INTRACELLULAR BACTERIA

TARGETED PHOTODYNAMIC
THERAPY – EFFECT OF
SENSITIZATION TIME
Saji George*, Shibi Mathew, Anil Kishen
Restorative Dentistry
Faculty of Dentistry
*Immunology programme
Department of microbiology
Contents

 Introduction
 Apical periodontitis
 Role of bacteria
 Photodynamic therapy
 Objective
 Materials and Methods
 Result and discussion
 Conclusion
 Biofilm in apical periodontitis

 Apical periodontitis- clinical manifestation of

immune
reaction to microorganisms persisting inside the
root
canal system (Nair 2006)

 Primary etiological agent- bacteria (Kakehashi et

al.,1965)

 Adhere to the dentinal walls , colonize the

surface,
form biofilms (Nair PNR , 1987)

 Apical periodontitis - biofilm induced disease

 Intracellular bacteria in immune cells of apical
periodontitis

 Lymphocytes, macrophages and plasma

cells form 4%-50% of cellular infiltrate
(Kawashima et al., 1996)

 Macrophages phagocytose, mostly kill,

bacteria reaching the periapical space.
 Intracellular bacteria surviving in
macrophages adjacent to the apical
foramen-periapical lesions (Walton et al. 1992;
Goldstein et al. 1981; Nair 1987).
(Nair , 1987)
 Biofilm-positive E. faecalis resistant to kill
by rat peritoneal macrophages than
biofilm-negative strains (Baldassarri et al. 2001; Gentry et
al. 1999).
 Bacterial growth modes that defies current antimicrobials

Biofilm
Community of
immobilized
bacteria in embedded
in self made matrix

Intracellular pathogens
Bacteria residing
inside host cells
 Photodynamic Therapy (PDT) as an antimicrobial agent

Photosensitizer + light oxygen Bacterial killing

PDT as an antimicrobial agent (Hamblin et al.,

2004)

In vitro and in vivo against Gram –ve and

Sensitized bacterial cell Damaged cell
+ve bacteria , multidrug resistant strains
(Embleton et al., 2002; Maisch et al., 2004)

A dual-stage application of
photosensitizer medium and
irradiation medium was standardized
for root canal disinfection
(George and Kishen 2007)
 Photodynamic therapy for eradicating intracellular bacteria

O’Riordan et al reported 80% reduction of

intracellular pathogen Mycobacterium
bovis using PDT(O’Riordan et al., 2006)

Subjecting the bacteria (E. faecalis ) and

mammalian cells (fibroblast cells)
simultaneously to PDT

y= 95.939e-
The rate of bacterial killing - faster than loss 0.0764x

of mammalian cells (George and Kishen 2007).

y= 137.48e-
1.0088x
The present study, evaluates the duration of sensitization time
on the elimination of internalized Enterococcus faecalis
from pre-monocyte cell line (THP1), using PDT.
 THP-1

50 µM methylene blue Irradiation (λ= 664 nm, 31.84J/cm2)

(0min, 5mins, 10 mins, 15mins) (1 minute)

Trypan-blue exclusion assay

Mitochondrial activity (MTT assay)

DNA (comet assay)

 PDT on internalized bacterial cells

HAP buffer +Antibiotics

Incubated for 4hr at 37ºC in
CO2 incubator

Lysed with
0.01%
Triton X100
Bacteria quantified
After 24 hrs .
PDT with 50 µM methylene blue
THP1 cell membrane integrity

Trypan-blue exclusion assay

Mitochondrial activity (MTT assay)

DNA (comet assay)

 Uptake of methylene blue by THP-1 cells

The association of MB with

THP1 cells increased with
increasing sensitization time
PDT induced THP1 cell membrane integrity and mitochondrial
activity

Gradual decrease in
membrane integrity (trypan
blue exclusion assay) as the
sensitization time increased

Mitochondrial activity (MTT

assay) showed no significant
change over the period of
sensitization
 1 2
DNA damage- Comet
assay
There was no

3 4

1. Control 5
2. H2O2
3. Irradiation
4. Sensitization
5. PDT
PDT induced THP1 cell membrane integrity and mitochondrial
activity

Viability of bacteria- linear

decrease.
PDT on THP1 cells with
intracellular bacteria showed
significant decrease in the
bacterial number as the
duration of sensitization was
increased (p<0.001).
100% elimination of bacteria
resulted in 70% THP1 cells
with damaged cell membrane.

Increased association of MB with THP1 at cell membrane could be

the reason for increased membrane damage
Increasing the duration of sensitization resulted in increased
photosensitizer uptake by THP1 cells.

The cell membrane integrity followed same pattern as the

photosensitizer uptake while effect on mitochondrial activity
and DNA integrity were minimal due to PDT.

PDT on THP1 cells with intracellular bacteria showed

significant decrease in the bacterial number as the
duration of sensitization was increased (p<0.001).

There was 100% elimination of bacteria with 70% of THP1

cells with damaged membrane
PDT could achieve selective killing of
intracellular bacteria with minimal vital
damage to mammalian cells
 Acknowledgments

NUS academic research fund -R-224-000-024-112

Faculty of Dentistry and the Department of Pathology

My supervisor and all my colleagues .

THANK YOU !!