scholarly journals Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment

2021 ◽  
Vol 11 ◽  
Author(s):  
Jiangqing Huang ◽  
Shengcen Zhang ◽  
Zhichang Zhao ◽  
Min Chen ◽  
Yingping Cao ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Plasmid Stability ◽  
Bioinformatic Analysis ◽  
Fitness Cost ◽  
Resistance Pattern ◽  
Carbapenem Resistant ◽  
Significant Difference ◽  
Union Hospital ◽  
Growth Assay ◽  
Medical University

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in blaKPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in blaKPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored blaKPC-2, blaSHV-182 and blaTEM-1B. Compared with KP135194, KP137060 harbored an additional blaNDM-5 positive plasmid. blaNDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that blaKPC-2- and blaNDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a blaNDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.

scholarly journals Colistin Susceptibility in Carbapenem Resistant Klebsiella Pneumoniae and their Ability of Biofilm Formation

2020 ◽  
pp. 517-527
Author(s):  
Sarab Murad Kadum
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Gene ◽  
Biofilm Formation ◽  
Rpob Gene ◽  
Resistance Rate ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Specific Primer ◽  
Carbapenem Resistant

A total of 157 clinical samples were collected from different clinical specimens (urine, sputum, blood, swabs, and cannula) from several hospitals in Iraq. Among the samples, 51 isolates (32.48%) of Klebsiella pneumoniae were identified according to morphologicaland cultural characteristics as well as the Enterosystem 18R test. Higher numbers of K. pneumoniae isolates were observed in urine samples (26, 52%) than the other samples, and in females (70.6%) than males (29.4%) (female: male ratio of about 2.4:1). Antibiotic susceptibility of K. pneumoniae against 12 commonly used antibiotics was determined through the disc-diffusion method. The results revealed a higher resistance rate in 51 isolates (100%) against Cephalexin, followed by Ceftazidime (50, 98%), while the lowest resistance rate (24, 47%) was against each of Imipenem and Meropenem. Also, the investigation of the minimum inhibitory concentration (MIC) of Colistin using E-test (strips) demonstrated that 33 isolates were resistance, as compared to 31 using the disk diffusion assay. DNA was extracted from K. pneumoniae isolates and molecularly tested using polymerase chain technique (PCR) with a specific primer and 108 bp product to detect the rpoB gene that represents this bacteria . Also, all of the 51 isolates of K. pneumoniae identified by the rpoB gene were detected for the expression of the Colistin drug resistance gene mgr-B , which was amplified (347 bp) using a specific primer. Colistin resistance gene mgr-B was amplified and sequenced from the twenty isolates. Only 6 isolates appeared with a single nucleotide substitution; G instead A, A instead G, C instead G and G instead C. Also, this study tested biofilm formation from K. pneumoniae isolates , using the microtiter plate method, in association with Colistin and Carbapenem resistant. The Colistin and Carbapenem resistance pattern was compared to the ability of biofilm-formation as weak formation versus strong and also, Multi-drug resistant isolates were more common among weak versus strong biofilm formers.

scholarly journals Aerobactin Seems To Be a Promising Marker Compared With Unstable RmpA2 for the Identification of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae: In Silico and In Vitro Evidence

2021 ◽  
Vol 11 ◽  
Author(s):  
Chaitra Shankar ◽  
Soumya Basu ◽  
Binesh Lal ◽  
Sathiya Shanmugam ◽  
Karthick Vasudevan ◽  
...  
Keyword(s):  
High Risk ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
In Silico ◽  
Fitness Cost ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Carbapenem Resistant ◽  
Antimicrobial Resistance Genes

BackgroundThe incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.MethodsTwenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.ResultsThe CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.ConclusionIncreasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

scholarly journals Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates with focus on antimicrobial resistance

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaoling Yu ◽  
Wen Zhang ◽  
Zhiping Zhao ◽  
Chengsong Ye ◽  
Shuyan Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Phenotypic Resistance ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Carbapenemase Gene ◽  
Long Read ◽  
Medical University

Abstract Background The enhancing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP)-mediated infections in Mengchao Hepatobiliary Hospital of Fujian Medical University in 2017 is the motivation behind this investigation to study gene phenotypes and resistance-associated genes of emergence regarding the CRKP strains. In current study, seven inpatients are enrolled in the hospital with complete treatments. The carbapenem-resistant K. pneumoniae whole genome is sequenced using MiSeq short-read and Oxford Nanopore long-read sequencing technology. Prophages are identified to assess genetic diversity within CRKP genomes. Results The investigation encompassed eight CRKP strains that collected from the patients enrolled as well as the environment, which illustrate that blaKPC-2 is responsible for phenotypic resistance in six CRKP strains that K. pneumoniae sequence type (ST11) is informed. The plasmid with IncR, ColRNAI and pMLST type with IncF[F33:A-:B-] co-exist in all ST11 with KPC-2-producing CRKP strains. Along with carbapenemases, all K. pneumoniae strains harbor two or three extended spectrum β-lactamase (ESBL)-producing genes. fosA gene is detected amongst all the CRKP strains. The single nucleotide polymorphisms (SNP) markers are indicated and validated among all CRKP strains, providing valuable clues for distinguishing carbapenem-resistant strains from conventional K. pneumoniae. Conclusions ST11 is the main CRKP type, and blaKPC-2 is the dominant carbapenemase gene harbored by clinical CRKP isolates from current investigations. The SNP markers detected would be helpful for characterizing CRKP strain from general K. pneumoniae. The data provides insights into effective strategy developments for controlling CRKP and nosocomial infection reductions.

