Abstract
Only two plasmid-mediated carbapenemases (KPC-2 and VIM-1) are reported in Klebsiella grimontii. Here, we report two blaKPC-3-positive isolates that were identified as K. oxytoca and E. coli by MALDI-TOF MS in the same rectal swab. Whole-genome sequencing indicated that K. oxytoca was actually K. grimontii of ST391, whereas E. coli was of ST10. In both, blaKPC-3 was carried by a pQil conjugative plasmid. The core-genome analysis identified additional blaKPC-positive K. grimontii strains from public databases, most of which were misidentified as K. oxytoca. Since K. grimontii represents an emerging reservoir of resistance traits, routine tools should improve their ability to detect this species.
Avoid common mistakes on your manuscript.
Klebsiella grimontii is an emerging pathogen associated with human infections and gut colonization that is frequently misidentified as Klebsiella oxytoca (e.g., implementing the matrix-assisted laser desorption ionization time of flight mass spectrometry, MALDI-TOF MS) [1, 2]. K. grimontii possesses a specific chromosomal β-lactamase gene (blaOXY-6) [3], but it can also acquire other antibiotic resistance genes (ARGs) via mobile genetic elements (MGEs). In particular, the recent reports of carbapenemase-producing K. grimontii possessing plasmid-mediated blaKPC-2 (China) and blaVIM-1 (Switzerland) are worrisome [2, 4]. Notably, very little is known about the K. grimontii ability to horizontally transfer such plasmids to other Enterobacterales.
In August 2020, following multiple hospitalizations caused by respiratory infections (starting in January with a respiratory syncytial virus bronchiolitis and including both methicillin-susceptible Staphylococcus aureus and Haemophilus influenzae), a 10-month old girl was admitted to a hospital based in Genoa (Italy) for the surgical management of grade 4 subglottic stenosis. During hospitalization, a KPC-producing Klebsiella pneumoniae strain (KPC-Kp) was detected from the tracheal aspirate and urine samples. In October 2020, the patient was discharged at home. One month later, the patient was admitted to the Alessandro Manzoni Hospital (Lecco, Italy) due to respiratory distress. At admission, the patient underwent a screening rectal swab for multidrug-resistant organisms that was directly streaked on different selective media including both a specific chromogenic medium for carbapenem-resistant Enterobacterales (Brilliance CRE Agar, Oxoid) and a MacConkey agar plate (bioMérieux) where disks of ertapenem (10 μg) and meropenem (10 μg) were placed. As a result, two carbapenem-resistant strains were routinely identified using VITEK 2 (bioMérieux) and MALDI-TOF MS (VITEK MS, bioMérieux; software version, v3.2 Database): Escherichia coli LC-1302–2020 (confidence value, 99.9%) and K. oxytoca LC-1303–2020 (confidence value, 99.9%). Notably, strain LC-1303–2020 was also identified as K. oxytoca (score 2.28) by using another MALDI-TOF MS apparatus [Bruker; FlexControl v3.4 (build 135); MBT Compass v4.1.100.10; BDAL RUO Library 10 (9607 MSPs)]. The infant was discharged after 2 weeks of hospitalization, where no infections due to carbapenem-resistant Enterobacterales were recorded.
Based on whole-genome sequencing (WGS) data and the Type (Strain) Genome Server (https://tygs.dsmz.de/), the E. coli species identification was confirmed, whereas K. oxytoca was actually a K. grimontii. Antimicrobial susceptibility testing performed using a broth microdilution GNX2F Sensititre panel (Thermo Fisher Scientific) indicated that both isolates were resistant to different classes of antibiotics and showed reduced susceptibility to carbapenems (Table S1).
WGS was performed combining NovaSeq 6000 (Illumina) and MinION (SQK-RBK004 library; FLO-MIN 106D R9 flow-cell; Oxford Nanopore Technologies) to generate complete genome assemblies (i.e., circular) with Unicycler v0.4.8 using the hybrid pipeline with default parameters as previously described [2, 5, 6]. The complete hybrid genomes were analyzed with the tools from the Center for Genomic Epidemiology (www.genomicepidemiology.org/). The genome assemblies of LC-1302–2020 (GenBank: CP091756-CP091761) and LC-1303–2020 (GenBank: CP091752-CP091755) are available under BioProject PRJNA801146.
E. coli LC-1302–2020 belonged to sequence type 10 (ST10) and its chromosome harbored the mdfA ARG. The strain also carried 5 plasmids, of which p1-LC-1302–2020-KPC3 (298.9 kb) of IncFIB(pQil) replicon sequence (also known as pQil) and possessing blaKPC-3, blaCTX-M-15, blaTEM-1B, ΔblaOXA-1-like, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id, dfrA14, qnrB1, sul2, and tet(A) ARGs (Fig. 1). K. grimontii LC-1303–2020 belonged to a new ST (ST391) since it carried a new infB allele (infB-54). Its chromosome harbored the blaOXY-6–4 gene. Three plasmids were also present, of which plasmid p1-LC-1303–2020-KPC3 (252.9 kb) carried an identical replicon sequence and ARGs as in p1-LC-1302–2020-KPC3 (Fig. 1).
