Abstract:

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.

Keywords:    Plasmonic photothermal therapy (PPTT); Gold nanorods (AuNRs); Klebsiella pneumoniae, pyogenic fistula, colony forming unit (CFU).

Introduction:

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) [3]. PPTT has been significant in the latter years in the practical medicine due to its ability of absorbing visible light (Ali, et al, 2016) [4]. 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) [7]. So, we can raise the antimicrobial potential of nanoparticles to lyse whole bacterial populations.

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) [8]. Klebsiella is a gram negative, non-motile, encapsulated, lactose fermenting, facultative anaerobe belonging to the Enterobacteriaceae family (Procop and Koneman, 2016) [9]. 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) [10]. 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) [15]. 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].

Biochemically typical 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) [18]. 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) [10]. ESβLs are plasmid-mediated class A enzymes commonly found in the family Enterobacteriaceae, mainly K. pneumoniae (Lin, Chin and Lee, 2005) [19]. 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) [21].

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).

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):

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) [23]. 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 successive sessions.

Fig 2:;   The steps of fistula infiltration along its pyogenic wall and intraluminal in multiple spots with AuNR at 7.5 nM for each 100 cm 3 concentration and then exposed to an 808 nm diode laser with power of 5.8 W/cm 2 and spot size about 5.6 mm 2 .

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 physical characters.

Bacteriological evaluation:

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 isolates :

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) [25]. 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; 2007) [26].

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) [27].

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) [15].

Statastical analysis :

Total bacterial count results are expressed as the mean ± SD of three petri dishes counting.

Histopathoilogical evaluation :

Histopathological examination :

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) [28] and examined under an Olympus microscope (Olympus, Japan).

Morphometric measurements:

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) [29].

Results :

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.

Fig 3:    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 Technique"

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).

Discussion :

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)[30], 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) [31], using surface plasmonic resonance-induced photo- activation of gold nanoparticles as bactericidal agents against methicillin-resistant Staphylococcus aureus (Mocan, et al, 2014) [32], 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) [22] and studying the action of bactericidal gold nanoparticles on Escherichia coli (Cui, et al, 2012) [33]. 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) [34]. 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) [35]. 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) [36, 37].

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) [39].

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 conditions.

Conclusion :

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.

References:

1. G. B. Kharkwal, S. K. Sharma, Y.-Y. Huang, T. Dai, and M. R. Hamblin (2011): “Photodynamic therapy for infections: clinical applications,” Lasers in Surgery and Medicine, vol. 43, no. 7, pp. 755–767, 2011.
2. S. K. Sharma, P. Mroz, T. Dai, Y.-Y. Huang, T. G. S. Denis, and M. R. Hamblin (2012): “Photodynamic therapy for cancer and for infections: what is the difference?” Israel Journal of Chemistry, vol. 52, no. 8-9, pp. 691–705, 2012.
3. R. Fekrazad, F. Khoei, N. Hakimiha, and A. Bahador (2013): “Photoelimination of Streptococcus mutans with two methods of photodynamic and photothermal therapy,” Photodiagnosis and Photodynamic Therapy, vol. 10, no. 4, pp. 626–631, 2013.
4. Ali, M. R. K., Ali, H. R., Rankin, C. R., and El-Sayed, M. A.(2016):Targeting heat shock protein 70 using gold nanorods enhances cancer cell apoptosis in low dose plasmonic photothermal therapy. Biomaterials 102, 1-8.
5. W.-C. Huang, P.-J. Tsai, and Y.-C. Chen (2007): “Functional gold nanoparticles as photothermal agents for selective-killing of pathogenic bacteria,” Nanomedicine, vol. 2, no. 6, pp. 777–787, 2007.
6. R. Sean Norman, J. W. Stone, A. Gole, C. J. Murphy, and T. L. Sabo-Attwood (2008): “Targeted photothermal lysis of the pathogenic bacteria, Pseudomonas aeruginosa, with gold nanorods,” Nano Letters, vol. 8, no. 1, pp. 302–306, 2008.
7. C.-B. Kim, D. K. Yi, P. S. S. Kim, W. Lee, and M. J. Kim (2009): “Rapid photothermal lysis of the pathogenic, bacteria, Escherichia coli using synthesis of gold nanorods,” Journal of Nanoscience and Nanotechnology, vol. 9, no. 5, pp. 2841–2845, 2009.
8. H. M. Lode, R. Stahlmann, and M. Kresken (2013): “Multiresistant pathogens—a challenge for clinicians,”Zentralblatt fur Chirurgie, vol. 138, no. 5, pp. 549–553, 2013.
9. Gary W. Procop and Elmer Koneman (2016): Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (7th Edition). Publisher Lippincott Williams & Wilkins, 2016. ISBN 1451116594, 9781451116595.
10. Sung-Sheng Tsai, Jui-Chu Huang, Szu-Tah Chen, Jui-Hung Sun, Chih-Ching Wang, Shu-Fu Lin, Brend Ray-Sea Hsu; Jen- Der Lin; Shu-Yu Huang; Yu-Yao Huang (2010): Characteristics of Klebsiella pneumoniae Bacteremia in Community-acquired and Nosocomial Infections in Diabetic Patients. Chang Gung Med J Vol. 33 No. 5 September-October 2010.
11. Bryan CS, Reynolds KL and Brenner ER. (1983): Analysis of 1,186 episodes of gram-negative bacteremia in non-university hospitals: the effects of antimicrobial therapy. Rev Infect Dis 1983; 5:629-38.
12. Yinnon AM, Butnaru A, Raveh D, Jerassy Z and Rudensky B. (1996): Klebsiella bacteraemia: community versus nosocomial infection. QJM 1996; 89:933-41.
13. Lee KH, Hui KP, Tan WC, Lim TK. (1994): Klebsiella bacteraemia: a report of 101 cases from National University Hospital, Singapore. J Hosp Infect 1994; 27:299-305.
14. Tsay RW, Siu LK, Fung CP, Chang FY. (2002): Characteristics of bacteremia between community-acquired and nosocomial Klebsiella pneumoniae infection: risk factor for mortality and the impact of capsular serotypes as a herald for community-acquired infection. Arch Intern Med 2002;162:1021-7.
15. Arti Kapil (2013): Ananthanarayan and Paniker’s Textbook of Microbiology (Ninth Edition). Published by Orient Black Swan Pvt. Ltd (2013). ISBN 10: 817371889X ISBN 13: 9788173718892.
16. Wang JH, Liu YC, Lee SS, Yen MY, Chen YS, Wann SR, Lin HH. (1998): Primary liver abscess due to Klebsiella pneumoniae in Taiwan. Clin Infect Dis 1998;26:1434-8.
17. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. (2007): Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis 2007;45:284-93.
18. Patrick R. Murray and Ken S. Rosenthal (2005): Review of Medical Microbiology (First Edition). Elsevier Health Sciences, 2005. ISBN 0323033253, 9780323033251
19. Lin RD, Chin YP, Lee MH. (2005): Antimicrobial activity of antibiotics in combination with natural flavonoids against clinical- extended-spectrum β-lactamase (ESβL)-producing Klebsiella pneumoniae. Phytother. Res. 2005; 19: 612- 617.
20. R. Hernandez-Delgadillo, D. Velasco-Arias, J. J. Martinez- Sanmiguel et al. (2013): “Bismuth oxide aqueous colloidal nanoparticles inhibit Candida albicans growth and biofilm formation,” International Journal of Nanomedicine, vol. 8, pp. 1645–1652, 2013.
