LETTER
Colistin resistance is primarily driven by the acquisition of the mobile colistin resistance gene (mcr) through horizontal gene transfer (1). mcr-1 is the most widespread mcr determinant globally and appears in 36 variants with varying frequencies (2). Rare mcr variants are instrumental in tracing the epidemiology of these genes and associated mobile genetic elements. Recently, the rare variant mcr-1.26 has been identified in 16 colistin-resistant extended-spectrum β-lactamase (ESBL)-producing and commensal Escherichia coli from poultry and a human clinical isolate in Germany (3, 4). Bioinformatic analysis revealed that these isolates shared a distant relationship but carried mcr-1.26 on highly similar IncX4 plasmids. This finding signified the establishment and dissemination of mcr-1.26-IncX4 plasmids within the poultry food chain and its transmission to humans.
Here, we report for the first time the presence of an mcr-1.26-IncX4 plasmid in a Klebsiella pneumoniae isolate 22-MO00052 (CVUAS 34108) obtained from pre-packaged raw turkey meat in Germany in 2022. Hybrid whole-genome sequencing (Illumina/ONT, Bioproject PRJNA1038782) revealed the location of mcr-1.26 on a 39.953-kb IncX4 plasmid (p22MO52B). The plasmid also carried the Tn2-associated beta-lactamase gene blaTEM-135 (99.88% nucleotide identity), but acquired additionally an IS91 family transposase distinguishing it from pEc200574 (Fig. 1A; Table 1) (3). A BlastN search showed that the IS91 family transposase is primarily found on plasmids of the Enterobacteriaceae family, associated with antibiotic resistance and virulence genes, creating a potential hotspot for acquiring additional pathogenicity genes (5, 6). p22MO52B exemplifies the ongoing evolution and adaption of mcr-1.26-IncX4 plasmids.
Fig 1.
Organization and interspecies transmission of resistance plasmids. (A) Comparison of the mcr-1.26-containing region of IncX4 plasmids between E. coli and K. pneumoniae isolated from poultry. (B) Interspecies transmission by conjugation of IncX4 plasmid and resulting phenotypic colistin resistance in transconjugants. Microbiological resistance profiles were determined using the broth microdilution method according to CLSI. Resistances in donor strain 22-MO00052, recipient strains (R), and corresponding transconjugants (T) are highlighted with a gray background. AK, amikacin; AMP, ampicillin; AZI, azithromycin; FOT, cefotaxime; TAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; GEN, gentamicin; MERO, meropenem; NAL, nalidixic acid; SMX, sulfamethoxazole; TET, tetracycline; TGC, tigecycline; and TMP, trimethoprim. (C) Schematic representation of the IncFIB plasmid p22MO52A.
TABLE 1.
| Feature | Chromosome | Plasmids | |
|---|---|---|---|
| ID | 22-MO00052 | p22MO52A | p22MO52B |
| Size (kb) | 5,347.601 | 118.898 | 39.953 |
| G + C content (%) | 57.24 | 50.83 | 43.36 |
| Molecule type | Circular | Circular (IncFIB, 98.93%) | Circular (IncX4, 100%) |
| Accession number | CP138466 | CP138467 | CP138468 |
| ORFse | 4,956 | 111 | 42 |
| Resistance determinants | +e | + | + |
| Aminoglycosides | –f | aph (6)-Id (100%)c | - |
| – | aph(3″)-Ib (100%)c | – | |
| Beta-lactams | blaSHV-27 (100%)c | – | – |
| – | blaTEM-135 (99.88%)c | blaTEM-135 (99.88%)c | |
| – | blaCTX-M-15 (100%)c | – | |
| Carbapenems | ompK37 (I70M),d ompK37 (I128K)d | – | – |
| Cephalosporins | ompK36 (N49S),d ompK36 (L59V),d ompK36 (T184P)d | – | – |
| Colistin | – | – | mcr-1.26 (100%)c |
| Disinfectants | oqxA (99.40%)c | – | – |
| oqxB (99.14%)c | – | – | |
| Fosfomycin | fosA (99.76%)c | – | – |
| Quinolone | – | qnrS1 (100%)c | – |
| Fluoroquinolone | acrR (P161R), acrR (G164A), acrR (F172S), acrR (R173G), acrR (L195V), acrR (F197I), acrR (K201M) | – | – |
| Sulphonamides | – | sul2 (100%)c | – |
| Trimethoprim | – | dfrA14 (100%)c | – |
| IS-elements | |||
| Insertion sequence | ISKpn1, IS100, IS911, ISEc30, ISEc52, ISKpn1 | ISKpn19, ISEc52, ISKpn26, IS26 | IS26, IS91 |
| Composite transposon | n.d.e | cn_5504_IS26 | n.d. |
| Unit transposon | n.d. | n.d. | Tn2 |
Nucleotide sequence identity is given for some elements in brackets.