Both plasmids were identical to each other (identity, 99.97%) and harbored the blaKPC-3 in the archetypal Tn4401a element [7]. However, p1-LC-1302–2020-KPC3 was ~ 40 kb larger, possibly due to a duplication event (Fig. S1). Similar duplications have been reported in other blaKPC-3-carrying pQil (blaKPC-3-pQil) plasmids in K. pneumoniae isolated from the same patient [8].
More importantly, both plasmids were closely related (coverage: 92–96%; identity: 99.25–100%; PLSDB Mash distribution plasmid search analysis) to two other deposited pQil plasmids hosted in K. pneumoniae: a blaKPC-2 plasmid (pJYC01A) from an outbreak in South Korea and a blaKPC-31 plasmid (pKpQIL_pKPN) recently isolated during a study in Italy (Fig. 1) [9, 10]. In this latter survey, it was also noted a high prevalence of high-risk ST512 KPC-Kp strains that possessed the pQil plasmid, suggesting the endemicity of this MGE [9]. Overall, these observations may indicate that K. grimontii cooperates with K. pneumoniae in the dissemination of such hyperepidemic multidrug resistance plasmids. It can be also speculated that the KPC-Kp strain colonizing the intestinal tract of the infant during the first hospitalization was the donor of the blaKPC-3-pQil plasmid to either E. coli LC-1302–2020 or K. grimontii LC-1303–2020. Unfortunately, such KPC-Kp strain was not available for further WGS analyses and plasmid-to-plasmid comparison.
To support our hypotheses, liquid conjugation experiments with the rifampicin-resistant E.coli recipient strain J53d-R1 were conducted at 37 °C for 16 h as previously done [2]. Transconjugants (TCs) were selected on MacConkey agar plates supplemented with rifampicin (50 mg/L) and ampicillin (100 mg/L). TCs showing reduced susceptibility to β-lactams and other classes of antibiotics were obtained (Table S1) with both donor strains. In particular, the conjugation efficiencies (average of 3 replicates) were: 1.2 × 10−4 for E. coli LC-1302–2020 and 1.8 × 10−7 for K. grimontii LC-1303–2020. The obtained TCs were blaKPC-positive according to a PCR performed as previously done [11]. These results confirm the ability of K. grimontii to transfer the blaKPC-3-pQil plasmid to other Enterobacterales, such as E. coli.
To further investigate the spread of the blaKPC-possessing K. grimontii (KPC-Kg) strains, a database search for other genomes (File S1) and core genome alignment were conducted as previously done (35′965 SNVs across 12 genomes; 88.1% average alignment) [2, 5, 12, 13]. As shown in Fig. 2, we further identified 3 blaKPC-3- and 8 blaKPC-2-positive genomes (mostly from North America) belonging to distinct STs. As expected, K. grimontii strain LC-1303–2020 was unique from all other KPC-Kg (range of ∆SNVs: 14′153–14′712). As well, read mapping of all other KPC-Kg against p1-LC-1303–2020-KPC3 confirmed that this pQil plasmid was not present in any of those 11 genomes (data not shown). Notably, as we have shown in our previous work exploring the spread of blaVIM-1-possessing K. grimontii, more KPC-Kg (mostly misidentified as K. oxytoca) in human and environmental sources have been identified since [2].
The identification of pQil replicon sequences in other deposited K. grimontii (Fig. 2) suggests an exchange, so far undetected, of this type of plasmids between closely related species (e.g., K. pneumoniae to K. grimontii). We also note that blaKPC-pQil plasmids have been reported worldwide in other species (e.g., E. coli and Klebsiella aerogenes) [7, 14]. Our conjugation experiment results and the finding of E. coli LC-1302–2020 demonstrated, in fact, that the horizontal transfer of the blaKPC-3-pQil plasmid between different species is possible and can favor the expansion of KPC-producing pathogens.
In conclusion, we reported the first blaKPC-3-carrying K. grimontii isolate. The strain was isolated from the gut of a patient concurrently with an E. coli carrying the same blaKPC-3-pQil conjugative plasmid. We also showed that other non-clonally related KPC-Kg possessing blaKPC-2/-3 were published and/or erroneously deposited in various databases as K. oxytoca [15].