21. A. R. Badireddy, R. Hernandez-Delgadillo, R. I. Sánchez- Nájera, S. Chellam, and C. Cabral-Romero (2014): “Synthesis and characterization of lipophilic bismuth dimercaptopropanol nanoparticles and their effects on oral microorganisms growth and biofilm formation,” Journal of Nanoparticle Research, vol. 16, no. 6, pp. 1–12, 2014.
22. Ali, Hala R., Moustafa RK Ali, Yue Wu, Salah A. Selim, Hazem FM Abdelaal, Essam A. Nasr, and Mostafa A. El-Sayed (2016): “Gold Nanorods as Drug Delivery Vehicles for Rifampicin Greatly Improve the Efficacy of Combating Mycobacterium Tuberculosis with Good Biocompatibility with the Host Cells.” Bioconjugate Chemistry (2016).
23. Ali, M. R., Snyder, B., & El-Sayed, M. A. (2012): Synthesis and optical properties of small Au nanorods using a seedless growth technique. Langmuir, 28(25), 9807-9815.
24. J.Gerald collee, Andrew G.Fraser, Barrie P. Marmion and Anthony Simmons. (2012): Mackie & Mc Cartney: Practical Medical Microbiology. 14 th edition. Churchill Livingstone: Elsevier.
25. Ma LC, Fang CT, Lee CZ, Shun CT and Wang JT. (2005): Genomic heterogeneity in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscess and metastatic infection. J Infect Dis 2005;192:117-28.
26. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. (2007): Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis 2007;45:284-93.
27. CLSI (2015): Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Twelfth Edition. CLSI document M02-A12. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
28. John D. Bancroft and Marilyn Gamble (2008): Theory and Practice of Histological Techniques (Sixth edition). Elsevier Health Sciences, 2008.
29. Valdinaldo Aragão de Melo, et al (2011): Effect of low level laser on sutured wound healing in rats. Acta Cirúrgica Brasileira - Vol. 26 (2) 2011; 129-134.
30. Juan Carlos Castillo-Martínez, Gabriel Alejandro Martínez- Castañón, Fidel Martínez-Gutierrez, Norma Veronica Zavala- Alonso, Nuria Patiño-Marín, Nereyda Niño-Martinez, V. Zaragoza-Magaña, and C. Cabral-Romero (2015): Antibacterial and Antibiofilm Activities of the Photothermal Therapy Using Gold Nanorods against Seven Different Bacterial Strains. Journal of Nanomaterials. Volume 2015 (2015), Article ID 783671, 7 pages. http://dx.doi.org/10.1155/2015/783671.
31. R. Sean Norman , John W. Stone , Anand Gole , Catherine J. Murphy and Tara L. Sabo-Attwood (2008): Targeted Photothermal Lysis of the Pathogenic Bacteria, Pseudomonas aeruginosa, with Gold Nanorods. Nano Lett., 2008, 8 (1), pp 302–306
32. Lucian Mocan, Ioana Ilie, Cristian Matea, Flaviu Tabaran, Ersjebet Kalman, Cornel Iancu And Teodora Mocan (2014): Surface Plasmon Resonance-Induced Photoactivation of Gold Nanoparticles As Bactericidal Agents Against Methicillin- Resistant Staphylococcus Aureus. International Journal Of Nanomedicine 2014:9 1453–1461.
33. Cui Y, Zhao Y, Tian Y, Zhang W, Lü X, Jiang X. (2012): The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials. 2012; 33(7):2327–2333.
34. Mohankandhasamy Ramasamy, Su Seong Lee, Dong Kee Yi and Kwangmeyung Kim (2014): Magnetic, optical gold nanorods for recyclable photothermal ablation of bacteria. J. Mater. Chem. B, 2014, 2, 981-988.
35. Pattani VP, Shah J, Atalis A, Sharma A, Tunnell JW. (2015): Role of apoptosis and necrosis in cell death induced by nanoparticle-mediated photothermal therapy. J Nanoparticle Res. 2015; 17(1):11.
36. Song AS, Najjar AM, Diller KR. (2014): Thermally induced apoptosis, necrosis, and heat shock protein expression in 3D culture. J Biomech Eng. 2014; 136(7).
37. Perez-Hernandez M, del Pino P, Mitchell SG, et al. (2015): Dissecting the molecular mechanism of apoptosis during photothermal therapy using gold nanoprisms. ACS Nano. 2015; 9(1):52–61.
38. M. A. MacKey, M. R. K. Ali, L. A. Austin, R. D. Near, and M. A. El-Sayed (2014): “The most effective gold nanorod size for plasmonic photothermal therapy: theory and in vitro experiments,” The Journal of Physical Chemistry B, vol. 118, no. 5, pp. 1319– 1326, 2014.
39. Moustafa R K Ali, Ibrahim M Ibrahim, Hala R Ali, Salah A Selim And Mostafa A El-Sayed (2016): Treatment of natural mammary gland tumors in canines and felines using gold nanorods-assisted plasmonic photothermal therapy to induce tumor apoptosis. International Journal of Nanomedicine 2016:11 4849–4863.