Prevalence and characteristics of ertapenem-mono-resistant isolates among carbapenem-resistant Enterobacterales in China

ABSTRACT

Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

KEYWORDS:

• Carbapenem-resistant Enterobacterales
• ertapenem
• carbapenemase
• antibiotic resistance
• molecular epidemiology

Introduction

Due to limited effective treatment options and high mortality, carbapenem-resistant Enterobacterales (CRE) infections have become an important global public health threat [1,2]. Currently, Enterobacterales strains that are resistant to any carbapenem agents, including meropenem, imipenem and ertapenem, or documented to produce carbapenemase are considered CRE [3]. For Enterobacterales, carbapenem resistance is mainly mediated by carbapenemase enzymes or by noncarbapenemase β-lactamase enzymes combined with impermeability [4].

Ertapenem belongs to group 1 carbapenems and is active against community-acquired gram-positive cocci and gram-negative rods, as well as anaerobes except Pseudomonas aeruginosa and Acinetobacter baumannii [5,6]. Ertapenem, possessing a large anionic side chain and larger size, is less stable to β-lactamases than other carbapenems: the MIC₉₀ for ertapenem increased by up to 8- or 16-fold in extended-spectrum β-lactamase (ESBL)- or AmpC-producing Enterobacterales isolates relative to wild-type isolates, whereas the MIC₉₀ for meropenem and imipenem showed no increase or at most a 2-fold increase [7–9]. Noncarbapenemase resistance mechanisms, including ESBLs and/or AmpC enzymes in combination with membrane permeability mutations, are the predominant mechanisms mediating ertapenem resistance in Enterobacterales [10]. In most cases, these strains are still susceptible or intermediate to meropenem and imipenem, namely, ertapenem-mono-resistant (ETP-mono-resistant) CRE strains [11]. Reports of ETP-mono-resistant CRE strains are emerging more frequently, with a low prevalence of carbapenemase genes in these strains [12,13]. Nonetheless, the presence of carbapenemases in ETP-mono-resistant CRE strains poses challenges to appropriate therapeutic options [12]. For instance, meropenem has been considered an effective treatment option for infections caused by meropenem-susceptible carbapenemase-producing CRE (CP-CRE) isolates, as demonstrated by significantly prolonged survival and valid clinical responses [14,15]. Conversely, another study reported a high clinical or microbiologic failure rate when a carbapenem was administered to patients with imipenem- or meropenem-susceptible CP-CRE infections [16].

In this study, we conducted a randomized matching investigation in a 1:1 ratio to explore the molecular epidemiological characteristics of ETP-mono-resistant CRE strains and to further characterize the CP-CRE isolates with ETP-mono-resistant phenotype.

Materials and methods

Ethics

This study was approved by the Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (20170301-3, 20200831-36).

Strain definition and study population

A CRE strain was defined as an isolate resistant to at least one carbapenem among meropenem, imipenem and ertapenem [3,17]. An ETP-mono-resistant strain was defined as an isolate resistant to ertapenem but susceptible or intermediate to any other carbapenem tested, and the MEM/IPM-resistant CRE strain was defined as an isolate resistant to meropenem and/or imipenem, regardless of its resistance to ertapenem.

A total of 1,354 CRE strains were collected from 102 hospitals in 31 provinces across China between 2018 and 2021. The species of the isolates were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry systems (Skyray Instrument, China). Depending on the species or species complex, an equivalent number of MEM/IPM-resistant CRE strains isolated during the same period were randomly selected as the matched group (MEM/IPM-resistant group).

