Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h(-1)) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T(90) (time until 90% of the population is inactivated) values (T(90, chemostat) = 2.66 h; T(90, batch) = 2.62 h), whereas T(90) for cells growing at a mu of 0.9 h(-1) was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (mu = 1.0 h(-1)) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (mu = 0.3 h(-1)), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.     

New water disinfection system using UVA light-emitting diodes

AIM: To evaluate the ability of high-energy ultraviolet A (UVA) light-emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation. METHODS AND RESULTS: We developed a new disinfection device equipped with high-energy UVA-LED. Inactivation of bacteria was determined by colony-forming assay. Vibrio parahaemolyticus, enteropathogenic Escherichia coli, Staphylococcus aureus and Escherichia coli DH5alpha were reduced by greater than 5-log(10) stages within 75 min at 315 J cm(-2) of UVA. Salmonella enteritidis was reduced greater than 4-log(10) stages within 160 min at 672 J cm(-2) of UVA. The formation of 8-hydroxy-2'-deoxyguanosine in UVA-LED irradiated bacteria was 2.6-fold higher than that of UVC-irradiated bacteria at the same inactivation level. Addition of mannitol, a scavenger of hydroxyl radicals (OH(*)), or catalase, an enzyme scavenging hydrogen peroxide (H(2)O(2)) to bacterial suspensions significantly suppressed disinfection effect of UVA-LED. CONCLUSION: This disinfection system has enough ability to inactivate bacteria and OH(*) and H(2)O(2) participates in the disinfection mechanism of UVA irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: We newly developed UVA irradiation system and found that UVA alone was able to disinfect the water efficiently. This will become a useful disinfection system.     

G-to-T transversions and small tandem base deletions are the hallmark of mutations induced by ultraviolet a radiation in mammalian cells

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm(2) produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage.     

Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing.

Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed "viable but nonculturable" (VNC) state. Initial difficulties in obtaining consistent reduction of rho-iodonitrotetrazolium violet (INT) by H. pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions. The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied. Representation and interpretation of these enhancement profiles were facilitated by digital image processing. Nonculturable cells of H. pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37 degrees C, while 99% of those produced at 4 degrees C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, alpha-ketoglutarate, or aspartate. Activity was detected at similar levels in cells with coccoid and spiral shapes. In contrast, only 1% of nonculturable cells of V. vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population. Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms. Nonculturable cells of H. pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as "bags of enzymes" remains to be established.     

Mutation and DNA modification in Salmonella exposed to N-nitrosodimethylamine under UVA- and sunlight-irradiation.

Previously, we reported that when Salmonella typhimurium and Escherichia coli were treated with N-nitrosodimethylamine (NDMA) under irradiation with ultraviolet-A (UVA), mutagenesis of the bacteria took place without externally added activation enzymes. We also observed the formation of O(6)-methylguanine (O(6)-meG), N(7)-methylguanine (N(7)-meG) and 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) in calf thymus DNA treated with NDMA plus UVA. In this study, we observed the mutagenicity of NDMA under irradiation of natural sunlight in S. typhimurium. Furthermore, we detected the formation of O(6)-meG, N(7)-meG and 8-oxodG in calf thymus DNA treated with NDMA plus simulated sunlight. Regarding the mutagenesis of S. typhimurium by NDMA plus UVA, we have now identified and quantified O(6)-meG formed in the genomic DNA of the bacteria under conditions of the mutagenesis.     

Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli

The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log(10) CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.     

Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water

The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m(-2)) at 40 degrees C. Viability assays (4',6'-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.     

Possible involvement of ERK 1/2 in UVA-induced melanogenesis in cultured normal human epidermal melanocytes

UV-induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m2 and examined the potential involvement of mitogen-activated protein kinases (MAPK) as UVA-responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal-related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c-Jun N-terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre-treated with N-acetyl-L-cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6-4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation-induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

In vivo UVA irradiation of mouse is more efficient in promoting pulmonary melanoma metastasis than in vitro

Background

We have previously shown in vitro that UVA increases the adhesiveness of mouse B16-F1 melanoma cells to endothelium. We have also shown in vivo that UVA exposure of C57BL/6 mice, i.v. injected with B16-F1 cells, increases formation of pulmonary colonies of melanoma. The aim of the present animal study was to confirm the previously observed in vivo UVA effect and to determine whether in vitro UVA-exposure of melanoma cells, prior the i.v. injection, will have an enhancing effect on the pulmonary colonization capacity of melanoma cells. As a second aim, UVA-derived immunosuppression was determined.

