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Physical damage to the bacteria using a combination of laser energy and absorbing nanoparticles.

INTRODUCTION

GNPs absorb light millions of times more than organic dye molecules. Almost 100% of the absorbed light is converted to heat by means of the previously described non-radiative properties. GNPs are highly photostable and biocompatible [1]. Low power lasers operate typically at powers of 100 milliwatts or less, and may produce energy in the visible spectrum (400-700nm wavelength), or in the ultraviolet (200-400nm), or near infrared regions (700-1500nm). At the present time, there are few purpose built low power lasers for the middle infrared (1500-4000nm) or far infrared regions (4000-15000nm). Rather, lasers operating in the middle and far-infrared regions are used in health care primarily for hard and soft tissue procedures [2,3].

Most E. coli strains are usually harmless members of human intestinal flora and other animals, but some strains have acquired virulence factors that contribute to their pathogenicity associated to important intestinal and extra-intestinal diseases [4,5].

Staphylococcus aureus (S. aureus) are Gram-positive bacteria, with diameters of 0.5-1.5 [micro]m and characterized by individual cocci, which divide in more than one plane to form grape-like clusters. S. aureus infection in human causes a wide variety of diseases in humans and animals, ranging in severity from a mild skin infection to more severe diseases, such as pneumonia and septicemia [6, 7].

Some alternative approaches for dealing involve the use of agents that cause physical or chemical damage to the bacterium. For example, photodynamic therapy (PDT) uses light in the attendance of a photosensitizer and oxygen to produce singlet of oxygen and causing a highly reactive bacterial damage.

Among the different nanostructures, gold nanoparticles, in different modifications, are the most capable candidates for such photothermal (PT) sensitizers because they are the strongest nanoabsorbers; This notion assumes that one uses convectional (i.e., solid spheres), relatively small (e.g., 10-40 nm) gold nanoparticles that can be conjugated with specific to selectively target the bacterium. These nanoparticles can attach to the bacterial surface individually or self-assemble under suitable surroundings into larger Nano clusters (Figure 1 a, b) [8].

Basic of laser:

The word of laser is an acronym of light amplification by stimulated emission of radiation [9] the light generated with a laser, in general, can be recognize in two types:

1. Continuous-wave [CW] laser: a constant flow of energy.

2. Pulsed laser: manifold discrete pulses.

The two types of lasers are basically different in format, light delivery, and implementation. A CW laser is generated by continuously pumping energy into the active medium to perform an equipoise between the number of atoms raised to the excited state and the number of photons emitted [10]. At such an equipoise, continuous laser output results. The interval of a CW laser pulse is approximately 0.25s. With this interval and with relatively constant power delivery to tissues, considerable thermal damage occurs. Examples of CW laser are the argon and CW dye laser used for vascular coagulation.

Pulsed lasers, in contrast, deliver high-energy beams in very short pulses in the range of milliseconds without the use of a shutter. Emissions are created when the pump is modulated to produce discrete laser pulses, which usually are broad and randomly shaped. Examples of pulsed laser are the copper vapor laser owing to a train of pulses at a frequency of 15000 pulses per second, which is so fast that the skin responds as it would do a continuous beam of light. Both types of lasers can be further modified to founder even shorter pulses, usually in the range of 10-250 ns, by using a method called as Q-switching. With this system, a large population inversion builds before emission is stimulated. This population inversion is accomplished by using a mechanical opaque shutter or by inserting a high-speed, electrically sensitive, polarizable optical shutter known as a pocket cell between the two mirrors of the laser. Q-switching lasers produce very short pulses at very high peak power. The Q-switching refers to the quality of the energy storage in the lasing medium, which is changed suddenly to produce a short, intense burst light [10,11].

Some properties of lasers beam:

Monochromaticity:

The light emitted by laser is vastly more monochromatic than that of any conventional monochromatic source. An inspection of a line emitted by the latter shows that it never perfectly sharp, but it spreads over a frequency range of the order of thousands of megacycles per second. A similar inspection of the light from a laser would reveal virtually no spreading at all. It must be noted, however that no light source, laser included, is capable of producing absolute monochromatic light [12,13].

Coherence:

Coherence denote that the light waves are in the identical phase. Laser light is much more coherent than congenital light. It is impossible to notice this property with our eyes and therefore its significant is sometimes over lacked. The incoherent waves have no correlation to each other. They don't start at the same point in time and space, nor do they have identical wavelength. On the other hand, coherent waves are locked together both in space and time. The coherence property of a laser ray is described by its ability to determine the appearance of interference fringes when reacting with a second laser beam emitted by the same laser at the same wavelength [14,15, 16].

Directivity:

The emanation in a narrow parallel beam, allows the transmission of light (whether visible, near infrared or near ultra violet) with the assist of an optical fiber of minuscule diameter (50 to 600 pm) [17].

The directivity is the property of the laser rays to be restricted within a beam which is propagating in space so that its cross section is practically not modified at small distances (of the order of some meters) [18].

