Antibacterial Effect of Amino Acid-Silver Complex Loaded Montmorillonite Incorporated in Dental Acrylic Resin.

Several dental materials contain silver for antibacterial effect, however the effect is relatively low. The reason for the lower antibacterial efficacy of silver is considered to be the fact that silver ions bind to chloride ions in saliva. To develop new effective silver antibacterial agents that can be useful in the mouth, we synthesized two novel amino acid (methionine or histidine)-silver complexes (Met or His-Ag) loaded with montmorillonite (Mont) and analyzed their antibacterial efficacy. At first the complexes were characterized using nuclear magnetic resonance (NMR), and amino acid-Ag complex-loaded Mont (amino acid-Ag-Mont) were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The antibacterial efficacy of these materials in dental acrylic resin was then investigated by bacterial growth measurement using a spectrophotometer. As controls, commercially available silver-loaded zeolite and silver-zirconium phosphate were also tested. Dental acrylic resin incorporating His-Ag-Mont strongly inhibited Streptococcus mutans growth. This was explained by the fact that His-Ag complex revealed the highest amounts of silver ions in the presence of chloride. The structure of the amino acid-Ag complexes affected the silver ion presence in chloride and the antibacterial efficacy. His-Ag-Mont might be used as antibacterial agents for dental materials.

Aesthetic Silver-Doped Octacalcium Phosphate Powders Exhibiting Both Contact Antibacterial Ability and Low Cytotoxicity.

Since the introduction of biomaterials, infection has been a serious problem in clinical operations. Although several studies have introduced hybrid materials of calcium phosphate and Ag0 nanoparticles (NPs) that exhibit antibacterial activity, released Ag+ ions and Ag0 NPs are highly cytotoxic and the materials require complex fabrication techniques such as laser irradiation. In this study, we introduce a simple one-pot synthesis method based on crystal-engineering techniques to prepare Ag+-substituted octacalcium phosphate (OCP-Ag) powder that simultaneously exhibits antibacterial activity, little change in color, and low cytotoxicity, thereby overcoming the shortcomings of calcium phosphate as a biomaterial. We used AgNO3-containing (NH4)2HPO4 aqueous solutions as reaction solutions in which Ag+ forms soluble complex [Ag(NH3)2]+ ions that are stable at Ag+ concentrations less than ∼30 mmol/L. Hydrolysis of soluble calcium phosphate in this solution led to pure OCP-Ag when the Ag+ concentration was less than ∼30 mmol/L. Crystallographic analysis showed that Ag+ substituted at the P5 PO4-conjugated sites and was uniformly distributed. When the concentration of Ag+ in the reaction solution was varied, the Ag+ content of the OCP-Ag could be controlled. The obtained OCP-Ag exhibited little color change or Ag+ release when immersed in various media; however, it exhibited contact antibacterial ability toward resident oral bacteria. The prepared OCP-Ag showed no substantial cytotoxicity toward undifferentiated and differentiated MC3T3-E1 cells in assays. Notably, when the Ag+ content in OCP-Ag was optimized (Ag: ∼1 at %), it simultaneously exhibited contact antibacterial ability, little color change, and low cytotoxicity.

Rechargeable anti-microbial adhesive formulation containing cetylpyridinium chloride montmorillonite.