Antimicrobial susceptibility testing

All the isolates were analysed for susceptibility to ertapenem, imipenem, meropenem, aztreonam, cefepime, ceftazidime, ceftazidime-avibactam, piperacillin-tazobactam, cefoperazone-sulbactam, levofloxacin, ciprofloxacin, tigecycline, fosfomycin, amikacin and polymyxin by agar dilution or microbroth dilution methods according to standard protocols [18]. For antibiotic susceptibility testing (AST) of meropenem, imipenem and ertapenem, the agar dilution method was uniformly performed. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 served as quality controls. US Food and Drug Administration (FDA) guidelines were used to interpret the MIC of tigecycline (susceptible, ≤ 2 mg/L and resistant, ≥ 8 mg/L). Clinical and Laboratory Standards Institute (CLSI) breakpoints were used to interpret the AST results of other antibiotics, while the susceptibility of cefoperazone/sulbactam was reported according to CLSI breakpoints for cefoperazone alone [18].

A spot assay was performed to determine bacterial sensitivity to meropenem as previously described [19], with some modifications. Briefly, the bacterial suspensions with an OD₆₀₀ nm value of 1.0 were 10-fold serially diluted. Then 10 µL of all suspensions were spotted on Mueller-Hinton agar with or without various concentrations of meropenem (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mg/L), respectively. Subsequently, the strains were cultured at 37 °C for 20 h.

Phenotypic detection of carbapenemase production

To evaluate the potency of carbapenem-hydrolysing activity, the original modified carbapenem inactivation method (mCIM) and semiquantitative mCIM with different initial inoculum were performed on ETP-mono-resistant CP-CRE strains according to CLSI standards and methods described previously [14,18]. In brief, a standard 1-µL loopful of bacteria from an overnight Mueller-Hinton agar plate with or without 16 mg/L meropenem was emulsified in 2 mL tryptic soy broth (TSB) and further diluted with TSB to 1:10, 1:100, 1:500 and 1:1000 suspensions. Next, a 10-µg meropenem disk was incubated in 2 mL of the respective suspension for 4 h at 37 °C and then placed on Mueller-Hinton agar that was previously inoculated with 0.5 McFarland E. coli ATCC 25922. Instead of categorical results (positive or negative), the zones of inhibition after incubation at 37 °C for 18 h were measured to estimate the potency of carbapenem-hydrolysing activity.

Whole-genome sequencing and sequence analysis

All isolates were subjected to whole-genome sequencing. The genomic DNA of strains was extracted by a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The Illumina HiSeq X-Ten (Illumina, San Diego, USA) platform was used for whole-genome sequencing. The raw reads were de novo assembled using Shovill v0.9.0 (https://github.com/tseemann/shovill). Prokka v1.11 was used to annotate the genome sequence [20]. CGE MLST 2.0 (https://cge.food.dtu.dk/services/MLST/) and mlst (https://github.com/tseemann/mlst) were used for multilocus sequence typing (MLST). The NCBI database (https://www.ncbi.nlm.nih.gov/pathogens/refgene/) was used to discover antimicrobial resistance genes (ARGs) with Abricate v1.0.0 (https://github.com/tseemann/abricate). Additionally, mutations in the promoter region of the chromosomal AmpC gene (bla_EC) were identified with AMRFinderPlus v3.11.26 [21].

Core genome analysis was performed using Panaroo v1.2.10 [22] as well as IQ-TREE v1.6.9 [23], and then the results were visualized using iTOL v6.8.1 (https://itol.embl.de/) [24]. Pairwise core single nucleotide polymorphism (SNP) distances were assessed by snp-dists v0.6.3 (https://github.com/tseemann/snp-dists).

OmpF/OmpK35- and OmpC/OmpK36-encoding genes with absence, frameshift, premature stop codon or truncation, as well as OmpK36 β-strand loop insertions, were defined as resistance-related mutations. To characterize these mutations, an in-house database of E. coli K-12 strain MG1655 ompC and ompF genes, as well as their respective homologs identified in Klebsiella pneumoniae species complex, Enterobacter cloacae complex, K. aerogenes, K. oxytoca complex and Salmonella enterica was established. Porin mutation analysis was then performed using BLASTn and BLASTp from BLAST + 2.5.0 [25] with reference to the in-house database.

