Novel Clinical Trial for Treatment of Infected Subcutaneous Fistula in
Female Griffon Dog using Gold Nano-Rod Assisted Near-Infrared Plasmonic
Haithem A.M. Farghali
, Hala R. Ali
, Moustafa R.K. Ali
and Ahmed H.A. Osman
Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
Department of Bacteriology and Immunology, Animal Health Research Institute (AHRI), Dokki, Giza, Egypt.
School of Chemistry and Biochemistry, Georgia Institute of Technology, and Laser Dynamics Laboratory, Atlanta, GA, USA.
Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
Raises in the occurrence of antibiotic resistant bacterial infections need novel methods for control. It is currently clear that a
nanotechnology-driven method using nanoparticles to target and terminate pathogenic bacteria can be positively applied. Plasmonic
photothermal therapy (PPTT) is a hopeful minimally-invasive antibacterial therapy as well as oncological treatment strategy where
plasmonic nanoparticles are used to transform near infrared light to localized heat to cause cell death, mainly through apoptosis and/
or necrosis. The objective of this work was to detect the clinical bactericidal activities of gold nanorods (AuNRs) using plasmonic
photothermal therapy (PPTT) against pathogenic extended spectrum Beta Lactamase (ESβL) Klebsiella pneumoniae as well as apoptotic
actions against pyogenic membrane of chronic subcutaneous fistula had no tendency to heal in clinically affected eight years old Griffon
female dog. The fistula was infiltrated along its pyogenic wall and intraluminal in multiple spots with AuNR at 7.5 nM concentration
and then exposed to an 808 nm diode laser with power of 5.8 W/cm2 and spot size about 5.6 mm2. The PPTT session was repeated
every two weeks for four successive sessions. The evaluations of the curative response were done through clinical, bacteriological
and histopathoilogical repetitive examinations. Clinically, the purulent discharge decreased in the quantity until it disappeared at the
third PPTT session and complete regression of the fistula occurred after the fourth PPTT application. Bacteriologically, Klebsiella
pneumoniae isolate showed dramatic reduction in colony forming unit (CFU) count after first session and was completely absent at
the fourth PPTT session. Regarding histopathology of the tissue specimens from infected fistula when the case was admitted to the
clinic, there was massive liquefied necrotic area infiltrated mainly by neutrophils represented as grade +3 and after first session of
PPTT, there was circumscribe necrotic area infiltrated by few numbers of neutrophils and represented as grade +1. After third session of
PPTT, complete absence of necrotic tissues was recorded and new fibrous connective tissue formation was observed. Altogether, these
outcomes powerfully recommend that AuNRs could be a motivating choice to control antibiotic resistant bacterial infections as well as
pyogenic conditions which have no tendency to heal.
Plasmonic photothermal therapy (PPTT); Gold nanorods (AuNRs); Klebsiella pneumoniae, pyogenic fistula, colony
forming unit (CFU).
Photodynamic therapy (PDT) is an active management to destroy
both eukaryote and prokaryote creatures (Kharkwal, Sharma,
Huang and Hamblin, 2011 and Sharma, Mroz, Dai, Huang, Denis
and Hamblin, 2012) [1, 2]. The plasmonic photothermal therapy
(PPTT) could be additional effective to eradicate microorganisms
through its local hyperthermia mechanism (Fekrazad, Khoei,
Hakimiha and Bahador, 2013) . PPTT has been significant
in the latter years in the practical medicine due to its ability of
absorbing visible light (Ali, et al, 2016) . At the same time, gold
nanoparticles with nonspherical shape, such as gold nanorods,
and polygonal particles have the ability of absorbing near-infrared
(NIR) light that is within a proper wavelength field for therapeutic
presentations. One of the prospective uses of gold nanoparticles
with NIR absorption abilities is in the hyperthermia. Hyperthermia
can be used as a potential tool to kill bacteria by joining the usage
of laser and functional gold nanoparticles. Just nanorods are
in contact with the bacterial cells and this is followed by laser
irradiation, the hyper-thermal action can speedily terminate the
