Surveillance and Characterization of Carbapenem-resistant Enterobacter cloacae Complex from China, 2015-2018

Surveillance and Characterization of Carbapenem-resistant Enterobacter cloacae Complex from China, 2015-2018 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Surveillance and Characterization of Carbapenem-resistant Enterobacter cloacae Complex from China, 2015-2018 Kun Ye, Yongqing Zhang, Xuemei Qiu, Liyan Ye, Yanning Ma, Jiyong Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5360835/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The carbapenem-resistant Enterobacter cloacae (CREC) has become a global health concern. However, our understanding of its epidemiological trends and resistance mechanisms still needs to be improved. Methods: 92 non-repetitive clinical CREC isolates were collected from 12 hospitals across China and identified using MALDI-TOF MS, rMLST, ANI, MASH, and dDDH. Minimal inhibitory concentrations were determined via broth microdilution. Resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder. Carbonyl cyanide 3-chlorophenylhydrazone suppression test and OmpC and OmpF overexpression test were conducted on 32 non-carbapenemase-producing (NCP) isolates. A phylogenetic tree was constructed with kSNP4 and visualized through iTOL. Results: Among the 92 CREC isolates, E. hormaechei was predominate (76 isolates), followed by E. kobei (five isolates). Within E. hormaechei , E. hormaechei subsp xiangfangensis was the most widespread subspecies. Sixty isolates produced carbapenemase, with bla NDM-1 identified in 36 isolates, bla KPC-2 in six isolates , and bla NDM-5 in six isolates. Two isolates harbored both bla NDM-1 and bla KPC-2 . Thirty-two isolates were NCP isolates, primarily due to carbonyl cyanide 3-chlorophenylhydrazone suppression. Conclusions: Among CREC in China, the bla NDM-1 was the dominant carbapenemase-coding gene. For NCP isolates, efflux pumps was the main reason for carbapenem resistance, and susceptibility could be restored in an antibiotic-free environment. Enterobacter cloacae complex carbapenemase carbapenem resistance Figures Figure 1 Figure 2 IMPORTANCE Our study relies on four years of surveillance data of CREC from 2015 to 2018 in twelve hospitals spread across eleven cities in China. The complex mechanisms underlying the emergence of CREC highlight the importance of surveillance. This study aims to provide a comprehensive analysis of the prevalence, antimicrobial resistance profiles, genomic characteristic. Most CREC strains belonged to ST78, followed by ST93 and ST171. There has been an increase in NDM (New Delhi metallo-β-lactamase)-producing ECC in various regions worldwide , with many bla NDM -carrying strains. The spread of high-risk clones of CREC has occurred across various regions of China, emphasizing the need for continued surveillance and targeted intervention strategies. To restore bacterial susceptibility, it is essential for healthcare institutions to enhance the rational use of antibiotics, adhering to the principles of “right time, right dose, right duration” when prescribing them.teristics, and plasmid replicons associated of CREC in China. INTRODUCTION Enterobacter cloacae complex (ECC) is a significant opportunistic pathogen responsible for a wide range of nosocomial infections, such as pneumonia, urinary tract infections, skin and soft tissue infections, and septicemia. Within the Enterobacteriaceae family, ECC ranks as a leading cause of hospital-acquired infections, following Klebsiella pneumoniae and Escherichia coli [ 1 ]. Traditionally, ECC comprises seven species: E. cloacae , E. hormaechei , E. asburiae , E. kobei , E. ludwigii , E. nimipressuralis , and E. mori . Among them, E. cloacae and E. hormaechei are the most frequently isolated from clinical specimens and are associated with hospital-acquired infections and outbreaks. Notably, E. hormaechei subsp. xiangfangensis has shown a significant correlation with clinical outcomes, displaying a robust association with clinical carbapenem-resistant ECC species [ 2 ]. The rise of multi-drug resistance (MDR), including carbapenem resistance, has become a global health concern. Data from the China Antimicrobial Surveillance Network (CHINET) indicate that carbapenem-resistant Enterobacter increased from 8.4% (219/2601) in 2015 to 11.6% (861/7417) by 2021. Carbapenem resistance is mainly driven by carbapenemase produced by carbapenem-resistant Enterobacteriaceaes (CRE) strains. Additionally, overexpression of AmpC [ 3 ], and the loss of outer membrane proteins OmpF and OmpC [ 4 ] also contribute to carbapenem resistance. Global surveillance has identified several epidemic sequence types (STs) within carbapenem-resistant Enterobacter cloacae (CREC). The most prevalent clones worldwide include ST171, ST74, ST120, ST66, ST78, ST108, ST114, ST92, ST265, ST93, and ST418 [ 5 ]. ST171 was first identified in western Pennsylvania and has since spread throughout the United States [ 6 ]. In China, the CRE strain producing NDM-1 was initially reported in the northwest and southwest regions and is associated with ST78 and ST88. Additionally, ST51, which carries bla NDM−1 , shows potential for becoming a predominant strain in China [ 7 , 8 ]. Our study relies on four years of surveillance data of CREC from 2015 to 2018 in twelve hospitals spread across eleven cities in China. The complex mechanisms underlying the emergence of CREC highlight the importance of surveillance. This study aims to provide a comprehensive analysis of the prevalence, antimicrobial resistance profiles, genomic characteristics, and plasmid replicons associated with CREC in China. MATERIALS AND METHODS Strain Collection and Identification A total of 92 non-repetitive clinical CREC isolates were collected from clinical samples of12 hospitals across China between 2015 and 2018. These strains were primary identified as ECC using MALDI-TOF MS (bioMérieux). Species and subspecies identification were confirmed through average nucleotide identity (ANI) analysis, digital DNA-DNA hybridization (dDDH), and MASH (Metric Approach to Haplotype Phylogeny). ANI was calculated using FastANI [ 9 ], MASH was determined through MinHash [ 10 ], and dDDH was conducted on the TYGS platform [ 11 ]. Antimicrobial Susceptibility Test Various antibiotics’ minimum inhibitory concentrations (MICs) were determined based on broth microdilution using the Biofosun® Gram-negative panels (Biofosun Biotech, Co., Ltd., Shanghai, China). The antibiotics tested included amikacin, piperacillin-tazobactam, tigecycline, polymyxin, sulfamethoxazole/trimethoprim, ceftazidime, cefotaxime, cefotaxime/clavulanic acid, ciprofloxacin, ertapenem, imipenem, and meropenem. Modified carbapenem inactivation (mCIM) and ethylenediamine tetraacetic acid (EDTA) carbapenem inactivation (eCIM) was used for detect novel enzyme type. The results were interpreted according to the CLSI M100-S24/M45-A2 guidelines. E. coli ATCC 25922 served as the quality control strain. Whole-Genome Sequencing Analysis Whole-genome sequencing was conducted utilizing the Illumina NovaSeq platform, with paired-end reads of 150 base pairs and an insert size of 350 base pairs. Read quality evaluation and low-quality reads filter were performed using Fastp [ 12 ]. Genome assembly was achieved using ABySS [ 13 ]. Resistance genes and plasmid replicons were identified. using ResFinder [ 14 ] and PlasmidFinder [ 15 ]. Gene annotation and environmental analysis were carried out using RAST [ 16 ] and Prokka [ 17 ]. Multi-locus sequence typing (MLST) was performed with PubMLST [ 18 ]. A whole-genome phylogenetic tree was constructed using kSNP4 [ 19 ] and visualized with iTOL [ 20 ]. Efflux Pump & Outer Membrane Proteins Determination For non-carbapenemase-producing (NCP) strains, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used to assess efflux pump activities[ 1 ]. Expression levels of outer membrane proteins of OmpF and OmpC were determined based on the logarithmic transformation of fold changes (FC-fold change), with a threshold of log 2 FC ≥ 2.0 indicating significant changes. NCP strains were cultured on antibiotic-free Columbia Blood Agar plates for ten generations. Afterward, antibiotic susceptibility was tested. MICs of the 10th generation (MIC After) were compared to those of the 1st generation (MIC Origin) to interpret changes in susceptibility. The chi-square test was used to compare changes in the sensitive rate. RESULTS Distribution of Clinical CREC Isolates A total of 92 clinical isolates of CREC were reported across various cities in China. The highest number of isolates was found in Beijing (n = 38), followed by Shanghai (n = 17), Chengdu (n = 13), Lanzhou (n = 5), Zhengzhou (n = 4), Xi'an (n = 4), Shenyang (n = 4), Urumqi (n = 4), Guiyang (n = 1), and Guangzhou (n = 1). The most prevalent specimen types were sputum (n = 27, 29.35%), urine (n = 16, 17.39%), drainage (n = 13, 14.13%), bile (n = 9, 9.78%), blood (n = 7, 7.61%), and secretions (n = 5, 5.43%). Notably, all drainage isolates were from Beijing. Species Identification of the Strains The species identified among the CREC isolates included E. hormaechei (n = 75), E. kobei (n = 6), E. asburiae (n = 4), E. chengduensis (n = 3), E. roggenkampii (n = 2), and E. cloacae (n = 2) (Fig. 1). Discrepancies in identification results were observed among different methods. (Table 1 ) (Fig. 2) The final identification, determined based on the National Center for Medical Culture Collection, classified most isolates as E. hormaechei . E. hormaechei was further separated into seven subspecies, identified in our study. They were further divided into three clades in the phylogenic tree. (Fig. 2) Clade A included E. hormaechei subsp hoffmannii (10.67%, 8/75), E. hormaechei subsp hormaechei (8.00%, 6/75), and E. hormaechei subsp oharae (6.67%, 5/75). Clade B included E. hormaechei subsp steigerwaltii (10.67%, 8/75), E. hormaechei YT2 (17.33%, 13/75), and E. hormaechei YT3 (5.33%, 4/75). Clade C included E. hormaechei subsp xiangfangensis (40.00%, 30/75). Nested within the subsp. clades, three strains (CDL005, CDL006, and SHL018) were identified as E hormaechei YT2 , and SYL001 was categorized as E. hormaechei subsp. hoffmannii , which did not align with this classification in the phylogenetic tree. (Table 2 ) Table 1 Strains with Divergent Identification Results Among Methods Strain MASH ANI PubMLST IR53030 E. kobei E. kobei E. cloacae IR5476 E. hormaechei E. hormaechei E. roggenkampii SHL020 E. asburiae E. asburiae E. hormaechei Table 2 The divergence identification of subsp. and statistical of subsp. E. ho YT2 E. ho subsp steigerwaltii E. ho subsp hoffmannii E. ho subsp hormaechei CDL005 99.13* 98.91 / / CDL006 99.14* 98.92 / / SHL018 98.90* 98.89 / / SYL001 / / 98.94* 98.78 Antimicrobial Susceptibility Tests for the Strains Approximately 98.91% (91/92) of the isolates exhibited MDR phenotypes, demonstrating resistance to three or more antimicrobial classes. All isolates retained susceptibility to tigecycline, and 22 isolates were resistant to polymyxin B. Epidemiological and Phylogenetic Analysis A phylogenetic tree constructed from the 92 isolates revealed six major clades (I-VI), consisting of 28, 25, 14, 4, 9, and 7 isolates, respectively. These isolates were distributed among 50 different STs. The most prevalent STs were ST78 (n = 8), followed by ST93 (n = 6), ST171 (n = 5), and ST418 (n = 5). The six novel STs (ST1588, ST1600, ST1609, ST1610, ST1617, ST1619) originated from various regions and predominantly harbored the bla NDM gene. Despite the widespread distribution of STs, localized outbreaks were suspected in certain areas, such as ST1001 in Chengdu and ST528 and ST182 in Beijing. (Fig. 1) The STs carrying bla NDM−1 were identified as ST78 (n = 2), ST1001, ST182, ST528, and ST231, widely distributed across the country. The STs carrying bla NDM−5 were distributed among ST1600, ST1619, ST125, ST1344, and ST993. The STs carrying only unique resistance genes of bla IMP−4 or bla IMP−26 were ST520, ST87, ST102, and ST66. The STs carrying bla KPC−2 were ST414, ST78, ST231, and ST190. The STs carrying bla OXA−48 were only ST418. The STs co-producing NDM-1 and KPC-2 were ST78 and ST231. Resistance Gene, Resistance Plasmid, and Resistance Genes Environment Analysis Among the 92 isolates, 32 isolates did not possess any known carbapenemase genes, and 60 isolates were classified as carbapenemase-producing E. cloacae complex (CPEC). Among these CPEC isolates, and 70.00% (42/60) produces NDM enzymes, with NDM-1 (n = 36) and NDM-5 (n = 6) being predominant. Additionally, 10.00% (6/60) produces IPM-4, 8.33% (5/60) produces IPM-26, 10.00% (6/60) produces KPC-2, 3.33% (2/60) produces OXA-48, and 1.67% (1/64) produces IPM-1. Moreover, two distinct isolates, ZZL001 and ZZL003, produce both NDM-1 and KPC-2. These two isolates were isolated from Zhengzhou, He’nan Province. In ZZL001, the bla NDM−1 gene is located on contig1, corresponding to the strain's chromosome, while bla KPC−2 is located on contig3, indicative of a plasmid-borne gene. In contrast, both bla NDM−1 and bla KPC−2 in ZZL003 are located within the plasmid. The genes encoding metallo-beta-lactamases (MBLs) were commonly identified in plasmids such as IncHl2, IncHl2A, IncX3, IncFlB (pECA), IncFll (pECLA), IncC, IncR, IncX5, IncL, and IncN. The bla NDM−1 gene was predominantly carried on plasmid IncX3 (n = 21), followed by IncHl2/IncHl2A (n = 20). The bla NDM−5 gene was commonly found on plasmid IncX3 (n = 6), and the bla IMP and bla KPC genes were detected on plasmids IncHl2/IncHl2A (n = 9). Multidrug Resistance G enes The CREC isolates harbored a range of multidrug-resistant genes, including those for aminoglycosides, trimethoprim, quinolones, macrolides, sulfonamides, fosfomycin, tetracyclines, colistin, phenicols, and rifampicin. Thirteen isolates were resistant to aminoglycosides and contained genes encoding 16S rRNA methylase. Fifty-three isolates were resistant to ciprofloxacin and carried aac and/or qnr genes. All isolates resistant to SXT carried the dfrA gene (n = 67). Approximately 20 CREC strains harbored the mcr-9 gene, including thirteen NDM-1-producing isolates. Their colistin sensitivity varied from ≤ 0.5 µg/ml to ≥ 16 µg/ml. The colistin sensitivities of the eleven strains carrying only the mcr-9 gene were either 1 µg/ml or 16 µg/ml. NCP Strains’ Mechanism In our study, 32 isolates did not possess carbapenemase genes, consisting of four E. asburiae and twenty-eight E. hormaechei . mCIM and eCIM showed no novel enzyme types. However, a significant proportion of these NCP strains demonstrated resistance to carbapenem. Specifically, 70.65% (65/92) of the isolates tested positive for extended-spectrum beta-lactamase ( ESBL ) genes, with TEM-1B being the most prevalent, found in 43.48% (40/92) of isolates. Moreover, 95.65% (88/92) of the isolates tested positive for AmpC genes, with ACT-16 being the most prevalent AmpC -producing isolate, accounting for 31.52% (29/92) of the total isolates. Collectively, 73.91% (68/92) of the isolates harbored both ESBL and AmpC genes. Among the 32 NCP strains, 16 were ertapenem-resistant, showing positive in the CCCP test. This resistance is attributed to the overexpression of efflux pumps. OmpC and OmpF overexpression were observed in 12 and 14 isolates, respectively, indicating an additional resistance mechanism. Nine isolates displayed resistance through the combined mechanism of both efflux pump overexpression and OmpC/OmpF overexpression (Supplement Table 1 ). Carbapenem resistance in Enterobacteriaceae commonly results from various interacting β-lactam resistance strategies, including the production of acquired carbapenemase, alterations in outer membrane permeability, significantly increased production of chromosomally encoded β-lactamases (with slight carbapenemase activity, i.e., AmpC), and/or active efflux. After ten generations of culture without antibiotic pressure, most NCP strains exhibited significant changes in their MICs and susceptibility to ertapenem, meropenem, and Ceftazidime and Avibactam. For example, the susceptibility to ertapenem was restored in 65.6% of strains, meropenem in 25%, and Ceftazidime and Avibactam in 90.625%. The susceptibility to ertapenem and Ceftazidime and Avibactam was more easily restored in an antibiotic-free environment. Additionally, although the susceptibility to imipenem and Ceftazidime and Avibactam slightly increased, it did not reach statistical significance (Table 3 ). Most strains exhibited resistance only to ertapenem, while remaining susceptible to imipenem and ertapenem, demonstrating low resistance. Table 3 Susceptibility changes and statistical analysis of ETP, IMP, MEM, CAV and TZP S% I% R% Changes of S% p Significance After Origin After Origin After Origin ETP 84.375% (27/32) 18.75% (6/32) 9.375% (3/32) 31.25% (10/32) 6.25% (2/32) 50% (16/32) ↑ < 0.0001 **** IMP 68.75% (22/32) 71.875% (23/32) 25% (8/32) 18.75% (6/32) 6.25% (2/32) 9.375% (3/32) ↓ 0.7844 ns MEM 96.875% (31/32) 71.875% (23/32) 3.125% (1/32) 12.5% (4/32) 0% (0/32) 15.625% (5/32) ↑ 0.006 ** CAV 93.75% (30/32) 3.125% (1/32) / / 6.25% (2/32) 96.875% (31/32) ↑ < 0.0001 **** TZP 6.25% (2/32) 9.375% (3/32) 18.75% (6/32) 25% (8/32) 75% (24/32) 65.625% (21/32) ↓ 0.6414 ns DISCUSSION ECC is an important pathogen capable