Download Development of Escherichia coli systemic infection model

Development of Escherichia coli systemic infection model for monitoring persisters and
antibiotic susceptibility
K. Kogermann1, M. Putrinś1, M. Lippus1, I. Smidt2, K. Truusalu2, I. Lutsar2, T. Tenson1
1 Institute of Technology, University of Tartu, Nooruse 1, Tartu 50411, Estonia;2Institute of Microbiology, University of Tartu, Ravila 19, Tartu 50411, Estonia
.
Dual-Fluorescent UPEC
Intraperitoneal (IP) infection
of mice (1.5x107 cells/ml)
Development of
systemic infection
6, 24 or 48 h
green fluorescent protein
IPTG inducible expression
reporter for cell division
CAM
Fig. 1.
UPEC CFT073
mCherry
Reporter
plasmid
IP liquid (CFU/ml)
IP liquid(CFU/g)
(CFU/ml)
Spleen
Spleen (CFU/g)
Kidney
Kidney /CFU/g)
(CFU/g)
Liver
Liver(CFU/g)
(CFU/g)
6
10
5
Isolation of bacterial cells
from different organs
10
Analyses of bacteria
10
4
10
3
2
10
Inoculum
Bacteria from mouse IP liquid
R2
R1
6h
24 h
1
10
Flow cytometry
CFU
Microscopy
Fig. 2.
In addition to traditional CFU plating, flow
cytometry
and
confocal
fluorescent
microscopy together with fluorescent reporter
protein dilution (with DAPI staining) were
used for monitoring UPEC persisters (Fig. 3).
mCherry
• red fluorescent protein
• constitutive epression
• helps to indentify bacterial cells
•
•
•
Results and Discussion
In the mouse systemic infection model,
bacteria disseminated from IP liquid to all
internal organs as well as to a bloodstream.
Infection levels after 6, 24 and 48 h were all
comparable (Fig. 4). Highest level of infection
was obtained in spleen.
Relative number of cells
Experimental design
Rel. number of cells
Introduction
Persisters are multidrug-tolerant bacteria that
are the likely cause for many of the infection
relapses. As opposed to the resistant cells,
persisters cannot grow in the presence of the
drug and they are genetically identical to the
drug-susceptible cell. Their existence has
been shown in several in vitro studies, but
their emergence during in vivo infections is
less reported. Uropathogenic Escherichia coli
(UPEC) is a main cause of urinary tract
infections, but it is also able to spread and
cause systemic infections.
Aims of the study
To develop a mouse systemic infection
model for monitoring UPEC persisters in vivo.
The model will be further used to reveal their
impact on antibiotic treatment efficacy.
Methods
We have investigated the faith of UPEC
CFT073 cells carrying a reporter plasmid
expressing two fluorescent reporter proteins
and chloramphenicol resistance (Fig. 1)
during in vivo systemic infection in C57bl/6J
(female, 8-10 weeks old) mice (Fig. 2).
Animal experiments were approved by
Estonian ethical committee for animal
experimentation (licence no 34 and 52 ).
0h
2h
3h
no GFP ind.
GFP fluorescence
Number of generations calculated from
GFP dilution is in a good correlation with
generations calculated from cell numbers.
Cell
numbers
GFP level
lacI
Fig. 3.
2h
3.0 gener.
3h
4.3 gener.
2.8 gener.
4.1 gener
6h
24 h
48 h
48 h
Data are shown for all tested mice in each
timepoint (n=3-10). Control mice (1xPBS)
showed no CFU, CFU of control mice with
unmarked bacteria were comparable with
marked ones.
Fig. 4.
Cell division analysis showed that most of
the bacteria (high GFP) had not divided
after 6 h of infection, however after 24 h,
extensive cell division was observed (Fig.
5). After 48 h no persister cells were
detected in mice. The presence of different
bacterial subpopulations were also verified
by fluorescent microscopy.
GFP fluorescence
Number of generations calculated from
GFP dilution:
R1
R2
6h
0.9
<6
Data for individual
timepoint are shown.
24 h
2.8
<6
mice
48 h
n.a.
<6
in
each
Fig. 5.
Conclusions
The developed mouse systemic infection model and single cell analysis methods revile the
location and frequency of potential UPEC persisters. Dividing and nondividing subpopulations of
bacteria were detected in different body sites during the time course of infection suggesting that
the heterogeneity of bacteria in vivo might influence the outcome of antibiotic treatment. The
reporter strain and mouse infection model are potentially very useful tools for determining both the
decrease of bacterial number due to the action of immune system and antibiotics, as well as
increase by cell division.
Copyright © 2015 Kogermann, K. Address: Nooruse 1, 50411 Tartu, Estonia; e-mail: [email protected]