Journal of Medical Microbiology (2013), 62, 467–473
DOI 10.1099/jmm.0.048843-0
Antimicrobial photodynamic therapy using visible
light plus water-filtered infrared-A (wIRA)
A. Al-Ahmad,1 C. Tennert,1 L. Karygianni,1 K. T. Wrbas,1,2 E. Hellwig1
and M. J. Altenburger1
1
Correspondence
Department of Operative Dentistry and Periodontology, Albert-Ludwigs-University, Freiburg,
Germany
Ali Al-Ahmad
[email protected]
Received 21 June 2012
Accepted 20 November 2012
2
Department of Endodontics, Centre for Operative Dentistry and Periodontology, University of
Dental Medicine and Oral Health, Danube Private University (DPU), Krems, Austria
The aim of this study was to investigate the effectiveness of antimicrobial photodynamic therapy
(APDT) using visible light together with water-filtered infrared-A (VIS+wIRA) to eradicate single
species of planktonic bacteria and micro-organisms during initial oral bacterial colonization in situ.
A broadband VIS+wIRA radiator with a water-filtered spectrum in the range 580–1400 nm was
used for irradiation. Toluidine blue (TB) was utilized as a photosensitizer at concentrations of 5,
10, 25 and 50 mg ml”1. The unweighted (absolute) irradiance was 200 mW cm”2 and it was
applied for 1 min. Planktonic cultures of Streptococcus mutans and Enterococcus faecalis were
treated with APDT. Salivary bacteria harvested by centrifugation of native human saliva were also
tested. In addition, initial bacterial colonization of bovine enamel slabs carried in the mouths of six
healthy volunteers was treated in the same way. Up to 2 log10 of S. mutans and E. faecalis were
killed by APDT. Salivary bacteria were eliminated to a higher extent of 3.7–5 log10. All TB
concentrations tested proved to be highly effective. The killing rate of bacteria in the initial oral
bacterial colonization was significant (P50.004) at all tested TB concentrations, despite the
interindividual variations found among study participants. This study has shown that APDT in
combination with TB and VIS+wIRA is a promising method for killing bacteria during initial oral
colonization. Taking the healing effects of wIRA on human tissue into consideration, this technique
could be helpful in the treatment of peri-implantitis and periodontitis.
INTRODUCTION
Although photodynamic therapy (PDT) is currently being
applied in cancer cell elimination (Allison & Sibata, 2010;
Ericson et al., 2008), more attention has been paid to its
application in antimicrobial treatment (Wood et al., 2006;
Donnelly et al., 2007; Maisch, 2007). Due to the growing
number of antibiotic-resistant micro-organisms, research is
focusing on PDT as an alternative chemotherapy modality
(Garcez et al., 2010; Kharkwal et al., 2011). Since PDT
causes damage to different parts of microbial cells and
affects different interaction pathways in the micro-organisms, the development of antibiotic resistance against PDT
can be excluded (Wainwright et al., 2010).
PDT impresses with its excellent target-cell specificity and
high efficiency. The photodynamic effect requires a
photosensitizer, suitable light and oxygen. The photosensitizer can be transferred to the high-energy triplet state and
Abbreviations: APDT, antimicrobial photodynamic therapy; BES, bovine
enamel slabs; PDT, photodynamic therapy; TB, toluidine blue; VIS, visible
light; wIRA, water-filtered infrared-A.
048843 G 2013 SGM
reacts with oxygen, producing singlet oxygen and other
radical species which themselves cause increased target cell
damage (Konopka & Goslinski, 2007). The photosensitizer
should be non-toxic and show antimicrobial activity only
after activation by illumination. In addition it should not
be bio-destructible. Light sources used in PDT should
produce low-power visible light at a specific wavelength (or
range of wavelengths) which itself does not harm human
tissues in the absence of the photosensitizer. Additionally,
light sources should be inexpensive, lightweight and highly
flexible (Konopka & Goslinski, 2007). Despite the fact that
the antimicrobial activity of LED light sources has been
intensively investigated in various studies (Omar et al.,
2008; Omar, 2010), the efficacy of visible light in
combination with water-filtered infrared-A (VIS+wIRA)
in killing bacteria has not previously been examined. This
combination delivers a broad-band light source which
could be used in combination with different photosensitizers. A significant reduction of the total bacterial load was
described previously with this combination for the
treatment of chronic wounds (von Felbert et al., 2008).
