Use of ultraviolet light in sterilization/ sanitization and emergence of resistant microbes

There are several methods of sterilization (killing all microbes) including physical (dry heat, steam, low-temperature plasma gas to sterilization, filtration, boiling or tyndallisation, flaming and microwave) chemical (hydrogen peroxide, vaporized hydrogen peroxide, ethylene oxide, chlorine dioxide gas, per-formic acid, phenol, and vaporized per-acetic acid) and radiation (UV rays, gamma rays or electron beams). Most sterilizing methods are directly dependent on exposure time and cleanliness. If the exposure time is less or the object to be sterilized is not clean then sterilization failure is imminent.

Here the method to be discussed is ultraviolet (UV) light (UV-C or simply UV) sterilization. On the basis of wavelength UV light rays are grouped into three, and all three are different functionality.

1.   The wavelength from 315 to 400 nanometres (nm) is known as UVA. It accounts for 90-95% of UV radiation reaching the Earth and has the least harmful effect on our skin. This light is used for the polymerization of UV adhesives, and UV-activated gels, and in fluorescent inspection processes.

2.   A bit smaller wavelength of UV light, 280 - 315 nm is called UVB. This is the most common cause of sunburn and skin cancer. It constitutes 5-10% of UV radiation on the Earth. However, it also has some uses too like curing UV inks and lacquers through its ability to eliminate surface tacks.

3.   Among UVs, UVC has the smallest wavelength, 100 - 280 nm but is the most damaging UV. However, it rarely reaches the earth's surface as it’s filtered out by our atmosphere. Ozone depletion in the environment due to numerous reasons may increase its proportion to reach the earth. It is generated from welding, torches, and some mercury bulbs. In practice, it is commonly used for sterilization and germicidal applications in different settings. The most preferred wavelength for sterilization purposes is 254nm wavelength. It can kill nearly 99.9% of microbes on phones, and other surfaces within a minute. However, the highest microbial inactivation rates are reported with 265 nm light (1) through damaging protein and nucleic acid of microbes.

 

Sterilization efficiency of UVC: Studies have shown that UVC light used in hospitals may cut transmission of nosocomial superbugs by 30% (2). Another study revealed that using UVC sanitizers (on devices where UVC sanitizers can be used) may nearly kill 99.99% of bacteria, fungi, and viruses that cause disease (3). One company (MicroLumix, https://microlumix.com/) claims that their devices (GermPass) can kill up to 99.99931% of harmful microbes within 7 seconds of exposure.  A study (1) has shown that the UV inactivation rate of antibiotic-resistant bacteria (ARB) is higher than the inactivation rate of antibiotic-sensitive bacteria (ASB).

Despite big claims on the utility of UV light sterilization, research has revealed the drawbacks of using UVC as sanitizers or in sterilization.

1.     Use of UV sanitizers can be dangerous on repeated use because microbes have a remarkable ability to evolve and adapt thus the emergence of UV-resistant microbes may populate the niche emptied by death of UV susceptible microbes. The microbes resistant to very strong antimicrobials are usually more resistant to commonly used antimicrobials (4-5). The most UV-resistant bacteria including Micrococcus luteus, Micrococcus radiophilus, einococcus radiodurans, Hymenobacter actinosclerus, and Chryseobacterium species strains can tolerate high levels of UV radiation for a long time, and also stands to other ionizing radiation (gamma rays). Other radiation-resistant bacteria are shown to emerge in Pseudomonadota, Bacillota, and Actinomycetota or Bacteroidota groups of microbes due to their fast adaptability. A recent study on UVC sterilization cabins (1) revealed that at least 30 types of potentially pathogenic bacteria were detected inside UVC sterilization cabins. Among UV-resistant bacteria most abundant were Staphylococcus, Kocuria, Micrococcus, and Pseudomonas. Of the UV-resistant bacteria, some are as Staphylococcus and Pseudomonas are known as the most common cause of nosocomial pathogens too (https://www.ncbi.nlm.nih.gov/books/NBK559312/#:~:text=Common%20Gram%2Dpositive%20organisms%20include,%5D%5B8%5D%5B9%5D).

2.   Due to the very limited penetrating power of UVC, it doesn’t deeply penetrate the eyes/ skin and therefore poses a little risk of cataracts or skin cancer. However, repeated exposure or long-term exposure may increase the risk of cataracts and skin cancer over time. 

