Simultaneous gut colonization by Klebsiella grimontii and Escherichia coli co-possessing the blaKPC-3-carrying pQil plasmid

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. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-022-04462-z.

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 (bla OXY-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 bla KPC-2 (China) and bla VIM-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 carbapenemresistant 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).
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 bla KPC-2 plasmid (pJYC01A) from an outbreak in South Korea and a bla KPC-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 bla KPC-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 bla KPC -positive according to a PCR performed as previously done [11]. These results confirm the ability of K. grimontii to transfer the bla KPC-3 -pQil plasmid to other Enterobacterales, such as E. coli.
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 bla KPC -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 bla KPC-3 -pQil plasmid between different species is possible and can favor the expansion of KPC-producing pathogens.
In conclusion, we reported the first bla KPC-3 -carrying K. grimontii isolate. The strain was isolated from the gut of a patient concurrently with an E. coli carrying the same bla KPC-3 -pQil conjugative plasmid. We also showed that other non-clonally related KPC-Kg possessing bla KPC-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 bla OXY ) or, alternatively, reporting the results as K. oxytoca complex [3].
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.
Code availability Not applicable.

Declarations
Ethics approval The anonymized case description has been carried out in accordance with the Declaration of Helsinki, as revised in 2013.

Competing interests
The authors declare no competing interests.
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