Such effects are potentially of great importance in dental
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467
A. Al-Ahmad and others
medicine, since VIS+wIRA is indicated for chronic pain
relief and is capable of increasing the tissue oxygen partial
pressure and stimulating metabolism (Hartel et al., 2006;
von Felbert et al., 2010; Maisch et al., 2007). Additionally,
due to its penetration properties, wIRA causes energy
transfers into subcutaneous tissue without overheating the
skin (Fuchs et al., 2004). Furthermore, it has been shown
that wIRA reduces pain in chronic wounds, warts, herpes,
scleroderma, gonarthrosis and spondylitis (Fuchs et al.,
2004; Hartel et al., 2006). Applying wIRA would enhance
the effects of PDT since heating has some synergistic effect
when applied together with PDT (Waldow et al., 1985;
Kelleher et al., 2003; Yanase et al., 2006). The use of
antimicrobial PDT (APDT) could therefore potentially
improve the tissue healing process in a number of dental
procedures.
The oral biofilm formed on teeth surfaces and soft gingival
tissue in the oral cavity acts as a trigger for dental caries,
periodontitis, gingivitis and peri-implantitis (Al-Ahmad
et al., 2010; Marsh et al., 2011). The effects of PDT on oral
bacteria and oral biofilms have mainly been studied on
single bacterial species biofilm generated in vitro. An
overview of such studies has been published by Konopka &
Goslinski (2007). The differences in the cell wall structure
of Gram-positive and Gram-negative bacteria lead to
variations in the binding ability of photosensitizing agents
to different bacterial surfaces. For example, the affinity of
negatively charged photosensitizers to Gram-negative
bacteria can be enhanced by modifying the sensitizer by
using cationic molecules such as chlorine e6 (Rovaldi et al.,
2000). This emphasizes the necessity for the identification
of an effective photosensitizer and a suitable light source,
which together are capable of eliminating various resistant
micro-organisms in the presence of a pronounced bacterial
diversity in the oral cavity.
In the oral cavity, bacteria can grow as single cells in
human saliva in concentrations up to 109 ml21 (Marsh &
Martin, 1999). Oral bacteria can also adhere to the enamel
surface or other restorative materials in the oral cavity.
After an initial phase of colonization, oral micro-organisms
multiply and aggregate to form the dental oral biofilm,
which consists of extracellular substances and a diverse
range of micro-organisms (Al-Ahmad et al., 2009; Marsh &
Martin, 1999). Hence, testing the efficacy of antimicrobial
oral therapy should include not only the mature dental
biofilm but also the planktonic micro-organisms and the
initial bacterial colonization.
The aim of the present study was to assess the effectiveness
of APDT in the eradication of oral micro-organisms which
initially adhered to bovine enamel slabs in situ. This is
believed to be the first time that the effect of VIS+wIRA
on initial bacterial oral biofilm has been tested in vivo. The
effectiveness of this specific photodynamic therapy was
previously examined in vitro using high concentrations of
Streptococcus mutans and Enterococcus faecalis. Since the
effects of wIRA on wound infections have been shown, it
468
could be expected that PDT using wIRA would have an
impact on oral microoganisms and oral initial colonization
METHODS
Light source and photosensitizer. A broad-band VIS+wIRA
radiator (Hydrosun 750 FS, Hydrosun Medizintechnik) with a 7 mm
water cuvette was used in this in situ assay. An orange filter, BTE31,
which provides more than a doubled weighted effective integral
irradiance regarding the absorption spectrum of protoporphyrin IX
compared to the common orange filter BTE595, was fitted to the
device.
The continuous water-filtered spectrum covers 570–1400 nm with
local minima at 970 nm, 1200 nm and 1430 nm due to the
absorption of water molecules (Piazena & Kelleher, 2010). The
unweighted (absolute) irradiance was 200 mW cm22 VIS+wIRA,
including approximately 48 mW cm22 VIS and 152 mW cm22 wIRA,
and the application time was 1 min.
