmic growth phase. Total RNA was isolated using a bacterial RNA extraction kit (Vazyme, Nanjing, China). Genomic DNA was removed, and cDNA was prepared using the HiScript III-RT SuperMix for qPCR kit (Vazyme, Nanjing, China). RT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with bla_KPC primers (Table S1) on a LightCycler 96 instrument (Roche). The relative transcription levels of bla_KPC in all strains were calculated using the 2^−ΔΔCT method, with rpoB gene as the internal reference [25]. The strain with the lowest bla_KPC expression served as the control. The average transcript levels were determined from at least three independent total RNA samples isolated from three separate microbial broth cultures of each strain.

Efflux pump inhibitor tests and growth curve analysis

The MICs of CAZ–AVI and IMI–REL in combination with carbonyl cyanide m-chlorophenylhydrazine (CCCP) at a concentration of 10 mg/L were determined. Compared to the MICs of CAZ–AVI and IMI–REL, a four-fold or greater decrease in MIC values after the addition of CCCP was considered significant.

To ensure that the CCCP added to the efflux pump inhibition test did not inhibit the growth of the strains, the growth curve of each strain was measured using LB broth with 10 mg/L CCCP [26]. An optical growth analyzer (BioTek Epoch2, USA) was used to monitor the growth of the KPC-Kp strains. Briefly, overnight cultures in LB broth were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard and then inoculated at a 1:1000 dilution in fresh LB broth. For each strain, 300 μl of inoculated LB broth was added to triplicate wells of a microplate. Three wells containing fresh medium served as blank controls. The cultures were incubated at 37 °C with continuous shaking for 24 h, and the OD600 was measured every 30 min. And GraphPad Prism 9 software were used to analyze the Growth curves.

Functional verification of new mutations

To verify the effect of new mutations in OmpK36 and PBP3 on antibiotics resistance, the functions of wild-type OmpK36 and PBP3 proteins were restored. Briefly, wild-type genes were amplified by PCR and the products were purified using a gel extraction kit (Takara, Tokyo, Japan) according to a previously described approach [10]. The target genes were inserted into the linearized plasmid pACYC184 or pBAD33 digested by EcoRI or BamHI, using the In-Fusion HD Cloning Kit (Takara, Tokyo, Japan) according to the manufacturer’s instruction.

The competent cells of the strains were prepared using 10% glycerol as previously described with a minor modification [27]. Briefly, the overnight culture of a single colony in 2 mL of LB broth was diluted into 100 mL of fresh LB broth and grown to early logarithmic phase (OD600 = 0.4–0.6) at 37 °C. Then the cells were harvested by centrifugation at 4000g for 10 min at 4 °C, washed with ice-cold 10% glycerol (performing on the ice), and the centrifugation and washing steps were repeated twice. Finally, the cells were resuspended in ice-cold 10% glycerol and divided into 90μL each tube (performing on the ice) and stored at −80 °C for future use. The recombinant vector was then introduced into the mutant strain competent cell via electroporation.

To verify the effect of new KPC mutations on antibiotics resistance, a pACYC184 plasmid carrying the mutant bla_KPC gene was constructed using the same approach and transformed into the engineered Escherichia coli strain DH5α.

An empty vector introduced into the mutant strain was used as a control, as well as DH5α. MIC assays were performed in triplicate using Mueller–Hinton broth microdilutions for mutant strains, revertant mutant strains, DH5α strains, and DH5α strains with the transferred plasmid. To ensure all the inserted genes in the plasmid express successfully, the strains that were electrotransferred into the plasmid were tested for the expression of the corresponding genes.

Statistical analysis

All data were analyzed using SPSS version 25.0. Categorical variables were reported as a percentage (%) and analyzed using Chi-square test. For bla_KPC expression, the standard normality of the original data and log₁₀-transformed data were evaluated using the Shapiro–Wilk test, with p > 0.05 indicating normality. Independent two-sample t-tests were conducted to determine the differences in bla_KPC expression between resistant and sensitive strains, and p < 0.05 considered to be statistically significant.

Results

Characteristics of bacteria strains and in vitro antimicrobial susceptibility

In total, 782 KPC-Kp strains were included, of which 344 (44.0%) were isolated from sputum samples, 147 (18.8%) were isolated from urine samples, 129 (16.5%) were isolated from blood samples, and 162 (20.7%) were isolated from other specimens. The most common serotypes among all strains were KL64 (n = 446, 57.0%) and KL47 (n = 235, 30%), followed by KL19 (n = 30, 3.9%) and other serotypes (such as KL25, KL24, KL124, and KL10, 9.1%).

Antimicrobial susceptibility data of the 782 KPC-Kp isolates against CAZ, CAZ–AVI, IMI, IMI–REL, TGC, and PB are listed in Table 1. The results showed that only 2.7% and 0.8% of KPC-Kp isolates were resistant to CAZ–AVI and IMI–REL, respectively, while almost all the KPC-Kp strains were resistant to IMI (99.7%) and CAZ (100%). The antimicrobial resistance rates of TGC and PB were 2.4% and 6.7%, respectively. The MIC₉₀ of CAZ–AVI was 8 mg/L, which was close to the MIC breakpoint (≥ 16/4 mg/L). More than half of the strains (57.3%) had MIC values distributed between 4 mg/L and 8 mg/L (Figure S1). The MIC₉₀ value of IMI–REL was 0.5 mg/L, which was relatively away from the MIC breakpoint (≥ 4/4 mg/L), and almost all strains (94.0%) had MIC values of 0.5 mg/L or below (Figure S1).

Table 1.

Antimicrobial susceptibility data of 782 KPC-Kp clinical isolates

Antibiotics^a	Breakpoints (mg/L)^b		Resistant (n,%)	MIC range (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)
Antibiotics^a	S	R	Resistant (n,%)	MIC range (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)
CAZ	≤ 4	≥ 16	782(100%)	32–128	> 128	> 128
CAZ–AVI	≤ 8/4	≥ 16/4	21(2.7%)	0.125–128	4	8
IMI	≤ 1	≥ 4	779(99.6%)	0.25–128	64	128
IMI–REL	≤ 1/4	≥ 4/4	6(0.8%)	0.125–16	0.25	0.5
TGC	≤ 2	≥ 8	19(2.4%)	0.25–32	1	4
PB	≤ 2	≥ 4	52(6.7%)	0.125–128	1	2

Open in a new tab

^aCAZ ceftazidime, CAZ–AVI ceftazidime–avibactam, IMI imipenem, IMI–REL imipenem–relebactam, TGC tigecycline, PB polymyxin

^bS sensitive, R resistant. The breakpoints of TGC were based on the US Food and Drug Administration standards

Comparison of characteristics of CAZ–AVI resistant and IMI–REL resistant isolates

A total of 22 KPC-Kp clinical isolates were found to be resistant to CAZ–AVI and/or IMI–REL. Among the 22 KPC-Kp strains, 21 (2.7%) were resistant to CAZ–AVI and six (0.8%) were resistant to IMI–REL (p = 0.004). Five of 22 KPC-Kp strains were resistant to both IMI–REL and CAZ–AVI. All 22 strains belonged to ST11, with 12, 9, and 1 strains belonging to the KL64, KL47, and KL10 serotypes, respectively (Table 2). Among the 22 strains, nine strains (40.9%) showed positive results in the string test. Particularly, both rmpA2 and iucA/B/C/D were detected in eight of the nine strains with positive string test, as shown in Table S2. The detailed molecular characteristics of each resistant strain are presented in Table S3.

Table 2.

