Open Access

Insights from the draft genome into the pathogenicity of a clinical isolate of Elizabethkingia meningoseptica Em3

  • Shicheng Chen1Email author,
  • Marty Soehnlen2,
  • Frances P. Downes3 and
  • Edward D. Walker1
Standards in Genomic Sciences201712:56

https://doi.org/10.1186/s40793-017-0269-8

Received: 16 March 2017

Accepted: 8 September 2017

Published: 16 September 2017

Abstract

Elizabethkingia meningoseptica is an emerging, healthcare-associated pathogen causing a high mortality rate in immunocompromised patients. We report the draft genome sequence of E. meningoseptica Em3, isolated from sputum from a patient with multiple underlying diseases. The genome has a length of 4,037,922 bp, a GC-content 36.4%, and 3673 predicted protein-coding sequences. Average nucleotide identity analysis (>95%) assigned the bacterium to the species E. meningoseptica. Genome analysis showed presence of the curli formation and assembly operon and a gene encoding hemagglutinins, indicating ability to form biofilm. In vitro biofilm assays demonstrated that E. meningoseptica Em3 formed more biofilm than E. anophelis Ag1 and E. miricola Emi3, both lacking the curli operon. A gene encoding thiol-activated cholesterol-dependent cytolysin in E. meningoseptica Em3 (potentially involved in lysing host immune cells) was also absent in E. anophelis Ag1 and E. miricola Emi3. Strain Em3 showed α-hemolysin activity on blood agar medium, congruent with presence of hemolysin and cytolysin genes. Furthermore, presence of heme uptake and utilization genes demonstrated adaptations for bloodstream infections. Strain Em3 contained 12 genes conferring resistance to β-lactams, including β-lactamases class A, class B, and metallo-β-lactamases. Results of comparative genomic analysis here provide insights into the evolution of E. meningoseptica Em3 as a pathogen.

Keywords

Draft genome Infections Elizabethkingia meningoseptica Human isolate

Introduction

Elizabethkingia meningoseptica , a Gram-negative, aerobic bacillus, belongs to the family Flavobacteriaceae within the phylum Bacteroidaeota [13]. Among the three clinically important Elizabethkingia species (including E. meningoseptica , Elizabethkingia anophelis and Elizabethkingia miricola ), E. meningoseptica has been intensively investigated for its pathogenicity [46]. Most of the E. meningoseptica infections are nosocomial, often transmitted in intensive care units [1, 7]. This bacterium survives in tap water, in disinfection fluid, on wet surfaces of sinks, in ventilators, hemodialysis equipment, catheters, and other medical apparatus. E. meningoseptica infection causes neonatal meningitis, nosocomial pneumonia, bacteremia, osteomyelitis, endocarditis, and skin infections [1, 4, 8]. Moreover, older (age > 65) and immunocompromised patients are more susceptible to infection; case-fatality rates have reached 50% [9].

Infections by E. meningoseptica are difficult to treat with antimicrobial agents due to multiple drug resistance [4]. Tetracycline, chloramphenicol, and β-lactams have been used to treat patients [10], but increasingly clinical isolates lack susceptibility to these antibiotics [11]. Analysis of the resistome in the related bacterium E. miricola revealed multiple drug resistance genes [12]. Some antibiotics effective against Gram-positive bacteria such as vancomycin, quinolones, tigecycline, and rifampin have been used for treating E. meningoseptica -infected patients, though the mechanism of action remains unclear [12, 13]. Also, the effectiveness of these antibiotics varied; many patients resolved infection but isolates showed high MICs in vitro, thus the relationship between MICs and clinical response was obscure [14]. Further genome analyses will elucidate the breadth of antibiotic susceptibility and resistance mechanisms in Elizabethkingia spp.

Differentiation of Elizabethkingia species using routine morphological and biochemical tests is difficult in clinical laboratories [14]. Comparison of 16S rRNA identity does not provide sufficient resolution to identify and separate these closely-related Elizabethkingia species [2, 14]. Characterization of Elizabethkingia species by MALDI-TOF mass spectrometry would facilitate it if species reference spectra were added to the database [14]. A limitation is that MALDI-TOF mass spectrometry is not available in many smaller clinical microbiology laboratories. Whole genome analysis facilitates the development of molecular diagnosis tools (such as single nucleotide polymorphisms) that can be potentially useful for small laboratories. In this study, we sequenced, annotated and analyzed a clinical E. meningoseptica genome, with the aim of providing a better understanding of antibiotic resistance and pathogenesis mechanisms in this pathogen, and of unveiling useful biosystematic molecular markers.