Long-term anti-bacterial effect is a desired ability of any dental material in combating tooth caries as one of the most common and widespread persistent diseases today. Among several cationic quaternary ammonium compounds with antiseptic properties, cetylpyridinium chloride (CPC) is often used in mouthrinses and toothpastes. In this study, we incorporated CPC in a soft phyllosilicate mineral (clay), referred to as montmorillonite (Mont), to enable gradual CPC release with rechargeability. Besides measuring CPC release and recharge, we examined the anti-bacterial effect, cytotoxicity and bonding effectiveness of five experimental adhesive formulations, prepared by adding 1 and 3 wt% CPC_Mont, 3 wt% Mont (without CPC), and 1 and 3 wt% CPC (without Mont) to the commercial adhesive Clearfil S3 Bond ND Quick ('C-S3B'; Kuraray Noritake). Strong inhibition of Streptococcus mutans biofilm formation by CPC_Mont adhesives was confirmed by optical density and SEM. CPC release from CPC_Mont adhesives was higher and lasted longer than from CPC adhesives, while CPC_Mont adhesives could also be recharged with CPC upon immersion in 2 wt% CPC. In conclusion, CPC_Mont technology rendered adhesives anti-bacterial properties with recharge ability, this without reducing its bonding potential, neither increasing its cytotoxicity. STATEMENT OF SIGNIFICANCE: Dental caries is one of the most prevalent chronic diseases in the population worldwide and is the major cause of tooth loss. In this study, we developed cetylpyridinium chloride (CPC) loaded montmorillonite (CPC-Mont) with a long-term antibacterial efficacy to prevent caries. CPC is an antibacterial agent approved by FDA, used as an OTC drug and contained in oral hygiene aids. CPC-Mont was incorporated in a dental adhesive to gradually release CPC. CPC_Mont technology rendered adhesives anti-bacterial properties with rechargeability, this without reducing its bonding potential, neither increasing its cytotoxicity.

Laser damage to marine plankton and its application to checking biofouling and invasion by aquatic species: a laboratory study.

In this laboratory study, the ability of low-power pulsed laser irradiation to kill planktonic organisms in a flowing water system was examined, thus, to test the possibility of using this technique as a water treatment strategy to reduce biofouling growth in condenser tubes of power plants and to reduce bioinvasion via the ballast water of ships. Two flow rates (4.6 and 9.0 l h(-1)) were tested on three planktonic organisms: two marine centric diatoms viz. Skeletonema costatum and Chaetoceros gracilis and a dinoflagellate, Heterocapsa circularisquama. A low-power pulsed laser irradiation at 532 nm with a fluence of 0.1 J cm(-2) from a frequency-doubled Nd:YAG laser was used as the irradiation source. The laser irradiation resulted in a heavy mortality of the test cells. The mortality observed was >90% for S. costatum and H. circularisqama and >70% for C. gracilis. The results suggest that laser irradiation has the potential to act as a water treatment strategy to reduce biofouling of condenser tubes in power plants as well as to reduce species invasion via the ballast water of ships.

In vitro laser ablation of natural marine biofilms.

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 +/- 27.8 microm (titanium) and 107.4 +/- 28.5 microm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm2). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.

In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength.

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.

Recolonization of laser-ablated bacterial biofilm.

The recolonization of laser-ablated bacterial monoculture biofilm was studied in the laboratory by using a flow-cytometer system. The marine biofilm-forming bacterium Pseudoalteromonas carrageenovora was used to develop biofilms on titanium coupons. Upon exposure to a low-power pulsed irradiation from an Nd:YAG laser, the coupons with biofilm were significantly reduced both in terms of total viable count (TVC) and area cover. The energy density used for a pulse of 5 ns was 0.1 J/cm(2) and the durations of irradiation exposure were 5 and 10 min. When placed in a flow of dilute ZoBell marine broth medium (10%) the laser-destructed bacterial film in a flow-cytometer showed significant recovery over a period of time. The flow of medium was regulated at 3.2 ml/min. The increase in area cover and TVC, however, was significantly less than that observed for nonirradiated control (t-test, P< 0.05). The coupons were observed for biofilm area cover and TVC at different intervals (3, 6, and 9 h) after irradiation. While the biofilm in the control coupon at the end of 9 h of exposure showed 95.6 +/- 4.1% cover, the 5- and 10-min irradiated samples after 9 h showed 60.3 +/- 6.5 and 37.4 +/- 12.1% area cover, respectively. The reduced rate of recolonization compared to control was thought be due to the lethal and sublethal impacts of laser irradiation on bacteria. This observation thus provided data on the online recolonization speed of biofilm, which is important when considering pulsed laser irradiation as an ablating technique of biofilm formation and removal in natural systems.