The action of laser lighton the tissue:

The most desired interaction is the absorption of the laser energy by the intended tissue. The amount of energy that is absorbed by the tissue depends on the tissue characteristics, such as pigmentation and water content, and on the laser wavelength and emission mode. In general, the shorter wavelengths (from about 5001000 nm) are readily absorbed in pigmented tissue and blood elements. Argon is highly attenuated by hemoglobin. Diode and Nd:YAG have a high affinity for melanin and less interaction with hemoglobin. The longer wavelengths are more interactive with water and hydroxyapatite. The largest absorption peak for water is just below 3000 nm, which is at the Er:YAG wavelength. Erbium is also well absorbed by hydroxyapatite. CO2 at 10.600 nm is well absorbed by water [19].

The assortment of communication techniques that may occur when applying laser light to biological matter is manifold. Definite biological material futures (optical and thermal property) and laser parameters (wavelength, exposure time, applied energy, focal spot size, and energy density and power density) contribute to this variety [20].

Photochemical interaction:

The physical processes involved in the interaction of a laser beam and a material are divided into three parts:

(1) Absorption of some of the laser beam energy.

(2) Transformation of this energy into chemical energy and/or into heat, and diffusion of heat away from the irradiated zone.

(3) Eventually, chemical reaction and/or phase transformation (in general, vaporization) [21].

Photochemical interaction mechanism play a significant role during photodynamic therapy (PDT).Frequently, biostimulation is also attributed to photochemical interactions:

Biostimulation:

Biostimulation is also attributed to photochemical interactions, which is believed to occur at very low irradiances, where the irradiation of cells at certain wavelengths can activate some of the native components and in this way specific biochemical reactions as well as whole cellular metabolism can be altered. Biostimulation can be used in wound healing and anti-inflammatory properties by red or near infrared light sources such as HeNe lasers or diode laser [19, 20].

Photothermal interaction:

The thermal effect of lasers on biological tissue is a complex process resulting from three distinct phenomena; conversion of light to heat, transfer of heat and the tissue reaction, which is related to the temperature and the heat in time, Depending on the duration and peak value of the tissue temperature achieved, different effects like coagulation, vaporization, carbonization, and melting may be distinguished [19].

Photoablation:

Photoablation consists in the spontaneous etching that occurs upon the absorption, at the material surface, of a pulse of laser, light, whose energy is greater than the ablation threshold value. The advantage of using UV light resides in the fact that the ablation is strictly confined to the volume that absorbs the energy. Owing to the high absorption coefficient in far-UV of many materials this volume is very small (of the order of a fraction of a micron in depth). In other words, the laser energy is, for most materials very densely absorbed at the surface. The ablative conditions are rapidly reached during the excitation pulse and the volatile products eventually formed are quickly propelled outside the solid [21,22, 23].

The aims of this study are:

1. Isolation and diagnosis of S. aureus and E. coli.

2. Study the effect of 504 nm laser with continuous wave (CW) on the biochemical characteristics of S. aureus and E. coli, without and with nanoparticles.

MATERIAL AND METHOD

This chapter is concerned with materials and methods, which are used in this investigation. The materials include equipment's, samples, chemical agent and lasers. While the methods include the method of isolation, identification and irradiation by lasers. All these steps can be summarized in the following experiment design:

Samples collection:

The samples were collected from Hella Surgical Hospital, and included seven samples, taken from different parts of the tools used in surgical operations as well as samples taken from patient's beds and equipments in the surgical operating room.

Isolation of bacteria:

Bacterial isolates were identified according to Berge's manual, which depends on biochemical and physiological characteristics of bacteria.

After treatment of the samples, biological transactions we noticed a clear satisfactory for bacterial isolates included types of gram-negative stain bacteria and the types of gram-positive stain bacteria. The bacteria E. coli are the most common among the species gram negative bacteria, and bacteria S. arueus was the most common among gram positive bacteria stain [4,9].

Identification of bacteria:

Collected samples transferred to laboratory by swabs media, then cultured on selective media to isolate the bacteria. The swabs were directly cultured onto eight soled media (Nutrient agar, chocolate agar, Blood agar Base, MacConky agar, Salamomoela-Shigella agar (S-S agar), Simmon's Citrate agar, Eosin Methalen Blue media, Manitol Salt agar). The plates were incubated aerobically and examined after 24-48 hours. All bacteria isolated were identified by routine laboratory method included morphological examination and biochemical test [24, 25].

A single colony was taken from each primaiy positive culture on Eosin Methylen Blue agar (EMB) and Manitol salt agar and it was identified depending on its morphology (shape, size, borders and texture) and then it was examined under microscope after staining it with Gram stain. After attaining, the biochemical tests were done on each isolate to complete the final identification. Eight specific biochemical testes were done to differentiate E. coli isolates and S. aureus from other bacteria [26,27].