Organism information

Classification and features

E. meningoseptica Em3 (Fig. 1) was isolated from a sputum sample from a patient with multiple underlying diseases and on life support. E. meningoseptica Em3 is Gram-negative, non-motile and non-spore-forming (Fig. 1 and Table 1). A taxonomic analysis was performed by comparing the 16S rRNA gene sequence to those in the GenBank (Fig. 2). The phylogenetic tree based on the 16S rRNA gene sequences indicated that strain Em3 was clustered within a branch containing other E. meningoseptica and departing from the clusters E. anophelis and E. miricola in the genus Elizabethkingia (Fig. 2). We further calculated the ANI and DDH values among the representative Elizabethkingia (Table 2). Our results showed that strain Em3 belongs to E. meningoseptica because of the high ANI (>95%, cutoff for species differentiation) and DDH (>70%, cutoff for species differentiation) values between strain Em3 and E. meningoseptica ATCC 13253 T [15].
Fig. 1

Demonstration of cell growth, pigment production and micrograph. a Em3 growing on SBA medium; b Demonstration of Em3 grown on SBA medium (control, up part) and MacConkey agar (low part). c Pigment production in Em3 grown on TSA agar. d Scanning electron microscopy image of Em3

Table 1

Classification and general features of E. meningoseptica Em3

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [37]

  

Phylum Bacteroidaeota

TAS [3]

  

Class Flavobacteriia

TAS [38]

  

Order Flavobacteriales

TAS [39]

  

Family Flavobacteriaceae

TAS [40]

  

Genus Elizabethkingia

TAS [2]

  

Species Elizabethkingia meningoseptica

TAS [2]

  

Strain Em3

TAS [2]

 

Gram stain

Negative

IDA

 

Cell shape

Rod

IDA

 

Motility

Non motile

IDA

 

Sporulation

Non-spore-forming

NAS

 

Temperature range

4–40 °C

IDA

 

Optimum temperature

37 °C

IDA

 

pH range; Optimum

4–10; 8

IDA

 

Carbon source

Heterotroph

IDA

 

Energy source

Varied; including glucose and mannitol

IDA

MIGS-6

Habitat

Human

NAS

MIGS-6.3

Salinity

Not determined

 

MIGS-22

Oxygen requirement

Aerobic

NAS

MIGS-15

Biotic relationship

Free-living

NAS

MIGS-14

Pathogenicity

Pathogen

NAS

MIGS-4

Geographic location

Michigan, USA

NAS

MIGS-5

Sample collection time

February, 6, 2016

NAS

MIGS-4.1

Latitude

42° 43′ 57″ N

NAS

MIGS-4.2

Longitude

84° 33′ 20″ W

NAS

MIGS-4.4

Altitude

Not reported

NAS

aEvidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [41]

Fig. 2

Phylogenetic tree displays the position of E. meningoseptica Em3 (shown in bold) relative to the other type strains of Elizabethkingia based on 16S rRNA. The phylogenetic tree was constructed by MEGA v. 7.0.14 using the Neighbor-Joining method [42]. The percentage of replicate trees where the associated taxa clustered together in the bootstrap test (500 replicates) is indicated next to the branches. The branch lengths are scaled to the same units as those of the evolutionary distances for inferring the phylogenetic tree. The accession numbers for 16 s rRNA sequences are listed in the parenthesis following selected bacteria: E. meningoseptica LMG 12279 (NR_115236), E. meningoseptica Che01 (KX774527), E. meningoseptica NBRC 12535 (NR_113592), E. meningoseptica ATCC 13253T (NR_115201), E. meningoseptica JL1 (JN201943), E. meningoseptica YMC R3259 (KP836320), E. meningoseptica Em3, E. meningoseptica 16H-201 M0546 (KX774526), E. miricola YMC R1459 (KP844567), E. miricola BM10T (CP011059), E. miricola EM_CHUV (CM003640), E. miricola YMC R1241 (KP836321), E. miricola ATCC 33958 (NZ_JRFN00000000), E. meningoseptica pp5b (GQ360070), E. anophelis NUHP1 (NUHP1_00209), E. anophelis PW2809 (NZ_CBYE010000032), E. anophelis 5.20 (EF426427), E. anophelis R26T (NR_116021), E. anophelis Endophthalmitis (JSAA01000000), Elizabethkingia endophytica JM-87T (NR_136481), Elizabethkingia endophytica F3201 (CP016374.1), E. anophelis B2D (NZ_JNCG00000000) and Riemerella anatipestifer ATCC 11845T (NC_017045)