Comparison of the characteristics of CAZ–AVI- and/or IMI–REL-resistant clinical isolates

Strains	MLST	Serotypes			String test	MIC (mg/L) range					Cross resistance (n,%)^a
	MLST	Serotypes				CAZ–AVI			IMI–REL		AMP	AZT	LEV	MIN	CIP	GM	AMK	TGC	PB
	ST11	KL47	KL64	KL10		4–8	16–32	64–128	≤ 1	≥ 4	AMP	AZT	LEV	MIN	CIP	GM	AMK	TGC	PB
Total (n = 22)	22	9	12	1	9	1	16	5	16	6	22, 100.0%	22, 100.0%	22, 100.0%	11, 50.0%	22, 100.0%	22, 100.0%	20, 91.0%	3, 13.7%	2, 9.1%
Only CAZ–AVI-resistant (n = 16)	16	6	9	1	7	0	12	4	16	0	16, 100.0%	16, 100.0%	16, 100.0%	9, 56.3%	16, 100.0%	16, 100.0%	16, 100.0%	3, 18.8%	1, 6.3%
CAZ–AVI- and IMI–REL-resistant (n = 5)	5	3	2	0	2	0	4	1	0	4	5, 100.0%	5, 100.0%	5, 100.0%	1, 20.0%	5, 100.0%	5, 100.0%	3, 60.0%	0, 0.0%	1, 20.0%
Only IMI–REL-resistant (n = 1)	1	0	1	0	0	1	0	0	0	2	1, 100.0%	1, 100.0%	1, 100.0%	1, 100%	1, 100.0%	1, 100.0%	1, 100.0%	0, 0.0%	0, 0.0%

Open in a new tab

The MIC results of tigecycline were determined according to the US Food and Drug Administration standard

^aAMP ampicillin, AZT aztreonam, LEV levofloxacin, MIN minocycline, CIP ciprofloxacin, GM gentamycin, AMK amikacin, TGC tigecycline, PB polymyxin B

All the resistant strains have cross-resistance to AMP, AZT, LEV, CIP, and GM but not to MIN, AMK, TGC, and PB (Table 2). The heatmap of resistance gene showed that a total of five varieties of Extended-Spectrum β-Lactamase (ESBL) genes, namely bla_CTX-M-65 (n = 15, 68.2%), bla_SHV-12 (n = 1, 4.5%), bla_SHV-158 (n = 2, 9.1%), bla_SHV-187 (n = 20, 90.1%) and bla_TEM-1 (n = 20, 90.1%), and an AmpC enzyme gene bla_CMY-2 (n = 1, 4.5%) were detected in 22 resistant strains (Figure S2). In addition, a total of four varieties of quinolone resistance genes were detected, including qnrD1 (n = 1, 4.5%), qnrS1 (n = 14, 63.6%), oqxA (n = 5, 22.7%) and oqxB (n = 5, 22.7%). And a total of ten varieties of aminoglycoside resistance genes were detected, including rmtB1 (n = 20, 90.9%), aadA2 (n = 16, 72.7%), ant(2 ″)-Ia (n = 4, 18.2%), aph(3 ")-Ib (n = 4, 18.2%), aph(6)-Id (n = 4, 18.2%), aph(3′)-Ia (n = 2, 9.1%), aadA1 (n = 1, 4.5%), aac(6′)-Ib-D181Y (n = 1, 4.5%), aac(3)-IIe (n = 1, 4.5%) and mph(A) (n = 1, 4.5%). One gene [tet (A), n = 12, 54.5%] related to TGC resistance was detected. And no gene related to PB resistance was detected (Figure S2).

Based on the differences in resistance to CAZ–AVI and IMI–REL, the 22 resistant strains were divided into three groups: only CAZ–AVI resistance, only IMI–REL resistance, and both CAZ–AVI and IMI–REL resistance. The molecular characteristics and cross-resistance of the different bacterial groups are shown in Table 2. The results suggested that among the 16 strains with only CAZ–AVI resistance, the serotypes of six strains were KL64, nine were KL47, and one was KL10. Seven of the sixteen strains (43.8%) tested positive in the string test. A total of 4 strains with high CAZ–AVI resistance levels (≥ 64 mg/L), and the cross-resistance rates of 16 strains to MIN, AMK, TGC, and PB were 56.3%,100%, 18.8%, and 6.3%, respectively. In addition, 2–5 varieties of aminoglycoside resistance genes were detected in these 16 strains. Of the five strains resistant to both CAZ–AVI and IMI–REL, the serotypes of three strains were KL47, two strains were KL64. Only one strain had a high level of resistance to CAZ–AVI, and two strains (40.0%) showed positive results in the string test. The cross-resistance rates of five strains to MIN, AMK, TGC, and PB were 20.0%, 60%, 0%, and 20%, respectively. In addition, 0–3 varieties of aminoglycoside resistance genes were detected in these five strains. One strain that was only resistant to IMI–REL belonged to KL64, and showed cross-resistance to both MIN and AMK. However, cross-resistance to TGC and PB was not detected. This was consistent with the results of the heatmap of resistance gene (Figure S2).

Mechanisms of antimicrobial resistance in CAZ–AVI- and/or IMI–REL-resistant strains

Analysis of mutation in antimicrobial resistance-related genes

To investigate the resistance mechanisms of KPC-Kp to CAZ–AVI and IMI–REL, we identified and compared the mutations in KPC, OmpK35/36, and PBP2/3 of strains resistant to CAZ–AVI and/or IMI–REL (Tables 3 and S4). Mutations in 40 KPC-Kp strains that sensitive to CAZ–AVI and IMI–REL were served as controls. All sensitive strains were selected based on the epidemic characteristics (with capsular serotype and MIC distribution primarily considered) of KPC-Kp in China. Briefly, we selected KPC-Kp strains that have similar capsular serotype distribution to the 22 resistant strains, possess diverse specimen sources, and were sensitive to CAZ–AVI and IMI–REL. The results revealed significant differences in mutations between the CAZ–AVI- and IMI–REL-resistant strains. Given that the same mutations in OmpK35 (a premature stop codon at amino acid position 63) and OmpK36 (134 to 135 GD insertion) were detected in all 22 resistant strains and 40 sensitive strains, only unique mutations are described in Table S4 and subsequent analyses.

Table 3.

Comparison of common antimicrobial resistance mechanisms in KPC-Kp resistant to CAZ–AVI and/or IMI–REL

Strains	Mutation^a					Increased expression of bla_KPC^b	Efflux pump inhibitor tests^c
Strains	KPC	OmpK35	OmpK36	PBP2	PBP3	Increased expression of bla_KPC^b	Efflux pump inhibitor tests^c
Only CAZ–AVI-resistant (n = 16)	D179Y (n = 3;729, CR059, CR063) A172V (n = 1;ZSS072)	–	–	–	–	n = 10 (CR059, CR063, ZSS072, ZSS048, 617, 430, 794, A398, A206, 795)	n = 2 (794,795)
CAZ–AVI- and IMI–REL-resistant (n = 5)	–	–	Q296*(n = 1;A125) K196E(n = 1;ZSS129) S125fs(n = 1;A301) Lack(n = 1;744)	–	D343N(n = 1;ZSS129)	n = 4 (CR067, ZSS129, A125, A301)	n = 2 (ZSS129,744)
Only IMI–REL-resistant (n = 1)	–		Lack(n = 1;478)	–	–	n = 0	n = 0

Open in a new tab

a”–” indicates that no mutations were detected in KPC or PBP2/3 and no additional mutations were detected in OmpK35/36, except for a common mutation (a premature stop codon at amino acid position 63 in OmpK35, 134 to 135 GD insertion in OmpK36)

^bIndependent two-sample t-tests were conducted to determine the differences in bla_KPC expression between resistant and sensitive strains, and p < 0.05 considered to be statistically significant

^cMICs of CAZ–AVI and IMI–REL in combination with CCCP at a concentration of 10 mg/L were determined. A four-fold decrease in the MIC after the addition of CCCP was considered significant

Of the 16 strains that were only resistant to CAZ–AVI, four strains (25%) were identified haboring mutant KPC, of which three strains (729, CR059, and CR063) had D179Y mutations and one (ZSS072) had a A172V mutation. Notably, the MIC values of these four KPC mutants were higher than 64 mg/L, thus accounting for 80% (4/5) of all strains with high levels of CAZ–AVI resistance. In addition, three strains with D179Y mutation were sensitive to IMI, whereas the strains with A172V mutation were resistant to IMI (Table S4). No additional mutations were found in OmpK35/36 or PBP2/3 in any of the 16 strains. Of the five KPC-Kp strains resistant to both CAZ–AVI and IMI–REL, additional mutations were detected in OmpK36 in four (80%) strains (ZSS129, K196E; A301, S125fs; A125, Q296*; and 744, lack). One of these strains (ZSS129) also harbored a PBP3 mutation (D343N). Among these five KPC-Kp strains, no other mutations were detected in KPC, PBP2, or OmpK35. In one KPC-Kp strain (478) that was only resistant to IMI–REL, a deletion of OmpK36 was identified and no other mutations were detected (Table 3).