The photosensitizing agent applied was toluidine blue (TB) (SigmaAldrich). TB was dissolved in 0.9 % saline solution to obtain
concentrations of 5, 10, 25 and 50 mg ml21. TB solutions were
freshly prepared and stored in a dark place prior to use. In saline
solution, TB has absorption maxima at 590 nm, 620 nm and 637 nm.
Absorption minima are also seen at 570 nm and 650 nm. The broadband light source used in this study allows optimal light absorption
by TB.
Planktonic bacterial strains and culture conditions. The
bacterial strains used to test the adequacy of the APDT,
Streptococcus mutans DSM 20523 and Enterococcus faecalis T9, were
obtained from Professor Dr J. Hübner, Department of Medical
Infectiology, University of Freiburg, Germany. In addition, unstimulated human saliva from a healthy 45-year-old volunteer who had not
used antibacterial mouthwashes and/or antibiotics for 6 months prior
to the start of the experiment was investigated. Streptococcus mutans,
Enterococcus faecalis and saliva were cultivated on Columbia blood
agar plates at 37 uC in an aerobic atmosphere with 5 % CO2. The
maintenance of the strains was as described previously (Al-Ahmad
et al., 2006). The overnight cultures of bacterial strains were prepared
in tryptic soy broth (Merck). Exponential-phase cells were used for
testing in the study. The unstimulated saliva and 8 ml cell suspensions
of each organism were centrifuged at 4000 g for 10 min. The
supernatants were then removed, the pellets washed in sterile 0.9 %
saline solution, and the centrifugation step repeated. After discarding
the supernatant, 8 ml 0.9 % saline solution was finally added.
Treatment of bacterial strains and salivary bacteria. The
bacterial concentration tested was up to 109 c.f.u. ml21. The bacterial
suspensions were tested with four different TB concentrations (5, 10,
25 and 50 mg ml21) as mentioned above. For irradiation, 1 ml of the
bacterial solution containing a given photosensitizer concentration
was placed into multiwell plates (24-well plate, Greiner bio-one) in
triplicate. Irradiation was applied at 37 uC for 1 min. All experiments
were conducted twice. After irradiation, a dilution series of the treated
bacterial solution was prepared and each dilution was streaked onto
Columbia blood agar and cultured at 37 uC in an aerobic atmosphere
with 5 % CO2. To quantify the c.f.u. a gel documentation system
(ChemidocXRS1, Bio-Rad) was used. The surviving c.f.u. were
compared with those of the untreated control.
Salivary bacteria, which served as controls, were washed and diluted
1 : 10 in 0.9 % sterile saline prior to the application of APDT as
described above. Four different TB concentrations were also utilized
following the same procedure as previously mentioned for the
bacterial strains.
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Journal of Medical Microbiology 62
APDT of initial oral bacterial colonization
BES
healthy volunteers participated in the study. Prior to the experiments,
a clinical oral examination was performed. The subjects showed no
signs of gingivitis or caries. Informed written consent had been given
by the participants in the study. The study design was reviewed and
approved by the Ethics Committee of the University of Freiburg (EK
63/07). Cylindrical bovine enamel slabs (BES) were prepared as
described in detail previously (Al-Ahmad et al., 2009). In brief, bovine
incisors were obtained from BSE-free and freshly slaughtered 2-yearold cattle for preparation of the BES. First, the teeth were separated
from their roots and prepared using a grinding unit (ExaktMikroschleifsystem, Exakt-Apparatebau). Cylindrical enamel slabs
(diameter 5 mm, height 1.5 mm, surface area 19.63 mm2) were
prepared from the labial surfaces of the bovine incisors. The final
grinding of the bovine enamel was carried out using a grinding
machine (Knuth-Rotor-3, Streuers) using sandpaper of 1200, 2400
and 4000 grids in decreasing order of grain size. The surface of the
enamel specimen was controlled by a light microscope (Leica Wild
M3Z).
Six enamel slabs were fixed on individual upper jaw splints with the
aid of a polysiloxane impression material (Aquasil Ultra, Dentsply
DeTrey), as previously described (Hannig et al., 2007). This ensured
that only the surface of the slabs was exposed to the oral cavity, as
most of the margins were completely covered by the impression
material (Fig. 1). The specimens were attached to the buccal surfaces
of the upper premolars and the first molar and held in the oral cavity
for 2 h. The volunteers carried the splint systems twice to deliver a
sufficient number of BES for the PDT tests that followed.