Methods

Mice were i.v. injected with B16-F1 cells into the tail vein and then immediately exposed to UVA. Alternatively, to study the effect of UVA-induced adhesiveness on the colonization capacity of B16-F1 melanoma, cells were in vitro exposed prior to i.v. injection. Fourteen days after injection, lungs were collected and the number of pulmonary nodules was determined under dissecting microscope. The UVA-derived immunosuppression was measured by standard contact hypersensitivity assay.

Results and Discussion

Obtained results have confirmed that mice, i.v. injected with B16-F1 cells and thereafter exposed to UVA, developed 4-times more of melanoma colonies in lungs as compared with the UVA non-exposed group (p < 0.01). The in vitro exposure of melanoma cells prior to their injection into mice, led only to induction of 1.5-times more of pulmonary tumor nodules, being however a statistically non-significant change. The obtained results postulate that the UVA-induced changes in the adhesive properties of melanoma cells do not alone account for the 4-fold increase in the pulmonary tumor formation. Instead, it suggests that some systemic effect in a mouse might be responsible for the increased metastasis formation. Indeed, UVA was found to induce moderate systemic immunosuppression, which effect might contribute to the UVA-induced melanoma metastasis in mice lungs.     

Possible Involvement of ERK 1/2 in UVA‐Induced Melanogenesis in Cultured Normal Human Epidermal Melanocytes

UV‐induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m² and examined the potential involvement of mitogen‐activated protein kinases (MAPK) as UVA‐responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal‐related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c‐Jun N‐terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre‐treated with N‐acetyl‐l ‐cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6‐4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation‐induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

UV irradiation increases ROS production via PKCdelta signaling in primary murine fibroblasts

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earth's surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.     

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA

Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. In conclusion: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Nitric oxide-mediated depletion of Langerhans cells from the epidermis may be involved in UVA radiation-induced immunosuppression.

Ultraviolet A (UVA) irradiation of the dorsal skin of mice reduced the contact hypersensitivity (CHS) response and the density of epidermal Langerhans cells (LC). The roles of nitric oxide (NO) and reactive oxygen species (ROS) in these biological effects of UVA were investigated. Topical application of N(G)-monomethyl-L-arginine acetate, an inhibitor of NO production, 2,2'-dipyridyl, an iron chelater, or 4-hydroxy-tempo, a superoxide dismutase mimicking agent, inhibited UVA-induced suppression of the CHS response. N(G)-monomethyl-L-arginine acetate but not the ROS inhibitors prevented UVA from reducing LC numbers in the epidermis. This suggests that NO but not ROS produced in response to UVA mediates a depletion of LC from the epidermis, probably by signaling these cells to migrate from the skin. This could be responsible for UVA-induced immunosuppression. UVA-induced ROS can also cause immunosuppression, but by a different mechanism. Agents that inhibit or modulate NO or ROS production may be useful for preventing damage caused by the UVA component of sunlight to the skin immune system.     

Retinyl palmitate is non-genotoxic in Chinese hamster ovary cells in the dark or after pre-irradiation or simultaneous irradiation with UV light

Retinyl palmitate (RP), an ingredient of cosmetic and medical skin-care preparations, has been reported to be photo-genotoxic/photo-clastogenic in mouse lymphoma cells (Tk locus) as well as in human Jurkat T-cells, as measured by use of the comet assay. Given that these results were obtained under exploratory conditions, we re-investigated the photo-genotoxicity of RP following a protocol consistent with current recommendations for photo-genotoxicity testing of drugs and chemicals. We tested RP in Chinese hamster ovary (CHO) cells in the dark (standard chromosome aberration test), under pre-irradiation (UVA irradiation of cells and subsequent treatment with RP) or simultaneous irradiation (irradiation of cells and RP together, standard photo-genotoxicity protocol) conditions. UVA irradiation was applied at 350 and 700 mJ/cm2 with the high UV dose targeted to produce a small increase in the incidence of structural chromosome aberrations (CA) in cells not treated with RP. RP was tested up to and above its limit of solubility in the culture medium (20-40 μg/mL). There was no overt cytotoxicity under dark or different irradiation conditions. Treatment of cells with RP in the dark, as well as treatment under pre- or simultaneous irradiation conditions failed to produce biologically significant increases in the incidence of CA, whereas the positive control substances 4-nitroquinolone and 8-methoxypsoralene produced significantly positive effects in the dark or under simultaneous irradiation, respectively. Overall, our results failed to confirm the reported positive photo-genotoxic effects, and suggest that they may have been due to the test conditions, i.e. high irradiation doses, high cytotoxicity or re-irradiation of photo-products. In conclusion, our data suggest that, under standard conditions for testing photo-genotoxicity, RP had no in vitro genotoxic or photo-genotoxic potential and is therefore unlikely to pose a local or systemic genotoxic or photo-genotoxic risk.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9-9.4J/cm2 of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.