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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.