3.   Photokeratitis (corneal inflammation) is the most common problem even after a few seconds of exposure to UVC. After photokeratitis,      you feel severe eye pain as you had sand in your eyes which may result in temporary blindness for one or more days. It is also known as snow blindness, arc eye, or welders’ eye.

4.   Some of the UV sanitizers produce ozone, thus enhancing respiratory illnesses particularly irritating to airways, initiating and escalating allergies and asthma in susceptible persons.

5.   Direct exposure of skin to UV lamps does not cause skin damage but signs like sunburns fading away in a week.

6.  After UV sterilization or disinfection you may smell (similar to burning hair or the pungent odor of rotten eggs or garlic) its effect because our nose is extremely sensitive (detects even at 1 ppb level) to molecules produced in the environment under effect of UV sanitizers. The Mercaptan (R-SH), a sulfur compound, produced in the UV disinfection process is the same compound usually added in natural gas for the detection of any leakage. A detectable amount of mercaptan (about 1 ppb) is produced when the dust load in the air is 50 μg/m³. However, 500 ppb exposure for 8 hours is said safe for human health by most of the safety guidelines. The dangers of using UV disinfection may be felt more in a polluted environment as the air quality index (AQI) of 100 of 2.5 µm particles means 35 µg/m³, 200 AQI of 2.5 µm particles means 125.4 µg/m³, 300 AQI 2.5 µm particles means 225.4 µg/m³. Further, 100 AQI  of 10 µm particles means 154 µg/m³, 200 AQI of 10 µm particles means 354 µg/m³ and 300 AQI of 10 µm particles means 424 µg/m³. This means in polluted areas this smell is destined to prevail after the use of UV disinfection. Though a small risk the smell may mask or may make you ignore the natural gas leakage and that may be a serious fire and health risk. If pollutants are organic carbon compounds they sharply impact the inactivation of microbes even at high fluences (high photon intensity) of UV (calculated with the formula, UV dose (μJ/cm²) = UV intensity (μW/cm²) × exposure time (seconds), 1 rad = 0.01 J/ Kg). 

7. To produce UVC, UV lamps are used, and similar to any other light lamp the rays emitted by UV sanitizer may lose their potency over time of use because of the natural decay making the UV sanitizers less or non-effective on designated surfaces/objects. Though it may be overcome by mentioning the expiry hours of use of the life of the lamp, our habit is not such as changing the UVC emitters frequently.

 

Mechanisms of radiation resistance in bacteria

      Different radiation-resistant bacteria (Deinococcus radiodurans, Rubrobacter radiotolerans, Kineococcus radiotolerans, Halobacterium salinarum, Thermococcus gammatolerans and Pyrococcus furious, and other microbes) use one of multiple mechanisms (6, 7) to have radiation-resistance, viz.

1.   More efficient DNA repair system:  Deinococcus radiodurans (grow under continuous 6 kilorads (60 Gy)/h radiation and may tolerate up to 1,500 kilorads while Escherichia coli stops growing and get killed by 6 kilorads/h or instantly with  100 to 200 kilorads) have special DNA repair enzymes or proteins (DdrA and DdrB) those quickly repair broken DNA strands (caused by radiations like UV or gamma rays), to mend any damage to their genetic code (DNA). 

2.   Possessing multiple DNA copies allows them to tolerate significant DNA damage and mutations because a functional copy can be restored from undamaged copies. Deinococcus radiodurans contains 8 to 10 haploid genome copies during exponential growth and 4 genome copies in the stationary phase, is a highly radiation-resistant bacteria said to be one of the reasons to make them radiation resistant. However, the role of multiple genome copies has been jeopardized by the fact that many radiation-sensitive bacteria also have multiple chromosome copies like Borrelia burgdorferi (causes Lyme disease, has 11 copies of a single linear chromosome, and Azotobacter vinelandii (up to 80 chromosomes per cell), are sensitive to UV damage. 

3.   Re-combinational repair system, utilizing homologous recombination pathways to repair DNA damage by using undamaged DNA as a template, though it is present in all known microbes the system is certainly different in radiation-resistant bacteria (Dianococcus possesses an unusual domain architecture in the RecQ helicase; RecD protein in Deinococcus is unusual as it contains an N-terminal region of about 200 amino acid residues made of three tandem predicted HhH DNA-binding domains; but it is also present in radiation susceptible B. subtilis and Chlamydia). 