Reverse transcription-quantitative PCR

E. coli strains containing bla_NDM-5 gene (n = 7 in each group) and K. pneumoniae species complex strains harbouring bla_KPC-2 (n = 12 in the ETP-mono-resistant group, n = 6 in the MEM/IPM-resistant group) in both groups were selected for reverse transcription-quantitative PCR (Table S3). Total RNA was extracted at the bacterial mid-exponential phase by E.Z.N.A. total RNA Kit I (Omega Bio-Tek, GA, USA). Reverse transcription and quantitative PCR were conducted using the PrimeScript RT reagent Kit and TB green Premix Ex Taq (TaKaRa, Beijing, China). Primers targeting bla_KPC, bla_NDM and housekeeping genes are listed in Table S4.

Statistical analysis

The analysis was performed with IBM SPSS 20.0 and GraphPad Prism 8. The chi-square test or Fisher’s exact test was used to test categorical variables, as appropriate. Student’s t test or the Mann‒Whitney U test was used to compare continuous variables according to distribution type. A P value <0.05 was considered statistically significant.

Data availability statement

Illumina sequencing data of isolates were deposited under projects in the NCBI database (accession numbers: PRJNA832474 and PRJNA983945).

Results

Microorganism profiles of ertapenem-mono-resistant CRE strains

In total, 18.8% (254/1354) of CRE strains were ETP-mono-resistant. In addition to one S. enterica isolate, the proportions of ETP-mono-resistant phenotype in the CRE population for different species were as follows: E. coli (37.4%, 123/329), K. pneumoniae species complex (11.2%, 104/930), E. cloacae complex (29.2%, 21/72), K. aerogenes (30.0%, 3/10) and K. oxytoca complex (18.2%, 2/11). Due to our randomized matching design, the species or species complex and corresponding amount in the MEM/IPM-resistant group were consistent with those in the ETP-mono-resistant group (Table 1).

Table 1. Isolate characteristics and comparative analysis of ertapenem-mono-resistant and matched groups.

	ETP-mono-resistant (n = 254), No. (%)	MEM/IPM-resistant (n = 254), No. (%)	P value
Bacterial species
Escherichia coli	123 (48.4)	123 (48.4)
Klebsiella pneumoniae species complex	104 (40.9)	104 (40.9)
Enterobacter cloacae complex	21 (8.3)	21 (8.3)
Klebsiella aerogenes	3 (1.2)	3 (1.2)
Klebsiella oxytoca complex	2 (0.8)	2 (0.8)
Salmonella enterica	1 (0.4)	1 (0.4)
Source			0.979^a
Stool	187 (73.6)	187 (73.6)
Sputum	23 (9.1)	23 (9.1)
Urine	14 (5.5)	15 (5.9)
Blood	12 (4.7)	16 (6.3)
Secretion	6 (2.4)	6 (2.4)
Shunt fluid	6 (2.4)	5 (2.0)
Others^b	3 (1.2)	1 (0.4)
Broncho-alveolar lavage	1 (0.4)	1 (0.4)
Catheter	1 (0.4)	0 (0.0)
Cerebrospinal fluid	1 (0.4)	0 (0.0)
ETP MIC (mg/L)			<0.001^c
<16	193 (76.0)	26 (10.2)
>16	9 (3.5)	188 (74.0)
Resistance rate^c
ETP	254 (100.0)	254 (100.0)
IPM	0 (0.0)	216 (85.0)	<0.001
MEM	0 (0.0)	241 (94.9)	<0.001
AZT	222 (87.4)	209 (82.3)	0.108
FEP	221 (87.0)	247 (97.2)	<0.001
CAZ	216 (85.0)	251 (98.8)	<0.001
CZA	32 (12.6)	139 (54.7)	<0.001
TZP	178 (70.1)	250 (98.4)	<0.001
CPS	214 (84.3)	251 (98.8)	<0.001
LEV	208 (81.9)	226 (89.0)	<0.05
CIP	234 (92.1)	233 (91.7)	0.871
TGC	12 (4.7)	17 (6.7)	0.339
FOS	28 (11.0)	68 (26.8)	<0.001
AMI	40 (15.7)	78 (30.7)	<0.001
POL	7 (2.8)	23 (9.1)	<0.01

^a Determined by Fisher’s exact test.

