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Background: Despite the great health significance of Legionella, there is only little information on their UV sensitivity. Besides Legionella pneumophila only L. longbeachae has been investigated so far. Methods: In this study L. rubrilucens has been spread on buffered charcoal yeast extract agar and irradiated with the 254 nm UV-C emission of a mercury vapor lamp. The disinfection success is measured by colony counting after incubation and comparison of the number of colonies on irradiated and unirradiated reference agar plates. Results: The average log-reduction dose is 1.08 mJ/cm² for free L. rubrilucens, which is at the lower end of the so far published Legionella log-reduction values, but all three Legionella species show similar UV-C sensitivities. Conclusion: The log-reduction dose of legionellae in amoebae has not been investigated, but with the observed high UV-C sensitivity for free Legionella, the idea of a future point-of-use disinfection by small UV-C LEDs in water-taps or shower heads appears to be realistic, even if legionellae are more resistant in amoebae.
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UV-C inactivation of Legionella rubrilucens
UV-C-Inaktivierung von Legionella rubrilucens
Background: Despite the great health significance of Legionella, there
is only little information on their UV sensitivity. Besides Legionella
pneumophila only L. longbeachae has been investigated so far.
Julian Schmid1
Katharina Hoenes1
Monika Rath1
Methods: In this study L. rubrilucens has been spread on buffered
charcoal yeast extract agar and irradiated with the 254 nm UV-C emis- Petra Vatter1
sion of a mercury vapor lamp. The disinfection success is measured by Martin Hessling1
colony counting after incubation and comparison of the number of
colonies on irradiated and unirradiated reference agar plates.
Results: The average log-reduction dose is 1.08 mJ/cm2for free
L. rubrilucens, which is at the lower end of the so far published Legion-
1 Ulm University of Applied
Sciences, Ulm, Germany
ella log-reduction values, but all three Legionella species show similar
UV-C sensitivities.
Conclusion: The log-reduction dose of legionellae in amoebae has not
been investigated, but with the observed high UV-C sensitivity for free
Legionella, the idea of a future point-of-use disinfection by small UV-C
LEDs in water-taps or shower heads appears to be realistic, even if le-
gionellae are more resistant in amoebae.
Keywords: Legionella, UV-C radiation, mercury vapor lamp, log-reduction
dose, point-of-use disinfection
Hintergrund: Trotz der großen gesundheitlichen Bedeutung von Legio-
nellen gibt es bisher nur wenige Untersuchungen zu ihrer UV-C-Empfind-
lichkeit. Außer Legionella pneumophila wurden bisher nur Ergebnisse
für L. longbeachae veröffentlicht.
Material und Methoden: In der hier vorgestellten Untersuchung wurden
Bakterienlösungen mit L. rubrilucens auf gepuffertem Aktivkohle-Hefe-
Extrakt verteilt und der 254 nm UV-C-Strahlung einer Quecksilberdampf-
lampe ausgesetzt. Der Desinfektionserfolg wird durch die Zählung von
Legionellen-Kolonien nach der Bebrütung ermittelt, indem die Anzahl
der Kolonien auf bestrahlten Agarplatten mit der Zahl an Kolonien auf
unbestrahlten Referenzplatten verglichen wird.
Ergebnisse: Die durchschnittlich notwendige UV-C-Strahlungsdosis für
eine Log-Stufen-Reduktion beträgt 1,08 mJ/cm2für planktonische
L. rubrilucens. Dieser Wert liegt im unteren Bereich der 4 bisher veröf-
fentlichten Legionellen-Studien und zeigt aber auch, dass alle 3 bisher
untersuchten Legionella-Spezies ähnliche UV-C-Empfindlichkeiten auf-
Schlussfolgerung: Die UV-C-Empfindlichkeit aller Legionellen-Spezies
scheint vergleichbar hoch zu sein. Selbst wenn Legionellen in Amöben
etwas unempfindlicher sein sollten, scheint die Idee einer zukünftigen
Point-of-Use-Desinfektion sehr realistisch. Kleine UV-C-LEDs in Wasser-
hähnen und Duschköpfen könnten Legionellen im schnell vorbeiströ-
menden Wasser signifikant inaktivieren. Der Einsatz einer solchen
Technik scheint insbesondere in Gesundheitseinrichtungen wie Kran-
kenhäusern oder Altenheimen sinnvoll.
Schlüsselwörter: Legionella, UV-C-Strahlung, Quecksilberdampflampe,
Log-Stufen-Reduktions-Dosis, Point-of-Use-Desinfektion
1/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Research Article
Figure 1: Absorption of a UV-C photon by DNA and formation of a thymine dimer
In 2014 about 7,000 cases of detected Legionnaires’
Disease were reported in the European Union [1] and the
number of unreported infections is probably much higher.
Legionnaires’ Disease is the most severe form of pneu-
monia infections caused by Gram-negative Legionella spp.