Table 2

Percentage of in silico DNA-DNA hybridization (DDH)a and average nucleotide identities (ANI)b among the selected Elizabethkingia genomes

 

E. meningoseptica EM3

E. anophelis

R26T [43]

E. meningoseptica

ATCC 13253T [44]

E. miricola

BM10T [45]

E. endophytica

JM-87T [46]

E. meningoseptica EM3

 

31.90

80.15

91.10

98.52

31.20

80.44

32.70

80.25

E. anophelis R26T

31.90

80.15

 

33.60

80.26

68.80

91.52

78.60

97.49

E. meningoseptica ATCC 13253T

91.10

98.52

33.60

80.26

 

31.40

80.26

33.30

80.41

E. miricola BM10T

31.20

80.44

68.80

91.52

31.40

80.26

 

68.70

91.41

E. endophytica JM-87T

32.70

80.25

78.60

97.49

33.30

80.41

68.70

91.41

 

Nucleotide sequences were downloaded from GenBank. The accession numbers for E. anophelis R26T, E. meningoseptica ATCC 13253T, E. miricola BM10T and E. endophytica JM-87T are NZ_ANIW01000001.1, NZ_ASAN01000001.1, NZ_CP011059.1 and NZ_CP016372, respectively

aIn silico DNA-DNA hybridization was calculated by using Genome-to-Genome Distance Calculator (GGDC) [47]. The percentage of DDH was shown on the top and bolded

bANI values were computed for pairwise genome comparison with using the OrthoANIu algorithm [48]. The percentage of ANI was shown on the bottom

The motility was tested on semi-TSA. The cells of strain Em3 are straight and rods and have a diameter of 0.7 μm and length of 24.0 μm. Strain Em3 grew on TSA, producing yellow pigment (Fig. 1). This bacterium also grew well on SBA with greyish discoloration around the colonies, showing it had the α-hemolytic activity (Fig. 1). E. meningoseptica Em3 did not grow on MacConkey agar, a finding consistent with strain-dependent growth on this medium; e.g., E. meningoseptica CCUG 214 T grew on MacConkey agar whereas other hospital-associated E. meningoseptica strains did not [2]. Of those strains growing on MacConkey agar, lactose was not utilized [2]. The optimal growth temperature for strain Em3 was 37 °C (Table 1). Carbon source, nitrogen source utilization and osmotic tolerance were assayed by incubating cells in Biolog GEN III microplates at 37 °C overnight (CA, USA). The results showed that E. meningoseptica Em3 did not tolerate 4% NaCl. E. meningoseptica Em3 utilized several carbon sources, including D-maltose, D-trehalose, D-gentibiose, D-melibiose, D-glucose, D-mammose, D-fructose, D-fucose, D-mannitol and D-glycerol. The ability to use D-melibiose can differentiate E. meningoseptica from E. anophelis and E. miricola [16]. The inability to grow on cellobiose or citrate was consistent with previous reports [16]. Moreover, E. meningoseptica Em3 utilized D-serine, L-alanine, L-aspartic acid, L-glutamic acid, L-histidine and L-serine when tested on Biolog GEN III microplates.

Extended feature descriptions

Phylogenetic analysis (Additional file 1: Figure S1) was further conducted by using 19 genomes with 1181 core genes per genome (22,439 in total). As expected, E. meningoseptica Em3 grouped together with the selected E. meningoseptica species and separated from the clusters E. anophelis , E. endophytica and E. miricola , a finding similar to the phylogenetic analysis based on 16 s rRNA sequences. Further, both trees (Fig. 1 and Additional file 1: Figure S1) show that species E. anophelis and E. endophytica are not separated well, which is consistent with previous reports [17].