The above results showed a clear distinction in the mutations of CAZ–AVI- and IMI–REL-resistant strains: KPC mutations only occurred in strains that were only resistant to CAZ–AVI and all additional mutations or deletions of OmpK36 were detected in strains resistant to both IMI–REL and CAZ–AVI, as well as in strains only resistant to IMI–REL.

Expression levels of bla_KPC

To explore the contribution of expression levels of bla_KPC to CAZ–AVI and IMI–REL resistance, bla_KPC expression of 22 CAZ–AVI- and/or IMI–REL-resistant and 40 sensitive strains (as controls) were identified.

The results showed that expression levels of bla_KPC were significantly higher in 14 of the 22 (63.6%) resistant strains compared with the 40 sensitive strains (p < 0.05). Among the 16 strains only resistant to CAZ–AVI and five strains resistant to both CAZ–AVI and IMI–REL, ten (62.5%) and four (80.0%) had higher bla_KPC expression level than the sensitive strains (both p < 0.05), respectively. The one KPC-Kp strain, which is resistant to IMI–REL only, did not show a higher bla_KPC expression level (Table 3).

In addition, expression level of bla_KPC was significantly higher in the group of resistant strains than sensitive strains, regardless of the CAZ–AVI (p < 0.001) or IMI–REL resistance (p = 0.031) (Fig. 1). The expression levels of bla_KPC in the CAZ–AVI- and IMI–REL-resistant strains were 1.83-fold and 1.71-fold higher than those in the sensitive strains, respectively. As the MIC value of CAZ–AVI for KPC-Kp increased, the expression level of bla_KPC in strains also tended to increase. For IMI–REL, when the MIC value was less than 8 mg/L, the expression of bla_KPC in strains tended to increase, and when the MIC value was higher than 8 mg/L, the expression level of bla_KPC in strains was lower than that in strains with MIC values of 2 and 4 mg/L (Fig. 1).

Fig. 1 — Correlation between expression levels of *bla*_KPC and CAZ–AVI or IMI–REL MICs. Comparison analysis of the expression levels of *bla*_KPC between A CAZ–AVI-sensitive (n = 40) and resistant (n = 21) or B IMI–REL-sensitive (n = 40) and resistant (n = 6) strains. Correlation between C CAZ–AVI MICs (n = 61) or D IMI–REL MICs (n = 46) and *bla*_KPC transcription levels. * and *** represent adjusted p values of < 0.05 and < 0.001, respectively, as determined by a independent two-sample t-test. S sensitive, R resistant

To further verify that CAZ–AVI and IMI–REL resistance was associated with the overexpression of the bla_KPC, we added AVI or REL at a fixed concentration of 8 mg/L. Susceptibility of the isolates to CAZ–AVI and IMI–REL was restored in 66.7% (14/21) and 100% of the isolates, respectively (Table S3). In addition, the MICs of 75%(15/20) and 100% (6/6) of the strains against CAZ–AVI and IMI–REL decreased by fourfold or more, respectively.

Role of efflux pumps in resistance

Efflux pump inhibitor tests were used to assess the contribution of efflux pumps to the resistance of KPC-Kp to CAZ–AVI and IMI–REL. The growth curves of all strains, with or without the efflux pump inhibitor CCCP, were measured to confirm that they were not affected by CCCP (Figure S3). The results showed that two (794 and 795) of the 16 KPC-Kp strains resistant only to CAZ–AVI showed a significant decrease in the MIC value of CAZ–AVI, and both decreased by fourfold. Among the five strains resistant to both CAZ–AVI and IMI–REL, the MIC value of one strain (ZSS129) to IMI–REL decreased by eightfold after adding efflux pump inhibitors; while, the MIC value of CAZ–AVI did not decrease. The MIC values of the other strain (744) to IMI–REL and CAZ–AVI decreased by fourfold. The MIC value of one strain only resistant to IMI–REL showed no significant change (Tables 3 and S5).

To clarify whether the efflux pump having effect on resistance of CAZ–AVI and IMI–REL was mainly related to β-lactam or β-lactamase inhibitors, the contribution of the efflux pump to CAZ and IMI resistance was evaluated. The results showed that the decrease in CAZ and IMI was similar to that in CAZ–AVI or IMI–REL after the addition of efflux pump inhibitors (Tables 3 and S5).

Effects of new mutations on bacterial resistance to CAZ–AVI and IMI–REL

To further explore the resistance mechanism of KPC-Kp against CAZ–AVI and IMI–REL, the contributions of newly discovered and unverified mutations in KPC, OmpK36, and PBP3 to the resistance of CAZ–AVI and IMI–REL were verified. To verify the contribution of the A172V mutation in KPC-2 to antimicrobial resistance, mutant and wild-type bla_KPC-2 were introduced into Escherichia coli DH5α and the MIC values were tested. The results are summarized in Table 4. After being transferred into wild-type bla_KPC-2, the MICs of DH5α for CAZ, CAZ–AVI, IMI, and IMI–REL increased significantly. Compared with wild-type bla_KPC-2, the A172V mutation caused the MICs of CAZ and CAZ–AVI increased by 2- and 16-fold, respectively; however, the MICs of IMI and IMI–REL did not change significantly.

Table 4.

Effect of the A172V mutation in KPC-2 on resistance of KPC-Kp to CAZ–AVI and IMI–REL

Transformed plasmid	Strain	MIC value of CAZ–AVI (mg/L)	MIC fold change (WT was used as control)	MIC value of CAZ (mg/L)	MIC fold change (WT was used as control)	MIC value of IMI–REL (mg/L)	MIC fold change (WT was used as control)	MIC value of IMI (mg/L)	MIC fold change (WT was used as control)
None	DH5α	< 0.03	< 0.5	< 2	< 0.06	< 0.03	< 0.25	< 0.03	< 0.03
pACYC184		< 0.03	< 0.5	< 2	< 0.06	< 0.03	< 0.25	< 0.03	< 0.03
pACKPCWT		0.06	1	32	1	0.125	1	4	1
pACKPCA172V		1	16	64	2	0.125	1	2	0.5

Open in a new tab

The lowest detection limit of concentrations of CAZ–AVI, CAZ, IMI–REL and IMI in antimicrobial susceptibility testing were 0.03 mg/L, 2 mg/L, 0.03 mg/L and 0.03 mg/L, respectively. The MIC values of these four antimicrobials to the parent strain DH5α and the strain DH5α carrying the plasmid pACYC184 are all below the lowest detect limitation. Therefore, the accurate fold changes of MIC values could not be calculated between these two strains and the control strain (DH5α carrying the plasmid pACKPCWT), respectively

WT wild type

To verify the contribution of the newly detected OmpK36 and PBP3 mutations to CAZ–AVI and IMI–REL resistance, wild-type OmpK36 and PBP3 were introduced into all three corresponding mutant strains (Table 5). The MIC values of all strains for CAZ–AVI and IMI–REL decreased by fourfold or more after being transferred into wild-type OmpK36, and the extent of the decline in MIC values of the same strain for CAZ–AVI and IMI–REL was basically the same. In addition, after being transferred into wild-type OmpK36, the MIC values of most mutant KPC-Kps for CAZ and IMI decreased to varying degrees, although the decline was significantly lower than those of CAZ–AVI and IMI–REL. For PBP3, when the mutant strain was transformed into the wild-type gene, there were no changes observed in the MIC of IMI–REL, IMI, CAZ–AVI, and CAZ (Table 5).

Table 5.