of causing a wide array of infections. Recently, it has emerged as one of the most prevalent nosocomial pathogens [ 21 ]. This research aims to deepen our understanding of key factors of CREC strains, such as the prevalence, antimicrobial resistance profiles, genomic characteristics, and plasmid carriage. This enhanced comprehension is intended to elucidate the transmission dynamics and molecular mechanisms facilitating the rapid acquisition and dissemination of antibiotic resistance genes within CREC. Most CREC isolates in this study were obtained from respiratory tract specimens, corroborating prior global findings on the prevalence of NDM-producing E. cloacae strains. Our data underscores the ubiquity of CREC in clinical settings and its potential to cause infections across multiple sites, with a notable proportion of samples sourced from the respiratory tract, urine, drainage fluids, and bile. Beijing, Shanghai, and Chengdu were the primary domestic locations for strain collection. The accurate and rapid identification of ECC remains challenging [ 22 ] due to the close genomic relationship among species. Molecular technologies such as 16S rRNA sequencing, MLST, rMLST, ANI, dDDH, and phylogenetic analysis offer diverse methods for species identification. Accurate identification is crucial for providing additional insights into clinical epidemiology. The presence of E. hormaechei subsp. xiangfangensis , associated with higher mortality, prolonged hospitalization, and increased resistance rate [ 23 ], underscores the importance of precise identification. The threshold for ANI to distinguish different species was set at 95% to ensure accurate identification of trends observed in the phylogenetic tree. However, four strains (IR5378, IR5382, IR5418, and IR5433)exhibited ANI values (94.271935%, 94.309982%, 94.238464%, and 94.273605%) below this threshold and were determined to be E. hormaechei by the National Center for Medical Culture Collection. Additionally, three strains showed different results with different identification methods, yet all were identified as E. hormaechei , consistent with the trend observed in the phylogenetic tree. In subspecies identification, a specific ANI threshold exists for each subsp. The setting of these thresholds depends on various factors, including the identification methods, species characteristics, and study purpose [ 24 ]. Different studies may employ different thresholds, which need to be determined based on specific experimental designs and data analysis. Some studies use ANI criteria to set species/subspecies boundaries, while others may rely on serological, molecular species delimitation methods, and biochemical methods. It is necessary to consider these factors comprehensively to achieve accurate subspecies identification. A total of 1,659 STs have been reported, further corroborating the genomic diversity of CREC, characterized by a high level of clonal diversity. These findings align with documented research in China [ 7 , 25 , 26 ] and other Asian countries, differing from those observed in the United States [ 27 , 28 ]. However, in our study, the predominant strain was E. hormaechei , distinct from other studies primarily focusing on E. hormaechei subsp. xiangfangensis . Moreover, except for ST1609 and ST1610, the other new isolates primarily carried the bla NDM gene. NDM-1, first described in 2009, has since spread globally and is the predominant mechanism of carbapenem resistance in CREC in China [ 29 ]. Our study revealed that 45.65% of CREC isolates produced NDMs, with NDM-1-producing strains being the predominant, causing outbreaks in two regions across the country [ 1 ]. The coexistence of bla NDM−1 and bla KPC−2 in two isolates suggests a concerning trend, as the simultaneous production of these major carbapenemases could lead to a higher resistance. The ease of gene transfer among unrelated bacterial species through plasmids highlights the risk of the spread of drug resistance. The study identified various STs of CREC, indicating the spread of the complex with subtype diversity. ESBL-producing CREC predominantly belonged to ST78, ST171 [ 5 ], and ST418, with ST78 carrying the ACT β-lactamase gene. The ST93 cluster was found to express various carbapenemases, including NDM-1. Multiple studies have linked the global spread of antimicrobial resistance genes in CREC to the emergence of high-risk bacterial clones [ 5 ]. ST418 mainly originates from Beijing and Shanghai, with no strain carrying NDM-1. ST1001, ST528, and ST182 all tested positive for NDM-1, indicating a small outbreak in Chengdu and Beijing. Several pieces of evidence have shown that high-risk bacterial clones have resulted from the global spread of antimicrobial resistance genes in CREC. Our studies indicate that the molecular mechanism of colistin resistance in these strains is independent of the mcr-9 gene [ 30 ]. The specific genes responsible for conferring colistin resistance have yet to be identified. Our findings suggest that the exceptional capability of CREC to acquire and spread antibiotic resistance across different classes, along with its ability to adapt to antibiotic pressure, has likely contributed to its widespread dissemination [ 30 ]. Notably, 32 isolates lacked carbapenemase genes. Coexistence of ESBL genes and OmpF and/or OmpC loss was observed in 22 isolates. Half of them showed positive results in the efflux pump suppression test, indicating potential co-resistance to carbapenems and tigecycline. The NCP strains belonged to 19 different STs, suggesting unique genetic characteristics. It is alarming that NCP strains’ susceptibility to ertapenem, meropenem, and Ceftazidime and Avibactam can be restored in an antibiotic-free environment. Antecedent carbapenem exposure as a risk factor for NCP [ 31 ] highlights the critical need to reduce antibiotic use to restore bacterial susceptibility in NCPECC strains. The misuse and overuse of antibiotics have led to the emergence and spread of antibiotic-resistant strains. Prolonged antibiotic exposure can induce resistance through various mechanisms, such as increased drug efflux. CREC harbors a diverse array of multi-genre antibiotic resistance genes, including those for aminoglycosides, fluoroquinolones, sulfonamides, trimethoprim, colistin, and other antibiotics. Limited genomic studies suggest that establishing successful clones and acquiring MDR phenotypes by diverse lineages may have played a significant role. The deregulation of ACT, the natural cephalosporinase of E. cloacae , is associated with reduced membrane permeability, contributing to carbapenem resistance [ 32 ]. The combination of ESBL or AmpC with mutations in porins is another significant mechanism [ 33 ]. A high percentage (93.10%) of isolates tested positive for ACT, with ACT-16 (33.70%, 31/92) being the most dominant. Of the 92 isolates tested, 78.12% (75/92) were positive for ESBLs, with TEM-1B being the most prevalent dominant at 81.52% (43/92). Research on CREC remains relatively scarce, with only a handful of hospital outbreaks having been documented. This lack of information makes it challenging to fully understand the specific genomic features of Enterobacteriaceae that facilitate their transmission and their recent epidemiological success. In summary, most CREC strains belonged to ST78, followed by ST93 and ST171. There has been an increase in NDM-producing ECC in various regions worldwide [ 34 ], with many bla NDM -carrying strains. The spread of high-risk clones of CREC has occurred across various regions of China, emphasizing the need for continued surveillance and targeted intervention strategies. To restore bacterial susceptibility, it is essential for healthcare institutions to enhance the rational use of antibiotics, adhering to the principles of “right time, right dose, right duration” when prescribing them. Declarations Ethics approval and consent to participate: This study did not require ethical approval. All strains were processed anonymously. No clinical data was collected during the study. Consent for publication: Not applicable. Availability of data and material: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding: This work has no funders. Competing interests: The authors declare that they have no competing interests. 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Front Microbiol, 2018. 9: p. 1549. Qiu, X., et al., Genome sequence-based species classification of Enterobacter cloacae complex: a study among clinical isolates. Microbiol Spectr, 2024. 12(6): p. e0431223. Hu, S., et al., Molecular eidemiology of carbapenem-resistant Enterobacter cloacae complex in a tertiary hospital in Shandong, China. BMC Microbiol, 2023. 