Laser impact on bacterial ATP: insights into the mechanism of laser-bacteria interactions.

The mechanisms of laser action on bacteria are not adequately understood. Here, an attempt has been made to study the fluctuation in ATP (adenosine triphosphate) concentration following laser irradiation from a pulsed Nd:YAG laser on a marine biofilm-forming bacterium Pseudoalteromonas carrageenovora. A stationary phase bacterial suspension (density 10(7-8) ml-1) was exposed to pulsed laser irradiations at a fluence of 0.1 J cm-2 (pulse width 5 ns, repetition rate 10 Hz) for different durations, ranging from 2 s to 15 min. The total viable count (TVC) and ATP concentration of the irradiated samples were determined immediately after the laser irradiation. While the maximum reduction in the TVC observed with respect to the control was 59% immediately after 15 min irradiation, the ATP concentration showed a reduction of about 86% for the same duration. The ATP concentration showed an abrupt reduction from 3 min of laser irradiation and continued to reduce significantly with increasing duration of irradiation. Thus, 3 min irradiation at a fluence of 0.1 J cm-2 is considered as an approximate threshold for ATP production in this bacterium. As the decreased level of ATP production continued, bacterial mortality resulted. The reduction in ATP production could be due to damage caused by the laser irradiations on bacterial metabolic processes such as cellular respiration.

Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system.

A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.

Lethal and sub-lethal impacts of pulsed laser irradiations on the larvae of the fouling barnacle Balanus amphitrite.

Laboratory experiments were conducted to study the impact of laser irradiation on the larvae of the fouling barnacle Balanus amphitrite. Research pertaining to fouling invertebrate larvae-laser interaction is sparse and, hence, data on this aspect were thought significant in order to consider pulsed low power laser irradiations as a possible future antifouling tool. Lethal and sub-lethal impacts of four very low laser fluences, viz. 0.013, 0.025, 0.05 and 0.1 J cm-2 for three different durations, viz. 2, 10 and 30 s were investigated. Three growth stages of barnacle larvae, viz. nauplii stage II, nauplii stage IV and cyprids were exposed to the mentioned laser fluences for different durations. While lethal impact was assessed immediately after and 1 d after irradiation, sub-lethal impacts were studied by monitoring the success rate of the irradiated nauplii in reaching the cyprid stage. In addition, the swimming speed of VIth stage nauplii after irradiation was studied. In the case of cyprids, in addition to the mortality measurement immediately after and 1 d after irradiation, the settlement rate was investigated. In all the above experiments, non-irradiated larvae served as controls. The results showed an increase in mortality with increasing laser fluence and duration of irradiation. Irradiation for 2 s resulted in significant mortality in nauplii, while it was less in the case of cyprids. In IInd stage nauplii, the mortality immediately after irradiation for 2 s varied from 14.8 +/- 2.12 to 97.1 +/- 4.1% for laser fluences of 0.013 and 0.1 J cm-2, respectively. However, in cyprids, the mortality immediately after irradiation for 2 s varied from 12.2 +/- 3 to 13.4 +/- 1.2% for fluences of 0.013 and 0.1 J cm-2, respectively. The mortality in IVth stage nauplii was less than that for IInd stage nauplii but more than that for cyprids. There was a significant increase in mortality with time after irradiation. The formation of cyprids from the irradiated larvae was significantly less than that observed for non-irradiated larvae. Also, the irradiated larvae showed a significantly slower swimming speed compared to the control samples. The settlement rate in cyprids was reduced significantly by the laser irradiation. This was true even for the lowest fluence and shortest period of irradiation tested. Thus, the results of the experiment showed that even a low power pulsed laser irradiation of 0.013 J cm-2 for 2 s can cause significant damage to fouling barnacle larvae.

