First Report of Kosakonia radicincitans Bacteraemia from Europe (Austria) - Identification and Whole-Genome Sequencing of Strain DSM 107547

Kosakonia radicincitans is a species within the new genus Kosakonia. Many strains of this genus have been isolated from plants, but some strains are assumed to act as facultative human pathogens. In this study, an in-depth analysis of a Kosakonia isolate from human blood was performed. The strain was originally isolated from blood and identified as a member of the Enterobacter cloacae complex, exhibiting an atypical result in susceptibility testing. Therefore, the genetic background was examined, including phylogenetic classification and screening for virulence factors. Using whole-genome sequencing, the isolate was identified as a K. radicincitans strain, revealing a virulence gene cluster for yersiniabactin biosynthesis in contrast to all other strains of the species. Whole-genome sequencing was the perfect method for identifying putative virulence factors of a particular Kosakonia strain and will help distinguish beneficial strains from pathogenic strains in the future. To our knowledge, this is the first report of Kosakonia-related bacteraemia from Europe.

of hospitalization. Four weeks after admission, the inflammation values increased, and she developed a fever. Two pairs of blood cultures were taken during the increase in inflammation parameters. One aerobic blood culture was positive for a bacterium initially identified as a member of the Enterobacter cloacae complex. Treatment with piperacillin/tazobactam (Pip/Taz) was initiated, and moxifloxacin was added later. Under treatment with this regime, the patient's condition improved, and her inflammation parameters declined, and after six weeks of hospitalization, she was discharged.

Results
Microbiological testing. Of the two sets of blood cultures obtained during the increase in inflammation parameters, one aerobic blood culture showed positive growth 20 hours after inoculation. Using Gram staining, the bacteria were shown to be gram-negative bacilli. Agar diffusion test and subcultures were performed and revealed the growth of grey-coloured colonies on blood agar and pink colonies on MacConkey agar. The agar diffusion test showed pan-susceptibility, with the exception of amoxicillin (Fig. 1, Suppl. Table 1). For identification, colonies from different agar media were subjected to MALDI-TOF (VITEK MS, bioMerieux), but the identification failed three times. Therefore, GN (gram-negative) and N196 automated biochemical testing was performed using the VITEK 2 system (bioMerieux), which yielded a 91% probability match with the Enterobacter cloacae complex. According to EUCAST (The European Committee on Antimicrobial Susceptibility Testing) guidelines, however, this genus was not found to be susceptible (intrinsic resistance to amoxicillin, amoxicillin/clavulanic acid and cefuroxime).