23(1): p. 177. Yan, Z., et al., Analysis of the transmission chain of carbapenem-resistant Enterobacter cloacae complex infections in clinical, intestinal and healthcare settings in Zhejiang province, China (2022-2023). Sci Total Environ, 2024. 920: p. 170635. Guh, A.Y., et al., Epidemiology of Carbapenem-Resistant Enterobacteriaceae in 7 US Communities, 2012-2013. Jama, 2015. 314(14): p. 1479-87. Wilson, B.M., et al., Carbapenem-Resistant Enterobacter cloacae in Patients from the US Veterans Health Administration, 2006-2015. Emerg Infect Dis, 2017. 23(5): p. 878-880. Yong, D., et al., Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother, 2009. 53(12): p. 5046-54. Xu, T., et al., Frequent convergence of mcr-9 and carbapenemase genes in Enterobacter cloacae complex driven by epidemic plasmids and host incompatibility. Emerg Microbes Infect, 2022. 11(1): p. 1959-1972. Marimuthu, K., et al., Antecedent Carbapenem Exposure as a Risk Factor for Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae and Carbapenemase-Producing Enterobacteriaceae. Antimicrob Agents Chemother, 2019. 63(10). Hu, J., et al., Molecular characteristics of global β-lactamase-producing Enterobacter cloacae by genomic analysis. BMC Microbiol, 2022. 22(1): p. 255. Jacoby, G.A., AmpC beta-lactamases. Clin Microbiol Rev, 2009. 22(1): p. 161-82, Table of Contents. Gartzonika, K., et al., High prevalence of clonally related ST182 NDM-1-producing Enterobacter cloacae complex clinical isolates in Greece. Int J Antimicrob Agents, 2023. 62(1): p. 106837. Additional Declarations No competing interests reported. Supplementary Files Supplementtable1MechanismDeterminationinNCPStrains.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5360835","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375415481,"identity":"4a2f5a79-06da-4704-b2c7-b22e22ad7378","order_by":0,"name":"Kun Ye","email":"","orcid":"","institution":"First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Ye","suffix":""},{"id":375415482,"identity":"ee37121c-794d-400b-acc7-9372fdcc5f97","order_by":1,"name":"Yongqing Zhang","email":"","orcid":"","institution":"Peking University Shougang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongqing","middleName":"","lastName":"Zhang","suffix":""},{"id":375415483,"identity":"31e5d78d-3f29-45d7-9633-ef41b27a34e5","order_by":2,"name":"Xuemei Qiu","email":"","orcid":"","institution":"First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Qiu","suffix":""},{"id":375415484,"identity":"662018d6-256d-4be4-a614-20a69bc546c7","order_by":3,"name":"Liyan Ye","email":"","orcid":"","institution":"First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liyan","middleName":"","lastName":"Ye","suffix":""},{"id":375415485,"identity":"04cce8ac-6006-4ae1-846a-3e69758e4b81","order_by":4,"name":"Yanning Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACHgaGBB4JBjb25sMPPhjY2BGvhY/nWJrhjIK0ZOK0gICcRI6BNM+HQ4wNhDTotp89JvFAxkKOTSItwdjG4AAzA/vhoxvwaTE7k5cmAXSYMRvP4wOPcwzu8DHwpKXdwKvlQI4ZSEtiGzvQlhyDZ8wMEjxm+LWcfwPWUt/GAPSLhcFhxgaCWm5AbElg4wBqYSBOyxtjC6AWwzZQIPcYpCWzEfTL+RzDmz976uTl24FR+eOPjR0/++FjeLWAAWMPEoeNoHIw+EGcslEwCkbBKBihAACv8kZ60eAMWQAAAABJRU5ErkJggg==","orcid":"","institution":"First Medical Center of Chinese PLA General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yanning","middleName":"","lastName":"Ma","suffix":""},{"id":375415486,"identity":"bd19fac9-3c60-4648-bb15-26f1bc8928bc","order_by":5,"name":"Jiyong Yang","email":"","orcid":"","institution":"First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiyong","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-10-30 11:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5360835/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5360835/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69216925,"identity":"a3b79cb3-b368-4843-a874-12189ff6d1fe","added_by":"auto","created_at":"2024-11-18 06:37:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234971,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5360835/v1/26f15a246eba51c67ab1aba2.png"},{"id":69216927,"identity":"f92aeb85-1c21-41b4-9c87-a453f6d93e11","added_by":"auto","created_at":"2024-11-18 06:37:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":202977,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5360835/v1/b1d7548f12813cdaa8290af1.png"},{"id":69637262,"identity":"7a87a889-295a-498f-9152-841fd27c8726","added_by":"auto","created_at":"2024-11-22 13:23:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1226732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5360835/v1/a81f565b-9470-4efe-8bb6-5c3794fc1083.pdf"},{"id":69216926,"identity":"82615c83-cebd-4aea-b179-de52ef367070","added_by":"auto","created_at":"2024-11-18 06:37:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29641,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementtable1MechanismDeterminationinNCPStrains.docx","url":"https://assets-eu.researchsquare.com/files/rs-5360835/v1/444faaca300d49e4f7b2d763.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Surveillance and Characterization of Carbapenem-resistant Enterobacter cloacae Complex from China, 2015-2018","fulltext":[{"header":"IMPORTANCE","content":"\u003cp\u003eOur study relies on four years of surveillance data of CREC from 2015 to 2018 in twelve hospitals spread across eleven cities in China. The complex mechanisms underlying the emergence of CREC highlight the importance of surveillance. This study aims to provide a comprehensive analysis of the prevalence, antimicrobial resistance profiles, genomic characteristic. Most CREC strains belonged to ST78, followed by ST93 and ST171. There has been an increase in NDM (New Delhi metallo-β-lactamase)-producing ECC in various regions worldwide , with many bla\u003csub\u003eNDM\u003c/sub\u003e-carrying strains. The spread of high-risk clones of CREC has occurred across various regions of China, emphasizing the need for continued surveillance and targeted intervention strategies. To restore bacterial susceptibility, it is essential for healthcare institutions to enhance the rational use of antibiotics, adhering to the principles of “right time, right dose, right duration” when prescribing them.teristics, and plasmid replicons associated of CREC in China.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex (ECC) is a significant opportunistic pathogen responsible for a wide range of nosocomial infections, such as pneumonia, urinary tract infections, skin and soft tissue infections, and septicemia. Within the Enterobacteriaceae family, ECC ranks as a leading cause of hospital-acquired infections, following \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Traditionally, ECC comprises seven species: \u003cem\u003eE. cloacae\u003c/em\u003e, \u003cem\u003eE. hormaechei\u003c/em\u003e, \u003cem\u003eE. asburiae\u003c/em\u003e, \u003cem\u003eE. kobei\u003c/em\u003e, \u003cem\u003eE. ludwigii\u003c/em\u003e, \u003cem\u003eE. nimipressuralis\u003c/em\u003e, and \u003cem\u003eE. mori\u003c/em\u003e. Among them, \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eE. hormaechei\u003c/em\u003e are the most frequently isolated from clinical specimens and are associated with hospital-acquired infections and outbreaks. Notably, \u003cem\u003eE. hormaechei\u003c/em\u003e subsp. \u003cem\u003exiangfangensis\u003c/em\u003e has shown a significant correlation with clinical outcomes, displaying a robust association with clinical carbapenem-resistant ECC species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rise of multi-drug resistance (MDR), including carbapenem resistance, has become a global health concern. Data from the China Antimicrobial Surveillance Network (CHINET) indicate that carbapenem-resistant \u003cem\u003eEnterobacter\u003c/em\u003e increased from 8.4% (219/2601) in 2015 to 11.6% (861/7417) by 2021. Carbapenem resistance is mainly driven by carbapenemase produced by carbapenem-resistant \u003cem\u003eEnterobacteriaceaes\u003c/em\u003e (CRE) strains. Additionally, overexpression of AmpC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the loss of outer membrane proteins OmpF and OmpC [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] also contribute to carbapenem resistance.