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البحث (1):

عنوان البحث:

Galleria
mellonella Infection Model Demonstrates High Lethality of ST69 and ST127
Uropathogenic E. coli

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تاريخ النشر:

 29/07/2014

موجز عن البحث:

Galleria
mellonella larvae are an alternative in vivo model for investigating
bacterial pathogenicity. Here, we examined the pathogenicity of 71 isolates
from five leading uropathogenic E. coli (UPEC) lineages using G. mellonella
larvae. Larvae were challenged with a range of inoculum doses to determine
the 50% lethal dose (LD50) and for analysis of survival outcome using
Kaplan-Meier plots. Virulence was correlated with carriage of a panel of 29
virulence factors (VF). Larvae inoculated with ST69 and ST127 isolates (104
colony-forming units/larvae) showed significantly higher mortality rates than
those infected with ST73, ST95 and ST131 isolates, killing 50% of the larvae
within 24 hours. Interestingly, ST131 isolates were the least virulent. We
observed that ST127 isolates are significantly associated with a higher
VF-score than isolates of all other STs tested (P 0.0001), including ST69
(P,0.02), but one ST127 isolate (strain EC18) was avirulent. Comparative
genomic analyses with virulent ST127 strains revealed an IS1 mediated
deletion in the O-antigen cluster in strain EC18, which is likely to explain
the lack of virulence in the larvae infection model. Virulence in the larvae
was not correlated with serotype or phylogenetic group. This study
illustrates that G. mellonella are an excellent tool for investigation of the
virulence of UPEC strains. The findings also support our suggestion that the
incidence of ST127 strains should be monitored, as these isolates have not
yet been widely reported, but they clearly have a pathogenic potential
greater than that of more widely recognised clones, including ST73, ST95 or
ST131.

المؤتمرات العلمية:

المؤتمر (1):

عنوان المؤتمر:

24 ECCMID (European Society of Clinical
Microbiology and Infectious Diseases)

تاريخ الإنعقاد:

10-13 May 2014

مكان
الإنعقاد:

Barcelona,
Spain

طبيعة المشاركة:

Poster
presentation

عنوان المشاركة:

Comparative
genomics reveals that O-antigen is essential for virulence of ST127 UPEC in
Galleria mellonella larvae

ملخص المشاركة:

Introduction

Escherichia coli is the major cause of
extraintestinal infections including urinary tract infection (UTI),
Gram-negative bacteremia and neonatal meningitis. Uropathogenic E. coli (UPEC) is the most frequent cause of
UTI, being responsible for up to 85% of community acquired UTI and 25% of
nosocomial UTI. Our previous studies suggest that ST127 is an important and
emerging lineage that has a high pathogenic potential. Surveillance of 29
virulence genes using PCR shows that ST127 isolates are significantly
associated with a high number of virulence genes (P= 0.0001). In
addition, an in vivo study using Wax moth larvae (Galleria
mellonella) showed that larvae inoculated with ST127 isolates (10⁴ colony-forming units) had
significantly higher mortality compared to other UPEC including those from
ST73 (P= 0.0024) and ST131 (P= 0.0004). However, an interesting
result to emerge from the data was the observation that one strain of ST127
(EC18) did not show any lethal effects, even for high inoculum doses (up to
2.33×10⁷ cfu/10µl).

Objectives:

Using comparative genomic analyses to identify the genetic basis
for UPEC ST127 EC18 non-lethality in the Wax moth model G.mellonella.

Methods:

The draft genome sequences of ST127, strains EC18 and EC41 were
sequenced by Illumina HiSeq 2000 and strain EC224 was sequenced by Roche 454
GS-FLX(Ti). Pair-wise whole genome comparison was performed using BLASTn of
UPEC strain 536, the only completely sequenced ST127 in the public databases,
against the three ST127 strains. Comparison and visualization were carried
out using Artemis comparison tool (ACT), BLAST Ring Image Generator (BRIG)
and Easyfig.

Results:

Comparative genomic revealed a deletion of the O-antigen gene
cluster. Evidence of bacterial insertion sequence IS1 was seen at the
position where the O-antigen deletion occurs in EC18. In addition, this work
has shown that four pathogenicity islands PAI I536, PAI II536, PAI III536 and
PAI V536 are highly variable between EC18, EC41 and EC224, when compared to
strain 536.