The most pathogenic Legionella species is Legionella
pneumophila that was identified in the first documented
Legionella outbreak in Philadelphia in 1976 [2]. Legion-
ellae infections often occur by inhaling aerosols derived
from contaminated water sources. The reservoirs of these
Gram-negative bacteria are natural surface water, and
especially drinking and cooling water systems [3]. There
are different approaches for clearance of legionellae in
these man-made water reservoirs but they usually do not
show long lasting effects leading to an omnipresent
danger of a Legionella contamination.
Among the well-known disinfection techniques is the ap-
plication of hot water with temperatures of about 70°C,
but it is technically difficult to reach these high tempera-
tures, especially in long pipe systems with lengths of 10 m
and more. Chemical disinfection, e.g. by chlorine com-
pounds, shows a reduced disinfection effect due to the
fact that legionellae are often incorporated in biofilms
and amoebae that protect the bacterium against direct
contact with disinfectants [4].
UV-C radiation was successfully applied for water disin-
fection already 100 years ago [5]. The radiation is usually
generated by mercury vapor lamps, which show strong
emission at a wavelength of 254 nm. The UV-C radiation
is absorbed by DNA, resulting in the formation of thymine
dimers [6] as illustrated in Figure 1. This DNA damage
hinders gene expression and DNA replication and should
lead to the death of the irradiated microorganisms. Unfor-
tunately UV-C disinfection offers no depot effect. Bacteria
that survived the irradiation can proliferate again on their
way through the water pipe system. Extremely aggravating
is the formation of biofilms on the pipe walls that are
capable of repeatedly releasing microorganisms including
Legionella that might get in direct contact with humans
without a further disinfection step.
With the recent developments of UV-C LEDs the idea of
a point-of-use disinfection with an UV-C LED directly in-
stalled in the water-tap or shower head becomes more
realistic. These LEDs are very small and offer only low
radiation intensities in the range of up to 90 mW [7],
which is about a factor of 100 below strong mercury vapor
lamps, but do not contain mercury and offer a longer life
time. The UV-C LED radiation power might be sufficient
for Legionella inactivation, because according to [8], [9],
[10], [11] the necessary log-reduction dose for
L. pneumophila is somewhere between 0.9 and
3.1 mJ/cm2and 1.4 mJ/cm2for L. longbeachae [9] – a
dose that could be reached within 0.03 s if a 90 mW UV-C
LED irradiates an area of 1 cm2.
The intention of this paper is to get more information on
necessary log-reduction doses for legionellae by perform-
ing experiments on L. rubrilucens and compare them with
the so far reported log-reduction doses. L. rubrilucens
was reported as co-pathogen in a case of Legionnaires’
Disease [12] but its UV-C sensitivity has not been inves-
tigated so far.
Materials and methods
The UV-C radiation was generated by a low pressure
mercury vapor lamp type TUV 8W FAM/10X25BOX of
Philips Lighting Holding B.V. (The Netherlands). It offers
2/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
Figure 2: Emission spectrum of the employed low pressure mercury vapor lamp
several emission lines in the UV and visible spectral
range, but the emission at 254 nm is by far the strongest
as can be seen in Figure 2. In a distance of 15 cm the
obtained 254 nm irradiance was 0.47 ± 0.05 mW/cm2
over an area of 10 x 30 cm2. It should be mentioned that
the total irradiance, including UV-B, UV-A and visible light
is slightly higher with about 0.65 mW/cm2but the disin-
fection contribution of these longer wavelength emissions
to the disinfection result is negligible with about 3% of
the impact of the 254 nm emission, calculated with the
spectral antimicrobial action spectra values given in [13]
for Escherichia coli, but it is assumed that other bacteria
show a similar behavior.
The bacteria investigated were L. rubrilucens (DSM
No. 11884) that were obtained as freeze-dried culture
from Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (Braunschweig, Germany) and cultiv-
ated in buffered yeast extract (BYE) broth [14]. For the
UV-C inactivation experiments solid buffered charcoal
yeast extract (BCYE) agar [14] was prepared in Petri
dishes with a diameter of 90 mm.
Bacterial suspensions of Legionella with an optical
density of 0.24 at 600 nm, that contained about
2.4 x 108CFU/ml in preliminary experiments, were further
diluted in phosphate-buffered saline to concentrations
below 10,000 CFU/ml. 100 µl of this samples were
carefully dispensed over the surface of the BCYE agar
The radiation experiments were performed with different
exposure times to achieve different irradiation doses. The
maximum irradiation time was 12 s, which resulted in
doses of up to 5.6 mJ/cm2. Afterwards the agar plates
were stored in an incubator at 37°C and high humidity
for 4 days before the plates were photographed for better
colony counting (see example in Figure 3) and the ob-
served colonies on irradiated agar plates were compared
to unirradiated reference plates. This procedure was
performed three times with three agar plates for each ir-
radiation dose and each run.
