1. Staphylococcus epidermidis is a Gram-positive bacterium
naturally found on human skin and an important opportunistic
pathogen linked with neonatal blood sepsis.
Preterm neonates are a highly susceptible patient group for
bacterial infections, due to their naive immune status and
the invasive procedures to which they are often subjected to
in neonatal ICU settings. Rapid detection of blood sepsis and
characterisation of the causative pathogen are critical first
steps to enable appropriate treatment and improved prognostic
outcomes.
As part of the ClouDx-i consortium, we aim to expand our
knowledge of circulating pathogenic strains linked with neonatal
blood sepsis to inform the continued development of new
molecular diagnostic assays.
INTRODUCTION
GENOME SEQUENCES OF SIX DIFFERENT STAPHYLOCOCCUS EPIDERMIDIS CLONES
Isolated Individually from Preterm Neonates Presenting with Sepsis at Edinburgh’s Royal Infirmary
M. Bekaerta,b
E. Cullenb
P. Walshb
B. Kellyb
A. O’Driscollc
X. Lub
C. Smithd
P. Dickinsond
K. Templetond,e
W. Alsheikhly Dantofte
A. Lucidf
R. Langleye
P. Ghazald,e
R. D. Sleatorf
a
Institute of Aquaculture, University of Stirling, UK
b
NSilico LifeScience Ltd., Cork, Ireland
c
Department of Computing, Cork Institute of Technology, Ireland
d
Division of Pathway Medicine, University of Edinburgh, UK
e
Microbiological Diagnostic Unit, Royal Infirmary, University of Edinburgh, UK
f
Department of Biological Sciences, Cork Institute of Technology, Ireland
We report results and implications of the establishment of
rapid genome assembly, plasmid detection and genome
comparison procedures of six individual S. epidermidis
strains, all done typically in less than 1 hour, isolated from
preterm neonates at the Royal Infirmary, Edinburgh, United
Kingdom, in 2014
WWW.CLOUDXI.EU
ClouDx-i is a major research project concerned with
developing host pathogen response biomarkers for
infection and is funded under the EU FP7 Marie Curie
Actions IAPP program.
CONTACT US
Positivity for blood sepsis and species identification of
each isolate was confirmed by classical microbiological
identification and characterisation techniques.
Isolates were grown overnight at 37°C on Luria broth
(LB) agar, and genomic DNA was isolated using Qiagen
genomic tips (Venlo, Limburg, Netherlands). Genomic
DNA fragments, ranging in size from 2 to 10 kb, were
generated by sonication. Fragments were subsequently
used to produce a non-size-selected genome library using
the Nextera mate-pair kit (Illumina, San Diego, CA). The
resulting libraries were then sequenced on an Illumina
MiSeq using MiSeq Reagent kit v3.
METHOD
CONCLUSION
Genomic sequence assembly, analysis, and automated
reporting were carried out using NSilico Simplicitytm
pipeline (https://simplicity.nsilico.com). Sequence
assembly was achieved using a de-novo assembly pipeline
based on the Spades 3.10 assembly tool, with k-mers
21, 33, 55, 77, 99, and 127 nucleotides in length. Each
genome was initially annotated with the Prokka and
the identified 16S rRNA genes were used to confirm the
species as S. epidermidis in each case. Each genome was
then screened using the Glimmer 3. The predicted Coding
Sequences (CDS) were compared to the Uniprot Trembl
database using BLASTp.
Strain Reads
Assembled
genome
Coverage
GC
content
Total
contigs
No.
CDS
Accession
number
NGS-ED-1107 2,599,368 2,599,368 bp 135.2x 32.0% 53 2,554 JZUK00000000
NGS-ED-1109 2,436,309 2,628,145 bp 114.6x 32.0% 51 2,252 JZUL00000000
NGS-ED-1110 4,476,721 2,628,994 bp 223.5x 32.0% 45 2,515 JZUM00000000
NGS-ED-1111 5,197,793 2,670,519 bp 163.5x 32.0% 44 2,583 JZUN00000000
NGS-ED-1117 1,607,705 2,596,186 bp 76.0x 32.0% 48 2,459 JZUO00000000
NGS-ED-1118 1,681,207 2,578,927 bp 94.1x 31.9% 48 2,444 JZUP00000000
Deaths from sepsis are on the rise due to growing antibiotic resistance which means previously harmless infections
can become fatal, Sepsis develops when an infection gets into the bloodstream and affects the whole body,
triggering a violent and potentially fatal immune response which can cause organ failure.