\u003c/p\u003e \u003cp\u003eGlobal surveillance has identified several epidemic sequence types (STs) within carbapenem-resistant \u003cem\u003eEnterobacter cloacae\u003c/em\u003e (CREC). The most prevalent clones worldwide include ST171, ST74, ST120, ST66, ST78, ST108, ST114, ST92, ST265, ST93, and ST418 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. ST171 was first identified in western Pennsylvania and has since spread throughout the United States [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In China, the CRE strain producing NDM-1 was initially reported in the northwest and southwest regions and is associated with ST78 and ST88. Additionally, ST51, which carries \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, shows potential for becoming a predominant strain in China [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study relies on four years of surveillance data of CREC from 2015 to 2018 in twelve hospitals spread across eleven cities in China. The complex mechanisms underlying the emergence of CREC highlight the importance of surveillance. This study aims to provide a comprehensive analysis of the prevalence, antimicrobial resistance profiles, genomic characteristics, and plasmid replicons associated with CREC in China.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain Collection and Identification\u003c/h2\u003e \u003cp\u003eA total of 92 non-repetitive clinical CREC isolates were collected from clinical samples of12 hospitals across China between 2015 and 2018. These strains were primary identified as ECC using MALDI-TOF MS (bioM\u0026eacute;rieux). Species and subspecies identification were confirmed through average nucleotide identity (ANI) analysis, digital DNA-DNA hybridization (dDDH), and MASH (Metric Approach to Haplotype Phylogeny). ANI was calculated using FastANI [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], MASH was determined through MinHash [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and dDDH was conducted on the TYGS platform [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntimicrobial Susceptibility Test\u003c/h3\u003e\n\u003cp\u003eVarious antibiotics\u0026rsquo; minimum inhibitory concentrations (MICs) were determined based on broth microdilution using the Biofosun\u0026reg; Gram-negative panels (Biofosun Biotech, Co., Ltd., Shanghai, China). The antibiotics tested included amikacin, piperacillin-tazobactam, tigecycline, polymyxin, sulfamethoxazole/trimethoprim, ceftazidime, cefotaxime, cefotaxime/clavulanic acid, ciprofloxacin, ertapenem, imipenem, and meropenem. Modified carbapenem inactivation (mCIM) and ethylenediamine tetraacetic acid (EDTA) carbapenem inactivation (eCIM) was used for detect novel enzyme type. The results were interpreted according to the CLSI M100-S24/M45-A2 guidelines. \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 served as the quality control strain.\u003c/p\u003e\n\u003ch3\u003eWhole-Genome Sequencing Analysis\u003c/h3\u003e\n\u003cp\u003eWhole-genome sequencing was conducted utilizing the Illumina NovaSeq platform, with paired-end reads of 150 base pairs and an insert size of 350 base pairs. Read quality evaluation and low-quality reads filter were performed using Fastp [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Genome assembly was achieved using ABySS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Resistance genes and plasmid replicons were identified. using ResFinder [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and PlasmidFinder [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Gene annotation and environmental analysis were carried out using RAST [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and Prokka [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Multi-locus sequence typing (MLST) was performed with PubMLST [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A whole-genome phylogenetic tree was constructed using kSNP4 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and visualized with iTOL [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEfflux Pump \u0026 Outer Membrane Proteins Determination\u003c/h3\u003e\n\u003cp\u003eFor non-carbapenemase-producing (NCP) strains, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used to assess efflux pump activities[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Expression levels of outer membrane proteins of OmpF and OmpC were determined based on the logarithmic transformation of fold changes (FC-fold change), with a threshold of log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026ge;\u0026thinsp;2.0 indicating significant changes. NCP strains were cultured on antibiotic-free Columbia Blood Agar plates for ten generations. Afterward, antibiotic susceptibility was tested. MICs of the 10th generation (MIC After) were compared to those of the 1st generation (MIC Origin) to interpret changes in susceptibility. The chi-square test was used to compare changes in the sensitive rate.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDistribution of Clinical CREC Isolates\u003c/h2\u003e \u003cp\u003eA total of 92 clinical isolates of CREC were reported across various cities in China. The highest number of isolates was found in Beijing (n\u0026thinsp;=\u0026thinsp;38), followed by Shanghai (n\u0026thinsp;=\u0026thinsp;17), Chengdu (n\u0026thinsp;=\u0026thinsp;13), Lanzhou (n\u0026thinsp;=\u0026thinsp;5), Zhengzhou (n\u0026thinsp;=\u0026thinsp;4), Xi'an (n\u0026thinsp;=\u0026thinsp;4), Shenyang (n\u0026thinsp;=\u0026thinsp;4), Urumqi (n\u0026thinsp;=\u0026thinsp;4), Guiyang (n\u0026thinsp;=\u0026thinsp;1), and Guangzhou (n\u0026thinsp;=\u0026thinsp;1). The most prevalent specimen types were sputum (n\u0026thinsp;=\u0026thinsp;27, 29.35%), urine (n\u0026thinsp;=\u0026thinsp;16, 17.39%), drainage (n\u0026thinsp;=\u0026thinsp;13, 14.13%), bile (n\u0026thinsp;=\u0026thinsp;9, 9.78%), blood (n\u0026thinsp;=\u0026thinsp;7, 7.61%), and secretions (n\u0026thinsp;=\u0026thinsp;5, 5.43%). Notably, all drainage isolates were from Beijing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpecies Identification of the Strains\u003c/h3\u003e\n\u003cp\u003eThe species identified among the CREC isolates included \u003cem\u003eE. hormaechei\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;75), \u003cem\u003eE. kobei\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;6), \u003cem\u003eE. asburiae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003eE. chengduensis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eE. roggenkampii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003eE. cloacae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;1). Discrepancies in identification results were observed among different methods. (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Fig.\u0026nbsp;2) The final identification, determined based on the National Center for Medical Culture Collection, classified most isolates as \u003cem\u003eE. hormaechei\u003c/em\u003e. \u003cem\u003eE. hormaechei\u003c/em\u003e was further separated into seven subspecies, identified in our study. They were further divided into three clades in the phylogenic tree. (Fig.\u0026nbsp;2) Clade A included \u003cem\u003eE. hormaechei\u003c/em\u003e subsp \u003cem\u003ehoffmannii\u003c/em\u003e (10.67%, 8/75), \u003cem\u003eE. hormaechei\u003c/em\u003e subsp \u003cem\u003ehormaechei\u003c/em\u003e (8.00%, 6/75), and \u003cem\u003eE. hormaechei subsp oharae\u003c/em\u003e (6.67%, 5/75). Clade B included \u003cem\u003eE. hormaechei\u003c/em\u003e subsp \u003cem\u003esteigerwaltii\u003c/em\u003e (10.67%, 8/75), \u003cem\u003eE. hormaechei YT2\u003c/em\u003e (17.33%, 13/75), and \u003cem\u003eE. hormaechei YT3\u003c/em\u003e (5.33%, 4/75). Clade C included \u003cem\u003eE. hormaechei\u003c/em\u003e subsp \u003cem\u003exiangfangensis\u003c/em\u003e (40.00%, 30/75). Nested within the subsp. clades, three strains (CDL005, CDL006, and SHL018) were identified as \u003cem\u003eE hormaechei YT2\u003c/em\u003e, and SYL001 was categorized as \u003cem\u003eE. hormaechei\u003c/em\u003e subsp. \u003cem\u003ehoffmannii\u003c/em\u003e, which did not align with this classification in the phylogenetic tree. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStrains with Divergent Identification Results Among Methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMASH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eANI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePubMLST\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIR53030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. kobei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. kobei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE. cloacae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIR5476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. hormaechei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. hormaechei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE. roggenkampii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHL020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. asburiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. asburiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE. hormaechei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe divergence identification of subsp. and statistical of subsp.