Discussion
Here, we report the first case of bacteraemia with K. radicincitans, previously known as Enterobacter radicincitans, in Europe. Some bacteria of the genus Enterobacter are major causes of human infections. However, the classification of Enterobacter species has changed rapidly in recent years. Several species were transferred to or excluded from this genus. In 2013, Enterobacter was divided into 5 new genera: Lelliottia, Pluralibacter, Kosakonia, www.nature.com/scientificreports www.nature.com/scientificreports/ Cronobacter and Enterobacter 9 . Kosakonia spp. (K. radicincitans, K. sacchari, K. oryzae, K. cowanii, K. arachidis) are usually known as plant growth-promoting bacteria, improving the yield and quality of fruits such as maize, radish, sugarcane or cabbage 1,2,10,11 . There are rare reports of Kosakonia spp. involved in human infections, but there are known species that can act as human pathogens, such as Kosakonia cowanii 3 . To date, there are no   www.nature.com/scientificreports www.nature.com/scientificreports/ epidemiological data on the occurrence of Kosakonia species in human samples. The first case of a human bloodstream infection with K. radicincitans was reported in a 61-year-old man with cholangiocarcinoma in Houston, TX, USA, in Dec. 2016 4 . Unfortunately, the authors of this report considered some common features of enteric bacteria to be virulence factors, and confused type IV secretion systems with type IV pili that are involved in several phenomena, not only pathogenicity 12 .
Comparison of the cases from Austria and the USA showed that both patients had problems in the bile duct system. The patient in the USA presented with cholangiocarcinoma and fever, and the patient in Austria presented with bile duct stenosis and fever. Nevertheless, with only two cases considered, this could also be pure coincidence.
Future genomic comparisons will show whether human pathogenic strains can be clearly distinguished from plant-associated strains of the same bacterial species based on true virulence factors, such as the syntenic yersiniabactin-like gene cluster encoding an iron, copper and nickel ion-chelating siderophore 13 , which has been solely found in strain DSM 107547 among Kosakonia spp. Such a finding simplifies the diagnosis of pathogenic bacteria significantly. The role of yersiniabactin (Ybt) in mediating the virulence of human pathogenic bacteria is unquestionable: (i) invasive enteric bacteria from the genera Yersinia, Escherichia, and Klebsiella secrete Ybt during human infection to combat host-mediated metal deficiencies 13 ; (ii) comparative genomics revealed that the list of horizontally transferred gene sets in Salmonella enterica is dominated by virulence factors and the yersiniabactin gene cluster 14 , suggesting an important role of this siderophore in human pathogenicity; and (iii) in addition to uropathogenic enterobacteria expressing Ybt 13 , it was shown very recently that all isolates of Klebsiella pneumoniae from infant blood or stool samples taken during outbreaks in neonatal intensive care units produced Ybt 15 .
Regarding the current Kosakonia infections, it must be considered that some infections with this organism were not correctly diagnosed previously. The most important concern is that diagnoses may be imprecise and may provide no or even false positive results: Kosakonia sp. yields no ID in MALDI-TOF analysis, and Vitek2 susceptibility testing suggests the presence of Enterobacter spp. A growing database for MALDI-TOF MS might solve this problem, as would the use of different MALDI-TOF systems. A second look at unsuitable germs and the resistance would also be useful with regard to the increased use of automation in many laboratories, where a specific pathogen is inevitably assigned to a stored antibiogram. We would like to motivate colleagues to take a closer look at such germs and publish the sequences of more of these isolates to facilitate further genomic comparisons of human samples and perhaps to test therapeutic options or antibiotic efficacy, as well as to obtain actual epidemiological data.
However, sequencing approaches for the identification of these bacterial species are recommended. Especially in our case, whole-genome sequencing is the perfect means for species identification and might help reveal new Kosakonia species in the future.
With improved diagnostic tools, researchers will be able to show whether infections with Kosakonia spp. are rare events indeed or occur more frequently than previously shown.

Methods
Strain. The Kosakonia radicincitans strain used in this study was archived at the DSMZ (German Collection of Microorganisms and Cell Cultures) as Kosakonia radicincitans strain DSM 107547.
Microbiological methods. For identification, colonies from different agar media were subjected to MALDI-TOF (VITEK MS -bioMerieux), and N196 automated biochemical testing was performed using the VITEK 2 system (bioMerieux).
Susceptibility testing was performed as recommended by the European Committee on Antimicrobial Susceptibility testing (EUCAST) 16 . Interpretation of zone diameters was performed according to EUCAST 2017.
Whole-genome sequencing. Whole-genome sequencing was performed using a combination of Pacific Biosciences long-read sequencing and Illumina short-read sequencing. For both sequencing runs, DNA was isolated using Qiagen Genomic-tip 100/G (Qiagen, Hilden Germany) according to the manufacturer's instructions.  21 . Samples were sequenced on a NextSeq ™ 500 instrument. Genome assembly was performed by applying the RS_HGAP_Assembly.3 protocol included in SMRT Portal version 2.3.0 using default parameters. The assembly revealed a circular bacterial chromosome and a plasmid, both with coverages of 110× . Both replicons were circularized, artificial redundancies at the ends of the contigs were removed and adjustment to dnaA (parA = soj) as the first gene was performed. Error correction was performed by mapping Illumina short reads onto the finished genome using Burrows-Wheeler Alignment (bwa 0.6.2) in paired-end (sample) mode using the default setting 22 with subsequent variant and consensus calling using VarScan 2.3.6 (parameters: mpi-leup2cns-min-coverage 10-min-reads2 6-min-avg-qual 20-min-var-freq. 0.8-min-freq-for-hom 0.75-p-value 0.01-strand-filter 1-variants 1-output-vcf 1) 23 . A consensus concordance of QV60 could be confirmed.
The genome sequence has been deposited at NCBI GenBank under accession nos. CP040392 and CP040393 for the circular chromosome (5,656,428 bp) and plasmid, respectively (118,312 bp: Table 1).