he was initially hospitalized with erysipelas in august 2015 and successfully treated with penicillin and dicloxacillin . on readmission in september 2015 , six days later , the patient felt increased pain , and his knee became warm and sore . these findings were interpreted to be symptoms of reactive arthritis caused by a urinary tract infection , and his drug treatment was changed to cefuroxime . the following day , a cloudy synovial fluid was aspired from his knee , so ciprofloxacin was added to his drug regimen . because of aggravating symptoms and increasing c - reactive protein levels , the patient s treatment was then changed to piperacillin with tazobactam , after which his symptoms improved . the patient was discharged with oral dicloxacillin and ciprofloxacin , and 4 weeks later he was well . an overnight culture of the synovial fluid from the patient s knee grew multidrug - resistant , gram - negative , nonmotile rods . maldi - tof ( maldi biotyper 3.1 , bruker daltonics microflex lt , database mbt db-5627 ) mass spectrometry identified the colonies as e. miricola ( score 2.105 ) . antimicrobial susceptibility testing showed that the isolate was susceptible to ciprofloxacin ( mic 0.25 mg / l ) and co - trimoxazole ( mic 0.125 mg / l ) ; intermediately susceptible to piperacillin / tazobactam ( mic 16 8 mg / l ) , and tigecycline ( mic 0.5 mg / l ) ; and resistant to all other drugs tested ( table ) . the imipenem / imipenem - edta etest ( biomrieux , marcy letoile , france ) for detection of metallo--lactamase was positive . the isolate contains antimicrobial resistance genes blab-4 , blagob-13 , and an extended - spectrum -lactamase , sharing 90.6% nucleotide identity to blacme-1 . antimicrobial susceptibility testing was performed by using etest ( biomrieux , marcy letoile , france ) according to european committee on antimicrobial susceptibility testing ( eucast ) guidelines ( http://www.eucast.org ) . for interpretation , non  species - related ( pharmacokinetics / pharmacodynamics ) breakpoints were used except for co - trimoxazole , in which stenotrophomonas malthophilia breakpoints were used , and for gentamicin and colistin , in which pseudomonas sp . for susceptibility testing purposes , the concentration of tazobactam was fixed at 4 mg / l . for susceptibility testing purposes , the concentration of clavulanic acid was fixed at 2 mg / l . to confirm the species identity and identify the antimicrobial resistance genes , we performed wgs of the isolate with the illumina miseq platform producing 2  150-bp paired - end reads by using the nextera xt library preparation kit ( illumina denmark aps , copenhagen , denmark ) . reads were assembled by using spades , which produced a 4.13-mbp genome termed hvh - wgs333 ( bioproject prjna335686 , biosample samn05450241 ) . we performed annotation of the genome using prokka ( 8) and used arg - annot ( 9 ) to identify 2 metallo--lactamase genes , namely blab-4 and blagob-13 , and an extended - spectrum -lactamase gene sharing 90.6% nucleotide identity with blacme-1 . the isolate was identified by eztaxon ( 10 ) as e. miricola on the basis of its 16s rrna gene with 99.69% similarity . however , single - nucleotide variant ( snv ) analysis by read alignment with bowtie2 and snv - calling with samtools comparing hvh - wgs333 and the gtc 862 e. miricola strain ( accession no . furthermore , analyzing the coverage breadth by using bedtools revealed that this isolate shared 80.5% of this e. miricola genome . because the isolate from this patient was not closely related to the e. miricola type strain , we performed a genus - level core genome analysis of 35 publicly deposited elizabethkingia strains from genbank to identify a more closely related isolate . each genome was annotated ( 8) and the core genome was approximated by roary ( 11 ) , yielding a 419,813-bp core genome of 426 genes . we constructed a phylogenetic tree of the core genome using raxml ( 12 ) with 100 bootstrapping replicates ( figure ) . the atcc 13253 e. meningoseptica strain did not cluster with the other e. meningoseptica strains and was the most distantly related elizabethkingia sp . analyzed . before wgs was commonly used , high levels of divergence were reported among e. meningoseptica ( formerly flavobacterium meningosepticum ) isolates , with 2 groups detected , ursing and bruun ( ub ) i and ubii ( 13 ) , with atcc 13253 belonging to ubi . our hvh - wgs333 isolate was most closely related to em_chuv , an endotracheal isolate submitted as e. miricola ; these 2 isolates are next most closely related to g4071 , which belongs to the ubii:2 ( 13 ) , and then to gtc 862 e. miricola . e. miricola atcc 33958 is most closely related to g4075 , a clinical blood culture e. meningoseptica in the ubii:3 group . strains belonging to groups ubi and ubii are phenotypically very similar , explaining why dna - dna hybridization ( ddh ) was previously used to distinguish these groups ( 14 ) . phylogenetic tree of elizabethkingia isolate from a patient with hospital - acquired septic arthritis in copenhagen , denmark 2015 compared with reference strains and in silico dna - dna hybridization ( ddh ) . bootstrapping support was implemented by running 100 replicates , with values > 70% indicated on branches . isolates assigned into ursing and bruun ( ub ) groups and subgroups are indicated in brackets following the isolate name . table at top right indicates the in silico ddh values ; black boxes indicate isolates with in silico ddh > 70% indicating they belong to the same species. the e. meningoseptica isolates nbrc 12535 , atcc13253 , and kc1913 seem to be identical strains . additionally , we performed in silico ddh ( 15 ) to other sequenced elizabethkingia isolates to determine whether our isolate could be defined as the same species ( i.e. , having a ddh value > 70% ) . the in silico ddh value is calculated by summing up the identities in matches between the genome sequences and dividing by the total match hit lengths . hvh - wgs333 only had an in silico ddh > 70% with em_chuv and g4071 , which clustered most closely together in the tree but was also closely related ( 67.1% ) to strain gtc 862 ( figure ) . a previous ddh study investigating the elizabethkingia genus found ddh values at 70c of 52% between ubii:2 and ubii:3 , 45% between ubii:2 and ubii:4 , and 36% between ubii:3 and ubii:4 ( 14 ) , in agreement with our in silico ddh results ( figure ) . our analyses support the identification of the 3 ubii subgroups 2 , 3 , and 4 as being 3 different elizabethkingia species , with ubii:2 being the closest related to the e. miricola type strain . the wide adoption of maldi - tof mass spectrometry for bacterial identification is likely to increase the detection of infections caused by elizabethkingia , and thus the number of infection reports . however , the applied maldi - tof database contained only 3 e. meningoseptica and 1 e. miricola isolates at the time of our study . identification on the basis of the 16s rrna gene corroborated our maldi - tof mass spectrometry identification of e. miricola . when exploring this further using wgs , we discovered our isolate shared 80.5% of the gtc 862 e. miricola genome and the in silico ddh value was 67.1% , just below the 70% species threshold . it thus appears that our isolate , along with em_chuv and g4071 , is closely related to the e. miricola species and that the taxonomic position of the ubii:3 and ubi:4 strains should be reconsidered . in light of our wgs analysis demonstrating considerable elizabethkingia genetic diversity , we suggest that the maldi - tof database needs to be updated and the nomenclature needs to be re - examined . wgs is proving to be a valuable tool for the correct identification of new species and detailed characterization of multidrug - resistant bacteria .
in a hospital - acquired infection with multidrug - resistant elizabethkingia , matrix - assisted laser desorption / ionization time - of - flight mass spectrometry and 16s rrna gene analysis identified the pathogen as elizabethkingia miricola . 
 whole - genome sequencing , genus - level core genome analysis , and in silico dna - dna hybridization of 35 elizabethkingia strains indicated that the species taxonomy should be further explored .