^b Others included body fluids and fibrous tissue.

^c Determined by Chi-square test.

Abbreviations: ETP-mono-resistant, resistant to only ertapenem among the carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem; ETP, ertapenem; IPM, imipenem; MEM, meropenem; AZT, aztreonam; FEP, cefepime; CAZ, ceftazidime; CZA, ceftazidime-avibactam; TZP, piperacillin-tazobactam; CPS, cefoperazone-sulbactam; LEV, levofloxacin; CIP, ciprofloxacin; TGC, tigecycline; FOS, fosfomycin; AMI, amikacin; POL, polymyxin.

In both groups, the most prevalent sources of strains were stool (73.6%), followed by sputum (9.1%), urine (ETP-mono-resistant: 5.5%; MEM/IPM-resistant: 5.9%) and blood (ETP-mono-resistant: 4.7%; MEM/IPM-resistant: 6.3%) (Table 1).

Carbapenems MIC distributions and antibiotic susceptibility profile

For ETP-mono-resistant strains, the MIC values of ertapenem ranged from 2 to 128 mg/L, with MIC₅₀ and MIC₉₀ values of 8 and 16 mg/L, respectively (Figure 1A). For both imipenem and meropenem in the ETP-mono-resistant group, the MIC₅₀ and MIC₉₀ were 0.5 and 2 mg/L, respectively (Figure 1B and C). For strains of the MEM/IPM-resistant group, ertapenem MIC values ranged from 4 to >128 mg/L (MIC₅₀= 32 mg/L and MIC₉₀> 128 mg/L), imipenem MIC values ranged from 0.25 to >128 mg/L (MIC₅₀= 8 mg/L and MIC₉₀= 64 mg/L) and meropenem MIC values ranged from 1 to >128 mg/L (MIC₅₀= 8 mg/L and MIC₉₀= 128 mg/L) (Figure 1A-C).

Figure 1. Carbapenem MIC distribution curves. (A, B and C) Carbapenem MIC distributions of strains in the two groups. (D, E and F) Carbapenem MIC distributions of strains in the two groups stratified by whether carbapenemase was produced. ETP, ertapenem; IPM, imipenem; MEM, meropenem; ETP-mono-resistant, resistant to only ertapenem among carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem; CRE, carbapenem-resistant Enterobacterales; CP-CRE, carbapenemase-producing CRE; non-CP-CRE, non-carbapenemase-producing CRE.

Although the ertapenem MIC values of both groups were ≥2 mg/L (CLSI resistant breakpoint for ertapenem), Figure 1 (A) shows a significant difference in the distribution of results for strains of different groups. For the ETP-mono-resistant group, the proportion of strains with an ertapenem MIC value >16 mg/L was 3.5%. While for the MEM/IPM-resistant group, the percentage for >16 mg/L was 74.0% (P < 0.001, Table 1). An ertapenem MIC value >16 mg/L may serve as an indicator to distinguish ETP-mono-resistant strains from MEM/IPM-resistant CRE strains.

Besides, ETP-mono-resistant strains were more likely to be susceptible or intermediate to β-lactams and β-lactam/β-lactamase inhibitor combinations (BLBLIs), including cefepime, ceftazidime, ceftazidime/avibactam, piperacillin/tazobactam and cefoperazone/sulbactam, compared with MEM/IPM-resistant strains (P < 0.05, Table 1). In addition, these strains exhibited increased susceptibility to levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05, Table 1).

