Abstract
Escherichia coli A382 was isolated in July 2024 from a positive blood culture obtained from the central venous catheter of a male patient undergoing chemotherapy at the Hospital of Volos, Thessaly, Greece. Whole-genome sequencing analysis revealed that the isolate A382 is E. coli belonging to the ST410 high-risk clone, which co-harbors the blaKPC-3 and blaSHV-182 genes on an IncX3 plasmid. It also harbors blaTEM-1 and has five replicons, as follows: IncX3, IncQ1, CoIRNAI, IncF1A, and IncFIB. Complete genome analysis revealed that E. coli A382 isolate carries a range of virulence factors (iutA, iucC, fimH, fdeC, yehA, yehD, yehC, yehB, cgs, ahha, ccI, hlyE, papC, irp2, fyuA, lpfA, and nlpl) and many other non-beta-lactam resistance determinants, including dfrA14 and sul2, but it is susceptible to aminoglycosides, nitrofurantoin, tigecycline, colistin and ceftazidime-avibactam. In conclusion in this study, we describe the phenotypic and genome characteristics of an extensively drug-resistant E. coli ST410.
Introduction
Escherichia coli is considered to be one of the most important pathogens for humans, as it causes a range of infections in humans, including urinary tract infections and other community-acquired infections [1]. The ability of E. coli to accumulate resistance genes, particularly those conferring resistance to beta-lactams, has led to increasing challenges in treatment. Among these resistance mechanisms, extended-spectrum beta-lactamases (ESBLs) such as CTX-M-15 are particularly prevalent [2]. However, the emergence of carbapenemase-producing E. coli has become a serious issue in the field of healthcare [3].
In addition to ESBLs, E. coli has also been increasingly associated with carbapenem resistance, primarily due to the acquisition of carbapenemase genes. Recent reports have shown the spread of blaOXA, blaNDM and the blaKPC genes among E. coli isolated from patients in different geographical regions [4]. Initially identified in Klebsiella pneumoniae, KPC-type enzymes have now spread to E. coli and to other Enterobacteriaceae, mainly through plasmids and transposons [5–7]. In recent years, the presence of KPC in the E. coli sequence type 131 strain has raised concerns [8]. In Greece, the emergence of E. coli type 410 with KPC-2 lactamase was reported by Efthymia Petinaki et al. in 2010 [9]. However, E. coli ST410 strain is known to be associated with ΟΧΑ, NDM and CTX-M-15 beta-lactamase production [10–12].
Beyond beta-lactamases, E. coli can harbor other resistance mechanisms, including aminoglycoside-modifying enzymes, which inactivate aminoglycosides, efflux pumps and mutations in target sites such as gyrA and parC, leading to fluoroquinolone resistance. Additionally, resistance to colistin a last-resort antibiotic, has emerged through modifications in the pmrAB and phoPQ regulatory systems, and by acquisition of the mcr-1 gene [10–13]. The accumulation of multiple resistance determinants within a single strain can lead to extensively drug-resistant (XDR) phenotypes, significantly narrowing the options for effective treatment [12, 13].
This study presents the phenotypic and genomic characterization of A382 an XDR E. coli ST410 strain isolated from a patient in Volos, Greece. Whole genome analysis of the A382 strain revealed multiple resistance genes to beta-lactams like blaKPC-3, blaCTX-M-15, and to other classes of antibiotics, like dfrA14 and sul2. It also revealed the presence of multiple virulence genes like iutA and iucC for the synthesis and transport of iron transferring molecule aerobactin.
Materials and methods
Collection of strain
The strain was recovered from a blood culture of a male patient who was receiving chemotherapy in the Hospital of Volos, Greece, representing an E. coli isolate fulfilling the phenotypic criteria for carbapenemase production.
Susceptibility testing
Minimum inhibitory concentrations (MICs) were determined using an automated method with a Vitek-2 system (Biomerieux). The MIC of ceftazidime-avibactam was determined with E-test (Biomerieux). The MIC of colistin was determined using the broth microdilution method. The interpretive criteria of the European Committee on Antimicrobial Susceptibility testing (EUCAST) were used (https://www.eucast.org/clinical_breakpoints (accessed on 14-07-2024)).
Whole-genome sequencing
For genome sequencing, total DNA was extracted using a PureLinkTM Quick Gel Extraction Kit (Life Technologies, CA, USA). Whole-genome sequencing was performed in a private laboratory in Greece (Cemia). Libraries were prepared using Ion Torrent technology and Ion Chef workflows (Thermo Scientific). Sequencing was performed using the S5XLS system and the analysis of primary data was conducted with the Ion Torrent Suite software (v.5.10.0). Resistance genes were identified using Resfinder-4.6.0. Mobile genetic elements were identified using MobileElementFinder-1.0.3. The core genome ST was identified using the cgMLSTFinder-1.2 Server. The replicons were identified using the PlasmidFinder-2.0 Server. The CH type was identified using the CHTyper-1.0 Server. CHTyper is a web tool for the subtyping of extra-intestinal pathogenic E. coli based on the fumC and fimH alleles [14]. The pathogenicity was predicted using PathogenFinder. Finally, the phylogroup was predicted using Clermon typing [15].