bacterial cells (Huang, Tsai and Chen, 2007 and Norman, Stone,
Gole, Murphy and Sabo-Attwood, 2008) [5, 6]. The situation of
the plasmon maximum is associated to the particle size (larger
diameter results in a longer wavelength) and the highest width to
size dispersion (Kim, Yi, Kim, Lee and Kim, 2009) . So, we
can raise the antimicrobial potential of nanoparticles to lyse whole
The growing progress of multidrug-resistant strains among
pathogenic microorganisms has come to be one of the most
significant difficulties in medicine worldwide (Lode, Stahlmann,
and Kresken, 2013) . Klebsiella is a gram negative, non-motile,
encapsulated, lactose fermenting, facultative anaerobe belonging
to the Enterobacteriaceae family (Procop and Koneman, 2016)
. Klebsiella pneumoniae (K. pneummiae), a member of the
human intestine flora, is commonly related to hospital-acquired
infection. Definite underlying syndromes such as malignancy,
cirrhosis, biliary tract disorders, diabetes mellitus (DM), and
alcoholism may damage an patient’s defense mechanisms and
raise the possibility of K. pneumoniae infection ( Sung-Sheng
Tsai, et al, 2010) . K. pneumoniae is the second most common
reason of gram-negative bacteremia after Escherichia coli (Bryan
et al, 1983 and Yinnon, et al, 1996) [11, 12]. K. pneumoniae
bacteremia leads to significant morbidity and mortality in common
populations (Lee, et al; 1994 and Tsay, et al; 2002) [13, 14]. It has
even replaced Escherichia coli in some centers as a nosocomial
pathogen. It causes pneumonia, urinary tract infection, other
pyogenic infections, septicemia, rarely diarrhea and hospital-
acquired infections (Arti Kapil, 2013) . Metastatic infections,
such as pyogenic brain abscess, meningitis, and endophthalmitis,
are the most important features of K. pneumoniae infections (Wang
et al, 1998 and Fang et al, 2007) [16, 17].
strains of Klebsiella pneumoniae are resistant to a wider range of
antibiotics than are most Escherichia coli strains. They are nearly
always naturally resistant to ampicillin (Murray and Rosenthal,
2005) . Resistance of Klebsiella to previously sensitive
antibiotics is also increasing in the recent years due to overuse
and misuse of antimicrobial agents and or natural causes. The
existence of enzymes of extended-spectrum β-lactamases (ESβLs)
producing organism that are resistant to virtually all β-lactam
antibiotics have been reported ( Sung-Sheng Tsai, et al, 2010)
. ESβLs are plasmid-mediated class A enzymes commonly
found in the family Enterobacteriaceae, mainly K. pneumoniae
(Lin, Chin and Lee, 2005) . The increase in ESβL-producing
organisms is sure to create significant therapeutic problems in the
future. The available treatment regimens for infections caused by
ESβL-producing bacteria are not always effective. Therefore, it is
necessary to discover new antimicrobial compounds against ESβL-
producing K. pneumoniae strains. Abundant improvements in
nanotechnology have delivered a hard basis for using nanoparticles
(NPs) in the battle against pathogenic microorganisms, containing
multidrug resistant bacteria (Hernandez-Delgadillo, et al; 2013,
Badireddy, et al; 2014 and Ali et al; 2016) [20, 21, 22]. Numerous
works have studied the effectiveness of PDT for disinfection of
oral pathogens; but the usage of PPTT against oral pathogens has
not been sufficient discovered (Badireddy, et al; 2014) .
In this work, the clinical bactericidal activities of gold nanorods
(AuNRs) plus laser (PPTT) against pathogenic antibiotic resistant
Klebsiella pneumoniae as well as apoptotic actions against
pyogenic membrane of chronic subcutaneous fistula had no
tendency to heal in clinically affected five years old Griffon female
dog were studied.
Materials and Methods :
This study was started at January 2016 when a case of eight years
old Griffon bitch admitted to the clinic of the department of surgery,
anesthesiology and radiology, faculty of veterinary medicine,
Cairo University suffering from chronic infected subcutaneous
fistula at the region of the left stifle fold (Fig. 1).
The affected eight years
old bitch at the time of admission
to the surgery clinic showed
infected fistula at the level of the
left stifle fold has no tendency to
heal since about one year.