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. ho YT2\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. ho subsp steigerwaltii\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE. ho subsp hoffmannii\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. ho subsp hormaechei\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDL005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.13*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDL006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.14*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHL018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.90*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSYL001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.94*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAntimicrobial Susceptibility Tests for the Strains\u003c/h3\u003e\n\u003cp\u003eApproximately 98.91% (91/92) of the isolates exhibited MDR phenotypes, demonstrating resistance to three or more antimicrobial classes. All isolates retained susceptibility to tigecycline, and 22 isolates were resistant to polymyxin B.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEpidemiological and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eA phylogenetic tree constructed from the 92 isolates revealed six major clades (I-VI), consisting of 28, 25, 14, 4, 9, and 7 isolates, respectively. These isolates were distributed among 50 different STs. The most prevalent STs were ST78 (n\u0026thinsp;=\u0026thinsp;8), followed by ST93 (n\u0026thinsp;=\u0026thinsp;6), ST171 (n\u0026thinsp;=\u0026thinsp;5), and ST418 (n\u0026thinsp;=\u0026thinsp;5). The six novel STs (ST1588, ST1600, ST1609, ST1610, ST1617, ST1619) originated from various regions and predominantly harbored the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e gene. Despite the widespread distribution of STs, localized outbreaks were suspected in certain areas, such as ST1001 in Chengdu and ST528 and ST182 in Beijing. (Fig.\u0026nbsp;1)\u003c/p\u003e \u003cp\u003eThe STs carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e were identified as ST78 (n\u0026thinsp;=\u0026thinsp;2), ST1001, ST182, ST528, and ST231, widely distributed across the country. The STs carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;5\u003c/sub\u003e were distributed among ST1600, ST1619, ST125, ST1344, and ST993. The STs carrying only unique resistance genes of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e or \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;26\u003c/sub\u003e were ST520, ST87, ST102, and ST66. The STs carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e were ST414, ST78, ST231, and ST190. The STs carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e were only ST418. The STs co-producing NDM-1 and KPC-2 were ST78 and ST231.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eResistance Gene, Resistance Plasmid, and Resistance Genes Environment Analysis\u003c/h2\u003e \u003cp\u003eAmong the 92 isolates, 32 isolates did not possess any known carbapenemase genes, and 60 isolates were classified as carbapenemase-producing \u003cem\u003eE. cloacae\u003c/em\u003e complex (CPEC). Among these CPEC isolates, and 70.00% (42/60) produces NDM enzymes, with NDM-1 (n\u0026thinsp;=\u0026thinsp;36) and NDM-5 (n\u0026thinsp;=\u0026thinsp;6) being predominant. Additionally, 10.00% (6/60) produces IPM-4, 8.33% (5/60) produces IPM-26, 10.00% (6/60) produces KPC-2, 3.33% (2/60) produces OXA-48, and 1.67% (1/64) produces IPM-1. Moreover, two distinct isolates, ZZL001 and ZZL003, produce both NDM-1 and KPC-2. These two isolates were isolated from Zhengzhou, He\u0026rsquo;nan Province. In ZZL001, the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e gene is located on contig1, corresponding to the strain's chromosome, while \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e is located on contig3, indicative of a plasmid-borne gene. In contrast, both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e in ZZL003 are located within the plasmid.\u003c/p\u003e \u003cp\u003eThe genes encoding metallo-beta-lactamases (MBLs) were commonly identified in plasmids such as IncHl2, IncHl2A, IncX3, IncFlB (pECA), IncFll (pECLA), IncC, IncR, IncX5, IncL, and IncN. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e gene was predominantly carried on plasmid IncX3 (n\u0026thinsp;=\u0026thinsp;21), followed by IncHl2/IncHl2A (n\u0026thinsp;=\u0026thinsp;20). The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;5\u003c/sub\u003e gene was commonly found on plasmid IncX3 (n\u0026thinsp;=\u0026thinsp;6), and the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e genes were detected on plasmids IncHl2/IncHl2A (n\u0026thinsp;=\u0026thinsp;9).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMultidrug Resistance\u003c/b\u003e G\u003cb\u003eenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe CREC isolates harbored a range of multidrug-resistant genes, including those for aminoglycosides, trimethoprim, quinolones, macrolides, sulfonamides, fosfomycin, tetracyclines, colistin, phenicols, and rifampicin. Thirteen isolates were resistant to aminoglycosides and contained genes encoding 16S rRNA methylase. Fifty-three isolates were resistant to ciprofloxacin and carried \u003cem\u003eaac\u003c/em\u003e and/or \u003cem\u003eqnr\u003c/em\u003e genes. All isolates resistant to SXT carried the \u003cem\u003edfrA\u003c/em\u003e gene (n\u0026thinsp;=\u0026thinsp;67). Approximately 20 CREC strains harbored the \u003cem\u003emcr-9\u003c/em\u003e gene, including thirteen NDM-1-producing isolates. Their colistin sensitivity varied from \u0026le;\u0026thinsp;0.5 \u0026micro;g/ml to \u0026ge;\u0026thinsp;16 \u0026micro;g/ml. The colistin sensitivities of the eleven strains carrying only the \u003cem\u003emcr-9\u003c/em\u003e gene were either 1 \u0026micro;g/ml or 16 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNCP Strains\u0026rsquo; Mechanism\u003c/h2\u003e \u003cp\u003eIn our study, 32 isolates did not possess carbapenemase genes, consisting of four \u003cem\u003eE. asburiae\u003c/em\u003e and twenty-eight \u003cem\u003eE. hormaechei\u003c/em\u003e. mCIM and eCIM showed no novel enzyme types. However, a significant proportion of these NCP strains demonstrated resistance to carbapenem. Specifically, 70.65% (65/92) of the isolates tested positive for extended-spectrum beta-lactamase (\u003cem\u003eESBL\u003c/em\u003e) genes, with \u003cem\u003eTEM-1B\u003c/em\u003e being the most prevalent, found in 43.48% (40/92) of isolates. Moreover, 95.65% (88/92) of the isolates tested positive for \u003cem\u003eAmpC\u003c/em\u003e genes, with \u003cem\u003eACT-16\u003c/em\u003e being the most prevalent \u003cem\u003eAmpC\u003c/em\u003e-producing isolate, accounting for 31.52% (29/92) of the total isolates. Collectively, 73.91% (68/92) of the isolates harbored both \u003cem\u003eESBL\u003c/em\u003e and \u003cem\u003eAmpC\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eAmong the 32 NCP strains, 16 were ertapenem-resistant, showing positive in the CCCP test. This resistance is attributed to the overexpression of efflux pumps. OmpC and OmpF overexpression were observed in 12 and 14 isolates, respectively, indicating an additional resistance mechanism. Nine isolates displayed resistance through the combined mechanism of both efflux pump overexpression and OmpC/OmpF overexpression (Supplement Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCarbapenem resistance in Enterobacteriaceae commonly results from various interacting β-lactam resistance strategies, including the production of acquired carbapenemase, alterations in outer membrane permeability, significantly increased production of chromosomally encoded β-lactamases (with slight carbapenemase activity, i.e., AmpC), and/or active efflux.\u003c/p\u003e \u003cp\u003eAfter ten generations of culture without antibiotic pressure, most NCP strains exhibited significant changes in their MICs and susceptibility to ertapenem, meropenem, and Ceftazidime and Avibactam. For example, the susceptibility to ertapenem was restored in 65.6% of strains, meropenem in 25%, and Ceftazidime and Avibactam in 90.625%. The susceptibility to ertapenem and Ceftazidime and Avibactam was more easily restored in an antibiotic-free environment. Additionally, although the susceptibility to imipenem and Ceftazidime and Avibactam slightly increased, it did not reach statistical significance (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Most strains exhibited resistance only to ertapenem, while remaining susceptible to imipenem and ertapenem, demonstrating low resistance.