Genome sequencing information

Genome project history

The genome of E. meningoseptica Em3 was selected for whole genome sequencing because of its association with pulmonary disease. Comparison of strain Em3 genome with other Elizabethkingia species may provide insights into the molecular basis of pathogenicity and metabolic features of this strain. The high-quality draft genome sequence was completed on August 1, 2016 and was deposited to GenBank as a Whole Genome Shotgun project under accession number MDTY00000000 and the Genome OnLine Database with ID Gp0172366 (Table 3).
Table 3

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

High-quality draft

MIGS-28

Libraries used

two paired-end 250 bp library

MIGS 29

Sequencing platforms

MiSeq-Illumina

MIGS 31.2

Fold coverage

50.0X

MIGS 30

Assemblers

SPAdes 3.9.0

MIGS 32

Gene calling method

NCBI Prokaryotic Genome, Annotation Pipeline

 

Locus Tag

BFF93_

 

Genbank ID

MDTY00000000.1

 

GenBank Date of Release

October 25, 2016

 

GOLD ID

Gp0172366

 

BIOPROJECT

PRJNA338129

MIGS 13

Source Material Identifier

CL16–200185

 

Project relevance

Clinical pathogen

Growth conditions and genomic DNA preparation

For genomic DNA isolation, E. meningoseptica Em3 (CL16–200185, Bureau of Laboratories, Michigan Department of Health and Human Services) culture was grown overnight in 25 mL LB medium at 37 °C with vigorous shaking. DNA was isolated using a Wizard Genomic DNA Purification Kit (Promega, Madison). The amount of genomic DNA was measured using a Nanodrop2000 UV-Vis Spectrophotometer (Thermo scientific) and Qubit DNA assay kit. DNA integrity was confirmed by agarose gel assay (1.5%, w/v).

Genome sequencing and assembly

NGS libraries were prepared using the Illumina TruSeq Nano DNA Library Preparation Kit. Completed libraries were evaluated using a combination of Qubit dsDNA HS, Caliper LabChipGX HS DNA and Kapa Illumina Library Quantification qPCR assays. Libraries were combined in a single pool for multiplex sequencing and the pool was loaded on one standard MiSeq flow cell (v2) and sequencing performed in a 2x250bp, paired end format using a v2, 500 cycle reagent cartridge. Base calling was done by Illumina Real Time Analysis [18] v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.8.4.

The Illumina data were assembled into contiguous sequences using SPAdes version 3.9.0 [19], then short contigs (<400 bp) were filtered out. The 11 contigs identified in this strain were therefore submitted to the NCBI database as a Whole Genome Shotgun project.

Genome annotation

Annotation of the 11 contigs was first done through the NCBI Prokaryotic Genome Automatic Annotation Pipeline [20]. The predicted CDSs were next translated and analyzed against the NCBI non-redundant database, iPfam, TIGRfam, InterPro, KEGG and COG. The RAST system was used to check the annotated sequences [21, 22]. Additional gene prediction and manual revision was performed by using the IMG/MER platform. E. meningoseptica Em3 genome is available in IMG (genome ID = 2,703,719,242).

Genome properties

The draft genome sequence is 4,037,922 bp long, 36.37% G + C rich and contains 11 scaffolds (Table 4). Of 3729 genes predicted, 3673 encoded proteins and 56 were RNAs. 2585 (69.32%) were assigned a putative function, while the other 1088 (30.68%) were designated as hypothetical proteins. The distribution of coding genes into general COG functional categories analyzed by IMG is listed in Table 5. Collectively, the genome features were similar to those in other sequenced E. meningoseptica (Additional file 2: Table S1).
Table 4

Genome statistics of E. meningoseptica Em3

Attribute

Value

% of total

Genome size (bp)

4,037,922

100

DNA coding (bp)

3,571,073

88.44

DNA G + C (bp)