Characterization of the core genome among ETP-mono-resistant and MEM/IPM-resistant CRE isolates

To analyse the correlation between the carbapenem-resistant phenotype and core genome characteristics, phylogenetic analysis was performed. The mean pairwise difference in core SNPs of ETP-mono-resistant E. coli strains was 62,668 SNPs (standard deviation [SD] = 23,964), while it was 64,509 SNPs (SD = 80,751) and 99,610 SNPs (SD = 106,369) for ETP-mono-resistant isolates of K. pneumoniae species complex and E. cloacae complex, respectively. These results indicated high genetic diversity and limited clonal dissemination of ETP-mono-resistant CRE strains (Figure 2 and Table S5).

Figure 2. Bacterial population structure. Maximum likelihood phylogenetic tree of E. coli (A), K. pneumoniae species complex (B) and E. cloacae complex (C) in the two groups. The branch label background corresponds to sequence types (STs), and the outer circle indicates the group. The predominant STs in the two groups are displayed. ETP-mono-resistant, resistant to only ertapenem among the carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem.

We observed that ETP-mono-resistant E. coli strains mainly belonged to sequence types ST648 (13.8%), ST405 (10.6%), ST2003 (8.9%) and ST38 (7.3%). E. coli strains in the MEM/IPM-resistant group were predominantly found in international high-risk clones, including ST167 (8.9%), ST410 (7.3%) and ST617 (5.7%) (Table 2). For K. pneumoniae species complex, the proportion of ST11 in the ETP-mono-resistant group was significantly decreased compared with that in the MEM/IPM-resistant group (23.1% vs. 55.8%, P < 0.001). Additionally, the percentage of ST11-KL64 (currently dominant CRKP type in China) was significantly reduced among ST11 K. pneumoniae strains in the ETP-mono-resistant group (37.5% vs. 62.1%, P < 0.05) (Table 2). The most predominant sequence type of E. cloacae complex isolates in the ETP-mono-resistant group was ST1053 (28.6%). In the MEM/IPM-resistant group, high-risk clones ST171 (19.0%) and ST78 (19.0%) E. cloacae complex isolates were the most common (Table 2). The above observations indicated that multilocus sequence types may be related to the ETP-mono-resistance phenotype, more specifically, ETP-mono-resistant CRE isolates were less likely to manifest as high-risk pandemic clones.

Table 2. Predominant sequence types by species in the two groups.

E. coli, No. (%)				K. pneumoniae species complex, No. (%)				E. cloacae complex, No. (%)
ETP-mono-resistant (n = 123)		MEM/IPM-resistant (n = 123)		ETP-mono-resistant (n = 104)		MEM/IPM-resistant (n = 104)		ETP-mono-resistant (n = 21)		MEM/IPM-resistant (n = 21)
ST648	17 (13.8)	ST167	11 (8.9)	*ST11	24 (23.1)	*ST11	58 (55.8)	ST1053	6 (28.6)	ST171	4 (19.0)
				*ST11-KL64	9 (37.5)	*ST11-KL64	36 (62.1)
ST405	13 (10.6)	ST410	9 (7.3)	ST15	11 (10.6)	ST15	11 (10.6)	ST171	3 (14.3)	ST78	4 (19.0)
ST2003	11 (8.9)	ST617	7 (5.7)	ST37	4 (3.8)	ST307	5 (4.8)	ST78	3 (14.3)	ST116	2 (9.5)
ST38	9 (7.3)	ST156	5 (4.1)	ST45	4 (3.8)	ST485	4 (3.8)	ST45	2 (9.5)	ST114	2 (9.5)

Abbreviations: ETP-mono-resistant, resistant to only ertapenem among the carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem; ST, sequence type; *, P < 0.05 (Chi-square test).

Carbapenem resistance determinants

Based on the genetic determinants associated with carbapenem resistance (excluding bla_EC in E. coli), the entire ETP-mono-resistant group could be categorized into five resistome groups. (i) harboured one of any known carbapenemase genes (n = 47), (ii) harboured any ESBL and/or AmpC gene, combined with porin mutations (n = 101), (iii) harboured any ESBL gene and/or AmpC gene without porin mutations (n = 96), (iv) showed the absence of any ESBL and AmpC gene but contained porin mutations (n = 6), (v) all of the above determinants were absent (n = 4). Notably, mutations in the promoter region of the chromosomal AmpC gene (bla_EC) that can increase gene expression, were not identified in ETP-mono-resistant E. coli isolates. Overall, these observations indicated that the ETP-mono-resistant phenotype was mainly mediated by noncarbapenemase mechanisms, potentially involving ESBL- and/or AmpC-producing as well as porin mutations.