Synthesis, Conjugation and characterization of Gold Nanorods
AuNRs with an average size of 25 (± 3) X 5 (± 0.8) nm (length X
width) were synthesized by seedless mediated growth in school of
Chemistry and Biochemistry, Georgia Institute of Technology, and
Laser Dynamics Laboratory, Atlanta, USA (Ali et al; 2012) .
Briefly, the AuNRs growth solution was prepared, which contains
100 mL of HAuCl4 (1.0 mM), 100 mL of CTAB (0.2 M), 5 mL of
AgNO3 (4.0 mM), HCl (160 μL, 37%), and 1.4 mL of ascorbic acid
(78.8 mM). Then, we injected 300 μL of NaBH4 (Sigma-Aldrich,
USA) to the growth solution and kept it undisturbed for 12 h for
the growth of AuNRs. After that, the extra toxic surfactant CTAB
were removed by centrifugation (19,000 rcf) the AuNRs solution
for 1 hour and redispersed in deionized (DI) water, followed by
a second centrifugation (14,000 rcf) for 30 min. The size of the
gold nanoparticles was measured by JEOL 100 CX transmission
electron microscopy (TEM) (JEOL Ltd., Tokyo, Japan) images by
calculating the average dimensions of 100 particles. The surface
plasmon resonance peak of the AuNRs was measured by UV−vis
spectroscopy. The molar concentrations of AuNRs were calculated
according to Beer’s law. For conjugation of AuNPs with poly
ethylene glycol thiol (SH-PEG, Mw=5000), SH-PEG solution (1
mM) was added into the AuNRs solutions (1 nM), followed by
incubation for 10 h at room temperature. The excess PEG was
removed by centrifugation.
Administration of PPTT :
The affected case was sedated using xylazine Hcl 2% in a dose
rate 1 mg/kg BWs via intramuscular route. Complete aseptic
preparation of the site of the infiltrations and laser application
was performed. The fistula was infiltrated along its pyogenic wall
and intraluminal in multiple spots with AuNR at 7.5 nM for each
100 cm3 concentration and then exposed to an 808 nm diode laser
with power of 5.8 W/cm2 and spot size about 5.6 mm2 (Fig. 2).
The dose per injection was measured based on the volume of the
fistula. The PPTT session was repeated every two weeks for four
The steps of fistula infiltration along its pyogenic
wall and intraluminal in multiple spots with AuNR at 7.5
nM for each 100 cm
concentration and then exposed to
an 808 nm diode laser with power of 5.8 W/cm
size about 5.6 mm
Clinical evaluation :
Clinical evaluation was done every PPTT session for eight weeks
until complete healing of the fistula to measure length and wide of
the fistula in addition to presence and quantity of discharge and its
Collection of samples
The pus samples were either aspirated by disposable syringes
or collected onto sterile cotton tipped swabs. The samples were
collected at the first time of the case admission for culture and
antibiotic sensitivity and then collected before each PPTT session
and five minutes after session for culture and total colony counts.
Characterization of bacterial isolates :
Pus was aseptically inoculated on to Blood and MacConkey agar
plates and incubated overnight at 370C. Klebsiella isolates were
identified by their morphology and biochemical characteristics
(Collee, et al; 2012 and Procop and Koneman, 2016) [24, 9].
Confirmatory test for ESβL-producing K. pneumoniae
The double-disc synergy and agar diffusion tests were used as
screening tools to detect ESβL-producing strains. In the double-
disc synergy test, the antibiotic discs (Oxoid) used were cefotaxime
(30 μg) and ceftazidime (30 μg) placed on Mueller-Hinton agar
adjacent to a co-amoxiclav disc (20μg amoxicillin plus 10μg
clavulanate). Reduced susceptibility to cefotaxime (30 g) and
Ceftriaxone (30 g) with zone sizes 27mm and 25mm respectively
were used as screening method for ESBL production (Fang, et al;
2005) . The agar diffusion test was performed according to
NCCLS guidelines12. A ≤5 mm increase in a zone diameter for
either ceftazidime/clavulanic acid (30 μg/ 10 μg) or cefotaxime/
clavulanic acid (30 μg/ 10 μg) versus its zone when tested alone
was taken as being indicative of ESβL-production (Fang, et al;
Antimicrobial susceptibility testing :
Was done for the isolates on Mueller-Hinton agar plates by Kirby-
Bauer disc diffusion method according to the CLSI guidelines
2015 (CLSI, 2015) .