1,468,714

36.37

DNA scaffolds

11

NA

Total genes

3729

100

Protein coding genes

3673

98.50

RNA genes

56

1.50

Pseudo genes

0

0

Genes in internal clusters

752

20.17

Genes with function prediction

2585

69.32

Genes assigned to COGs

1993

53.45

Genes with Pfam domains

2740

73.48

Genes with signal peptides

452

12.12

Genes with transmembrane helices

818

21.94

CRISPR repeats

0

0

Table 5

Number of genes associated with general COG functional categories

Code

Value

%age

Description

J

186

8.58

Translation, ribosomal structure and biogenesis

A

0

0

RNA processing and modification

K

170

7.84

Transcription

L

91

4.20

Replication, recombination and repair

B

0

0

Chromatin structure and dynamics

D

21

0.97

Cell cycle control, Cell division, chromosome partitioning

V

81

3.74

Defense mechanisms

T

82

3.78

Signal transduction mechanisms

M

184

8.49

Cell wall/membrane biogenesis

N

10

0.46

Cell motility

U

17

0.78

Intracellular trafficking and secretion

O

110

5.08

Posttranslational modification, protein turnover, chaperones

C

106

4.89

Energy production and conversion

G

120

5.54

Carbohydrate transport and metabolism

E

184

8.49

Amino acid transport and metabolism

F

60

2.77

Nucleotide transport and metabolism

H

134

6.18

Coenzyme transport and metabolism

I

96

4.43

Lipid transport and metabolism

P

153

7.06

Inorganic ion transport and metabolism

Q

39

1.80

Secondary metabolites biosynthesis, transport and catabolism

R

203

9.37

General function prediction only

S

105

4.85

Function unknown

1736

46.55

Not in COGs

Insights from the genome sequence

Elizabethkingia bacteria cause sepsis, bacteremia, meningitis or respiratory tract infections in hospitalized patients, indicating that they have the ability to colonize host tissues, suppress the host immune response, and disrupt erythrocytes to obtain nutrients and propagate in the host bloodstream [1, 13, 14]. Genome analysis showed that E. meningoseptica Em3 carried a gene (BFF93_RS1398) encoding a hemagglutinin protein. Hemagglutinins as adhesins are required for virulence in bacterial pathogens [23]. Hemagglutins facilitate infection via adherence to epithelial cell lines from the human respiratory tract in Bordetella pertussis [24]. Darvish et al. showed that filamentous hemagglutinin adhesins were crucial for bacterial attachment and subsequent cell accumulation on target substrates [25]. An in vitro biofilm assay showed that, compared to the mosquito isolate E. anophelis Ag1, clinical isolates E. meningoseptica Em3 and E. miricola Emi3 formed a higher amount of biofilm (Fig. 3). Furthermore, E. meningoseptica Em3 had better ability to form biofilm than did E. miricola Emi3. The capacity for strain Em3 to form biofilm was further exemplified by discovery of an operon involved in curli biosynthesis and assembly (BFF93_RS03755, BFF93_RS03760, BFF93_RS03765, BFF93_03725 and BFF93_RS03775). In vitro studies demonstrated that curli fibers participated in bacterial adhesion to target cell surfaces, cell aggregation, as well as biofilm formation [26, 27]. Moreover, some studies showed that curli mediated host cell attachment and invasion in vivo [28]. Curli were involved in inducing the host inflammatory response [29]. It should be noted that this curli synthesis operon is present in E. meningoseptica while it is absent in E. miricola . Further experiments are warranted to test if the curli gene cluster contributed to biofilm formation in strain Em3 because biofilm formation may involve other genes.
Fig. 3

In vitro biofilm assay in the selected Elizabethkingia sp. The cells were first cultured by shaking in TSB at 37 °C overnight. The cell density was adjusted to the same OD at 600 nm (0.1). 200 μl of cells were placed on 96-well plates for 24 h. The biofilm assay was carried out using crystal blue staining [49]. Values are mean values for single measurements from eight independent cultures. The error bars are standard deviations. The statistical test was the Student’s t-test. The asterisk indicates a significant difference compared to biofilm formation in E. anophelis Ag1 (p < 0.05).

A cytolysin encoding gene (BFF93_RS16990) was found in the strain Em3 genome, whose product belonged to a thiol-activated, CDC family [30]. CDC as a virulence factor is widely distributed among Gram-positive, opportunistic pathogens [31]. For example, Streptococcus pyogenes utilized pore-forming CDC to translocate a protein into eukaryotic cells [32]. Disruption of expression of a hemolysin (CDC) gene in the intracellular pathogen Listeria monocytogenes reduced virulence in mice, showing that CDC was critical for full virulence [33]. Furthermore, perfringolysin, a CDC toxin, has cytotoxicity and leukostasis activities, allowing the cells to escape from macrophage phagosomes during Clostridium perfringens gas gangrene [34]. Only a few CDCs have been found in Gram-negative bacteria [31], and this is the first report of CDC genes in E. meningoseptica . It should also be noted that this cytolysin gene is located immediately downstream of hmuY, which together comprise part of an iron metabolism gene cluster. Such gene organization was only seen in E. meningoseptica . This CDC protein sequence in strain Em3 shared 87%, 83% and 81% identity to that in E. meningoseptica ATCC 13253, E. meningoseptica B2D and E. meningoseptica NBRC 12535, respectively. It is interesting that it did not have close identity to that in E. meningoseptica FMS-007 (48%) and E. meningoseptica 502 (48%); it was absent in an E. meningoseptica strain associated with endophthalmitis [35]. Similarly, it was not conserved in E. anophelis (identity ranging from 0 to 50%) and absent in all E. miricola species. Such observations may stress that a diverse pathogenesis process exists in various E. meningoseptica and other Elizabethkingia species.