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSusceptibility changes and statistical analysis of ETP, IMP, MEM, CAV and TZP\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eS%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eI%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eR%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChanges of S%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSignificance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAfter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAfter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAfter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eETP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84.375% (27/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.75% (6/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.375% (3/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.25% (10/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25% (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50% (16/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e****\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68.75% (22/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.875% (23/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25% (8/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.75% (6/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25% (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.375% (3/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.7844\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96.875% (31/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.875% (23/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.125% (1/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.5% (4/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0% (0/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.625% (5/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93.75% (30/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.125% (1/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.25% (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e96.875% (31/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e****\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTZP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.25% (2/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.375% (3/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.75% (6/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25% (8/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75% (24/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65.625% (21/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.6414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eECC is an important pathogen capable of causing a wide array of infections. Recently, it has emerged as one of the most prevalent nosocomial pathogens [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This research aims to deepen our understanding of key factors of CREC strains, such as the prevalence, antimicrobial resistance profiles, genomic characteristics, and plasmid carriage. This enhanced comprehension is intended to elucidate the transmission dynamics and molecular mechanisms facilitating the rapid acquisition and dissemination of antibiotic resistance genes within CREC.\u003c/p\u003e \u003cp\u003eMost CREC isolates in this study were obtained from respiratory tract specimens, corroborating prior global findings on the prevalence of NDM-producing \u003cem\u003eE. cloacae\u003c/em\u003e strains. Our data underscores the ubiquity of CREC in clinical settings and its potential to cause infections across multiple sites, with a notable proportion of samples sourced from the respiratory tract, urine, drainage fluids, and bile. Beijing, Shanghai, and Chengdu were the primary domestic locations for strain collection.\u003c/p\u003e \u003cp\u003eThe accurate and rapid identification of ECC remains challenging [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] due to the close genomic relationship among species. Molecular technologies such as 16S rRNA sequencing, MLST, rMLST, ANI, dDDH, and phylogenetic analysis offer diverse methods for species identification. Accurate identification is crucial for providing additional insights into clinical epidemiology. The presence of \u003cem\u003eE. hormaechei\u003c/em\u003e subsp. \u003cem\u003exiangfangensis\u003c/em\u003e, associated with higher mortality, prolonged hospitalization, and increased resistance rate [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], underscores the importance of precise identification.\u003c/p\u003e \u003cp\u003eThe threshold for ANI to distinguish different species was set at 95% to ensure accurate identification of trends observed in the phylogenetic tree. However, four strains (IR5378, IR5382, IR5418, and IR5433)exhibited ANI values (94.271935%, 94.309982%, 94.238464%, and 94.273605%) below this threshold and were determined to be \u003cem\u003eE. hormaechei\u003c/em\u003e by the National Center for Medical Culture Collection. Additionally, three strains showed different results with different identification methods, yet all were identified as \u003cem\u003eE. hormaechei\u003c/em\u003e, consistent with the trend observed in the phylogenetic tree.\u003c/p\u003e \u003cp\u003eIn subspecies identification, a specific ANI threshold exists for each subsp. The setting of these thresholds depends on various factors, including the identification methods, species characteristics, and study purpose [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Different studies may employ different thresholds, which need to be determined based on specific experimental designs and data analysis. Some studies use ANI criteria to set species/subspecies boundaries, while others may rely on serological, molecular species delimitation methods, and biochemical methods. It is necessary to consider these factors comprehensively to achieve accurate subspecies identification.\u003c/p\u003e \u003cp\u003eA total of 1,659 STs have been reported, further corroborating the genomic diversity of CREC, characterized by a high level of clonal diversity. These findings align with documented research in China [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and other Asian countries, differing from those observed in the United States [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, in our study, the predominant strain was \u003cem\u003eE. hormaechei\u003c/em\u003e, distinct from other studies primarily focusing on \u003cem\u003eE. hormaechei\u003c/em\u003e subsp. \u003cem\u003exiangfangensis\u003c/em\u003e. Moreover, except for ST1609 and ST1610, the other new isolates primarily carried the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e gene.\u003c/p\u003e \u003cp\u003eNDM-1, first described in 2009, has since spread globally and is the predominant mechanism of carbapenem resistance in CREC in China [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our study revealed that 45.65% of CREC isolates produced NDMs, with NDM-1-producing strains being the predominant, causing outbreaks in two regions across the country [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The coexistence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e in two isolates suggests a concerning trend, as the simultaneous production of these major carbapenemases could lead to a higher resistance. The ease of gene transfer among unrelated bacterial species through plasmids highlights the risk of the spread of drug resistance.