Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay

Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.     

Extensive photodimerization of non-adjacent pyrimidines

In a prior study we found that non-adjacent thymidyl residues in the single-stranded alternating copolymer poly[d(G-T)] are subject to photodimerization by germicidal lamp irradiation (lambda max 254 nm). The maximum yield of this photoproduct was 1% of the total thymine of poly[d(G-T)]. We now report that dimer formation in this polymer is increased to 10 to 40% thymine as dimer between non-adjacent pyrimidines, using near-ultraviolet irradiation (lambda max 310 nm) with or without acetone triplet-sensitization. As previously observed for 254 nm irradiation, dimer formation was nearly absent in double-stranded poly[d(G-T).d(C-A)]. These observations extend prior findings by demonstrating high-yield dimerization between non-adjacent pyrimidines via direct irradiation at environmentally relevant wavelengths (greater than or equal to 280 nm), and are potentially relevant to the mechanism of the ultraviolet light-induced targeted -1 frameshift mutation.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Photomodification of blood by laser and ultraviolet radiation: a comparative study

The efficiency of in vivo blood irradiation by a laser light source (λ = 632.8 and 670 nm) and a mercury lamp (UV light, λ = 254 nm) was compared. Absorption spectra, gas content, oxyhemoglobin content, hemoglobin oxygen saturation, concentrations of lactate and glucose were studied for both irradiated and control samples. Hemoglobin was assumed to be the primary photoacceptor of light radiation for the indicated wavelengths. No substantial differences have been found between the effects of laser and non-laser irradiation. We conclude that the biological impact of the procedure is related to photoinduced changes in hemoglobin oxygen saturation.     

Thermal mitigation of Pseudomonas aeruginosa biofilms

Bacterial biofilms infect 2–4% of medical devices upon implantation, resulting in multiple surgeries and increased recovery time due to the very great increase in antibiotic resistance in the biofilm phenotype. This work investigates the feasibility of thermal mitigation of biofilms at physiologically accessible temperatures. Pseudomonas aeruginosa biofilms were cultured to high bacterial density (1.7?×?10⁹ CFU cm^?2) and subjected to thermal shocks ranging from 50°C to 80°C for durations of 1–30 min. The decrease in viable bacteria was closely correlated with an Arrhenius temperature dependence and Weibull-style time dependence, demonstrating up to six orders of magnitude reduction in bacterial load. The bacterial load for films with more conventional initial bacterial densities dropped below quantifiable levels, indicating thermal mitigation as a viable approach to biofilm control.     

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.     

Inactivation of Pseudomonas aeruginosa biofilms formed under high shear stress on various hydrophilic and hydrophobic surfaces by a continuous flow of ozonated water

Abstract

The inactivation of Pseudomonas aeruginosa biofilms grown on glass under high shear stress and exposed to a range of dissolved ozone concentrations (2, 5 and 7?ppm) at 10 and 20?min was investigated. The regression equation, log reduction (biofilm)?=?0.64?+?0.59×(C – 2)?+?0.33×(T – 10), described the dependence of biofilm inactivation on the dissolved ozone concentration (C, ppm) and contact time (T, min). The predicted D-values were 11.1, 5.7 and 2.2?min at 2, 5 and 7?ppm, respectively. Inactivation of biofilms grown on various surfaces was tested at a single dissolved ozone concentration of 5?ppm and a single exposure time of 20?min. Biofilms grown on plastic materials showed inactivation results similar to that of biofilms on glass, while biofilms grown on ceramics were statistically significantly more difficult to inactivate, suggesting the importance of utilizing non-porous materials in industrial and clinical settings.     

Thermostable xylanase inhibits and disassembles Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms are problematic and play a critical role in the persistence of chronic infections because of their ability to tolerate antimicrobial agents. In this study, various cell-wall degrading enzymes were investigated for their ability to inhibit biofilm formation of two P. aeruginosa strains, PAO1 and PA14. Xylanase markedly inhibited and detached P. aeruginosa biofilms without affecting planktonic growth. Xylanase treatment broke down extracellular polymeric substances and decreased the viscosity of P. aeruginosa strains. However, xylanase treatment did not change the production of pyochelin, pyocyanin, pyoverdine, the Pseudomonas quinolone signal, or rhamnolipid. In addition, the anti-biofilm activity of xylanase was thermally stable for > 100 days at 45°C. Also, xylanase showed anti-biofilm activity against one methicillin-resistance Staphylococcus aureus and two Escherichia coli strains.     

Negative pressure wound therapy reduces the motility of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and enhances wound healing in a rabbit ear biofilm infection model

Pseudomonas aeruginosa motility, virulence factors and biofilms are known to be detrimental to wound healing. The efficacy of negative pressure wound therapy (NPWT) against P. aeruginosa has been little studied, either in vitro or in vivo. The present study evaluated the effect of negative pressure (NP) on P. aeruginosa motility in vitro, and the effect of NPWT on virulence factors and biofilms in vivo. P. aeruginosa motility was quantified under different levels of NP (atmospheric pressure, ? 75, ? 125, ? 200 mmHg) using an in vitro model. Swimming, swarming and twitching motility were significantly inhibited by NP (? 125 and ? 200 mmHg) compared with atmospheric pressure (p = 0.05). Virulence factors and biofilm components were quantified in NPWT and gauze treated groups using a rabbit ear biofilm model. Biofilm structure was studied with fluorescence microscopy and scanning electron microscopy. Additionally, viable bacterial counts and histological wound healing parameters were measured. Compared with the control, NPWT treatment resulted in a significant reduction in expression of all virulence factors assayed including exotoxin A, rhamnolipid and elastase (p = 0.01). A significant reduction of biofilm components (eDNA) (p = 0.01) was also observed in the NPWT group. The reduction of biofilm matrix was verified by fluorescence- and scanning electron-microscopy. NPWT lead to better histologic parameters (p = 0.01) and decreased bacterial counts (p = 0.05) compared with the control. NPWT treatment was demonstrated to be an effective strategy to reduce virulence factors and biofilm components, which may explain the increased wound healing observed.     

Modeling bioaffinity-based targeted delivery of antimicrobials to Escherichia coli biofilms using yeast microparticles. Part II: Parameter evaluation and validation

The design of bioaffinity-based targeted delivery systems for biofilm inactivation may require a comprehensive understanding of physicochemical and biochemical properties of biobased antimicrobial particles and their interactions with biofilm. In this study, Escherichia coli biofilm inactivation by chlorine-charged yeast microparticles was numerically simulated, and the roles of chemical stability, binding affinity, and controlled release of this targeted delivery system were assessed using this numerical simulation. The simulation results were experimentally validated using two different types of yeast microparticles. The results of this study illustrate that chorine stability achieved by yeast microparticles was a key factor for improved biofilm inactivation in an organic-rich environment (>6 additional log reduction in 20 min compared to the free chlorine treatment). Moreover, the binding affinity of yeast microparticles to E. coli biofilms was another key factor for an enhanced inactivation of biofilm, as a 10-fold increase in binding rate resulted in a 4.2-fold faster inactivation. Overall, the mechanistic modeling framework developed in this study could guide the design and development of biobased particles for targeted inactivation of biofilms.     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.     

Survival of spacecraft-associated microorganisms under simulated martian UV irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m(-2) of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.     

Roles of PprA,IrrE, and RecA in the resistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H₂O₂ and quantification of H₂O₂ in plant extracts revealed that H₂O₂ accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.