Thymine dissociation and dimer formation: A Raman and synchronous fluorescence spectroscopic study.

In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in order to detect thymine dimer formation. The thymine dimer formation, as a function of irradiation dose, was determined by Raman spectroscopy. In addition, the formation of a mutagenic (6-4) photoproduct was identified by its synchronous fluorescence spectrum. Our spectroscopic data suggest that the rate of conversion of thymine to thymine dimer decreases after 20 min of UV irradiation, owing to the formation of an equilibrium between the thymine dimers and monomers. However, the formation of the (6-4) photoproduct continued to increase with UV irradiation. In addition, the Raman spectra of nonirradiated and irradiated calf thymus DNA were recorded, and the formation of thymine dimers was detected. The spectroscopic data presented make it possible to determine the mechanism of thymine dimer formation, which is known to be responsible for the inhibition of DNA replication that causes bacteria inactivation.

A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

In situ detection of live-to-dead bacteria ratio after inactivation by means of synchronous fluorescence and PCA.

The determination of live and dead bacteria is of considerable significance for preventing health care-associated infection in hospitals, field clinics, and other areas. In this study, the viable (live) and nonviable (dead) bacteria in a sample were determined by means of their fluorescence spectra and principal component analysis (PCA). Data obtained in this study show that it is possible to identify bacteria strains and determine the live/dead ratio after UV light inactivation and antibiotic treatment, in situ, within minutes. In addition, synchronous fluorescence scans enable the identification of bacterial components such as tryptophan, tyrosine, and DNA. Compared with the time-consuming plating and culturing methods, this study renders a means for rapid detection and determination of live and dead bacteria.

Synergistic reaction of silver nitrate, silver nanoparticles, and methylene blue against bacteria.

In this paper we describe the antibacterial effect of methylene blue, MB, and silver nitrate reacting alone and in combination against five bacterial strains including Serratia marcescens and Escherichia coli bacteria. The data presented suggest that when the two components are combined and react together against bacteria, the effects can be up to three orders of magnitude greater than that of the sum of the two components reacting alone against bacteria. Analysis of the experimental data provides proof that a synergistic mechanism is operative within a dose range when the two components react together, and additive when reacting alone against bacteria.

Rationale and mechanism for the low photoinactivation rate of bacteria in plasma.

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S-H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.

Risk of Contamination in Assembled vs Disassembled Instruments in Hip Arthroplasty Surgery.

BACKGROUND: Periprosthetic joint infection (PJI) is one of the most common causes of revision total hip arthroplasty (THA) and associated with higher costs, prolonged pain, and worse clinical outcomes. Many factors have been linked to increased infection rates, one being the operative equipment and instrumentation used during the surgical procedure. With few arthroplasty instruments designed for complete disassembly and increasingly complex instrument designs, this study seeks to understand the effect that instrument disassembly plays on infection using disassembled and assembled standard femoral broach handles (BHs). METHODS: Two BHs, not designed for disassembly, were modified and then contaminated in the disassembled state with Geobacillus stearothermophilus vegetative-form bacteria and spores. Using both flash and standard sterilization cycles, the BHs were steam sterilized in the disassembled or assembled state and then analyzed for remaining bacteria and spores. RESULTS: At all target locations after either a flash sterilization cycle or a standard sterilization cycle, complete eradication of both the vegetative-form and spore-form of G stearothermophilus was achieved. CONCLUSION: This study demonstrates that adequate decontamination of the tested BHs can be achieved after steam sterilization in either the disassembled or assembled state, without an increased risk of infection transmission.

The low photo-inactivation rate of bacteria in human plasma II. Inhibition of methylene blue bleaching in plasma and effective bacterial destruction by the addition of dilute acetic acid to human plasma.