Besides a CDC gene in strain Em3 genome, we found that there was a gene encoding the hemolysin with a CBS domain (BFF93_RS14485). Hemolysin can be possibly secreted and involved in lysis of the erythrocytes [35]. The predicted amino acid sequence was conserved in most E. meningoseptica strains (> 90%). Further examination of hemin-degrading/transporter/utilization proteins led to a discovery of the gene cluster including SAM-dependent methyltransferases (BFF93_RS02055), iron ABC transporter (BFF93_RS02045), hemin-degrading protein (hmuS, BFF93_RS02060), hemin importer ATP-binding protein (BFF93_RS02050) and iron-regulated protein (BFF93_RS02065). Furthermore, there was a gene encoding a hemin receptor (BFF93_RS03140).

Elizabethkiniga infections can be fatal in immune-compromised patients if appropriate antibiotic therapy is delayed or the antimicrobial treatment is not properly provided [9, 14]. However, Elizabethkiniga spp. are multi-drug resistant [4, 13]. The prediction results by CARD and RAST (Table 6) showed that there are at least 31 genes involved in antibiotic resistance including antibiotic inactivation enzymes and related efflux pumps in E. meningoseptica Em3. Many of them are possibly involved in mupirocin, vancomycin, β-lactam, aminocoumarin, elfamycin, isoniazid, tetracycline and fluoroquinolone resistance (Table 6). Several drugs used to treat Elizabethkiniga-infected patients in the past are not effective anymore [4], which agrees with recent resistome assays in clinical E. meningoseptica isolates [12]. Genes associated with resistance to β-lactams, aminoglycosides, tetracycline, vancomycin, and chloramphenicol, reported here in strain Em3, are present in most of the studied Elizabethkingia spp. (Table 6). Remarkably, at least 12 β-lactam resistance genes encoding MBL fold metallo-hydrolases, metallo-β-lactamases and β-lactamases (class A and B) were found in E. meningoseptica Em3 genome (Table 6). Alternatively, antibiotics such as ciprofloxacin, minocycline, trimethoprim-sulfamethoxazole, rifampin and novobiocin may remain effective due to absence of relevant antibiotic resistance genes in Elizabethkingia sp. [36]. Therefore, a combination of antimicrobial tests and resistome analysis, combined with rapid identification of infections, will contribute to efficient management for E. meningoseptica infections in the future.
Table 6

Antibiotic genes prediction

   

E. meningoseptica

E. anophelis

E. miricola

Locus number

Gene in Em3

Putative function

Em3

502

R26

NUHP1

ATCC 33958

EM_CHUV

  

β-lactam

      

BFF93_RS01220

bla GOB-13

Class B carbapenemase BlaGOB-13

+

+

+

+

+

+

BFF93_RS04805

β-lactamase

+

+

+

+

+

+

BFF93_RS05700

β-lactamase (EC 3.5.2.6)

+

+

+

+

+

+

BFF93_RS07625

bla ACME

β-lactamase (BlaACME) VEB-1-like

+

+

+

+

+

+

BFF93_RS06860

bla B

BJP β-lactamase

+

+

+

+

+

+

BFF93_RS09265

MBL fold metallo-hydrolase

+

+

+

+

+

+

BFF93_RS06995

β-lactamase (EC 3.5.2.6)

+

+

+

+

+

+

BFF93_RS14540

β-lactamase

+

+

+

+

+

BFF93_RS12085

β-lactamase (EC 3.5.2.6)

+

+

+

+

+

+

BFF93_RS12510

MBL fold metallo-hydrolase

+

+

+

+

+

+

BFF93_RS14000

blaB-9

Class B carbapenemase BlaB-9

+

+

+

+

+

+

BFF93_RS01365

β-lactamase (EC 3.5.2.6)

+

+

+

+

+

+

  

Sulfonamide

      