Compared with the MEM/IPM-resistant group, the positive detection rate of carbapenemases in the ETP-mono-resistant group was significantly reduced (18.5% vs. 80.3%, P < 0.001) (Figure 3A, Table S2). bla_NDM was the most commonly detected carbapenemase gene, followed by bla_KPC in both groups. For Klebsiella spp. in these two groups, serine carbapenemases, especially bla_KPC, were mainly detected. For E. coli and other species (E. cloacae complex and S. enterica), metallo-β-lactamase, specifically bla_NDM, was the most common carbapenemase gene in both groups (Figure 3B, Table S2). These were correlated with higher susceptibility to ceftazidime/avibactam of Klebsiella spp. than E. coli and other species in the MEM/IPM-resistant group (Table S1).

Figure 3. Carbapenem resistance genes in the two groups. (A) Comparison of β-lactamase positive rates and porin mutation rates. ETP-mono-resistant, resistant to only ertapenem among the carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem; ESBLs, extended-spectrum β-lactamases; bla_EC genes in E. coli were excluded for the analysis of AmpC cephalosporinases; OmpF/OmpK35- and OmpC/OmpK36-encoding genes with absence, frameshift, premature stop codon or truncation, as well as OmpK36 β-strand loop insertions, were defined as resistance-related mutations; a Chi-square test was performed to determine statistical significance. (B) Carbapenemase genotypes of different species in the two groups. Klebsiella spp. were K. pneumoniae species complex (n = 104), K. aerogenes (n = 3) and K. oxytoca complex (n = 2) strains, while others included E. cloacae complex (n = 21) and S. enterica (n = 1) strains in each group.

In terms of ESBLs, AmpC (excluding bla_EC in E. coli) and porin mutations, the positive detection rates in the ETP-mono-resistant group did not significantly differ from those observed in the MEM/IPM-resistant group (ESBLs: 81.5% vs. 75.2%, P = 0.085; AmpC: 24.8% vs. 23.2%, P = 0.678; porin mutations: 44.5% vs. 50.4%, P = 0.183) (Figure 3A, Table S2). Therefore, it is probable that the presence of carbapenemase was the primary factor accounting for the difference in carbapenem-resistant phenotypes between the two groups.

Characteristics of carbapenemase-producing ETP-mono-resistant isolates

There were 47 unique CP-CRE isolates in the ETP-mono-resistant group, among which, 17 isolates harboured bla_KPC, 20 isolates harboured bla_NDM, 7 isolates harboured bla_IMP-4, 2 isolates harboured bla_OXA-181 and 1 isolate harboured bla_VIM-1 (Figure 3B, Table S2). Except for two strains carrying bla_KPC-2 variants (bla_KPC-160 and bla_KPC-14) that lacked carbapenemase activity, the results of the standard mCIM method for 45 CP-CRE strains in the ETP-mono-resistant group were positive, which indicated functional carbapenemases.

NDM-5 and KPC-2 expression levels were further quantified in selected E. coli and K. pneumoniae species complex isolates, respectively (Table S3). We found that bla_NDM-5 and bla_KPC-2 expression levels were significantly lower in the ETP-mono-resistant group than in the MEM/IPM-resistant group (P < 0.05) (Figure 4B and C). Besides, the positive detection rate of ESBLs in ETP-mono-resistant CP-CRE isolates was significantly decreased compared with CP-CRE isolates in the MEM/IPM-resistant group (57.4% vs. 74.0%, P < 0.05) (Figure 4A). For porin mutations, in contrast to the high prevalence detected in MEM/IPM-resistant CP-CRE isolates, only 12.8% (6/47) of ETP-mono-resistant CP-CRE isolates had at least one ompF/ompK35 or ompC/ompK36 mutation (12.8% vs. 42.6%, P < 0.001), while two isolates harbouring bla_KPC-2 variants had ompF/ompK35 and ompC/ompK36 double mutations (Figure 4A). These indicated that the absence of ESBLs and porin mutations, as well as low expression levels of carbapenemase genes, may be involved in the ETP-mono-resistant phenotype of CP-CRE strains.