Irradiation of the samples:

(50) ml of the diluted bacterial suspension was transferred to sterile Eppendroff tube and exposed to laser light at 30 mints, another Eppendroff tube also contains (50) ml of the diluted bacterial suspension was not exposed to laser light in order to keep it as a control.

Results of irradiation by laser without nanoparticles:

Figure 3 illustrates the changes in the biochemical characteristics E. coli and S. aureus when it irradiated with 504 nm laser (CW mode) at (30) minutes exposure time. After irradiation of bacteria with laser (CW mode) high absorption were observed. Two different molecular mechanisms may be explain the effect of this laser on the metabolism of E. Coli and S. Aureus, first possibility is the photochemical mechanism due to the absorption of laser light by certain chromophores [14]. The second possible interaction mechanism is the thermal mechanism; a noticeable fraction of the excitation energy is inevitably (as is certain to happen) converted to heat, which causes a local transient increase in the temperature of the absorbing chromospheres. Any appreciable time averaged heating of the sample can be prevented by controlling the irradiation intensity and dose appropriately. However, there is still the possibility of localized transient (lasting only for a short time) heating of absorbing chromophores. The local transient rise in temperahrre of absorbing biomolecules may cause structural (e.g. conformational) changes and biochemical activity such as activation or inhibition of enzymes [28,29].

Results of irradiation biy laser 504 nm with nanoparticles:

Nanomedicine involves utilization of nanotechnology for the benefit of human health and wellbeing. The use of nanotechnology in various sectors of therapeutics has revolutionized the field of medicine where nanoparticles of dimensions ranging between 1-100 nm are designed and used for diagnostics, therapeutics and as biomedical tools for research). It is now possible to provide therapy at a molecular level with the help of these tools, thus treating the disease and assisting in study of the pathogenesis of disease. Gold nanoparticles strongly absorb laser irradiation; this absorbed energy transforms quickly into heat, which causes damage to the bacterium through local overheating effects. If many nanoparticles attach to the bacterial surface, there will be multiple damage sites with possible overlapping thermal spots from the particles within nanoclusters [8].

Discussion and conclusion:

Discussion:

This chapter is concerned with the results of the bacteriological examinations as well as these of laser irradiation. Laser irradiation tests involved examination of S. aureus species sensitivity to laser light, the effect of exposure time and the energy density. Effect of laser light on the bacteria was examined in term of absorption of laser light.

The discussion was based on a number of recent studies of the interaction of ultraviolet and visible lasers with the biological systems, as well as to illustrate every possible mechanism expected in the photoinactivation process.

Effect of exposure time:

For bacterial isolates, exposure times of (here, 30 min). figurer 3 shows the curve of E. coli and Figure 4 show the curve of S. aureus after the exposure to (504nm) laser light. It is clear that the absorption of cells decrease with increasing the exposure time to laser light, but the absorption decreased after few minutes after it reaches maximum absorption, then, absorption decreases was observed for the residual exposure times. Sensitivity to laser light:

Sensitivity to laser light:

The different species of E. Coli and S. aureus were different in their resistance to the laser light. This variation may be because the genus E. Coli and S. aureus is genetically heterogeneous. This conclusion is confirmed by the DNA base composition. At the species level, E. Coli and S. aureus isolates, considered to belong to a certain species according to the phenotypic classification, have been shown to differ in their DNA base compositions far more than expected, indicating the genetic unrelatedness of such strains.

This heterogenesity is of high importance because the primary target for the lethal effects of UV-radiation on living organisms is the DNA via the creation of different lesions or breaks in the DNA strands. However, inside cells, the DNA is found in close association with other macromolecules particularly proteins. The absorption of near UV-light is known to induce pyrimidine dimers and single strand breakage that have lethal effects on the cell [30,31]. It was expected that the heterogenesity in radiation resistance may be closely related to the heterogenesity in the genetic content of the bacterial isolates since the UV-light from laser affect cell survival directly by direct DNA damage by means of different mechanisms, and indirectly by means of different Chromophores that can absorb UV-light, lead to inhibition of DNA replication processes or inhibition of some metabolic pathways. The types and quantities of these chromospheres are also under genetic control, varying from one species to the other as well as from strain to others [32].

Conclusion:

1. The results investigation that E. coli and S. aureus can be killed by 504 nm laser after having been irradiated.

2. Irradiation of bacteria by 504 nm laser at 30 minutes exposure time, lead to change in the biochemical characteristics of the bacteria.

3. The effect was increasing when nanoparticles utilizing, by using the same of period.

ARTICLE INFO

Article history:

Received 28 September 2015

Accepted 15 November 2015

Available online 24 November 2015

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Sadiq Hassan

University of Babylon, Physics Of Laser College of Science for Women. Box.10. Hilla. Iraq

Corresponding Author: Sadiq Hassan, University of Babylon, Faculty of Science for Women, Laser Physis Department Box.10. Hilla. IRAQ. Phone: +964 7829869891; E-mail: sadiq.hassan74@yahoo.com
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Publication:Advances in Environmental Biology
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Date:Nov 1, 2015
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