Effects of mutations in Ompk36 and PBP3 in clinical KPC-KP isolates on resistance to CAZ–AVI and IMI–REL

Transformed plasmid	Strain	MIC value of CAZ–AVI (mg/L)				MIC value of CAZ (mg/L)				MIC value of IMI–REL (mg/L)				MIC value of IMI (mg/L)
Transformed plasmid	Strain	Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC fold change^a	Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC fold change^a	Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC fold change^a	Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC fold change^a
pACOmpk36	ZSS129(K196E)	16	16	4	4	> 2048	> 2048	> 2048	–	4	4	1	4	256	256	256	1
pACOmpk36	A125(Q296*)	128	128	8	16	2048	2048	1024	2	16	16	1	16	256	256	64	4
pACOmpk36	A301(S125fs)	32	32	4	8	2048	2048	1024	2	16	16	1	16	512	512	128	4
pBADPBP3	ZSS129(D343N)	16	16	16	1	> 2048	> 2048	> 2048	–	4	4	4	1	256	256	256	1

Open in a new tab

^aMIC fold change, MIC of Mutant strain + empty vector/MIC of mutant strain complemented with wild-type gene

“–”means incalculable

Q296*, a specific amino acid mutation, means that the glutamine (Q) at the 296th position in the amino acid sequence is replaced by a stop codon (*)

Discussion

KPC is the most common carbapenemase worldwide, particularly in Klebsiella pneumoniae strains isolated in China [2]. ST258 is the dominant CRKP clone in Europe, where KPC (46.0%) and OXA-48-like (39.0%) carbapenemases are commonly identified in the CRKP strains, and even in some regions, OXA-48-like (such as OXA-181) carbapenemases present a predominance (75.0%) [28]. However, in contrast to that in Europe, ST11 is the most prevalent CRKP clone in China and they mainly produced KPC (89.4%) with fewer produced OXA-48-like carbapenemases (1.8%) [29, 30]. Therefore, based on the epidemiological characteristics of CRKP in China, to provide guidance for clinical usage of antibiotics and offer insights for the development of new antimicrobial agents, this study investigated the resistance of KPC-Kp isolates from patients admitted to hospitals in China against CAZ–AVI and IMI–REL and preliminarily compared the underlying resistant mechanisms of CAZ–AVI and IMI–REL. As reported, CAZ–AVI has a good effect on inhibiting OXA-181, while IMI–REL has a poor inhibitory effect on OXA-181 [31]. This may have led to the result that IMI–REL (resistance rate: 0.8%) showed superior inhibitory effects in vitro on CRKP compared to CAZ–AVI (resistance rate: 2.7%) in our study.

In this study, both CAZ–AVI and IMI–REL effectively inhibited KPC-Kp, which is consistent with previous studies [7]. However, 2.7% of isolates were resistant to CAZ–AVI. The MIC₅₀ of CAZ–AVI was close to the breakpoint (MIC ≥ 16/4 mg/L), and the majority of strains had MIC values between 4 and 8 mg/L, indicating reduced sensitivity to CAZ–AVI. This finding highlights the need for monitoring the emergence of CAZ–AVI resistance. The resistance rate of KPC-Kp against IMI–REL was less than 1%, and the MIC₉₀ was far lower than the breakpoint (MIC ≥ 4/4 mg/L), indicating that IMI–REL can inhibit KPC-Kp effectively without showing decreases in sensitivity. The study suggest that both CAZ–AVI and IMI–REL are good choices for the clinical treatment of KPC-Kp, with IMI–REL having a better inhibitory effect potentially (p = 0.004). Although previous studies have indicated that some CAZ–AVI-resistant strains can restore sensitivity to IMI [32], our results show that most CAZ–AVI-resistant strains exhibit high-level resistance to IMI, which further limits the options.

In our study, the most common serotypes were KL64 and KL47 among 782 KPC-Kp strains in our study, consistent with previous reports indicating that ST11-KL64 and ST11-KL47 have become the predominant clones in CRKP clinical isolates [30]. Also, all CAZ–AVI- and IMI–REL-resistant strains belonged to ST11, of which KL47 and KL64 were the most common serotypes. Our results suggest that the potential of CAZ–AVI and IMI–REL resistance in these clones should not be underestimated. We found that 40.9% of the CAZ–AVI- and IMI–REL-resistant strains tested positive in the string test. Notably, these strains with positive string test generally carried both rmpA2 and iucA/B/C/D virulence genes, suggesting they were hypervirulent [33, 34]. Further attention should be paid to the emergence of these resistant and potentially hypervirulent strains. Furthermore, while most antibiotics showed no inhibitory effect on CAZ–AVI- and IMI–REL-resistant strains, our findings suggest that there are still some antibiotics that can be considered for clinical treatment. TGC and PB exhibited inhibition against most resistant strains, MIN inhibited approximately half of the resistant strains, and AMK showed inhibitory effects against some of the strains resistant to both CAZ–AVI and IMI–REL.

Our results confirmed that mutations in KPC contribute differently to the antimicrobial resistance of CAZ–AVI and IMI–REL. Specifically, KPC mutations may be associated with high levels of antimicrobial resistance against CAZ–AVI but not IMI–REL. We also demonstrated for the first time that A172V mutations in KPC could lead to CAZ–AVI resistance while maintaining IMI resistance. Among the CAZ–AVI-resistant strains, all strains with mutant KPC had a high level of resistance to CAZ–AVI. The most common mutation observed was D179Y, which is also widely reported around the world [35, 36]. Previous study has demonstrated that the D179Y mutation in KPC leads to CAZ–AVI resistance but restores the sensitivity of the strains to IMI [37], which consistent with our results. The mutation A172V was found during the induction of CAZ–AVI resistance in CAZ–AVI-sensitive KPC-Kp strains in vitro in previous studies [11], suggesting that it is likely to be a potentially high-occurring mutation. Notably, we confirmed that the A172V mutation in KPC not only confers resistance to CAZ–AVI but also maintains resistance to IMI. Combined with the potentially high incidence of A172V mutations in KPC, the A172V mutation may pose a greater threat to public health. Thus, further research should be conducted on this mutation in the future. Additionally, compared with wild-type KPC, the increase in the MIC value against CAZ–AVI caused by the A172V mutation was much higher than that against CAZ, suggesting that the mutation mainly affects the activity of AVI rather than CAZ. In contrast, no KPC mutations were detected in the IMI–REL-resistant strains in our study. Only one study has mentioned that mutant KPC may cause resistance to IMI–REL [13], whereas most studies investigating the mechanism of IMI–REL resistance have not detected KPC mutations [38, 39], which aligns with our results and suggests that KPC mutations are not the primary mechanism of IMI–REL resistance.

Mutations in OmpK36 were found to be the main mechanisms of IMI–REL resistance, and these mutations also confers resistance to CAZ–AVI simultaneously. OmpK36 serves as the major porin in Klebsiella pneumoniae that facilitates antibiotic influx [40]. Mutations in OmpK36 may prevent antibiotics from entering cells, thus leading to antimicrobial resistance. Our findings showed that the same mutation in OmpK36 (134 to 135 GD insertion) was detected in all the resistant and sensitive KPC-Kp strains, suggesting that this mutation is not the main cause of resistance to CAZ–AVI and IMI–REL. Previous studies have identified this common mutation in CRKP, which is consistent with our results [41]. Of the six IMI–REL-resistant strains, additional mutations of OmpK36 were detected in five strains (80%), suggesting that these additional mutations of OmpK36 may be the key factors contributing to IMI–REL resistance.

Previous studies have speculated that OmpK36 mutations may be associated with IMI–REL resistance; however, it has not been confirmed [42]. We found three new mutations in OmpK36 and further confirmed that these mutations could lead to CAZ–AVI and IMI–REL resistance in KPC-Kp. After restoring the function of wild-type OmpK36, the MIC of CAZ–AVI and IMI–REL decreased in a similar extent. However, the extent of the decline in the MIC values of CAZ and IMI was significantly lower than that of CAZ–AVI and IMI–REL, suggesting that this mechanism may have a similar contribution to the resistance of CAZ–AVI and IMI–REL and may affect the entry of CAZ, AVI, IMI, and REL into cells.

Our study suggests that the D343N mutation in PBP3 does not confer resistance in KPC-Kp strains against CAZ–AVI and IMI–REL. Studies have demonstrated that a four-amino-acid insertion after residue 333 in PBP3 can significantly augment CAZ–AVI resistance in Escherichia coli [11, 43]. The single point mutation observed in PBP3 during this study did not affect the resistance to CAZ–AVI, possibly because this mutation did not result in a significant change in the protein’s conformation or function. However, there is no research can confirm that PBP3 mutations can lead to resistance to IMI–REL currently, because in fact, CAZ exhibits the strongest inhibitory effect on PBP3 in Klebsiella pneumoniae, while IMI has weaker affinity towards PBP3 and stronger affinity towards PBP2 [35]. Thus, further researches are required to determine whether other mutations in PBP3 can result in resistance to IMI–REL.