Pulsed laser irradiation impact on two marine diatoms Skeletonema costatum and Chaetoceros gracilis.

The ability of pulsed laser irradiations to cause damage on the biofouling organisms is recently being investigated. If this technique is employed in industries such as power generation wherein a large quantity of water is being used for the cooling purpose, many organisms other than the targeted would get affected. In this study, we have investigated the damage caused by the pulsed laser irradiations from an Nd:YAG laser (fluence 0.1J/cm(2)) for varying durations such as 2, 5, 10, 30, 60 and 300 s on two marine diatom species namely Skeletonema costatum and Chaetoceros gracilis. Upon exposure to low power laser irradiations, these diatom species showed mortalities between 52.6+/-9.3% to 97.7+/-3.1% in the case of S. costatum and 57.8+/-2.5% to 98.9+/-0.6% in the case of C. gracilis for 2 and 300 s of irradiations, respectively. The mortality increased with the increase in the duration of laser irradiation. The estimation of the chlorophyll a concentration in the irradiated samples showed a considerable reduction varying between 9.8% and 57% in C. gracilis and 3% and 70.3% in S. costatum for 2 and 300 s of irradiations, respectively. The laser-survived cells grew as the non-irradiated (control) samples. C. gracilis frustules were broken by the laser whilst, the cell materials were drained out of the frustules in the case of S. costatum. The study therefore showed that the low power pulsed laser irradiations could cause significant damage on the two species of planktonic diatoms.

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system.

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.

Inhibition of bacterial attachment by pulsed Nd:YAG laser irradiations: an in vitro study using marine biofilm-forming bacterium Pseudoalteromonas carrageenovora.

The effect of low mean power laser irradiations with short pulse duration from an Nd:YAG (neodymium-doped yttrium aluminium garnet) laser on a marine biofilm-forming bacterium, Pseudoalteromonas carrageenovora, was investigated in the laboratory. Laser-irradiated bacteria were tested for their ability to attach on nontoxic titanium nitride (TiN) coupons with nonirradiated bacteria as the reference. Two durations of irradiation were tested, 10 and 15 min. Bacterial attachment was monitored after 20 min, 40 min, and 1 h of irradiation. The average laser fluence used for this study was 0.1 J/cm(2). The area of attachment of the irradiated bacteria was significantly less than the reference for both durations of irradiation. The growth of irradiated bacteria showed a longer lag phase than the nonirradiated sample, mainly due to mortality in the former. The bacterial mortality observed was 23.4 +/- 0.71 and 48.6 +/- 6.5% for 10- and 15-min irradiations, respectively. Thus, the results show that low-power pulsed laser irradiations resulted in a significant bacterial mortality and a reduced bacterial attachment on nontoxic hard surfaces.

Impact of pulsed Nd:YAG laser on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora: significance of physiological status.

The impact of pulsed Nd:YAG (neodymium-doped yttrium/aluminium garnet) laser irradiation on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora during two growth stages (log phase and stationary phase) and under two stresses (reduced temperature and nutrient limitation) was investigated. Bacteria were exposed to a laser fluence of 0.1 J x cm(-2) for 5, 10, and 15 min with a peak power of 20 MW x cm(-2), a pulse width of 5 ns, and an average power of 1 W x cm(-2) with a repetition rate of 10 Hz. The mortality of bacteria immediately after the irradiation as well as after a set period of time was determined. Mortality was higher among log-phase bacteria (72%) than bacteria in the stationary phase (51%) and those grown under nutrient limitation (51%). Bacteria grown at reduced temperature had a mortality of 49%. However, the differences in cell density of log-phase, stationary-phase, nutrient-limited, and low-temperature irradiated samples compared with controls after 5 h of incubation were 96, 93, 94, and 86%, respectively. The mortality values suggest that the same laser fluence has different degrees of effectiveness, depending on the physiological state of the bacteria.