Treatment of adhered bacteria. Each volunteer carried an
individual upper jaw splint to which six BES were attached for 2 h.
This procedure was carried out twice in each volunteer. After
exposure in the oral cavity, each specimen was rinsed with 0.9 %
sterile saline solution for 30 s. Two specimens of a total of six BES per
participant served as a control and the remaining four BES were
treated with APDT. Four different TB concentrations (5, 10, 25 and
50 mg ml21) were tested on each of the two control specimens which
were initially colonized by oral bacteria. A total of 12 control BES was
examined for each TB concentration. For APDT treatment the BES
were placed into multiwell plates (24-well plate, Greiner bio-one) in
duplicate. The radiation was applied for 1 min at 37 uC. The adherent
bacteria were subsequently quantified by determination of c.f.u. as
described elsewhere in detail (Hannig et al., 2007). Briefly, the back
side of the slabs consisting of dentine as well as their margins were
brushed off with sterile small swab pellets and the BES were placed
into sterile tubes with 1 ml 0.9 % saline, vortexed and then treated in
an ultrasonic bath on ice for 30 s. The suspension from the untreated
BES (control) was then serially diluted up to 1 : 103 in 0.9 % saline; the
preparation of a similar dilution series for the treated BES was not
necessary. The c.f.u. were then determined.
Statistical analysis. For statistical analysis the Mann–Whitney U
Test (IBM SPSS Statistics 19) was used to detect significant differences
between the tested TB concentrations and the control adhesion targets
with respect to the c.f.u. values of the initial bacterial colonization.
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RESULTS
As shown in Fig. 2, 98 % of S. mutans cells were killed by
each of the TB concentrations ranging from 5 to 50 mg l21.
Only 1 % of E. faecalis cells survived after APDT,
irrespective of the TB concentration. This high bacterial
eradication rate of the APDT corresponds to 1.7–2 log10
steps of the original treated bacterial culture.
Salivary bacteria were eliminated to a higher extent of 3.7–
5 log10, which corresponds to a reduction rate higher than
99.99 % of the original salivary bacterial counts (8.6 log10).
All TB concentrations used were highly effective (Fig. 3).
Considering the initially adhered oral bacteria (Fig. 4), the
untreated control revealed a log10 c.f.u. value of 4.3
(median 4.2). The log10 c.f.u. of the adhered bacteria after
APDT was 1.8±1.2 (median 1.7) after treatment with
50 mg ml21 TB, 1.4±1.5 (median 1.1) with 25 mg ml21 TB,
1.2±1.2 (median 1.2) with 10 mg ml21 TB and 3.1±0.5
(a)
log10(c.f.u. ml–1)
Subjects and specimens for oral bacterial colonization. Six
10
8
6
4
2
0
(b)
log10(c.f.u. ml–1)
BES
Fig. 1. Front view of an individual upper jaw
splint with six BES attached to the buccal
surfaces of both premolars and first molar. The
specimens were fixed in small cavities with
silicone impression material.
C
50
25
10
5
TB concentration (µg ml–1)
C
50
25
10
5
TB concentration (µg ml–1)
10
8
6
4
2
0
Fig. 2. Eradication rates of (a) S. mutans and (b) E. faecalis after
the application of APDT using VIS+wIRA at four TB concentrations, plus a negative control (C). Data shown are means±SD
(n56).
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469
6.0
9
8
7
6
5
4
3
2
1
0
log10 (c.f.u. ml–1)
log10(c.f.u. ml–1)
A. Al-Ahmad and others
4.0
*
3.0
2.0
1.0
0.0
C
50
25
10
TB concentration (µg ml–1)
C
5
Fig. 3. C.f.u (log10) values of salivary bacteria without treatment
(negative control, C) and after APDT using VIS+wIRA at four TB
concentrations. Data shown are means±SD (n56).
(median 3.3) with 5 mg ml21 TB. The differences between
all tested TB concentrations and the untreated control were
significant (P50.004) despite the interindividual variations
among the volunteers involved in the study as shown by
the high standard deviations of the c.f.u. numbers
obtained. This antimicrobial activity of all tested TB
concentrations is of great clinical importance, since it
corresponds to an elimination rate of more than 90 %.