Methylene blue (MB) and other photo-sensitizer molecules have been recognized as effective means for the inactivation of bacteria and other pathogens owing to their ability to photo-generate reactive oxygen species (ROS) including singlet oxygen. These reactive species react with the membrane of the bacteria causing their destruction. However, the efficiency of MB to destroy bacteria in plasma is very low because the MB 660 nm absorption band, that is responsible for the ROS generation, is bleached. The bleaching of MB, in plasma, is caused by the attachment of a hydrogen atom to the central ring nitrogen of MB, which destroys the ring conjugation and forms Leuco-MB which does not absorb in the 600 nm region. In this paper we show that addition of dilute acetic acid, ∼10(-4) M, to human plasma, prevents H-atom attachment to MB, allowing MB to absorb at 660 nm, generates singlet oxygen and thus inactivates bacteria. The mechanism proposed, for preventing MB bleaching in plasma, is based on the oxidation of cysteine to cystine, by reaction with added dilute acetic acid, thus eliminating the availability of the thiol hydrogen atom which attaches to the MB nitrogen. It is expected that the addition of acetic acid to plasma will be effective in the sterilization of plasma and killing of bacteria in wounds and burns.

Viruses associated with pneumonia in adults.

Viral pneumonia, which is typically associated with disease in childhood, is increasingly recognized as causing problems in adults. Certain viruses, such as influenza virus, can attack fully immunocompetent adults, but many viruses take advantage of more-vulnerable patients. The latter include patients receiving immunosuppressive therapy and elderly subjects, particularly those residing in long-term care facilities. The range of viruses producing pneumonia in adults includes common agents, such as varicella-zoster virus and influenza virus, as well as respiratory syncytial virus, human metapneumovirus, adenoviruses, picornaviruses, and coronaviruses. The roles played by other agents, such as rhinoviruses and human bocaviruses, in pneumonia are still under study. While therapy for most of theses agents, at least in adults, has not yet been fully clarified, it is reasonable to assume antivirals may work in certain situations if they are introduced early enough in the course of infection.

Inactivation of bacteria in plasma.

The photo-inactivation rate of bacteria by methylene blue, MB, was found to be significantly lower in plasma than in water, saline, and PBS solutions. The spectroscopic data and ultrafast time resolved transient spectra and kinetics presented show that methylene blue photo-bleaches faster and to a larger degree in plasma and the MB excited singlet and triplet state populations in plasma are much lower in plasma than in water and PBS solutions. The optical density, OD, of MB in plasma was found to decrease by ~50% after a minute of illumination with 661 nm light, while under identical conditions the OD in PBS solution decreased by only 1%. Based on these data and the effect of the plasma proteins on MB photochemistry, a mechanism is proposed that accounts for the low inactivation rate of bacteria in plasma.

Comparison of Bovine and Carp Fish Visual Pigment Photo-Intermediates at Room Temperature.

This paper presents room temperature nanoseconds to milliseconds time-resolved spectra and kinetics of the intermediate states and species of bovine and carp fish rhodopsin visual pigments, which also contained ~5% cone pigments. The nanoseconds to milliseconds range cover all the major intermediates in the visual phototransduction process except the formation of bathorhodopsin intermediate which occurs at the femtosecond time scale. The dynamics of these visual pigment intermediates are initiated by excitation with a 532 nm nanosecond laser pulse. The recorded differences between bovine and carp rhodopsin time-resolved spectra of the formation and decay kinetics of their intermediates are presented and discussed. The data show that the carp samples batho intermediate decays faster, nearly by a factor of three, compared to the bovine samples. The formation and decay spectra and kinetics of rhodopsin outer segments and extracted rhodopsin inserted in buffer solution were found to be identical, with very small differences between them in the decay lifetimes of bathorhodopsin and formation of lumirhodopsin.

Effect of pH on methylene blue transient states and kinetics and bacteria photoinactivation.

We have measured directly by time-resolved spectroscopy the transient spectra and kinetics of the methylene blue (MB) excited singlet and triplet state as a function of pH from a few picoseconds to several microseconds. The data show that the acidic triplet state (3)MBH(2+) is the protonated analogue of the basic (3)MB(+). It is also shown that the singlet oxygen formation quantum yield is much higher in basic than in acidic media. The transient spectra and their kinetics suggest that because pH exerts a large influence in singlet oxygen and radical formation, it may also be important in bacteria inactivation. Therefore, we performed experiments, which showed that the rate of gram-positive and gram-negative bacteria inactivation at pH 9 is 3-25 times higher than the rate at pH 5.