Conclusion:

UPEC display clone specific characteristics such as
virulence-associated traits and variable metabolic activity and growth rates.
This study illustrates that GM larvae can be used as a novel model host to
investigate the virulence of UPEC strains. O-antigen biosynthesis is an
important virulence determinant for UPEC, which may be significant in the
ability of EC41 and EC224 to kill the Wax moth modelG.mellonella. The outcome of this analysis will
give considerable insight into the association of different E. coli virulence genes with lethality in
the experimental model of G.
mellonella larvae
infection. This work may also identify novel virulence genes for further
analysis.

المؤتمر (2):

Society
for General Microbiology Annual Conference

عنوان المؤتمر:

14-17 April 2014

تاريخ الإنعقاد:

Liverpool,
UK

مكان
الإنعقاد:

Poster
presentation

طبيعة المشاركة:

Genome
Sequence Determination and Comparative Genomics of Uropathogenic E. coli ST127

عنوان المشاركة:

Uropathogenic E. coli (UPEC) is the most frequent cause of UTI,
being responsible for up to 85% of community acquired UTI and 25% of
nosocomial UTI.   An in vivo study
using Galleria mellonella showed that larvae inoculated with ST127 isolates
(104 colony-forming units) had significantly higher mortality compared to
other UPEC including those from ST73 (P≤ 0.0024) and ST131 (P≤ 0.0004).
However, an interesting result to emerge from the data was the observation
that one strain of ST127 (EC18) did not show any lethal effects, even for
high inoculum doses (up to 2.33×107 cfu/10µl).

Using comparative genomic analyses to identify the genetic basis
for UPEC ST127 EC18 non-lethality in the Wax moth model G.mellonella. The
genomic analyses revealed a major deletion in the O-antigen cluster, which is
likely to explain the lack of virulence in the larvae. Evidence of bacterial
insertion sequence IS1 was seen at the position where the O-antigen deletion
occurs in EC18.

This study illustrates that G.mellonella can be used to
investigate the virulence of UPEC strains. The outcome of this analysis will
give considerable insight into the association of different E. coli virulence
genes with lethality in the experimental model of G. mellonella larvae
infection.

ملخص المشاركة:

المؤتمر (3):

عنوان المؤتمر:

23th European Congress of Clinical
Microbiology and Infectious Diseases, Berlin, Germany,

تاريخ الإنعقاد:

27–30 April 2013

مكان
الإنعقاد:

Berlin, Germany

طبيعة المشاركة:

Poster presentation

عنوان المشاركة:

Investigating the virulence of Uropathogenic E. coli (UPEC) using
Galleria mellonella larvae

ملخص المشاركة:

Introduction:

Uropathogenic
E. coli (UPEC) is recognized as the most frequent cause of urinary tract
infection (UTI), being responsible for up to 85% of community acquired UTI
and 25% of nosocomial UTI. According to previous studies a number of lineages
of UPEC such as ST131, ST73 and ST127 are consistently detected and can
represent a real threat to health. Galleria mellonella larvae have recently
been introduced as an alternative in vivo model to mammalian systems and here
we are using it to investigate the pathogenicity of different lineages of
UPEC.

Objectives:

UPEC
lineages equipped with a variety of virulence factors and the goal of this
study is to understand the pathogenicity and the behaviour of different
sequence types (STs) of UPEC ST131, ST73 and ST127 using Galleria mellonella
larvae as in vivo model.

Methods:

Larvae
were challenged with range of inoculum doses of different UPEC sequence types
to determine 50% lethal dose (LD50) and the infected larvae survival outcome
was compared by Kaplan-Meier survival analysis. Results: Larvae inoculated
with ST127 isolates (104 colony-forming unit) shows significant high
mortality rate than ST131 (P<= 0.0004) and ST73 (P<= 0.0024) which kill
50% of the larvae after 24 hours. Surveillance of 29 virulence genes shows
that ST127 isolates significantly association with high VF-score (P<=
0.0001). Although LD50 of ST131 and ST73 isolates shows no significant
difference, ST131 isolates appears to be significantly associated with low
FV-score (P< 0.0001) compared to ST73.

Conclusion:

The
results of this study illustrated that the larvae could be used to
investigate the virulence of different UPEC strains. Compression of genomic
sequences of different STs of UPEC may lead to deeper understanding of the
virulence factors, and thus, the Galleria mellonella is a useful in vivo
model to investigate UPEC virulence.