4.   Desiccation resistance genes: The direct relevance of desiccation resistance and radiation resistance is not lucid, but most of the radiation resistance microbes stop multiplication and stop their metabolic activities as soon as they are exposed to radiation for a substantially long time, and may have the danger of internal desiccation. Trehalose plays a major role in the desiccation resistance in E. coli and other bacteria including Deinococcus. However, Deinococcus has two additional genes for trehalose metabolism, maltooligosyl trehalose synthase (DR0463), and trehalohydrolase (DR0464) which might be important for their survival. 

5.   Production of enzymatic antioxidants: Reactive oxygen species (ROS) are generated on exposure to ionizing radiation on the absorption of photons by small molecules like water, and surrounding cellular bio-macromolecules. The ROS reacts with macromolecules of cellular contents of microbes, including DNA, RNA, and proteins to disrupt their normal functioning. To counter the effect of ROS microbes produce enzymes like superoxide dismutase, catalase, and peroxidases to scavenge and neutralize ROS (8) besides accumulating free radical scavengers like glutathione, carotenoids, pyrroloquinoline-quinone, deinoxanthin, and bacillithiol to generate protection from oxidative damage.

6.   Nucleoid structure: Bacteria having highly condensed DNA, irrespective of the shape of the nucleoid, is said to be more radiation resistant, as D. radiodurans maintains a highly condensed and organized nucleoid structure, even during their replication in exponential growth. The nucleoid structure of other radioresistant species of Dianococcus  (D. deserti, D. geothermalis) and Rubrobacter radiotolerans also maintain highly compact DNA. The compactness of DNA may passively contribute to radioresistance by preventing the dispersion of free DNA ends and maintaining the continuity of the genome even when it is fragmented (9). 

7.   Intracellular cation balance: Though it is not clearly understood how cation balance imparts radiation resistance to microbes, it is evident that cancer cells use this mechanism to counter the effect of radiation (10). Maintaining specific ion concentrations (Mn++) in the cytoplasm may contribute to radiation resistance by stabilizing cellular structures and scavenging radicals. 

 

References

1.   Molina-Menor E., et al. Ecology and resistance to UV light and antibiotics of microbial communities on UV cabins in the dermatology service of a Spanish hospital. Sci Rep. 2023; 13: 14547. https://doi.org/10.1038/s41598-023-40996-8

2.   Anderson D.J., et al. Enhanced terminal room disinfection and acquisition and infection caused by multidrug-resistant organisms and Clostridium difficile (the Benefits of Enhanced Terminal Room Disinfection study): A cluster-randomised, multicentre, crossover study. Lancet. 2017; 389(10071):805-814. doi:10.1016/S0140-6736(16)31588-4

3.   Mackenzie D. Ultraviolet light fights new virus. Engineering (Beijing). 2020; 6(8):851-853. doi:10.1016/j.eng.2020.06.009.

4.   Álvarez-Molina A. et al. Selection for antimicrobial resistance in foodborne pathogens through exposure to UV light and non-thermal atmospheric plasma decontamination techniques. Appl. Environ. Microbiol. 2020; 86(9), e00102-e120.  doi: 10.1128/AEM.00102-20.

5.   Vadhana P., et al. MexAB-OprM efflux pump of Pseudomonas aeruginosa offers tolerance to carvacrol: A herbal antimicrobial agent. Front Microbiol. 2019; 10:2664. doi:10.3389/fmicb.2019.02664

6.   Cho C., et al. Characterization of radiation-resistance mechanism in Spirosoma montaniterrae DY10^T in terms of transcriptional regulatory system. Sci Rep. 2023; 13, 4739. doi: 10.1038/s41598-023-31509-8.

7.   Makarova K.S., et al.  Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol. Molecular Biol Reviews. 2001; 65(1), 44. doi: 10.1128/MMBR.65.1.44-79.2001.

8.   Smith T.A., et al. Radioprotective agents to prevent cellular damage due to ionizing radiation. J Translational Medicine. 2017; 15, 232.  doi: 10.1186/s12967-017-1338-x.

9.   Levin-Zaidman S., et al. Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science. 2003; 299: 254–256.

10.            Heise N., et al. Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells. Cell Physiol. Biochem. 2010; 26, 597–608. doi: 10.1159/000322327.