We also found that the expression of bla_KPC was significantly higher in CAZ–AVI- and IMI–REL-resistant strains than in sensitive strains, suggesting that the overexpression of bla_KPC is a co-mechanism of resistance of CAZ–AVI and IMI–REL. Such overexpression is widely observed in CAZ–AVI- and IMI–REL-resistant strains. The expression levels of bla_KPC increased as the MIC of CAZ–AVI increased, suggesting that an increase in expression of bla_KPC may play an important role in most strains with decreased sensitivity to CAZ–AVI. Previous studies have also observed an increase in bla_KPC expression in CAZ–AVI-resistant strains, with a decreased sensitivity to CAZ–AVI [44]. For IMI–REL, when the MIC was less than 8 mg/L, the expression of bla_KPC increased with increasing MIC values. However, when the MIC was higher than 8 mg/L, the expression level of bla_KPC was slightly lower than that in strains with MIC of 2 or 4 mg/L. This may be because complete resistance to IMI-REL primarily related to OmpK36 mutations and the strain may reduce the expression of bla_KPC to reduce the fitness costs. Therefore, additional strains should be collected for further verification. In our study, increasing concentrations of AVI and REL were able to reduce the MIC value of CAZ–AVI and IMI–REL, which further demonstrates that the current β-lactamase inhibitor is not sufficient to inhibit the increased expression of bla_KPC.

Efflux pumps were considered as an insignificant mechanism of resistance to CAZ–AVI or IMI–REL because the addition of efflux pump inhibitors in 21 and 6 strains resistant to CAZ–AVI and IMI–REL only led to a significant decrease in the MIC values of 3 and 2 strains, respectively. Previous studies have also reported that the overexpression of efflux pumps is one of the resistance mechanisms against IMI and CAZ but not the main mechanism [45, 46]. The addition of efflux pump inhibitors led to substantially lower MICs of CAZ and IMI compared with those of CAZ–AVI or IMI–REL, suggesting that efflux pumps may only be effective for CAZ and IMI but not for AVI or REL.

In addition, a total of five varieties of ESBL genes and one ampC gene were detected in 22 CAZ–AVI and/or IMI–REL-resistant KPC-Kp strains in our study. However, no correlation between these genes and resistance to CAZ–AVI and/or IMI–REL-resistant has been observed in this study. Xiong L et al. previously summarized that mutations in certain genes of ESBL and AmpC enzyme (such as bla_CTX-M-14 and bla_CTX-M-15) contributed to resistance to CAZ–AVI [46]. However, to our knowledge, no studies have reported the association between the detected ESBL and ampC genes in our study and the increased resistance to CZA–AVI or/and IMI–REL. We propose it is highly unlikely that the detected ESBL and ampC genes in our study increased the resistance to CZA–AVI or/and IMI–REL. Further exploration is needed in future studies.

Nonetheless, Our study presented several limitations. First, given the diversity of the sample sources, capsular serotypes and drug-resistant phenotypes of strains collected in our study, while we have done our best to exclude excessive outbreak strains among the 782 KPC-Kp strains to avoid a skew of this study, it cannot be accurately guaranteed that there were no outbreak strains in the 782 KPC-Kp strains. Second, the KPC-Kp strains in our study were only collected from three hospitals in Guangzhou, China, future studies should include more hospitals located in more cities to improve the generalizability of the findings.

In conclusion, our research shows that both CAZ–AVI and IMI–REL can effectively inhibit KPC-Kp. However, IMI–REL may had superior inhibitory effects on KPC-Kps in vitro than CAZ–AVI, and the increase in CAZ–AVI resistance rate needs further attention. We found that various resistance mechanisms did not contribute equally to resistance to CAZ–AVI and IMI–REL. Mutations in KPC may be associated with high-level resistance to CAZ–AVI; mutations in OmpK36 may represent the main factor underlying IMI–REL resistance, and confers the resistance to CAZ–AVI simultaneously; bla_KPC upregulation may play an important role in both CAZ–AVI and IMI–REL resistance. Notably, we identified and verified the effect of some new mutation sites on CAZ–AVI and IMI–REL resistance and demonstrated for the first time that the A172V mutation of KPC confers resistance to CAZ–AVI while maintaining resistance to IMI.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 53 KB)^{(52.8KB, docx)}

Supplementary file2 (DOCX 96 KB)^{(96.4KB, docx)}

Supplementary file3 (DOCX 463 KB)^{(462.9KB, docx)}

Supplementary file4 (DOCX 17 KB)^{(16.8KB, docx)}

Supplementary file5 (DOCX 18 KB)^{(17.8KB, docx)}

Supplementary file6 (DOCX 20 KB)^{(20.2KB, docx)}

Supplementary file7 (DOCX 20 KB)^{(19.7KB, docx)}

Supplementary file8 (DOCX 22 KB)^{(22.2KB, docx)}

Acknowledgements

Thanks for MSD China provided relebactam powder.

Abbreviations

CAZ–AVI: Ceftazidime–avibactam
IMI–REL: Imipenem–relebactam
CRKP: Carbapenem-resistant Klebsiella pneumoniae
KPCs: Klebsiella pneumoniae Carbapenemases
KPC-Kp: KPC-producing Klebsiella pneumoniae
WGS: Whole-genome sequencing
AMP: Ampicillin
AZT: Aztreonam
LEV: Levofloxacin
MIN: Minocycline
CIP: Ciprofloxacin
GM: Gentamycin
AMK: Amikacin
TGC: Tigecycline
CLSI: Clinical and laboratory standardization institute
MLST: Multilocus sequence typing
CCCP: Carbonyl cyanide m-chlorophenylhydrazine
ESBL: Extended-spectrum β-lactamase

Author contributions

C.Z., Y.G. and Z.L. initiated and designed the study. Y.G., L.Y., J.W., Y.Z. and J.L. performed the experiments and analyses. C.Z., Y.G., L.Y., J.W., Y.L., N.H. and J.C. collected and provided the bacterial isolates. Y.G. wrote the manuscript with input from X.Y., Y.W., S.X., Z.L. and C.Z.. Y.G., J.W., L.Y., Y.Z. and Z.L. finished the revised manuscript. All of the authors reviewed the manuscript.

Funding

This work was supported by the State Key Project of Research and Development Plan (grant number 2024YFE0106200) and High-level Scientific Research Project of People’s Hospital of Yangjiang (grant number G2021003).

Data Availability

All sequences of the strains included in this study are available under BioProject accession number PRJNA1019939.

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Yingyi Guo, Likang Yao, Jiong Wang, Yan Zhang are co-first author.

Contributor Information

Zhiwei Lin, Email: 422156321@qq.com.

Chao Zhuo, Email: chaosheep@gzhmu.edu.cn.