The K. pneumoniae strain 22-MO00052 harbored three additional plasmids that did not carry resistance genes: p22MO52C [accession: CP138469, Col RNAI (89.26%), 5.631 kb, 47.38% GC, six ORFs], p22MO52D [accession: CP138470, Col440I (97.37%), 3.631 kb, 44.20% GC, three ORFs], and p22MO52E [accession: CP138471, Col MG828 (94.27%), 2.967 kb, 52.85% GC, two ORFs].
Acquired resistance determinants.
Mutations are predicted to play a role in phenotypic carbapenem and cephalosporin resistance.
n.d., not detected; +, present; and ORF, open reading frame.
"-" in the Table means "not present".
Filter mating experiments of p22MO52B using K. pneumoniae 22-MO00052 as a donor demonstrated an mcr-1.26 transfer frequency of 4.7 × 102 to E. coli, 2.3 × 101 to K. pneumoniae, and 1.1 × 103 to Salmonella enterica Typhimurium, confirming its potential for interspecies transmission (Fig. 1B). While the notification of mcr genes in klebsiellae is not new, studies focusing on emerging mcr variants, specifically mcr-1.26, have provided valuable insights into the origin and dynamics of mcr-associated colistin resistance development. mcr-1.26 serves as a suitable indicator to shed light on the spread of resistance genes across various bacterial hosts and One Health compartments.
In addition to p22MO52B, the K. pneumoniae isolates carried a 118.898-kb multidrug-resistance plasmid p22MO52A belonging to the incompatibility group IncFIB (Fig. 1C). p22MO52A had no similarity to any other plasmid in the NCBI database (accessed 18 October 2023) and carried a 24.4-kb integron with seven resistance genes, conferring resistance to four classes of antibiotics (Table 1). The integron contained a second blaTEM-135 gene (99.88% nucleotide identity), but unlike p22MO52B, this gene was not linked to Tn2. The integron was associated with an ISKpn19 element and was identical to the integrons of the IncY and IncFIB plasmids from E. coli (LR999865.1, host Branta leucopsis) and K. pneumoniae (CP084503, host Capra aegagrus hircus), respectively (accessed 13 November 2023).
The isolate belongs to ST716 [KL 110 (unknown capsule type, not serologically defined), O1/O2v1 (O2a O-type)], which has been associated with human infections (Pathogenwatch, https://pathogen.watch/, accessed 07 November 2023). In addition to the acquired resistance genes, 22-MO00052 carried chromosomal mutations in ompK36, ompK37, and acrR, which are predicted to contribute to carbapenem, cephalosporin, and fluoroquinolone resistance, respectively (Table 1) (7–10). Besides carbapenem, the isolate exhibited phenotypic resistance toward cephalosporins and fluoroquinolones, which could be mediated by both chromosomal mutations and acquired resistance genes (Fig. 1B). K. pneumoniae is an [ESKAPE(E)] microorganism, known for its excessive exchange of genetic information with the environment and other bacteria, including mcr-1.26, as an adaptation to varying selective pressures in different ecosystems (11). K. pneumoniae is also a nosocomial pathogen for which novel therapeutic approaches are needed, as recognized by the World Health Organization.
