A case of blaNDM-1-positive Salmonella Kottbus, Denmark, November 2020

We present a case of carbapenemase-producing blaNDM-1-positive Salmonella Kottbus in an 82-year-old Danish man. The blaNDM-1 was also identified in Escherichia coli and Citrobacter freundii in the same patient on the same 43 kb IncN2 plasmid, suggesting in vivo inter-species plasmid transfer. A NCBI BLAST analysis of the plasmid (pAMA003584_NDM-1) identified 12 highly similar plasmids, all originating from east and south-east Asia. This case could be the first confirmed case of blaNDM-1-positive Salmonella not related to travel outside Europe.

We present a case of carbapenemase-producing blaNDM-1-positive Salmonella Kottbus in an 82-year-old Danish man. The blaNDM-1 was also identified in Escherichia coli and Citrobacter freundii in the same patient on the same 43 kb IncN2 plasmid, suggesting in vivo inter-species plasmid transfer. A NCBI BLAST analysis of the plasmid (pAMA003584_ NDM-1) identified 12 highly similar plasmids, all originating from east and south-east Asia. This case could be the first confirmed case of blaNDM-1-positive Salmonella not related to travel outside Europe.
We present a case of an NDM-1 carbapenemase-producing S. Kottbus, isolated in a Danish man who did not have travel history outside of Europe.

Case report
The patient was an 82-year-old man with a recent diagnosis of inoperable lung cancer with no option for chemotherapy. In November 2020, he was hospitalised because of intermittent fever, abdominal pain, and diarrhoea for several weeks. On examination, he had tachycardia (112 beats/minute) and a body temperature of 37.4°C. Laboratory findings showed leucocytosis (34.1 × 10 9 /L; norm: 3.5-10.0) and elevated C-reactive protein (265 mg/L; norm: < 8.0). A computed tomography scan with contrast revealed bowel wall thickening in the left colon, suggestive of an underlying inflammatory or infectious condition. A stool sample taken on the day of admission was positive for Clostridioides difficile toxin B (Xpert C. difficile BT, Cepheid, Sunnyvale, California, United States (US)) and oral metronidazole treatment was initiated. In another stool sample also from the day of admission, Salmonella was isolated by routine methods and antimicrobial susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standardised disk diffusion method for Enterobacteral es (Breakpoint table v10.0) [7]. Surprisingly, the isolate was resistant to meropenem (inhibition zone diameter: 11mm) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) [8] was positive for NDM. The stool sample was also plated on chromID CARBA SMART agar (bioMérieux, Marcy l'Etoile, France), showing growth of Escherichia coli and Citrobacter freundii; both isolates were NDM-positive with the NG-Test CARBA 5. The diarrhoea symptoms subsided after a few days and the patient recovered. On day 8, he was discharged and treatment with oral metronidazole continued for a total of 10 days. After 2 weeks, he died at his home.
A review of the patient's records clarified that, in September 2020, he had been at a resort on the Ionian Sea, Greece, for a week-long holiday. The patient and his partner stayed at an all-inclusive hotel; both had an onset of diarrhoea 2 days after arrival. The patient had many loose stools so he visited an outpatient clinic where he received intravenous rehydration and antibiotic therapy with oral cefuroxime. After the patient returned to Denmark, the diarrhoea symptoms subsided but the patient was hospitalised a few days later with kidney failure; a rectal swab taken as part of screening procedures for multidrug-resistant bacteria were negative. Throughout September and October, the patient was hospitalised for chronic kidney failure, and was eventually diagnosed with inoperable lung cancer. He was dialysed and received different antibiotic regimens, including oral metronidazole for recurrent diarrhoea. However, a stool sample for enteric pathogenic bacteria was not taken until the final hospital stay in November.

Serotyping and genomic analysis at the National Reference Laboratory
In 2018, the Danish Health Authority added carbapenemase-producing organisms (CPO) to the list of notifiable bacteria, and the Danish National Reference Laboratory (Statens Serum Institut (SSI)) carries out whole genome sequencing of all CPO isolates. At SSI, the isolate from the patient was serotyped as S. Kottbus based on the Kauffmann-White-Le Minor scheme, which was later verified using the sequence data to predict the serotype.
A National Center for Biotechnology Information (NCBI) Basic Local Alignment Tool (BLAST) analysis of pAMA003584_NDM-1 identified 12 highly similar plasmids, all originating from east and south-east Asia (Table 1).

Ethical Statement
A signed informed consent for publication from the deceased's partner was obtained before submitting.

Discussion
In the European Union (EU)/European Economic Area (EEA), the notification rate of NTS was 20 cases per 100,000 inhabitants in 2019 [11]. In Denmark, the notification rate for NTS was almost identical (19.3 cases/100,000 inhabitants) in 2019, while the lowest rates were reported by Cyprus, Greece, Ireland, Italy, Portugal, and Romania (≤ 7.1 cases/100,000 inhabitants) [11].
Here we present the first confirmed case of blaNDM-1-positive S. Kottbus not related to travel outside Europe. The blaNDM-1 gene was also identified in E. coli and C. freundii on the same 43 kb IncN2 plasmid, suggesting an inter-species transfer of the blaNDM-1-carrying IncN2 plasmid in vivo.
The first NDM-producing NTS case, published in 2011, was a 60-year-old American man who was transferred from India to a hospital in the US where blaNDM-1-positive Salmonella Senftenberg was isolated from a perirectal surveillance culture [12]. Following this, other blaNDM-1-positive human NTS cases were described in connection to India, Pakistan and China, as reviewed by Fernández et al [6]. and Salmonella Typhimurium (VIM-producing) were identified, whereas no NDMproducing Salmonella were reported [14].
In the case presented here, we identified three bacteria in the same patient, all of which harboured the same carbapenem-resistance gene. Carbapenemase acquisition by an NTS from other Enterobacteriaceae in immunocompromised patients in a healthcare context has been suggested [6]. According to the patient's partner, other guests at the resort also had diarrhoea; we can speculate that this patient may have eaten contaminated food at the resort in Greece, but the exact source of the blaNDM-1 plasmid remains unknown. Furthermore, we can only hypothesise which of the three bacteria first obtained the blaNDM-1 plasmid. Denmark has a low prevalence of carbapenemase-producing bacteria [15] and the fact that S. Kottbus is very rare in Denmark also suggests that the three bacteria had 'spent time together' in vivo. During the autumn, the patient was hospitalised and diagnosed with terminal lung cancer. He received different antibiotic regimens so, on the other hand, we cannot rule out an acquisition of the IncN2 plasmid by one of the three bacteria in Denmark.

Conclusions
We describe the first case of blaNDM-1-positive S. Kottbus located on a 43 kb IncN2-plasmid from an 82-year-old man with terminal lung cancer detected in Denmark. The plasmid was also found in E. coli and C. freundii from the same patient, suggesting horizontal gene transfer. The patient had no known travel history outside Europe and could be the first confirmed case of blaNDM-1-positive Salmonella not related to travel outside Europe. Our finding underscores the importance of remaining vigilant for the potential risk of emerging resistance strains. In sum, we find any potential spread of NDM-1-producing NTS worrisome and emphasize the need for antimicrobial resistance surveillance in Europe, especially in countries where NDM-producing Enterobacteriaceae is spreading.