Figure 4. Characteristics of carbapenemase-producing ETP-mono-resistant isolates. (A) Comparison of carbapenem resistance genes. ESBLs, extended-spectrum β-lactamases; bla_EC genes in E. coli were excluded for the analysis of AmpC cephalosporinases; OmpF/OmpK35- and OmpC/OmpK36-encoding genes with absence, frameshift, premature stop codon or truncation, as well as OmpK36 β-strand loop insertions, were defined as resistance-related mutations; ETP-mono-resistant, resistant to only ertapenem among the carbapenems tested; MEM/IPM-resistant, resistant to meropenem and/or imipenem; P values were calculated by Chi-square test. (B) bla_NDM-5 transcription comparison in E. coli between ETP-mono-resistant (n = 7) and MEM/IPM-resistant (n = 7) isolates. (C) bla_KPC-2 transcription comparison in K. pneumoniae species complex between ETP-mono-resistant (n = 12) and MEM/IPM-resistant (n = 6) isolates. All experiments were repeated three times from three different RNA preparations. The data presented are the mean with standard deviation (SD). C_T, cycle threshold; P values were calculated by unpaired t test. (D) Growth of 10⁷ CFU/mL bacterial inoculum of ETP-mono-resistant isolates in the presence or absence of various concentrations of meropenem. MEM, meropenem. (E) mCIM tests of carbapenemase-producing ETP-mono-resistant isolates and E. coli ATCC 25922 with different initial inoculum. Std, standard; mCIM, modified carbapenem inactivation method.

To assess the growth of ETP-mono-resistant isolates in the presence of meropenem, a spot assay was performed for selected isolates. The results showed that 10⁷ CFU/mL bacterial inoculum of ETP-mono-resistant CP-CRE isolates, including NDM-producing, KPC-producing, IMP-producing and VIM-producing isolates, grew at meropenem concentrations eightfold above the corresponding MIC values. This phenomenon was not observed in OXA-181-producing and non-carbapenemase-producing CRE (non-CP-CRE) isolates (Figure 4D). Bacteria grown on the agar plate were further subjected to mCIM with different dilutions of a standard 1-µL loop. We observed a relatively increased meropenem-hydrolysing activity of bacteria from the 16 mg/L meropenem agar plate compared with corresponding bacteria from the agar plate without antibiotic, although no significant difference was observed in the VIM-producing isolate (Figure 4E). This indicated that the growth of carbapenemase-producing ETP-mono-resistant isolates at high meropenem concentrations may be attributed to their rapidly enhanced carbapenem-hydrolysing ability.

Discussion

In this study, we systematically reported the molecular epidemiological characteristics of ETP-mono-resistant CRE strains that have unique phenotypic and genotypic features from MEM/IPM-resistant CRE strains, including distinct ertapenem MIC distributions; higher susceptibility to β-lactams, BLBLIs, levofloxacin, fosfomycin, amikacin and polymyxin; high genetic diversity; and a lower positive rate of carbapenemase genes.