ACKNOWLEDGMENTS
This work was supported by the German Federal Ministry of Health (https://www.rki.de/DE/Content/Institut/OrgEinheiten/Abt1/FG13/guecci.html) (BfR grant number 60-0103-08.P105 [project acronym GÜCCI]), JPIAMR project KLEOPATRA (01KI2302A), and the German Federal Institute for Risk Assessment (BfR grant numbers 1322-648 and 1322-820). The work of J.A.H. was supported by the European Joint Project (EJP) Full_Force funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement no. 773830.
Contributor Information
Ulrike Binsker, Email: ulrike.binsker@bfr.bund.de.
Rafael Vignoli, Instituto de Higiene, Montevideo, Uruguay.
REFERENCES
- 1. Umair M, Hassan B, Farzana R, Ali Q, Sands K, Mathias J, Afegbua S, Haque MN, Walsh TR, Mohsin M. 2023. International manufacturing and trade in colistin, its implications in colistin resistance and one health global policies: a microbiological, economic, and anthropological study. Lancet Microbe 4:e264–e276. doi: 10.1016/S2666-5247(22)00387-1 [DOI] [PubMed] [Google Scholar]
- 2. Nang SC, Li J, Velkov T. 2019. The rise and spread of mcr plasmid-mediated polymyxin resistance. Crit Rev Microbiol 45:131–161. doi: 10.1080/1040841X.2018.1492902 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Binsker U, Oelgeschläger K, Neumann B, Werner G, Käsbohrer A, Hammerl JA. 2023. Genomic evidence of mcr-1.26 IncX4 plasmid transmission between poultry and humans. Microbiol Spectr 11:e0101523. doi: 10.1128/spectrum.01015-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Neumann B, Rackwitz W, Hunfeld KP, Fuchs S, Werner G, Pfeifer Y. 2020. Genome sequences of two clinical Escherichia coli isolates harboring the novel colistin-resistance gene variants mcr-1.26 and mcr-1.27. Gut Pathog 12:40. doi: 10.1186/s13099-020-00375-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Garcillán-Barcia MP, de la Cruz F. 2002. Distribution of IS91 family insertion sequences in bacterial genomes: evolutionary implications. FEMS Microbiol Ecol 42:303–313. doi: 10.1111/j.1574-6941.2002.tb01020.x [DOI] [PubMed] [Google Scholar]
- 6. Soliman AM, Ramadan H, Zarad H, Sugawara Y, Yu L, Sugai M, Shimamoto T, Hiott LM, Frye JG, Jackson CR, Shimamoto T. 2021. Coproduction of Tet(X7) conferring high-level tigecycline resistance, fosfomycin fosa4, and colistin mcr-1.1 in Escherichia coli strains from chickens in Egypt. Antimicrob Agents Chemother 65. doi: 10.1128/AAC.02084-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Legese MH, Asrat D, Mihret A, Hasan B, Mekasha A, Aseffa A, Swedberg G. 2022. Genomic epidemiology of carbapenemase-producing and colistin-resistant Enterobacteriaceae among sepsis patients in Ethiopia: a whole-genome analysis. Antimicrob Agents Chemother 66:e0053422. doi: 10.1128/aac.00534-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Ruan Y, Li M, Wang D, Duan J, Zhang H, Zhou Y. 2024. Characteristics of non-carbapenemase producing carbapenem-resistant Klebsiella pneumoniae from a tertiary hospital in China. J Infect Dev Ctries 18:106–115. doi: 10.3855/jidc.17779 [DOI] [PubMed] [Google Scholar]
- 9. Uz Zaman T, Aldrees M, Al Johani SM, Alrodayyan M, Aldughashem FA, Balkhy HH. 2014. Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia. Int J Infect Dis 28:186–192. doi: 10.1016/j.ijid.2014.05.021 [DOI] [PubMed] [Google Scholar]
- 10. Schneiders T, Amyes SGB, Levy SB. 2003. Role of AcrR and ramA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore. Antimicrob Agents Chemother 47:2831–2837. doi: 10.1128/AAC.47.9.2831-2837.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Wyres KL, Holt KE. 2018. Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria. Curr Opin Microbiol 45:131–139. doi: 10.1016/j.mib.2018.04.004 [DOI] [PubMed] [Google Scholar]