Resonance Raman Spectra for the In Situ Identification of Bacteria Strains and Their Inactivation Mechanism.

The resonance Raman spectra of bacterial carotenoids have been employed to identify bacterial strains and their intensity changes as a function of ultraviolet (UV) radiation dose have been used to differentiate between live and dead bacteria. In addition, the resonance-enhanced Raman spectra enabled us to detect bacteria in water at much lower concentrations (∼108 cells/mL) than normally detected spectroscopically. A handheld spectrometer capable of recording resonance Raman spectra in situ was designed, constructed, and was used to record the spectra. In addition to bacteria, the method presented in this paper may also be used to identify fungi, viruses, and plants, in situ, and detect infections within a very short period of time.

Determination of live:dead bacteria as a function of antibiotic treatment.

Antibiotics are drugs that react against, kill, or inhibit the growth of bacteria. The method most often employed to evaluate the effectiveness of an antibiotic to kill bacteria requires at least 16 to 24 h for bacterial incubation. The requirement of long periods of time for the determination of the number of bacteria still alive after antibiotic treatment, may, in many cases, be detrimental to the patient's health. In addition, with increasing of bacterial antibiotic resistance, the need to utilize methods for distinguishing between live and dead bacteria within a short period of time after treatment with antibiotic agents, is becoming more crucial. To that effect, we have utilized a hand-held double monochromator to record in situ and within minutes the synchronous and normal fluorescence spectra of bacteria and other species. The fluorescence spectra of bacterial components such as tryptophan, tyrosine and DNA are clearly displayed. In addition, principal component analysis, PCA, makes it possible to display live and dead bacteria separately and determine the ratio of live:dead bacteria before and after treatment with antibiotics.

Hand-held synchronous scan spectrometer for in situ and immediate detection of live/dead bacteria ratio.

The design, construction, and operation of a hand-held synchronously scanned, excitation-emission, double monochromator spectrometer is described. Data show that it is possible to record and display within minutes the fluorescence spectra and ratio of live/dead bacteria in situ. Excitation emission matrix contour plots display clearly bacteria fluorescence spectra before and after UV inactivation, respectively. The separation of the fluorescence band maxima of molecular components, such as tryptophan, tyrosine, and DNA, may be distinguished in the diffused fluorescence spectra of bacteria and mixtures.

Fiber based pathogen photoinactivating system.

A new fiber based instrument described, which can be inserted through the nose, mouth, or other means into the body, has the potential to: (a) detect and image infected areas inside the body; (b) deliver drugs only onto the infected areas inside and outside the human body; (c) remove the excess drugs; (d) use the same fiber for both imaging and illumination with high intensity light of wavelengths varying from deep UV to IR to induce photoreactions of the drug with the pathogen of the infected area. This compact system utilizes much smaller quantities of drugs and radiation dose than conventional methods, eliminates the need for intravenous injection, and greatly decreases the toxic effects of drugs and irradiation to the human body including swelling, inflammation, nausea, fever, and sunlight sensitivity that lasts for about 30 days.

Accurate delivery of chemicals and intense light on infected areas only for targeted therapy inside the body.

Bacteria and virus deactivation is performed inside and on the skin of the body using the novel device described in this paper. This device delivers on the infected spot only the needed amount of drug and light necessary for therapy, while the excess is siphoned out, thus eliminating the deleterious affects that maybe caused by intravenous injection. A charged coupled device provides means for visual monitoring of the therapeutic reaction and an LED or laser diode supplies the intense light for the photochemical deactivation. The spectra and kinetics of the photochemical reaction that generate the reactive species, such as 1O2 and OH radicals that are responsible for pathogen deactivation, are presented. Topical therapy experiments on New Zealand rabbits are described.