Results and discussion
The isolate exhibited resistance to cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, meropenem, ertapenem, trimethoprim-sulfamethoxazole and ciprofloxacin; it exhibited sensitivity to aminoglycosides, nitrofurantoin, tigecycline, colistin and ceftazidime-avibactam (Table 1).
Susceptibility of E. coli ST410 strain A382 to antibiotics
| Antimicrobial | MIC (mg L−1) |
| Ampicillin | ≥32 |
| Ampicillin/Sulbactam | ≥32 |
| Amoxycillin/Clavulanic Acid | ≥32 |
| Piperacillin | ≥128 |
| Piperacillin/Tazobactam | ≥128 |
| Cefuroxime | ≥64 |
| Cefuroxime Axetil | ≥64 |
| Ceftriaxone | 8 |
| Ceftazidime | 16 |
| Aztreonam | ≥64 |
| Ceftazidime/Avibactam | 1 |
| Cefotaxime | 4 |
| Ertapenem | 2 |
| Imipenem | ≥16 |
| Meropenem | ≥16 |
| Amikacin | ≤2 |
| Gentamycin | ≤1 |
| Tobramycin | ≤1 |
| Ciprofloxacin | ≥4 |
| Levofloxacin | ≥8 |
| Moxifloxacin | ≥8 |
| Tigecycline | ≤0.5 |
| Nitrofurantoin | ≤16 |
| Colistin | 0.5 |
| Trimethoprim | ≥16 |
| Trimethoprim/Sulfomethoxazole | ≥320 |
Genotyping of E. coli A382 indicated that it has a genome size of 4,872,137 bp and G+C content of 57.16% (Table 2). The isolate belonged to the sequence type (ST) ST410, according to the MLST allelic profile of Achtman's scheme, which uses the sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). The E. coli of ST410 lineage has presented increasing worldwide spread and, due to its association with multiple antibiotic determinants and its efficient escalation in healthcare settings, it is now considered a highly successful pandemic clone compared to ST131. This international clone has shown a global context in 14 countries encompassing Europe, North and South America, Asia, and Africa [16]. In Greece the E. coli of ST410 lineage appeared in 2010 [9]. The persistence of this lineage in 2024 with the acquisition of blaKPC-3 gene is of great concern.
Genome of E. coli ST410 strain A382
| Parameter | Values |
| # contigs | 98 |
| # contigs (≥0 bp) | 124 |
| # contigs (≥1,000 bp) | 82 |
| Largest contig | 357,858 |
| Total length | 4,872,137 |
| Total length (≥0 bp) | 4,879,386 |
| Total length (≥1,000 bp) | 4,860,675 |
| N50 | 147,993 |
| N90 | 33,229 |
| auN | 148,317 |
| L50 | 12 |
| L90 | 39 |
| GC (%) | 50.62 |
| Mismatches | |
| #N's per 100 kbp | 0 |
| #N's | 0 |
The detection of fimH demonstrated that the isolate carries the fimH24 allele, resulting in the clonotyping CH4-24 type. The fimH24 subtype has been described by Roer et al. as a successful sub-clonal lineage among ST410 E. coli strains [14].
According to Clermon typing, the strain belongs to phylogroup C. This phylogroup is correlated with hemolytic uremic syndrome in humans [15].
The genome of E. coli A382 demonstrated an MDR genotype, carrying genes conferring resistance to aminoglycosides (aph(6)-Id, aph(3″)), sulfonamides (sul2), trimethoprim (dfrA14), macrolides (mph(A)), tetracyclines (tet(B)), and chloramphenicol (catA1). In addition to blaKPC-3, Resfinder identified blaSHV-182 and blaTEM-1B (Table 3). Also, it carried point mutation in genes parC, gyrA conferring resistance to fluoroquinolones, glpT conferring resistance to fosfomycin.