Virulence-associated characteristics of carbapenem-resistant Klebsiella pneumoniae in hospital-acquired infections: results from a hospital in central China

2019 ◽  
Author(s):  
OUYANG Pengwen ◽  
Bin JIANG ◽  
Juan WANG ◽  
Na PENG ◽  
Jianrong YE ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Central China ◽  
Capsular Antigen ◽  
Hospital Acquired Infections ◽  
Carbapenem Resistant ◽  
Positive Strain ◽  
Significant Difference ◽  
Hospital Acquired

Abstract Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) have been a clinically significant pathogen worldwide, but related reports about their virulence features in hospital-acquired infections (HAI) are pretty lacking.Methods CRKP causing HAI were continuously collected in 2018 from a hospital in central China. Isolates identification and antimicrobial susceptibility test were done using VITEK-2 compact system or MALDI-TOF MS. String test, multilocus sequence typing, carbapenemase genes, virulence genes and capsular antigen genes detection were conducted to understand their phenotype and genetic background. As well as case datas were collected and compared to assess their virulence characteristics.Results A total of 62 isolates of CRKP from 62 patients with HAI were collected. 41 carbapenemase genestic-confirmed hypervirulent Klebsiella pneumoniae (CR-hvKP) and 21 carbapenem resistant non-hypervirulent Klebsiella pneumoniae (CR-NhvKP) were screened out. Most CRKP causing HAI were ST11 KPC-2 producing strains and maily causing pneumonia. Only for blaKPC-2 there was a significant difference between CR-hvKP and CR-NhvKP (p<0.001). No significant difference of the two group strains in resistance against amikacin, trimethoprim-sulfamethoxazoleare, cefepime, ceftazidime, imipenem, piperacillin-tazobactam, colistin and tigecycline were found except levofloxacin (p<0.001), and all strains showed sensitive to tigecycline and colistin. In the CR-hvKP group, IucA (64.5%) were the most commonly detected virulence gene, followed by iroN (48.4%), prmpA2 (30.6%) and prmpA (4.8%), only 1 (2.4%) capsular serotype positive strain and 2 (4.9%) hypermucoviscosity phenotype strains were detected, while no hypermucoviscosity phenotype or capsular antigen gene positive strain was detected in the CR-NhvKP group. And there was no significant difference between the two groups in age, types of infection, departmental distribution, survival time or the final outcome of infection.Conclusion ST11 KPC-2-producing Klebsiella pneumoniae are most prevalent CRKP in HAI. Virulence gene espacially iucA has a high proportion and worth paying attention to. Hypermucoviscous phenotype and virulence-associated capsular serotype in CRKP both have a low prevalence. CRKP harboring virulence genes have a higher expression of KPC-2 and less sensitive to levofloxacin than those harboring no virulence gene, and there is no significant difference for virulence manifestations between the two groups.

scholarly journals 494. Fitness Cost of mcr-1-Mediated Colistin Resistance in Carbapenemase-Producing Klebsiella pneumoniae

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S241-S241
Author(s):  
Yi-Tsung Lin ◽  
Yi-Hsiang Cheng ◽  
Sheng-Hua Chou ◽  
Ying-Chi Huang ◽  
Liang Chen
Keyword(s):  
Growth Rate ◽  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Plasmid Stability ◽  
Fatal Case ◽  
Fitness Cost ◽  
Whole Genome ◽  
Colistin Resistance ◽  
The Impact