The condition causes ~37,000 deaths in the UK in 2015, figure that will rise if antibiotic resistance is not tackled.
Potential genes
ADHESION
atlE N N N N N N
dltA Y Y Y Y Y Y
tagO Y Y Y Y Y Y
fbe N N N N N N
BIOFILM
ica operon Y Y Y Y N Y
luxS Y Y Y Y Y Y
sarA Y Y Y Y Y Y
MUTATOR STATUS
LexA N N N N Y N
mutS (IS256) N N N N N N
sigB Y Y Y Y Y Y
rsbU Y Y Y Y Y Y
VIRULENCE
psm-gamma N N N N N N
sepA N N N N N N
sepI pathogenicity island N N N N N N
ecp N N N N N N
agr (QS system) N N N N N N
ANTIBIOTIC RESISTANCE
sccMec N N N N N N
blaZ N Y Y N N N
mecA Y Y Y Y Y Y
VanA N N N N N N
mprF Y Y Y Y Y Y
yycFG N N N N N N
cfr N N N N N N
rpoB Y Y Y Y Y Y
psk41 (Plasmid) N N N N N N
NGS-ED-1107
NGS-ED-1118
NGS-ED-1117
NGS-ED-1110
NGS-ED-1109
NGS-ED-1111
The presence or absence of genes was compared to the S. epidermidis
ATCC 12228 strain this was followed up by looking for genes that are
relevant for adhesion, biofilm formation, virulence and Antibiotic
resistance.
NGS-ED-1117NGS-ED-1109NGS-ED-1111NGS-ED-1110NGS-ED-1118NGS-ED-1107
Figure 5 - Genome comparison. The six genomes were aligned using Mauve. The main
conservation islands are reported for each strain at scale. An island is a region of the
alignment where one genome has a sequence element that one or more others lack and
is presumably homologous and internally free from genomic rearrangement.
Figure 1 - Staphylococcus epidermidis. Left panel: Colonies in a substrat
(skin). Right panel. Model of a colony
Credit: (c) Center for Disease and Prevention
Figure 2 - Assembled Staphylococcus epidermidis Genome atlas. (From the
outer circle inward, coding regions are marked on the first two rings. The third
ring shows the CG skew, with sharp changes in skew occurring at the origin
and terminus of replication. The innermost graph shows local CG content
measured in a sliding window as a black plot
Figure 3 - Assembly and Annotation Pipeline: Simplicitytm
implementation. Screenshot
of the Web Interface of the Pipelines (A) Data submission. (B) Advanced parameters.
(C) Processing the results. (D) Example of details results. Alignment against a reference
genome if order compare the overall de novo assembly.
 Quick turn over for acute medecine
 Extensive reporting for research & development
Genome annotation
 Gene detection




Similarity searches (BLAST)
Virulence database search
Phylogeny reconstruction
Chromosome view
Editable report
Genome announcements draft
. . .
Genome assembly
 Genome mappingde novo assembly
  Sequence variations
Prophage detection
. . .
Plasmid discovery
Prophage detection
Species identification
Assembly finishing
. . .
Read cleaning
 Quality filtering
 Contamination removal
 Trimming and adapter clipping
Figure 4 - Simplicitytm
Assembly and Annotation Pipeline. We design and develop
several assembly and annotation pipleline in order to assemble and annotate a genome
from the raw reads in less than 60 minutes.
www.nsilico.com www.stir.ac.uk www.ed.ac.uk www.cit.ie
info@nsilico.com michaelbekaert@stir.ac.uk p.ghazal@ed.ac.uk roy@sleator@cit.ie
A B
C D