Overall, our findings emphasize the importance of correctly identifying K. grimontii because it represents an emerging reservoir of ARGs threatening our antibiotic armamentarium. As long as MALDI-TOF MS databases are not updated to correctly identify this pathogen, we recommend achieving species identification by using molecular methods (e.g., sequencing of blaOXY) or, alternatively, reporting the results as K. oxytoca complex [3].
Data Availability
The genome assemblies of strains LC-1302–2020 (GenBank: CP091756-CP091761) and LC-1303–2020 (GenBank: CP091752-CP091755) are available under BioProject PRJNA801146.
Code availability
Not applicable.
References
Passet V, Brisse S (2018) Description of Klebsiella grimontii sp. nov. Int J Syst Evol Microbiol 68:377–381
Campos-Madueno EI, Moser AI, Risch M, Bodmer T, Endimiani A (2021) Exploring the global spread of Klebsiella grimontii isolates possessing blaVIM-1 and mcr-9. Antimicrob Agents Chemother 65:e0072421
Yang J, Long H, Hu Y, Feng Y, McNally A, Zong Z (2022) Klebsiella oxytoca complex: update on taxonomy, antimicrobial resistance, and virulence. Clin Microbiol Rev 35:e0000621
Liu L, Feng Y, Hu Y, Kang M, Xie Y, Zong Z (2018) Klebsiella grimontii, a new species acquired carbapenem resistance. Front Microbiol 9:2170
Campos-Madueno EI, Moser AI, Jost G, Maffioli C, Bodmer T, Perreten V et al (2022) Carbapenemase-producing Klebsiella pneumoniae strains in Switzerland: human and non-human settings may share high-risk clones. J Glob Antimicrob Resist 28:206–215
Moser AI, Campos-Madueno EI, Sendi P, Perreten V, Keller PM, Ramette A et al (2021) Repatriation of a patient with COVID-19 contributed to the importation of an emerging carbapenemase producer. J Glob Antimicrob Resist 27:267–272
Pitout JD, Nordmann P, Poirel L (2015) Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother 59:5873–5884
Stohr J, Verweij JJ, Buiting AGM, Rossen JWA, Kluytmans J (2020) Within-patient plasmid dynamics in Klebsiella pneumoniae during an outbreak of a carbapenemase-producing Klebsiella pneumoniae. PLoS One 15:e0233313
Carattoli A, Arcari G, Bibbolino G, Sacco F, Tomolillo D, Di Lella FM et al (2021) Evolutionary trajectories toward ceftazidime-avibactam resistance in Klebsiella pneumoniae clinical isolates. Antimicrob Agents Chemother 65:e0057421
Song JE, Jeong H, Lim YS, Ha EJ, Jung IY, Jeong W et al (2019) An outbreak of KPC-producing Klebsiella pneumoniae linked with an index case of community-acquired KPC-producing isolate: epidemiological investigation and whole genome sequencing analysis. Microb Drug Resist 25:1475–1483
Endimiani A, Carias LL, Hujer AM, Bethel CR, Hujer KM, Perez F et al (2008) Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrob Agents Chemother 52:2680–2682
Brilhante M, GobeliBrawand S, Endimiani A, Rohrbach H, Kittl S, Willi B et al (2021) Two high-risk clones of carbapenemase-producing Klebsiella pneumoniae that cause infections in pets and are present in the environment of a veterinary referral hospital. J Antimicrob Chemother 76:1140–1149
Campos-Madueno EI, Bernasconi OJ, Moser AI, Keller PM, Luzzaro F, Maffioli C et al (2020) Rapid increase of CTX-M-producing Shigella sonnei isolates in Switzerland due to spread of common plasmids and international clones. Antimicrob Agents Chemother 64:e01057–20
Chen L, Chavda KD, Melano RG, Jacobs MR, Koll B, Hong T et al (2014) Comparative genomic analysis of KPC-encoding pKpQIL-like plasmids and their distribution in New Jersey and New York Hospitals. Antimicrob Agents Chemother 58:2871–2877
Cooper A, Carter C, McLeod H, Wright M, Sritharan P, Tamber S et al (2021) Detection of carbapenem-resistance genes in bacteria isolated from wastewater in Ontario. FACETS 6:569–591
Funding
Open access funding provided by University of Bern. This work was supported by the Swiss National Science Foundation (SNF) Grant No. 192514 (to AE). Edgar I. Campos-Madueno is a PhD student (2021–2024) supported by SNF.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethics approval
The anonymized case description has been carried out in accordance with the Declaration of Helsinki, as revised in 2013.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Campos-Madueno, E.I., Mauri, C., Meroni, E. et al. Simultaneous gut colonization by Klebsiella grimontii and Escherichia coli co-possessing the blaKPC-3-carrying pQil plasmid. Eur J Clin Microbiol Infect Dis 41, 1087–1091 (2022). https://doi.org/10.1007/s10096-022-04462-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10096-022-04462-z