Abstract Background The emergence of mobile colistin resistance gene mcr-1, a plasmid-borne polymyxin resistance mechanism, in carbapenem-resistant Klebsiella pneumoniae is an alarming concern. However, previous studies showed that the acquisition of mcr-1 was associated with a significant biological fitness cost in K. pneumoniae. We aimed to study the impact of mcr-1 on the biological fitness in clinical carbapenemase-producing K. pneumoniae strains. Methods Clinical carbapenemase-producing K. pneumoniae strains were collected consecutively at the Taipei Veterans General Hospital between November 2017 and December 2018. The strain positive for mcr-1 was subjected to whole-genome sequencing to delineate its genomic features. Escherichia coli J53 strain was used as the recipient strain in plasmid conjugation assay and the transconjugants were selected with sodium azide and colistin. Plasmid stability was tested by serial passaging in antibiotic-free LB broth for 28 days. The growth rate was compared between the parental mcr-1-bearing strain and the plasmid-cured strain. Results One ST11 strain isolated from a fatal case with bacteremia (KP2509) was found to harbor blaKPC-2, blaOXA-48, and mcr-1. This strain was resistant to colistin (MIC=8 mg/L) and imipenem (MIC≥16 mg/L). Whole-genome sequencing of KP2509 showed that mcr-1, blaKPC-2 and blaOXA-48 were located on an IncHI-FIB type plasmid of 319 Kb, an IncFII type plasmid of 96 Kb, and an IncL type plasmid of 64 Kb, respectively. Conjugation efficiency of mcr-1-bearing plasmid was 2.24 × 10–4, and the colistin MIC of E. coli J53 transconjugant increased from 0.5 to 8 mg/L. The mcr-1-bearing plasmid in KP2509 showed high plasmid stability, and only ~1% were lost after 27-day passages. The resulting plasmid-cured strain (PC-KP2509) was susceptible to colistin (MIC=0.5 mg/L) and had a similar growth rate to that of parental mcr-1-bearing strain KP2509. Conclusion We identified an ST11 K. pneumoniae strain carrying blaKPC-2, blaOXA-48, and mcr-1 genes causing a fatal bacteremia. The large mcr-1-bearing plasmid confers a moderate level of colistin resistance but without significant biological fitness cost in carbapenemase-producing K. pneumoniae, which could result in a serious threat clinically. Disclosures All authors: No reported disclosures.

scholarly journals Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae

2018 ◽  
Vol 73 (6) ◽  
pp. 1604-1610 ◽  
Author(s):  
Sue C Nang ◽  
Faye C Morris ◽  
Michael J McDonald ◽  
Mei-Ling Han ◽  
Jiping Wang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Lipid A ◽  
Plasmid Stability ◽  
Fitness Cost ◽  
Infection Model ◽  
Competition Assay ◽  
In Vivo Growth ◽  
The Impact

Abstract Objectives The discovery of mobile colistin resistance mcr-1, a plasmid-borne polymyxin resistance gene, highlights the potential for widespread resistance to the last-line polymyxins. In the present study, we investigated the impact of mcr-1 acquisition on polymyxin resistance and biological fitness in Klebsiella pneumoniae. Methods K. pneumoniae B5055 was used as the parental strain for the construction of strains carrying vector only (pBBR1MCS-5) and mcr-1 recombinant plasmids (pmcr-1). Plasmid stability was determined by serial passaging for 10 consecutive days in antibiotic-free LB broth, followed by patching on gentamicin-containing and antibiotic-free LB agar plates. Lipid A was analysed using LC–MS. The biological fitness was examined using an in vitro competition assay analysed with flow cytometry. The in vivo fitness cost of mcr-1 was evaluated in a neutropenic mouse thigh infection model. Results Increased polymyxin resistance was observed following acquisition of mcr-1 in K. pneumoniae B5055. The modification of lipid A with phosphoethanolamine following mcr-1 addition was demonstrated by lipid A profiling. The plasmid stability assay revealed the instability of the plasmid after acquiring mcr-1. Reduced in vitro biological fitness and in vivo growth were observed with the mcr-1-carrying K. pneumoniae strain. Conclusions Although mcr-1 confers a moderate level of polymyxin resistance, it is associated with a significant biological fitness cost in K. pneumoniae. This indicates that mcr-1-mediated resistance in K. pneumoniae could be attenuated by limiting the usage of polymyxins.

Colonization of carbapenem-resistant Klebsiella pneumoniae in a sink-drain model biofilm system

2020 ◽  
pp. 1-9
Author(s):  
Maria Burgos-Garay ◽  
Christine Ganim ◽  
Tom J.B. de Man ◽  
Terri Davy ◽  
Amy J. Mathers ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Community Analysis ◽  
Model System ◽  
Model Systems ◽  
Microbial Consortia ◽  
Healthcare Facilities ◽  
Biofilm Reactors ◽  
Carbapenem Resistant ◽  
Significant Difference ◽  
Biofilm Development