DISCUSSION
APDT has become an interesting research topic in the field
of medical microbiology, not only due to the increasing
antibiotic resistance of various micro-organisms but also
because of its healing properties on sensitive human
tissues. Up until now, light sources using light-emitting
diodes have been frequently utilized in APDT. The
encouraging results showing pain reduction in patients
with multiple actinic keratoses when VIS+wIRA was
applied (von Felbert et al., 2010) inspired the present study.
The effectiveness of this specific light source was investigated for its efficacy against Streptococcus mutans and
Enterococcus faecalis in vitro as well as against initial oral
bacterial colonization in vivo. S. mutans was chosen since it
is considered to be a principal causative agent of dental
caries (Beighton et al., 1991; Al-Ahmad et al., 2006; Yoo et
al., 2011). E. faecalis is frequently isolated in secondary
endodontic infections and is considered to be capable of
withstanding unfavourable environmental conditions in
obturated root canals (Schirrmeister et al., 2009; Zhu et al.,
2010). Apart from testing the effects of APDT on
planktonic bacteria it is necessary to study the effectiveness
of new antimicrobial therapeutic modalities against
adherent bacteria in biofilms, due to the higher antibiotic
resistance of micro-organisms embedded within biofilms
(Costerton et al., 1999). Moreover, a study in which the
presence of a high bacterial diversity is investigated would
deliver more realistic results concerning the clinical
adequacy of APDT. To date however, most studies using
APDT have been conducted either on single species in
470
5.0
25
10
5
50
TB concentration (µg ml–1)
Fig. 4. Boxplots depicting the log10 values of adhered bacteria of
the 2-h-old initial oral colonization without treatment (negative
control, C) and after APDT using VIS+wIRA at four TB
concentrations (n512).
planktonic culture or on single-species biofilm generated in
vitro. In the present study, the effectiveness of APDT using
VIS+wIRA on multispecies biofilm in the initial oral
bacterial colonization was tested, we believe for the first
time. Furthermore, this appears to be the first time that
VIS+wIRA has been assessed as a light source for APDT.
The APDT had a much higher effect on salivary bacteria
than on the planktonic cultures. A possible explanation for
this could be the antimicrobial ingredients of human saliva,
which adhere to bacterial cells and could cause a synergistic
effect with the photosensitizers. However, some new
photosensitizers should also be tested in future studies.
TB is known to be an effective photosensitizer which is
capable of damaging cell membranes during APDT, thus
leading to the eradication of bacterial cells (Wakayama
et al., 1980). Zanin et al. (2006) used TB and irradiation of
85.7 J cm22 to kill in vitro-generated single-species biofilm
of S. mutans and other oral streptococci. The elimination
rates of the bacteria were found to vary between 95 and
99.9 %. In a similar in vitro study of Bevilacqua et al.
(2007), S. mutans was completely eliminated and biofilm
formation was prevented by APDT using 100 mg TB ml21
and LED light at 2.18 J cm22. However, favourable
antimicrobial effects can be achieved at a much lower TB
concentration of 5 mg ml21 according to the outcome of
our research. In a recent project, Souza et al. (2010)
succeeded in killing E. faecalis in vitro using APDT with
methylene blue and TB as photosensitizers. The authors
failed however to reveal whether they had found any
significant differences concerning the efficacy of the two
photosensitizers. This suggests that methylene blue could
probably also be combined with VIS+wIRA as the light
source. In addition, Sharma et al. (2008) applied APDT on
an in vitro-formed biofilm of Staphylococcus aureus and
Staphylococcus epidermidis, while also choosing TB as the
photosensitizer and a diode laser (25 J cm22) as the light
source. Among the main results, APDT accomplished a
significant inactivation of the biofilm of these clinically
relevant staphylococci, a finding that was confirmed by the
broad-spectrum efficacy of TB demonstrated in the present
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Journal of Medical Microbiology 62
APDT of initial oral bacterial colonization
study. Furthermore, Kömerik et al. (2003) showed that TBmediated lethal photosensitization of Porphyromonas
gingivalis, which results in decreased bone loss, is possible
in vivo. These findings suggest that APDT, in particular
using VIS+wIRA, may result in fewer undesired side
effects and a strong stimulation of the healing of human
tissue and may be useful as an alternative approach for the
antimicrobial treatment of periodontitis.