Monitoring bacterial count using :
“Spread Plate Technique”
Irrigation of the fistula using fixed volume (10 ml) of sterile distilled
water was done before and five minutes after each session. The
collected samples were subjected to series of dilution. A volume
of 0.1 ml was pipetted out from the appropriate desired dilution
series onto the center of each of three agar plate. Each sample was
spread evenly over the surface of the agar using the sterile glass
spreader and carefully the Petri dish was rotated underneath at
the same time. The plate was incubated at 37°C for 24 hours. The
colony forming unit (CFU) value was calculated of the sample.
Once the count of the colonies was recorded, it was multiplied by
the appropriate dilution factor to determine the number of CFU/
mL in the original sample (Arti Kapil, 2013) .
Statastical analysis :
Total bacterial count results are expressed as the mean ± SD of
three petri dishes counting.
Histopathoilogical evaluation :
Tissue specimens from infected fistula was collected, fixed in 10
% neutral buffered formalin, processed and embedded in Paraffin
wax, sectioned at 4 μm and stained with Hematoxylin and Eosin
(Bancroft and Gamble, 2008)  and examined under an Olympus
microscope (Olympus, Japan).
The intensity of the inflammatory response was assessed as
following: +1 (inflammatory cells representing less than 10%
of the cell population observed within the wound area), +2
(inflammatory cells representing in between 10 and 50% of the cell
population observed within the wound area) and +3 (inflammatory
cells representing more than 50% of the cell population observed
within the wound area). Light-microscopic histo-quantitation
was performed by counting PMN leukocytes in tissue sections
per high-power field (x40 objective, x10 ocular) with a binocular
microscope in three adjacent areas (Valdinaldo et al, 2011) .
The affected bitch was admitted to the surgery clinic at
January 2016 with history of infected fistula at the level of the left
stifle fold since about one year. At the first time of fistula evolution,
the owner observed doughy swelling at the lateral aspect of the left
stifle which increased in the size rapidly and then opened at the
proximal level of the left stifle fold discharging serosanguineous
fluid. The obtained fluid was examined for bacterial culture,
isolation, identification and antibiotic sensitivity. The isolated
microorganism was extended spectrum Beta Lactamase (ESβL)
producing Klebsiella pneumonia and was only sensitive to
Imipenem antibiotic. Total blood picture evaluations showed
marked neutrophilia. Serum glucose, liver and kidney functions
were within normal values. The pervious treatments were daily
dressing using hydrogen peroxide, povidine iodine, fucidine
ointment and application of drain. Systemic Imipenem antibiotic
injection in a dose rate (5 mg/kg) every 8 hours intravenously was
used. The dog showed mild response to the antibiotic as slight
reduction in the fistula discharge. After five successive days of
antibiotic administration, the affected bitch suffered from nausea,
vomiting and diarrhea which recorded as antibiotic side effects.
Blood analysis was performed and revealed marked neutrophilia
and elevation in the serum createnine. .
The veterinarian who
followed the case decided to stop antibiotic and continue the
topical treatment only. After two weeks of topical treatment,
reexamination of the blood parameters was done which showed
significant reduction of the serum createnine and the marked
neutrophilia was still present. There was no evidence of clinical
improvement of the fistula after continues local daily dressing for
two months. Cauterization of the fistula tract was applied using
silver nitrate which resulted in slight reduction of the discharge
for two weeks and then increased gradually by time. Repeating of
the cauterization was done without any significant improvement
of the case. The decision of application of PPTT trail was taken
after agreement of the owner. Isolation and identification of the
causative microorganism and antibiotic sensitivity were done
before trail which revealed the same result (extended spectrum
Beta Lactamase (ESβL) producing Klebsiella pneumonia sensitive
only to Imipenem antibiotic). Total colony count (using Spread
Plate Technique) was done and used as method of monitoring the
bactericidal effect of PPTT.