\u003c/p\u003e \u003cp\u003eThe study identified various STs of CREC, indicating the spread of the complex with subtype diversity. ESBL-producing CREC predominantly belonged to ST78, ST171 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and ST418, with ST78 carrying the ACT β-lactamase gene. The ST93 cluster was found to express various carbapenemases, including NDM-1. Multiple studies have linked the global spread of antimicrobial resistance genes in CREC to the emergence of high-risk bacterial clones [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. ST418 mainly originates from Beijing and Shanghai, with no strain carrying NDM-1. ST1001, ST528, and ST182 all tested positive for NDM-1, indicating a small outbreak in Chengdu and Beijing. Several pieces of evidence have shown that high-risk bacterial clones have resulted from the global spread of antimicrobial resistance genes in CREC. Our studies indicate that the molecular mechanism of colistin resistance in these strains is independent of the \u003cem\u003emcr-9\u003c/em\u003e gene [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The specific genes responsible for conferring colistin resistance have yet to be identified. Our findings suggest that the exceptional capability of CREC to acquire and spread antibiotic resistance across different classes, along with its ability to adapt to antibiotic pressure, has likely contributed to its widespread dissemination [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, 32 isolates lacked carbapenemase genes. Coexistence of ESBL genes and OmpF and/or OmpC loss was observed in 22 isolates. Half of them showed positive results in the efflux pump suppression test, indicating potential co-resistance to carbapenems and tigecycline. The NCP strains belonged to 19 different STs, suggesting unique genetic characteristics.\u003c/p\u003e \u003cp\u003eIt is alarming that NCP strains\u0026rsquo; susceptibility to ertapenem, meropenem, and Ceftazidime and Avibactam can be restored in an antibiotic-free environment. Antecedent carbapenem exposure as a risk factor for NCP [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] highlights the critical need to reduce antibiotic use to restore bacterial susceptibility in NCPECC strains. The misuse and overuse of antibiotics have led to the emergence and spread of antibiotic-resistant strains. Prolonged antibiotic exposure can induce resistance through various mechanisms, such as increased drug efflux.\u003c/p\u003e \u003cp\u003eCREC harbors a diverse array of multi-genre antibiotic resistance genes, including those for aminoglycosides, fluoroquinolones, sulfonamides, trimethoprim, colistin, and other antibiotics. Limited genomic studies suggest that establishing successful clones and acquiring MDR phenotypes by diverse lineages may have played a significant role. The deregulation of ACT, the natural cephalosporinase of \u003cem\u003eE. cloacae\u003c/em\u003e, is associated with reduced membrane permeability, contributing to carbapenem resistance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The combination of ESBL or AmpC with mutations in porins is another significant mechanism [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A high percentage (93.10%) of isolates tested positive for ACT, with ACT-16 (33.70%, 31/92) being the most dominant. Of the 92 isolates tested, 78.12% (75/92) were positive for ESBLs, with TEM-1B being the most prevalent dominant at 81.52% (43/92). Research on CREC remains relatively scarce, with only a handful of hospital outbreaks having been documented. This lack of information makes it challenging to fully understand the specific genomic features of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e that facilitate their transmission and their recent epidemiological success.\u003c/p\u003e \u003cp\u003eIn summary, most CREC strains belonged to ST78, followed by ST93 and ST171. There has been an increase in NDM-producing ECC in various regions worldwide [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with many \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e-carrying strains. The spread of high-risk clones of CREC has occurred across various regions of China, emphasizing the need for continued surveillance and targeted intervention strategies. To restore bacterial susceptibility, it is essential for healthcare institutions to enhance the rational use of antibiotics, adhering to the principles of \u0026ldquo;right time, right dose, right duration\u0026rdquo; when prescribing them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e This study did not require ethical approval. All strains were processed anonymously. No clinical data was collected during the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work has no funders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We would like to thank KunYe, Yongqing Zhang, Xuemei Qiu, Liyan Ye,\u0026nbsp;Yanning Ma\u003csup\u003e\u0026nbsp;\u003c/sup\u003e, Jiyong Yang for their assistance with this work.\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhang, Y., et al., Epidemiology of Carbapenem-Resistant Enterobacteriaceae Infections: Report from the China CRE Network. Antimicrob Agents Chemother, 2018. 62(2).\u003c/li\u003e\n \u003cli\u003eWu, W., et al., Precise Species Identification by Whole-Genome Sequencing of Enterobacter Bloodstream Infection, China. Emerg Infect Dis, 2021. 27(1): p. 161-169.\u003c/li\u003e\n \u003cli\u003eSeeberg, A.H., R.M. Tolxdorff-Neutzling, and B. Wiedemann, Chromosomal beta-lactamases of Enterobacter cloacae are responsible for resistance to third-generation cephalosporins. Antimicrob Agents Chemother, 1983. 23(6): p. 918-25.\u003c/li\u003e\n \u003cli\u003ePag\u0026egrave;s, J.M., M. Masi, and J. Barbe, Inhibitors of efflux pumps in Gram-negative bacteria. 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Microbiol Spectr, 2022. 10(6): p. e0216022.\u003c/li\u003e\n \u003cli\u003eJain, C., et al., High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun, 2018. 9(1): p. 5114.\u003c/li\u003e\n \u003cli\u003eOndov, B.D., et al., Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol, 2016. 17(1): p. 132.\u003c/li\u003e\n \u003cli\u003eMeier-Kolthoff, J.P. and M. G\u0026ouml;ker, TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun, 2019. 10(1): p. 2182.\u003c/li\u003e\n \u003cli\u003eChen, S., Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. Imeta, 2023. 2(2): p. e107.\u003c/li\u003e\n \u003cli\u003eJackman, S.D., et al., ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter. 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Int J Antimicrob Agents, 2023. 62(1): p. 106837.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Enterobacter cloacae complex, carbapenemase, carbapenem resistance","lastPublishedDoi":"10.21203/rs.3.rs-5360835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5360835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: The carbapenem-resistant \u003cem\u003eEnterobacter cloacae\u003c/em\u003e (CREC) has become a global health concern. However, our understanding of its epidemiological trends and resistance mechanisms still needs to be improved.\u003c/p\u003e\n\u003cp\u003eMethods: 92 non-repetitive clinical CREC isolates were collected from 12 hospitals across China and identified using MALDI-TOF MS, rMLST, ANI, MASH, and dDDH. Minimal inhibitory concentrations were determined via broth microdilution. Resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder. Carbonyl cyanide 3-chlorophenylhydrazone suppression test and OmpC and OmpF overexpression test were conducted on 32 non-carbapenemase-producing (NCP) isolates. A phylogenetic tree was constructed with kSNP4 and visualized through iTOL.\u003c/p\u003e\n\u003cp\u003eResults: Among the 92 CREC isolates, \u003cem\u003eE. hormaechei\u003c/em\u003e was predominate (76 isolates), followed by \u003cem\u003eE. kobei\u003c/em\u003e (five isolates). Within \u003cem\u003eE. hormaechei\u003c/em\u003e, \u003cem\u003eE. hormaechei\u003c/em\u003e subsp\u003cem\u003e xiangfangensis\u003c/em\u003e was the most widespread subspecies. Sixty isolates produced carbapenemase, with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e identified in 36 isolates, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e in six isolates\u003cem\u003e, \u003c/em\u003eand\u003cem\u003e bla\u003c/em\u003e\u003csub\u003eNDM-5\u003c/sub\u003e in six isolates. Two isolates harbored both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e. Thirty-two isolates were NCP isolates, primarily due to carbonyl cyanide 3-chlorophenylhydrazone suppression.\u003c/p\u003e\n\u003cp\u003eConclusions: Among CREC in China, the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e was the dominant carbapenemase-coding gene. For NCP isolates, efflux pumps was the main reason for carbapenem resistance, and susceptibility could be restored in an antibiotic-free environment.\u003c/p\u003e","manuscriptTitle":"Surveillance and Characterization of Carbapenem-resistant Enterobacter cloacae Complex from China, 2015-2018","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 06:37:16","doi":"10.21203/rs.3.rs-5360835/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dff2abf7-03b1-4574-827b-82a9dcd1982b","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-12T12:53:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 06:37:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5360835","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5360835","identity":"rs-5360835","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}