Abstract Background: Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella pneumoniae (CPKP). Methods: We investigated the colonization of a biofilm consortia by CPKP in a model system simulating a sink-drain P-trap. Centers for Disease Control (CDC) biofilm reactors (CBRs) were inoculated with microbial consortia originally recovered from 2 P-traps collected from separate patient rooms (designated rooms A and B) in a hospital. Biofilms were grown on stainless steel (SS) or polyvinyl chloride (PVC) coupons in autoclaved municipal drinking water (ATW) for 7 or 28 days. Results: Microbial communities in model systems (designated CBR-A or CBR-B) were less diverse than communities in respective P-traps A and B, and they were primarily composed of β and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with either K. pneumoniae ST45 (ie, strain CAV1016) or K. pneumoniae ST258 KPC+ (ie, strain 258), and samples were collected over 21 days. Under most conditions tested (CBR-A: SS, 7-day biofilm; CBR-A: PVC, 28-day biofilm; CBR-B: SS, 7-day and 28-day biofilm; CBR-B: PVC, 28-day biofilm) significantly higher numbers of CAV1016 were observed compared to 258. CAV1016 showed no significant difference in quantity or persistence based on biofilm age (7 days vs 28 days) or substratum type (SS vs PVC). However, counts of 258 were significantly higher on 28-day biofilms and on SS. Conclusions: These results suggest that CPKP persistence in P-trap biofilms may be strain specific or may be related to the type of P-trap material or age of the biofilm.

scholarly journals Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates with focus on antimicrobial resistance

2019 ◽  
Author(s):  
Xiaoling Yu ◽  
Wen Zhang ◽  
Zhiping Zhao ◽  
Chengsong Ye ◽  
Shuyan Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Phenotypic Resistance ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Carbapenemase Gene ◽  
Long Read ◽  
Medical University

Abstract Ba ckgrou nd The enhancing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP)-mediated infections in Mengchao Hepatobiliary Hospital of Fujian Medical University in 2017 is the motivation behind this investigation to study gene phenotypes and resistance-associated genes of emergence regarding the CRKP strains. In current study, seven inpatients are enrolled in the hospital with complete treatments. The carbapenem-resistant K. pneumoniae whole genome is sequenced using MiSeq short-read and Oxford Nanopore long-read sequencing technology. Prophages are identified to assess genetic diversity within CRKP genomes. Results The investigation encompassed eight CRKP strains that collected from the patients enrolled as well as the environment, which illustrate that bla KPC-2 is responsible for phenotypic resistance in six CRKP strains that K . pneumoniae sequence type (ST11) is informed. The plasmid with IncR, ColRNAI and pMLST type with IncF[F33:A-:B-] co-exist in all ST11 with KPC-2-producing CRKP strains. Along with carbapenemases, all K. pneumoniae strains harbor two or three extended spectrum β-lactamase (ESBL)-producing genes. f osA gene is detected amongst all the CRKP strains. The single nucleotide polymorphisms (SNP) markers are indicated and validated among all CRKP strains, providing valuable clues for distinguishing carbapenem-resistant strains from conventional K. pneumoniae . Co nclusions ST11 is the main CRKP type, and bla KPC-2 is the dominant carbapenemase gene harbored by clinical CRKP isolates from current investigations. The SNP markers detected would be helpful for characterizing CRKP strain from general K. pneumoniae. The data provides insights into effective strategy developments for controlling CRKP and nosocomial infection reductions.

scholarly journals Curcumin-meropenem synergy in carbapenem resistant Klebsiella pneumoniae curcumin-meropenem synergy

2021 ◽  
Author(s):  
Dumrul Gülen ◽  
Birol Şafak ◽  
Berna Erdal ◽  
Betül Günaydın
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Treatment Options ◽  
Resistance Pattern ◽  
Carbapenem Resistant ◽  
Efflux Pump Inhibition ◽  
Disk Method

Background and Objectives: The frequency of multiple resistant bacterial infections, including carbapenems, is increasing worldwide. As the decrease in treatment options causes difficulties in treatment, interest in new antimicrobials is increasing. One of the promising natural ingredients is curcumin. It is known to be effective in bacteria such as Pseudomonas aeruginosa, Escherichia coli, Burkholderia pseudomallei through efflux pump inhibition, toxin inhibition and enzymes. However, because its bioavailability is poor, it seffectiveness occurs in combination with antibiotics. In the study, the interaction of meropenem and curcumin in carbapenemase producing strains of Klebsiella pneumoniae was tested. Materials and Methods: Thirty-nine Klebsiella pneumoniae isolates, resistant to meropenem, were used in this study. From those 15 MBL, 6 KPC, 17 OXA-48 and 1 AmpC resistance pattern were detected by combination disk method. Meropenem and Curcumin MIC values were determined by liquid microdilution. Checkerboard liquid microdilution was used to determine the synergy between meropenem and curcumin. Results: Synergistic effects were observed in 4 isolates producing MBL, 3 isolates producing KPC, 4 isolates producing OXA-48, and 1 isolates producing AmpC (totally 12 isolates) according to the calculated FICI. No antagonistic effects were observed in any isolates. Conclusion: Curcumin was thought to be an alternative antimicrobial in combination therapies that would positively contribute to the treatment of bacterial infection. The effectiveness of this combination should be confirmed by other in vitro and clinical studies.

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