Resistance genes, point mutations, virulence genes and replicons in E.coli ST410 strain A382
| 1. Antibiotic resistance genes |
| 1a. β-lactams |
| blaKPC-3, blaTEM-1B, blaSHV-182, blaSHV-159, blaSHV-158 |
| 1b. other antibiotics |
| dfrA14 trimethoprim |
| sul2 sulfamethoxazole |
| mph(A) erythromycin |
| catA1 chloramphenicol |
| tet(B) tetracycline |
| 2. Point mutations |
| parC, parE, gyrA, gyrB, acrR, acrB quinolone |
| rpoB rifamycin |
| glpT_E448K fosfomycin |
| pmrB_Y358N colistin |
| 3. Tolerance to antiseptics: |
| sitABCD hydrogen peroxide |
| 4. Virulence-associated genes: |
| iutA aerobactin receptor synthesis; iucC aerobactin synthesis |
| sitA Iron transport protein, irp2 high-molecular-weight protein 2 non-ribosomal peptide synthetase, fyuA siderophore receptor |
| fimH type 1 fimbriae, papC outer membrane usher P fimbriae, lpfA long polar fimbriae |
| fdeC intimin like adhesin |
| yehA, yehC, yehD, yehB outer membrane lipoprotein, YHD fimbrial cluster |
| cgsA curlin major subunit |
| hha hemolysin expression regulator, hlyE Avian E. coli hemolysin, ccI cloacin |
| nlpl lipoprotein precursor |
| 5. Resistance to heavy metals: |
| terC tellurium iron resistance protein |
| 6. Replicons: |
| IncX3, ΙncQ1, CoIRNAI, IncF1A, and IncFIB |
The presence of many virulence genes in the genome of E. coli A 382 indicates that it is an extraintestinal pathogen.
Five replicons were detected as follows: IncX3, incQ1, CoIRNAI, InCF1A, and IncFIB. The blaKPC-3-carrying plasmid IncX3 has been acquired by E. coli ST410, which is known to be associated with CTX-M, KPC-2, and NDM production [16–20]. The fusion of blaKPC-3 in a common pathogen, such as E. coli, has been earlier reported [13]. The core genome of E. coli A382 belongs to ST114429. The input organism was predicted as a human pathogen, with a probability of being a human pathogen of 0.874.
Conclusions
This study documented the acquisition of blaKPC-3 by E. coli belonging to ST410. It is the first time that a blaKPC-3 gene in an E. coli strain carried by transposon Tn4401 on an IncX3 plasmid has been reported in Greece. The persistence of this lineage in Greece since its first appearance in 2010 [9] indicates insufficient infection control in healthcare settings.
These findings, which suggest that the KPC-3-encoding transposon Tn4401 was acquired by an IncX3 replicon, highlight the continued need for molecular surveillance of multidrug-resistant pathogens. They also emphasize the growing clinical significance of the IncX3 plasmid family.
Author contributions
M.C. Conceptualization, writing, corrections-suggestions; P.T. Writing—original draft preparation, laboratory testing; MA.K. Writing and Corrections; S.M. Suggestions-corrections. All authors have read and agreed to the published version of the manuscript. Writing-review and editing M.C.
Funding
This research received no external funding.
Institutional review board statement
The study protocol was approved by the Ethics Committee of Hospital of Volos.
Informed consent statement
Not applicable due to the retrospective nature of this study.
Data availability statement
The whole-genome shotgun sequence for the E. coli strain A382 was deposited in Genbank under the BioProject PRJNA1146892 accession number.
Conflicts of interest
The authors declare no conflicts of interest.
References
- 1.↑
Marin J, Clermont O, Royer G, Mercier-Darty M, Decousser JW, Tenaillon O, et al. The population genomics of increased virulence and antibiotic resistance in human commensal Escherichia coli over 30 Years in France. Appl Environ Microbiol 2022; 88: e0066422. https://doi.org/10.1128/aem.00664-22.
- 2.↑
Pournaras S, Ikonomidis A, Kristo I, Tsakris A, Maniatis AN. CTX-M enzymes are the most common extended-spectrum beta-lactamases among Escherichia coli in a tertiary Greek hospital. J Antimicrob Chemother 2004; 54(2): 574–5. https://doi.org/10.1093/jac/dkh323.
- 3.↑
Doi Y. Treatment options for carbapenem-resistant gram-negative bacterial infections. Clin Infect Dis 2019; 69(Suppl. 7): S565–S575. https://doi.org/10.1093/cid/ciz830.
- 4.↑
Tsilipounidaki K, Florou Z, Skoulakis A, Fthenakis GC, Miriagou V, Petinaki E. Diversity of bacterial clones and plasmids of NDM-1 producing Escherichia coli clinical isolates in Central Greece. Microorganisms 2023; 11(2): 516. https://doi.org/10.3390/microorganisms11020516.
- 5.↑
Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis 2009; 9: 228–236. https://doi.org/10.1016/s1473-3099(09)70054-4.
- 6.
Leavitt A, Chmelnitsky I, Ofek I, Carmeli Y, Navon-Venezia S. Plasmid pKpQIL encoding KPC-3 and TEM-1 confers carbapenem resistance in an extremely drug-resistant epidemic Klebsiella pneumoniae strain. J Antimicrob Chemother 2009; 65: 243–248. https://doi.org/10.1093/jac/dkp417.