Characterization of AuNRs (length 25 ± 3, width 5 ± 0.8 nm). (A) TEM image with 100
nm scale bar. (B) UV-vis absorbance spectra showing the SPR peaks of AuNRs before and after
conjugation with PEG.
Clinical evaluation :
Clinical evaluation was done every PPTT session for eight weeks until complete healing of the fistula. Length and wide of the fistula
were recorded each time (Fig. 4) (Table 1). Presence and quantity of discharge as well as its physical characters were detected (Fig.
5&6) (Table 2).
Microbiological evaluation :
Klebsiella isolates were identified by their morphology and
biochemical characteristics. Morphology of Klebsiella identified
were large dome shaped colonies on blood agar and lactose
fermenting mucoid colonies on MacConkey agar. In gram
staining, gram negative, short, plump, straight rods were seen .The
biochemical characters identified were positive Voges-Proskauer
test, positive citrate utilization test, positive urease test, acid and
abundant gas production from glucose, lactose, sucrose, maltose
and mannitol sugar fermentation tests
Confirmatory test for ESβL-producing K.
pneumoniae isolates :
The double-disc synergy and agar diffusion tests which were used
as screening tools confirmed isolation of ESβL-producing strain.
Antimicrobial susceptibility testing :
Antimicrobial susceptibility testing revealed that the isolated
bacteria was sensitive only to Imipenem antibiotic.
Monitoring bacterial count using “Spread Plate
The colony forming units were recorded before and five minutes
after each session as shown in figure (7) and table (3).
Histopathoilogical evaluation :
The tissue specimens from infected fistula were taken before treatment revealed congested blood capillaries and massive liquefied
necrotic area infiltrated mainly by neutrophils represented as grade +3 (inflammatory cells representing more than 50% of the cell
population observed within the wound area) per high-power field (x40 objective, x10 ocular) Fig. (8). On other side, after treatment by
laser and Nano-gold tissue section showed circumscribe necrotic area infiltrated by few numbers of neutrophils and represented as grade
+1 (inflammatory cells representing less than 10% of the cell population observed within the wound area) per high-power field (x40
objective, x10 ocular) Fig. (9). The healed wound characterized by a reconstitution of the regular epidermal lining, reduction of fibrinous
exudation and a more organized and a denser type of connective tissue indicating that a new epidermis had formed completely. Large,
wavy collagen fibers interspersed with blood vessels were seen. Superficial tissues had numerous small vessels, while larger blood
vessels were visible in the deep tissues Fig. (10).
In the present study, there were two challenges; the first was to
achieve bactericidal effect of PPTT against ESβL Klebsiella
pneumoniae and the second was enhancement of fistula healing
without evidence of hyperthermia side effects on the tissues.
Regarding first one, plasmonic photothermal therapy has been
extensively investigated (in vitro) as an alternative technique
in the biomedical area like determination of its bactericidal
and antibiofilm activities against different strains of oral
microorganisms (Castillo-Martínez, et al, 2015), using of
covalently primary antibodies linked gold nanorods to selectively
destroy the pathogenic Gram-negative bacterium, Pseudomonas
aeruginosa after exposure to near-infrared radiation (Norman, et
al, 2008) , using surface plasmonic resonance-induced photo-
activation of gold nanoparticles as bactericidal agents against
methicillin-resistant Staphylococcus aureus (Mocan, et al, 2014)
, developing a drug delivery platform where gold nanorods
(AuNRs) are conjugated to rifampicin (RF), which is released after
the uptake into macrophage cells (RAW264.7) (Ali, et al, 2016)
 and studying the action of bactericidal gold nanoparticles
on Escherichia coli (Cui, et al, 2012) . In addition to that, a
new antibacterial gold nanorod (GNR) conjugated magnetic
nanoparticle (MNP) composite (GNR–MNP) was synthesized
successfully for the eradication of antibiotic resistant nosocomial
pathogens in water to improve the water quality (Ramasamy, et
al, 2014) . There are no available lectures about using PPTT
against bacterial infection in vivo.
The second challenge in this work was healing acceleration of
the fistula (which had history of no tendency to heal) without risk
of hyperthermia complications on the tissues. PPTT can induce
cell death primarily by two pathways: necrosis and apoptosis
(Pattani, et al, 2015) . During necrosis, the heat induced from
PPTT disrupts the plasma membrane causing the cytoplasmic
components to leak out and inflammation to occur within the cell.