APDT could also play a key role as an alternative
antimicrobial modality in endodontic therapy, as suggested
by Silva Garcez et al. (2006). The authors compared the
capability of 0.5 % NaOCl versus PDT with azulene to
eradicate E. faecalis recovered from infected root canals in
vitro. It was reported that APDT with azulene achieved a
significantly higher reduction of the resistant E. faecalis
population when compared to NaOCl. Although azulene
was not tested in our study, it should be considered as a
potential effective photosensitizer in combination with
VIS+wIRA.
Microbial biofilm, which mainly contains bacteria up to
500 times more resistant to antimicrobials than their
planktonic counterparts, is a challenging target for the
most modern antibacterial agents (Costerton et al., 1999).
The application of APDT using methylene blue to eradicate
dental plaque bacteria revealed that micro-organisms in
oral biofilms are less affected by APDT than bacteria in the
planktonic phase (Fontana et al., 2009). However, the
authors stated that the antibacterial effect of APDT on oral
biofilm bacteria was stronger than that found after
antibiotic treatment under similar conditions. Such a
limitation of antimicrobial agents in the elimination of
biofilms was illustrated in one of our earlier studies, where
chlorhexidine failed to eradicate an in vitro-formed biofilm
of S. mutans (Al-Ahmad et al., 2008). This emphasizes the
need for improved antimicrobial treatments such as APDT.
Other research groups have applied APDT using erythrosine as the photosensitizer, which is already available for
use in the mouth, and has been used to eradicate an in
vitro-generated S. mutans biofilm (Wood et al., 2006). This
combination proved to be effective against the in vitro
biofilm of S. mutans. The latter reports are quite
promising, since they suggest that the effective application
of non-toxic and biocompatible photosensitizers in future
APDT studies could be possible. Moreover, chlorine e6
proved to be a more effective photosensitizer than TB when
combined with VIS+wIRA as a light source in order to kill
E. faecalis according to the results of our recent project
(data not shown). Further studies are needed to confirm
the high elimination rates of APDT for salivary bacteria
and the initial oral bacterial colonization as well. It has
been already shown that APDT using a diode laser as light
source and phenothiazine as photosensitizer effectively
reduced Streptococcus mutans within a layer of 10 mm in an
in vitro biofilm (Schneider et al., 2012). Furthermore,
Nastri et al. (2010) reported the elimination of different
periopathogens and the photoinactivation of an in vitro
Aggregatibacter actinomycetemcomitans biofilm using
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APDT with TB and a diode laser. Hence, an impact of
APDT using wIRA and TB could be assumed, but has to be
further studied.
Alhough the present study highlighted the effectiveness of
APDT in eliminating the initial oral bacterial colonization,
an impact on mature oral biofilm formed in vivo is also a
necessity if this procedure is to be applied clinically. Hence,
the applicability of wIRA within the oral cavity has to be
established in order to conduct further in vivo clinical
studies. Nevertheless, the limited ability of different
antimicrobial agents, including antibiotics and disinfectants, to eradicate biofilm microbiota will encourage
microbiologists to pay more attention to the application
of APDT in this field of research with the aim of
developing more effective treatments.
In conclusion, this study has shown that APDT using the
combination of TB and VIS+wIRA is an effective method
which is capable of killing bacteria within the initial oral
bacterial colonization. Taking the healing effects of wIRA
on human tissue into consideration as well, this technique
could prove to be helpful in the treatment of periimplantitis and periodontitis, although this would need to
be evaluated within the framework of more clinical trials.
Moreover, the broad-band light source consisting of
VIS+wIRA has a high potential for use in combination
with other photosensitizers.
ACKNOWLEDGEMENTS
The authors thank Gabriele Braun for excellent technical help. This
study was supported by the Swiss Dr Braun Science Foundation and
in part by the German Research Foundation (DFG, AL 1179/1-1). The
authors acknowledge the assistance of Debra Kelleher in the
preparation of this manuscript.
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