BFF93_RS00125

dhfR

Dihydrofolate reductase DHFR

+

+

+

+

+

+

BFF93_RS17395

Bifunctional deaminase-reductase

+

+

+

+

+

+

BFF93_RS00125

dhfR

Dihydrofolate reductase DHFR

+

+

+

+

+

+

BFF93_RS17395

Bifunctional deaminase-reductase protein

+

+

+

+

+

+

BFF93_RS14765

folP

Dihydropteroate synthase FolP (EC 2.5.1.15)

+

+

+

+

+

+

  

Tetracycline

      

BFF93_RS08380

tetA

Tetracycline efflux protein TetA

+

+

+

+

+

+

BFF93_RS07335

Transmembrane efflux protein

+

+

+

+

+

+

BFF93_RS12745

Antibiotic transporter

+

+

+

+

+

+

  

Macrolide

      

BFF93_RS00370

lolD

Macrolide resistance, ABC transporter

+

+

+

+

+

+

BFF93_RS05670

emrB

Erythromycin resistance, EmrB/QacA

+

+

+

+

BFF93_RS05670

emrB

Erythromycin resistance, EmrB/QacA

+

+

+

+

+

+

BFF93_RS10830

emrB

Erythromycin resistance, EmrB/QacA

+

+

+

+

+

+

BFF93_RS03320

Erythromycin esterase

+

+

+

+

+

+

  

Quinolone

      

BFF93_RS04670

gyrA

DNA gyrase GyrA subunit A (T83S)

+

+

+

+

+

+

BFF93_RS09245

gyrB

DNA gyrase GyrB subunit A (M437 L)

+

+

+

+

+

+

BFF93_RS08895

parE

DNA topoisomerase IV subunit B (M437F/A473L)

+

+

+

+

+

+

  

Aminoglycoside

      

BFF93_RS10790

ant-6

Aminoglycoside 6-adenylyltransferase

+

+

+

+

+

+

  

Chloramphenicol

      

BFF93_RS14765

catB

Chloramphenicol acetyltransferase CatB

+

+

+

+

+

+

BFF93_RS04080

bcr/cflA

Bcr/CflA efflux pump

+

+

+

+

+

+

“+” or “-” indicates the presence or absence of genes in the selected Elizabethkingia

Conclusions

The draft genome sequence of E. meningoseptica Em3 isolated from a sputum sample in a patient was sequenced, annotated and described. We found that E. meningoseptica Em3 had novel genes encoding thiol-activated cholesterol-dependent cytolysin, curli and heme metabolism related proteins, showing that E. meningoseptica Em3 may be a causative agent. Our results also indicated that E. meningoseptica might be resistant to β-lactam antibiotics due to the production of diverse MBLs and β-lactamases. Furthermore, these β-lactamase encoding genes were also found in other Elizabethkingia species, indicating that Elizabethkingia species were important reservoirs of novel β-lactamase genes. Comparative genomics is a crucial approach in the discovery of novel virulence determinants in Elizabethkingia species. Genome-based approaches contribute to develop novel genetic markers for future molecular diagnosis of Elizabethkingia infections.

Abbreviations

ANI: 

Average nucleotide identities

CDC: 

Cholesterol-dependent cytolysin

IMG/MER: 

Integrated Microbial Genomes and Microbiome Samples Expert Review

MALDI-TOF: 

Matrix assisted laser desorption-ionisation time-of-flight

MBL: 

Metallo-β-lactamase

MIC: 

Minimum inhibitory concentration

SBA: 

Sheep blood agar

TSA: 

Solid trypticase soy agar

Declarations

Acknowledgements

We acknowledge Dr. Jiannong Xu at the New Mexico State University for the provision of E. anophelis Ag1.

Funding

This project was funded by NIH grant R37 AI21884.

Authors’ contributions

SC assembled the genome sequence, analyzed the genome data in public databases for genes of interest and wrote the manuscript. MS performed the experiments and acquired the data. FD wrote and revised the manuscript. EW analyzed the data and wrote the manuscript. All authors read approved final the manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Authors’ Affiliations

(1)
Department of Microbiology and Molecular Genetics, Michigan State University, 2215 Biomedical and Physical Sciences Building
(2)
Michigan Department of Health and Human Services, Bureau of Laboratories
(3)
Biomedical Laboratory Diagnostics Program, Michigan State University

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Copyright

© The Author(s). 2017