However, apoptosis is a highly regulated cell death pathway and
would thus be a cleaner way for eliminating cancer cells. Therefore,
modulating PPTT to trigger apoptosis would be more favorable
in clinical studies. It has been reported that different intracellular
locations or shapes of NPs regulate the switch between necrosis
and apoptosis (Song, et al, 2014 and Perez-Hernandez, et al, 2015)
From these backgrounds, the decision of application of PPTT
trail for treatment of chronic extended spectrum Beta Lactamase
(ESβL) Klebsiella pneumoniae infected subcutaneous fistula was
taken after agreement of the case owner who was frustrated from
the longtime of hopeless treatments.
For the bactericidal purpose, the nanorods used in the present
work were of 28 × 8
nm in size. That was described previously
as the most effective size in the plasmonic photothermal heat
generation against bacterial and cancer cells (MacKey, et al, 2014
and Castillo-Martínez, et al, 2015) [38, 30]. The previous in vitro
bactericidal studies irradiated 1 mL of GNP solution at various
concentrations placed in a glass cuvette with approximately one cm
bottom surface for 10 minutes, using an 808 or 810 nm wavelength
laser with variable output power (up to 2 W/cm2 diode) which
was fixed above the surface, approximately 2 cm2, in a vertical
position and this dispersion reached a temperature up to 70°C
(Mocan, et al, 2014 and Castillo-Martínez, et al, 2015) [32, 30].
These conditions are not logically acceptable among the clinical
level as this temperature will lead to tissue burn.
In the present work, the fistula was infiltrated along its pyogenic
wall and intraluminal in multiple spots with AuNR at 7.5 nM
concentration and then exposed to an 808 nm diode laser with
power of 5.8 W/cm2 and spot size about 5.6 mm2. Each spot
irradiated for 2 minutes. In a previous study of PPTT effect on
canine and feline natural mammary gland tumors, it was able to
generate cancer cell apoptosis as a favorable cell death mechanism
(rather than necrosis) in vitro and in vivo by optimizing AuNRs-
PPTT’s conditions as they directly injected the Pegylated AuNRs
to the mammary tumors of each canine/feline with optimized PPTT
dosages to enable a slow cancer cell apoptosis which adjusting the
photothermic temperature to 44°C (Ali, et al, 2016) .
To achieve complete treatment of the infected fistula, the PPTT
session was repeated every two weeks for four successive
sessions. The evaluations of the curative response were done
through clinical, microbiological and histopathoilogical repetitive
examinations. Clinically, the fistula dimensions were decreased
gradually by time which indicated the good rate of granulation
tissue formation filling fistula cavity. The purulent discharge
decreased in the quantity and change in physical characters until it
disappeared after the third PPTT session and complete regression
of the fistula occurred after the fourth PPTT application.
Bacteriologically, Klebsiella pneumoniae isolate showed dramatic
reduction in colony forming unit (CFU) count after first session
and was completely absent at the fourth PPTT session. One of the
most interesting finding was the aggressive reduction in CFU five
minutes after each session which indicated rapid action of AuNRs-
PPTT on bacterial cells.
Regarding histopathology of the tissue specimens from infected
fistula when the case was admitted to the clinic, there was
massive liquefied necrotic area infiltrated mainly by neutrophils
represented as grade +3 and two weeks after first session of PPTT,
there was circumscribe necrotic area infiltrated by few numbers
of neutrophils and represented as grade +1. Two weeks after third
session of PPTT, complete absence of necrotic tissues was recorded
and new fibrous connective tissue formation was observed.
There were no clinical side effects recorded on the case during
and after treatment. From the obtained data, the AuNRs-PPTT
application gave very good bactericidal and healing responses
especially when compared with specific antibiotic administration
and traditional daily dressing or even cauterization.
Further studies are required on the molecular and genetic levels to
detect the actual mode of action and micro-environmental changes
during AuNRs-PPTT application on the clinically infected
These outcomes powerfully recommend that AuNRs could be a
motivating choice to control antibiotic resistant bacterial infections
as well as pyogenic conditions which have no tendency to heal.
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