- 7.
Venditti C, Fortini D, Villa L, Vulcano A, D'Arezzo S, Capone A, et al. Circulation of blaKPC-3-carrying IncX3 plasmids among Citrobacter freundii isolates in an Italian hospital. Antimicrob Agents Chemother 2017; 61: e00505–17. https://doi.org/10.1128/aac.00505-17.
- 8.↑
Gong L, Tang N, Chen D, Sun K, Lan R, Zhang W, et al. A nosocomial respiratory infection outbreak of carbapenem-resistant Escherichia coli ST131 with multiple transmissible blaKPC–2 carrying plasmids. Front Microbiol 2020; 11: 2068. https://doi.org/10.3389/fmicb.2020.02068.
- 9.↑
Mavroidi A, Miriagou V, Malli E, Stefos A, Dalekos GN, Tzouvelekis LS, et al. Emergence of Escherichia coli sequence type 410 (ST410) with KPC-2 β-lactamase. Int J Antimicrob Agents 2012; 39: 247–250. https://doi.org/10.1016/j.ijantimicag.2011.11.003.
- 10.↑
Hu X, Yang L, Dong N, Lin Y, Zhang L, Wang X, et al. Dissemination of blaNDM-5 in Escherichia coli through the IncX3 plasmid from different regions in China. Microb Drug Resist 2022; 28: 453–460. https://doi.org/10.1089/mdr.2021.0202.
- 11.
Wu W, Feng Y, Tang G, Qiao F, McNally A, Zong Z. NDM Metallo-β-lactamases and their bacterial producers in health care settings. Clin Microbiol Rev 2019; 32: e00115–18. https://doi.org/10.1128/cmr.00115-18.
- 12.↑
Dias JB, Soncini JGM, Cerdeira L, Lincopan N, Vespero EC. MDR Escherichia coli carrying CTX-M-24 (IncF[F-:A1:B32]) and KPC-2 (IncX3/IncU) plasmids isolated from community-acquired urinary trainfection in Brazil. Braz J Infect Dis 2022; 26: 102706. https://doi.org/10.1016/j.bjid.2022.102706.
- 13.↑
Sadek M, Saad AM, Nordmann P, Poirel L. Genomic characterization of an extensively drug-resistant extra-intestinal pathogenic (ExPEC) Escherichia coli clinical isolate Co-producing two carbapenemases and a 16S rRNA methylase. Antibiotics 2022; 11: 1479. https://doi.org/10.3390/antibiotics11111479.
- 14.↑
Roer L, Johannesen TB, Hansen F, Stegger M, Tchesnokova V, Sokurenko E, et al. CHTyper, a web tool for subtyping of extraintestinal pathogenic Escherichia coli based on the fumC and fimH alleles. J Clin Microbiol 2018; 56: e00063–18. https://doi.org/10.1128/jcm.00063-18.
- 15.↑
Beghain J, Bridier-Nahmias A, Le Nagard H, Denamur E, Clermont O. ClermonTyping: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping. Microb Genom 2018; 4: e000192. https://doi.org/10.1099/mgen.0.000192.
- 16.↑
Roer L, Overballe-Petersen S, Hansen F, Schønning K, Wang M, Røder BL, et al. Escherichia coli sequence type 410 is causing new international high-risk clones. mSphere 2018; 3: e00337–18. https://doi.org/10.1128/msphere.00337-18.
- 17.
Kaur H, Singh I, Modgil V, Singh N, Mohan B, Taneja N. Genome sequence of pan drug- resistant enteroaggregative Escherichia coli belonging to ST38 clone from India, an emerging EAEC/UPEC hybrid pathotype. Indian J Med Microbiol 2024; 49: 100606. https://doi.org/10.1016/j.ijmmb.2024.100606.
- 18.
Wang CH, Siu LK, Chang FY, Tsai YK, Huang LY, Lin JC. Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. coli strains. BMC Microbiol 2024; 24(1): 109. https://doi.org/10.1186/s12866-024-03259-8.
- 19.
Chen L, Peirano G, Kreiswirth BN, Devinney R, Pitout JDD. Acquisition of genomic elements were pivotal for the success of Escherichia coli ST410. J Antimicrob Chemother 2022; 77: 3399–3407. https://doi.org/10.1093/jac/dkac329.
- 20.
Pitout JDD, Peirano G, Matsumura Y, DeVinney R, Chen L. Escherichia coli sequence type 410 with carbapenemases: a paradigm shift within E. coli toward multidrug resistance. Antimicrob Agents Chemother 2024; 68(2): e0133923. https://doi.org/10.1128/aac.01339-23.