To date, few studies have assessed the proportion of ETP-mono-resistant CRE strains in the total CRE population. A large, geographically diverse study in the US found that 62.2% of CRE cases were resistant only to ertapenem among carbapenems [12], while another study conducted in China found that 7.4% of CRKP strains were resistant to ertapenem but susceptible to other carbapenems [13]. In our study, 18.8% of CRE strains were ETP-mono-resistant. Only 2.4% of ETP-mono-resistant CRE isolates possessed any carbapenemase gene in the US study [12], while none of the strains resistant to ertapenem but susceptible to other carbapenems contained a carbapenemase gene in the study conducted in China [13]. In our study, 18.5% of ETP-mono-resistant strains were positive for carbapenemase genes. Due to global and endemic differences in CRE epidemiology, data from other countries are less likely to be related to that from China. For instance, the proportion of CP-CRE strains among ETP-mono-resistant CRE strains may be higher in a country where OXA-48-like carbapenemases are predominant, such as Spain, due to their relatively weak carbapenem-hydrolysing activity [26,27]. To our knowledge, our study is the first large-scale, multi-centre study of ETP-mono-resistant CRE in China. The data presented here extend the findings in China and draw attention to the relatively prevalent ETP-mono-resistant CRE strains.

Our findings supported the idea that the phenotype of resistance to only ertapenem among carbapenems was mainly mediated by noncarbapenemase mechanisms, while the absence of ESBLs and porin mutations, as well as the reduced expression of carbapenemase genes may contribute to the ETP-mono-resistant phenotype of CP-CRE strains. Further studies are needed to explore and confirm the mechanism underlying the low expression level of carbapenemase genes as they had the same upstream promoter regions.

Previous studies have reported that the application of meropenem induces the amplification of bla_KPC gene causing hyperresistance [28], and revealed a high failure when a carbapenem was used to treat imipenem- or meropenem-susceptible KPC-producing CRE infections [16]. According to 2023 IDSA treatment guidance, meropenem or imipenem is the recommended treatment for infections caused by ertapenem-resistant, meropenem- and imipenem-susceptible CRE isolates without carbapenemase production. While cefiderocol or new BLBLIs such as ceftazidime-avibactam are preferred treatment options for CP-CRE infections [29]. Our study provided evidence that ETP-mono-resistant CP-CRE isolates, including NDM-producing, KPC-producing and IMP-producing isolates, can enhance carbapenem-hydrolysing ability and survive under higher meropenem concentrations. This indicated the possibility of clinical and/or microbiologic failure with meropenem monotherapy in ETP-mono-resistant CRE infections, emphasizing the importance of carbapenemase testing, which is also meaningful for the application of new BLBLIs. As for OXA-181-producing isolates, further research is needed to determine if meropenem remains valid for treatment. Additionally, our study revealed that ETP-mono-resistant CRE strains exhibited increased susceptibility to β-lactams, BLBLIs, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant CRE strains. It is crucial for clinical laboratories to perform susceptibility testing for these antibiotics to ensure appropriate treatment for ETP-mono-resistant CRE infection.

From the previous perspective of infection control, non-CP-CRE strains rarely spread clonally and cause epidemic outbreaks, indicating that they may not pose the same dissemination and infection control risk as CP-CRE strains [30,31]. Our results demonstrated that ETP-mono-resistant CRE isolates were less likely to be high-risk pandemic clones and less likely to harbour carbapenemase genes. This indicates that the resources employed for CRE infection control based on antibiotic resistance phenotypes, such as the ETP-mono-resistant phenotype, may be overused or wasted. Whether the definition of CRE should be modified remains questionable, but it is important to emphasize the concept of carbapenemase-producing strains and the detection of carbapenemase.

In conclusion, our study revealed that ETP-mono-resistant strains were relatively prevalent, and had unique molecular epidemiological characteristics compared with MEM/IPM-resistant CRE strains. Notably, ETP-mono-resistant CP-CRE isolates exhibited rapidly enhanced carbapenem-hydrolysing ability and survived at high meropenem concentrations. Caution should be exercised in the administration of meropenem as a treatment option for ETP-mono-resistant CRE infections, while the detection of carbapenemase should be prioritized. Further investigations are warranted to inform the optimal treatment options for ETP-mono-resistant CRE infections.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was funded by the National Natural Science Foundation of China [grant no 81830069], the Natural Science Foundation of Zhejiang Province [grant no LY22H190001] and the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang [grant no 2021R01012].