References

1.Hu FP, Guo Y, Zhu DM, Wang F, Jiang XF, Xu YC, et al. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005–2014. Clin Microbiol Infect. 2016;22(Suppl 1):S9-14. 10.1016/j.cmi.2016.01.001. [DOI] [PubMed] [Google Scholar]
2.CHINETS. Chinet 2020 bacterial drug resistance monitoring results (full year). http://www.chinets.com/Document. Accessed 22 Feb 2021.
3.Xu L, Sun X, Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann Clin Microbiol Antimicrob. 2017;16(1):18. 10.1186/s12941-017-0191-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Zhang Y, Wang Q, Yin Y, Chen H, Jin L, Gu B, et al. Epidemiology of carbapenem-resistant Enterobacteriaceae infections: report from the China CRE network. Antimicrob Agents Chemother. 2018;62(2):e01882-e1917. 10.1128/AAC.01882-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Yin D, Wu S, Yang Y, Shi Q, Dong D, Zhu D, et al. Results from the China antimicrobial surveillance network (CHINET) in 2017 of the in vitro activities of ceftazidime–avibactam and ceftolozane-tazobactam against clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2019;63(4):e02431-e2518. 10.1128/AAC.02431-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Yang Q, Zhang H, Yu Y, Kong H, Duan Q, Wang Y, et al. In vitro activity of imipenem/relebactam against Enterobacteriaceae isolates obtained from intra-abdominal, respiratory tract, and urinary tract infections in China: study for monitoring antimicrobial resistance trends (SMART), 2015–2018. Clin Infect Dis. 2020;71(Suppl 4):S427–35. 10.1093/cid/ciaa1519. [DOI] [PubMed] [Google Scholar]
7.Paterson DL, Ko WC, Von Gottberg A, Mohapatra S, Casellas JM, Goossens H, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. Clin Infect Dis. 2004;39(1):31–7. 10.1086/420816. [DOI] [PubMed] [Google Scholar]
8.Lucasti C, Vasile L, Sandesc D, Venskutonis D, McLeroth P, Lala M, et al. Phase 2, dose-ranging study of relebactam with imipenem-cilastatin in subjects with complicated intra-abdominal infection. Antimicrob Agents Chemother. 2016;60(10):6234–43. 10.1128/AAC.00633-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Yusuf E, Bax HI, Verkaik NJ, van Westreenen M. An update on eight “new” antibiotics against multidrug-resistant gram-negative bacteria. J Clin Med. 2021;10(5):1068. 10.3390/jcm10051068. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Mashaly ME, Mashaly GE. Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility. Iran J Microbiol. 2021;13(6):785–92. 10.18502/ijm.v13i6.8080. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Guo Y, Liu N, Lin Z, Ba X, Zhuo C, Li F, et al. Mutations in porin LamB contribute to ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae. Emerg Microbes Infect. 2021;10(1):2042–51. 10.1080/22221751.2021.1984182. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Gaibani P, Giani T, Bovo F, Lombardo D, Amadesi S, Lazzarotto T, et al. Resistance to ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam in gram-negative MDR Bacilli: molecular mechanisms and susceptibility testing. Antibiotics (Basel). 2022;11(5):628. 10.3390/antibiotics11050628. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Findlay J, Rens C, Poirel L, Nordmann P. In vitro mechanisms of resistance development to imipenem–relebactam in KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2022;66(10): e0091822. 10.1128/aac.00918-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, et al. wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol. 2013;51(12):4073–8. 10.1128/JCM.01924-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Jolley KA, Maiden MC. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinform. 2010;10(11):595. 10.1186/1471-2105-11-595. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Thirty-one informational supplement. 31th ed; 2021. pp. M100S.
17.The Food and Drug Administration. Tigecycline–injection products [EB/OL]. 2023. https://www.fda.gov/drugs/development-resources/tigecycline-injection-products. Accessed 8 Apr 2023.
18.Silva FA, Conde-Felipe M, Rosario I, Ferrer O, Real F, Déniz S, et al. Susceptibility of Malassezia pachydermatis to aminoglycosides. Mycoses. 2017;60(12):796–9. 10.1111/myc.12665. [DOI] [PubMed] [Google Scholar]
19.Trinetta V, Magossi G, Allard MW, Tallent SM, Brown EW, Lomonaco S. Characterization of Salmonella enterica isolates from selected U.S. swine feed mills by whole-genome sequencing. Foodborne Pathog Dis. 2020;17(2):126–36. 10.1089/fpd.2019.2701. [DOI] [PubMed] [Google Scholar]
20.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
21.Seeimann T. Mlst Github. https://github.com/tseemann/mlst. Accessed 22 Feb 2021.
22.Yao L, Liu N, Guo Y, Zhuo C, Yang X, Wang Y, et al. Comparison of hypervirulent and non-hypervirulent carbapenem-resistant Acinetobacter baumannii isolated from bloodstream infections: mortality, potential virulence factors, and combination therapy in vitro. Antibiotics (Basel). 2024;13(9):807. 10.3390/antibiotics13090807. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Olawoye IB, Frost SDW, Happi CT. The bacteria genome pipeline (BAGEP): an automated, scalable workflow for bacteria genomes with Snakemake. PeerJ. 2020;27(8): e10121. 10.7717/peerj.10121. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Sun L, Li H, Wang Q, Liu Y, Cao B. Increased gene expression and copy number of mutated blaKPC lead to high-level ceftazidime/avibactam resistance in Klebsiella pneumoniae. BMC Microbiol. 2021;21(1):230. 10.1186/s12866-021-02293-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC Bioinform. 2006;22(7):85. 10.1186/1471-2105-7-85. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Naha S, Sands K, Mukherjee S, Roy C, Rameez MJ, Saha B, et al. KPC-2-producing Klebsiella pneumoniae ST147 in a neonatal unit: clonal isolates with differences in colistin susceptibility attributed to AcrAB-TolC pump. Int J Antimicrob Agents. 2020;55(3): 105903. 10.1016/j.ijantimicag.2020.105903. [DOI] [PubMed] [Google Scholar]
27.Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, et al. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol. 2018;84(23):e01834-e1918. 10.1128/AEM.01834-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Budia-Silva M, Kostyanev T, Ayala-Montaño S, Bravo-Ferrer Acosta J, Garcia-Castillo M, Cantón R, et al. International and regional spread of carbapenem-resistant Klebsiella pneumoniae in Europe. Nat Commun. 2024;15(1):5092. 10.1038/s41467-024-49349-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Liao W, Liu Y, Zhang W. Virulence evolution, molecular mechanisms of resistance and prevalence of ST11 carbapenem-resistant Klebsiella pneumoniae in China: a review over the last 10 years. J Glob Antimicrob Resist. 2020;23:174–80. 10.1016/j.jgar.2020.09.004. [DOI] [PubMed] [Google Scholar]
30.Hu F, Pan Y, Li H, Han R, Liu X, Ma R, et al. Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study. Nat Microbiol. 2024;9(3):814–29. 10.1038/s41564-024-01612-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Ferous S, Anastassopoulou C, Pitiriga V, Vrioni G, Tsakris A. Antimicrobial and diagnostic stewardship of the novel β-lactam/β-lactamase inhibitors for infections due to carbapenem-resistant Enterobacterales Species and Pseudomonas aeruginosa. Antibiotics (Basel). 2024;13(3):285. 10.3390/antibiotics13030285. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Cano Á, Guzmán-Puche J, García-Gutiérrez M, Castón JJ, Gracia-Ahufinger I, Pérez-Nadales E, et al. Use of carbapenems in the combined treatment of emerging ceftazidime/avibactam-resistant and carbapenem-susceptible KPC-producing Klebsiella pneumoniae infections: report of a case and review of the literature. J Glob Antimicrob Resist. 2020;22:9–12. 10.1016/j.jgar.2019.11.007. [DOI] [PubMed] [Google Scholar]
33.Russo TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J Clin Microbiol. 2018;56(9):e00776-e818. 10.1128/JCM.00776-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Harada S, Aoki K, Yamamoto S, Ishii Y, Sekiya N, Kurai H, et al. Clinical and molecular characteristics of Klebsiella pneumoniae isolates causing bloodstream infections in Japan: occurrence of hypervirulent infections in health care. J Clin Microbiol. 2019;57(11):e01206-e1219. 10.1128/JCM.01206-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Alsenani TA, Viviani SL, Kumar V, Taracila MA, Bethel CR, Barnes MD, et al. Structural characterization of the D179N and D179Y variants of KPC-2 β-lactamase: Ω-loop destabilization as a mechanism of resistance to ceftazidime–avibactam. Antimicrob Agents Chemother. 2022;66(4): e0241421. 10.1128/aac.02414-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zhang P, Shi Q, Hu H, Hong B, Wu X, Du X, et al. Emergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China. Clin Microbiol Infect. 2020;26(1):124.e1-124.e4. 10.1016/j.cmi.2019.08.020. [DOI] [PubMed] [Google Scholar]
37.Di Bella S, Giacobbe DR, Maraolo AE, Viaggi V, Luzzati R, Bassetti M, et al. Resistance to ceftazidime/avibactam in infections and colonisations by KPC-producing Enterobacterales: a systematic review of observational clinical studies. J Glob Antimicrob Resist. 2021;25:268–81. 10.1016/j.jgar.2021.04.001. [DOI] [PubMed] [Google Scholar]
38.Gomis-Font MA, Cabot G, López-Argüello S, Zamorano L, Juan C, Moyá B, et al. Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones. J Antimicrob Chemother. 2022;77(4):957–68. 10.1093/jac/dkab496. [DOI] [PubMed] [Google Scholar]
39.Johnston BD, Thuras P, Porter SB, Anacker M, VonBank B, Vagnone PS, et al. Activity of imipenem–relebactam against carbapenem-resistant Escherichia coli isolates from the United States in relation to clonal background, resistance genes, coresistance, and region. Antimicrob Agents Chemother. 2020;64(5):e02408-e2419. 10.1128/AAC.02408-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Wong JLC, David S, Sanchez-Garrido J, Woo JZ, Low WW, Morecchiato F, et al. Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory OmpK36 mRNA secondary structure. Proc Natl Acad Sci U S A. 2022;119(38): e2203593119. 10.1073/pnas.2203593119. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Cui X, Shan B, Zhang X, Qu F, Jia W, Huang B, et al. Reduced ceftazidime–avibactam susceptibility in KPC-producing Klebsiella pneumoniae from patients without ceftazidime–avibactam use history - a multicenter study in China. Front Microbiol. 2020;23(11):1365. 10.3389/fmicb.2020.01365. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Balabanian G, Rose M, Manning N, Landman D, Quale J. Effect of porins and bla_KPC expression on activity of imipenem with relebactam in Klebsiella pneumoniae: can antibiotic combinations overcome resistance? Microb Drug Resist. 2018;24(7):877–81. 10.1089/mdr.2018.0065. [DOI] [PubMed] [Google Scholar]
43.Zhang Y, Kashikar A, Brown CA, Denys G, Bush K. Unusual Escherichia coli PBP 3 insertion sequence identified from a collection of carbapenem-resistant Enterobacteriaceae tested in vitro with a combination of ceftazidime-, ceftaroline-, or aztreonam-avibactam. Antimicrob Agents Chemother. 2017;61(8):e00389-e417. 10.1128/AAC.00389-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Zhu Y, Chen J, Shen H, Chen Z, Yang QW, Zhu J, et al. Emergence of ceftazidime- and avibactam-resistant Klebsiella pneumoniae carbapenemase-producing Pseudomonas aeruginosa in China. mSystems. 2021;6(6):e0078721. 10.1128/mSystems.00787-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Hassuna NA, Darwish MK, Sayed M, Ibrahem RA. Molecular epidemiology and mechanisms of high-level resistance to meropenem and imipenem in Pseudomonas aeruginosa. Infect Drug Resist. 2020;30(13):285–93. 10.2147/IDR.S233808. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Xiong L, Wang X, Wang Y, Yu W, Zhou Y, Chi X, et al. Molecular mechanisms underlying bacterial resistance to ceftazidime/avibactam. WIREs Mech Dis. 2022;14(6): e1571. 10.1002/wsbm.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 53 KB)^{(52.8KB, docx)}

Supplementary file2 (DOCX 96 KB)^{(96.4KB, docx)}

Supplementary file3 (DOCX 463 KB)^{(462.9KB, docx)}

Supplementary file4 (DOCX 17 KB)^{(16.8KB, docx)}

Supplementary file5 (DOCX 18 KB)^{(17.8KB, docx)}

Supplementary file6 (DOCX 20 KB)^{(20.2KB, docx)}

Supplementary file7 (DOCX 20 KB)^{(19.7KB, docx)}

Supplementary file8 (DOCX 22 KB)^{(22.2KB, docx)}

Data Availability Statement

All sequences of the strains included in this study are available under BioProject accession number PRJNA1019939.

[CR1] 1.Hu FP, Guo Y, Zhu DM, Wang F, Jiang XF, Xu YC, et al. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005–2014. Clin Microbiol Infect. 2016;22(Suppl 1):S9-14. 10.1016/j.cmi.2016.01.001. [DOI] [PubMed] [Google Scholar]

[CR2] 2.CHINETS. Chinet 2020 bacterial drug resistance monitoring results (full year). http://www.chinets.com/Document. Accessed 22 Feb 2021.

[CR3] 3.Xu L, Sun X, Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann Clin Microbiol Antimicrob. 2017;16(1):18. 10.1186/s12941-017-0191-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Zhang Y, Wang Q, Yin Y, Chen H, Jin L, Gu B, et al. Epidemiology of carbapenem-resistant Enterobacteriaceae infections: report from the China CRE network. Antimicrob Agents Chemother. 2018;62(2):e01882-e1917. 10.1128/AAC.01882-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Yin D, Wu S, Yang Y, Shi Q, Dong D, Zhu D, et al. Results from the China antimicrobial surveillance network (CHINET) in 2017 of the in vitro activities of ceftazidime–avibactam and ceftolozane-tazobactam against clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2019;63(4):e02431-e2518. 10.1128/AAC.02431-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Yang Q, Zhang H, Yu Y, Kong H, Duan Q, Wang Y, et al. In vitro activity of imipenem/relebactam against Enterobacteriaceae isolates obtained from intra-abdominal, respiratory tract, and urinary tract infections in China: study for monitoring antimicrobial resistance trends (SMART), 2015–2018. Clin Infect Dis. 2020;71(Suppl 4):S427–35. 10.1093/cid/ciaa1519. [DOI] [PubMed] [Google Scholar]

[CR7] 7.Paterson DL, Ko WC, Von Gottberg A, Mohapatra S, Casellas JM, Goossens H, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. Clin Infect Dis. 2004;39(1):31–7. 10.1086/420816. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Lucasti C, Vasile L, Sandesc D, Venskutonis D, McLeroth P, Lala M, et al. Phase 2, dose-ranging study of relebactam with imipenem-cilastatin in subjects with complicated intra-abdominal infection. Antimicrob Agents Chemother. 2016;60(10):6234–43. 10.1128/AAC.00633-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.Yusuf E, Bax HI, Verkaik NJ, van Westreenen M. An update on eight “new” antibiotics against multidrug-resistant gram-negative bacteria. J Clin Med. 2021;10(5):1068. 10.3390/jcm10051068. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Mashaly ME, Mashaly GE. Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility. Iran J Microbiol. 2021;13(6):785–92. 10.18502/ijm.v13i6.8080. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Guo Y, Liu N, Lin Z, Ba X, Zhuo C, Li F, et al. Mutations in porin LamB contribute to ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae. Emerg Microbes Infect. 2021;10(1):2042–51. 10.1080/22221751.2021.1984182. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Gaibani P, Giani T, Bovo F, Lombardo D, Amadesi S, Lazzarotto T, et al. Resistance to ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam in gram-negative MDR Bacilli: molecular mechanisms and susceptibility testing. Antibiotics (Basel). 2022;11(5):628. 10.3390/antibiotics11050628. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Findlay J, Rens C, Poirel L, Nordmann P. In vitro mechanisms of resistance development to imipenem–relebactam in KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2022;66(10): e0091822. 10.1128/aac.00918-22. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, et al. wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol. 2013;51(12):4073–8. 10.1128/JCM.01924-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Jolley KA, Maiden MC. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinform. 2010;10(11):595. 10.1186/1471-2105-11-595. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Thirty-one informational supplement. 31th ed; 2021. pp. M100S.

[CR17] 17.The Food and Drug Administration. Tigecycline–injection products [EB/OL]. 2023. https://www.fda.gov/drugs/development-resources/tigecycline-injection-products. Accessed 8 Apr 2023.

[CR18] 18.Silva FA, Conde-Felipe M, Rosario I, Ferrer O, Real F, Déniz S, et al. Susceptibility of Malassezia pachydermatis to aminoglycosides. Mycoses. 2017;60(12):796–9. 10.1111/myc.12665. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Trinetta V, Magossi G, Allard MW, Tallent SM, Brown EW, Lomonaco S. Characterization of Salmonella enterica isolates from selected U.S. swine feed mills by whole-genome sequencing. Foodborne Pathog Dis. 2020;17(2):126–36. 10.1089/fpd.2019.2701. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]

[CR21] 21.Seeimann T. Mlst Github. https://github.com/tseemann/mlst. Accessed 22 Feb 2021.

[CR22] 22.Yao L, Liu N, Guo Y, Zhuo C, Yang X, Wang Y, et al. Comparison of hypervirulent and non-hypervirulent carbapenem-resistant Acinetobacter baumannii isolated from bloodstream infections: mortality, potential virulence factors, and combination therapy in vitro. Antibiotics (Basel). 2024;13(9):807. 10.3390/antibiotics13090807. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Olawoye IB, Frost SDW, Happi CT. The bacteria genome pipeline (BAGEP): an automated, scalable workflow for bacteria genomes with Snakemake. PeerJ. 2020;27(8): e10121. 10.7717/peerj.10121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Sun L, Li H, Wang Q, Liu Y, Cao B. Increased gene expression and copy number of mutated blaKPC lead to high-level ceftazidime/avibactam resistance in Klebsiella pneumoniae. BMC Microbiol. 2021;21(1):230. 10.1186/s12866-021-02293-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC Bioinform. 2006;22(7):85. 10.1186/1471-2105-7-85. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Naha S, Sands K, Mukherjee S, Roy C, Rameez MJ, Saha B, et al. KPC-2-producing Klebsiella pneumoniae ST147 in a neonatal unit: clonal isolates with differences in colistin susceptibility attributed to AcrAB-TolC pump. Int J Antimicrob Agents. 2020;55(3): 105903. 10.1016/j.ijantimicag.2020.105903. [DOI] [PubMed] [Google Scholar]

[CR27] 27.Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, et al. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol. 2018;84(23):e01834-e1918. 10.1128/AEM.01834-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Budia-Silva M, Kostyanev T, Ayala-Montaño S, Bravo-Ferrer Acosta J, Garcia-Castillo M, Cantón R, et al. International and regional spread of carbapenem-resistant Klebsiella pneumoniae in Europe. Nat Commun. 2024;15(1):5092. 10.1038/s41467-024-49349-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Liao W, Liu Y, Zhang W. Virulence evolution, molecular mechanisms of resistance and prevalence of ST11 carbapenem-resistant Klebsiella pneumoniae in China: a review over the last 10 years. J Glob Antimicrob Resist. 2020;23:174–80. 10.1016/j.jgar.2020.09.004. [DOI] [PubMed] [Google Scholar]

[CR30] 30.Hu F, Pan Y, Li H, Han R, Liu X, Ma R, et al. Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study. Nat Microbiol. 2024;9(3):814–29. 10.1038/s41564-024-01612-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR31] 31.Ferous S, Anastassopoulou C, Pitiriga V, Vrioni G, Tsakris A. Antimicrobial and diagnostic stewardship of the novel β-lactam/β-lactamase inhibitors for infections due to carbapenem-resistant Enterobacterales Species and Pseudomonas aeruginosa. Antibiotics (Basel). 2024;13(3):285. 10.3390/antibiotics13030285. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Cano Á, Guzmán-Puche J, García-Gutiérrez M, Castón JJ, Gracia-Ahufinger I, Pérez-Nadales E, et al. Use of carbapenems in the combined treatment of emerging ceftazidime/avibactam-resistant and carbapenem-susceptible KPC-producing Klebsiella pneumoniae infections: report of a case and review of the literature. J Glob Antimicrob Resist. 2020;22:9–12. 10.1016/j.jgar.2019.11.007. [DOI] [PubMed] [Google Scholar]

[CR33] 33.Russo TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J Clin Microbiol. 2018;56(9):e00776-e818. 10.1128/JCM.00776-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Harada S, Aoki K, Yamamoto S, Ishii Y, Sekiya N, Kurai H, et al. Clinical and molecular characteristics of Klebsiella pneumoniae isolates causing bloodstream infections in Japan: occurrence of hypervirulent infections in health care. J Clin Microbiol. 2019;57(11):e01206-e1219. 10.1128/JCM.01206-19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Alsenani TA, Viviani SL, Kumar V, Taracila MA, Bethel CR, Barnes MD, et al. Structural characterization of the D179N and D179Y variants of KPC-2 β-lactamase: Ω-loop destabilization as a mechanism of resistance to ceftazidime–avibactam. Antimicrob Agents Chemother. 2022;66(4): e0241421. 10.1128/aac.02414-21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Zhang P, Shi Q, Hu H, Hong B, Wu X, Du X, et al. Emergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China. Clin Microbiol Infect. 2020;26(1):124.e1-124.e4. 10.1016/j.cmi.2019.08.020. [DOI] [PubMed] [Google Scholar]

[CR37] 37.Di Bella S, Giacobbe DR, Maraolo AE, Viaggi V, Luzzati R, Bassetti M, et al. Resistance to ceftazidime/avibactam in infections and colonisations by KPC-producing Enterobacterales: a systematic review of observational clinical studies. J Glob Antimicrob Resist. 2021;25:268–81. 10.1016/j.jgar.2021.04.001. [DOI] [PubMed] [Google Scholar]

[CR38] 38.Gomis-Font MA, Cabot G, López-Argüello S, Zamorano L, Juan C, Moyá B, et al. Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones. J Antimicrob Chemother. 2022;77(4):957–68. 10.1093/jac/dkab496. [DOI] [PubMed] [Google Scholar]

[CR39] 39.Johnston BD, Thuras P, Porter SB, Anacker M, VonBank B, Vagnone PS, et al. Activity of imipenem–relebactam against carbapenem-resistant Escherichia coli isolates from the United States in relation to clonal background, resistance genes, coresistance, and region. Antimicrob Agents Chemother. 2020;64(5):e02408-e2419. 10.1128/AAC.02408-19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Wong JLC, David S, Sanchez-Garrido J, Woo JZ, Low WW, Morecchiato F, et al. Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory OmpK36 mRNA secondary structure. Proc Natl Acad Sci U S A. 2022;119(38): e2203593119. 10.1073/pnas.2203593119. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR41] 41.Cui X, Shan B, Zhang X, Qu F, Jia W, Huang B, et al. Reduced ceftazidime–avibactam susceptibility in KPC-producing Klebsiella pneumoniae from patients without ceftazidime–avibactam use history - a multicenter study in China. Front Microbiol. 2020;23(11):1365. 10.3389/fmicb.2020.01365. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Balabanian G, Rose M, Manning N, Landman D, Quale J. Effect of porins and bla_KPC expression on activity of imipenem with relebactam in Klebsiella pneumoniae: can antibiotic combinations overcome resistance? Microb Drug Resist. 2018;24(7):877–81. 10.1089/mdr.2018.0065. [DOI] [PubMed] [Google Scholar]

[CR43] 43.Zhang Y, Kashikar A, Brown CA, Denys G, Bush K. Unusual Escherichia coli PBP 3 insertion sequence identified from a collection of carbapenem-resistant Enterobacteriaceae tested in vitro with a combination of ceftazidime-, ceftaroline-, or aztreonam-avibactam. Antimicrob Agents Chemother. 2017;61(8):e00389-e417. 10.1128/AAC.00389-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Zhu Y, Chen J, Shen H, Chen Z, Yang QW, Zhu J, et al. Emergence of ceftazidime- and avibactam-resistant Klebsiella pneumoniae carbapenemase-producing Pseudomonas aeruginosa in China. mSystems. 2021;6(6):e0078721. 10.1128/mSystems.00787-21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Hassuna NA, Darwish MK, Sayed M, Ibrahem RA. Molecular epidemiology and mechanisms of high-level resistance to meropenem and imipenem in Pseudomonas aeruginosa. Infect Drug Resist. 2020;30(13):285–93. 10.2147/IDR.S233808. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Xiong L, Wang X, Wang Y, Yu W, Zhou Y, Chi X, et al. Molecular mechanisms underlying bacterial resistance to ceftazidime/avibactam. WIREs Mech Dis. 2022;14(6): e1571. 10.1002/wsbm.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Comparison of the in vitro activities and resistance mechanisms against imipenem–relebactam and ceftazidime–avibactam in clinical KPC-producing Klebsiella pneumoniae isolated in China

Yingyi Guo

Likang Yao

Jiong Wang

Yan Zhang

Chuyue Zhuo

Yijing Wang

Xu Yang

Jiahui Li

Nanhao He

Jiakang Chen

Yexin Lin

Shunian Xiao

Zhiwei Lin

Chao Zhuo

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Introduction

Materials and methods

Bacterial collection

Antimicrobial susceptibility testing

Whole-genome sequencing (WGS) and bioinformatic analysis

Determination of blaKPC expression levels

Efflux pump inhibitor tests and growth curve analysis

Functional verification of new mutations

Statistical analysis

Results

Characteristics of bacteria strains and in vitro antimicrobial susceptibility

Table 1.

Comparison of characteristics of CAZ–AVI resistant and IMI–REL resistant isolates

Table 2.

Mechanisms of antimicrobial resistance in CAZ–AVI- and/or IMI–REL-resistant strains

Analysis of mutation in antimicrobial resistance-related genes

Table 3.

Expression levels of blaKPC

Fig. 1.

Role of efflux pumps in resistance

Effects of new mutations on bacterial resistance to CAZ–AVI and IMI–REL

Table 4.

Table 5.

Discussion

Supplementary Information

Acknowledgements

Abbreviations

Author contributions

Funding

Data Availability

Declarations

Conflict of interest

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Determination of bla_KPC expression levels

Expression levels of bla_KPC