Results and discussion
The disinfection results are presented in Table 1 and
Figure 4. For higher irradiation doses the measured ratios
of surviving bacteria follow a straight line in this half
logarithmic diagram, which is equivalent to an exponential
decrease for increasing UV-C doses. But this straight line
doesn’t pass the expected 100%-point (1.0 E+00) for
0 mJ/cm2irradiation. This well-known phenomenon is
called “shoulder effect” [15]. The UV-C sensitivity is lower
for lower doses and increases asymptotically for higher
doses. Lower sensitivity means a higher necessary irradi-
ation dose to reach a log-reduction (log-reduction: de-
crease by a factor of ten). The lowest irradiation dose of
1.86 mJ/cm2in Table 1 led to 4.3% or 4.3 E-02 surviving
bacteria. This is equivalent to a log-reduction dose of
1.36 mJ/cm2. But if the calculation is based on the
highest applied irradiation dose of 5.58 mJ/cm2and
0.0007% or 7.0 E-06 surviving bacteria the average log-
reduction dose for L. rubrilucens becomes 1.08 mJ/cm2.
These results are in good agreement with published
L. pneumophila and L. longbeachae log-reduction doses
of 0.9 and 1.6 mW/cm2measured by [8], [9], [10].
3/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
Figure 3: Example of a BCYE agar plate with visible colonies of
after incubation
Table 1: Applied UV-C irradiation doses, average bacterial concentrations after irradiation for each run, average reference
concentrations for each run and ratio of surviving bacteria (averaged over all three runs)
4/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
Figure 4: Bacterial reduction for
L. rubrilucens
during UV-C irradiation. Error bars depict the standard deviation of the average
of three independent measurements.
It has to be mentioned that the here applied incubation
durations of 4 days at 37°C after irradiation were rather
short compared to several other published incubation
times. Antopol and Ellner incubated their irradiated
samples for 4–5 days at 35°C [8], Oguma et al. for 7 days
[10] and Cervero-Arago et al. even for 10 days at 37°C
[9]. It is theoretically conceivable that legionellae are still
in a viable but nonculturable (VBNC) state [16] after
4 days, and over the course of time could recover by dark
repair and photo reactivation mechanisms [17] and
subsequently form colonies again. This would reduce the
real UV sensitivity or increase the log reduction dose.
However, there is no general rule for Legionella incubation
periods. The international DIN-ISO standard “Water
quality – Enumeration of Legionella” [18] gives different
incubation times depending on the application. In the
case of samples, which are not clear for the presence of
legionellae, a 7–10 day incubation should be carried out
but for confirmation of legionella suspicion samples
should be incubated for a maximum of 5 days. The Aus-
tralian Legionella Control Guidelines indicate that the in-
cubation period may take up to 10 days, but the average
is between 5–7 days [19].
It is to be expected that in the presented experiments,
no difference in the number of colonies caused by VBNC
legionellae would have been determined between 4 and
10 days of incubation. The first reason for this assumption
is the observation that legionellae in the VBNC state seem
to need amoebae for their recovery [20] that were not
present here in any case. The second reason is the fact
that even 10 days is a rather short period compared to
the 145 days and more, in which legionellae could stay
in a VBNC state according to Alleron et al. [21].
Very important is the fact that legionellae in the VBNC
state – at least in an guinea pig animal model – do not
lead to infections [20]. Therefore, they probably play a
subordinate role during the immediate period after irradi-
So the observed log-reduction dose of about
1.08 mW/cm2for L. rubrilucens should not be influenced
by the shorter incubation time and the result consolidates
the impression that different Legionella species show a
similar high sensitivity to UV-C radiation. Legionellae in
amoebae required almost twice the UV-C dose of free le-
gionellae [9], but this is still a high sensitivity resulting in
log-reduction doses that are applicable with UV-C LEDs
within fractions of a second. This supports the idea of a
future point-of-use water disinfection by small UV-C LEDs
that are integrated in water-taps and shower heads and
irradiate the pre-passing water at least in healthcare in-
stitutions like hospitals or nursing homes.
This work was financially supported by the German Fed-
eral Ministry of Economics and Technology within the ZIM
project “Clean Spring” (grant number KF2186208CR4).
Competing interests
The authors declare that they have no competing in-
5/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
1. European Centre for Disease Prevention and Control.
Legionnaires’ disease in Europe, 2014. Stockholm: ECDC; 2016.
DOI: 10.2900/585125
2. Brenner DJ, Steigerwalt AG, McDade JE. Classification of the
Legionnaires' disease bacterium: Legionella pneumophila, genus
novum, species nova, of the family Legionellaceae, familia nova.
Ann Intern Med. 1979 Apr;90(4):656-8. DOI: 10.7326/0003-
3. Steinert M, Hentschel U, Hacker J. Legionella pneumophila: an
aquatic microbe goes astray. FEMS Microbiol Rev. 2002
Jun;26(2):149-62. DOI: 10.1111/j.1574-6976.2002.tb00607.x
4. Kim BR, Anderson JE, Mueller SA, Gaines WA, Kendall AM.
Literature review--efficacy of various disinfectants against
Legionella in water systems. Water Res. 2002 Nov;36(18):4433-
44. DOI: 10.1016/S0043-1354(02)00188-4
5. Henri V, Helbronner A, de Recklinghausen M. Stérilisation de
grandes quantités d'eau par les rayons ultraviolets. Compt Rend
Acad Sci. 1910;150:932-4.
6. Wacker A, Dellweg H, Weinblum D. Strahlenchemische
Veränderung der Bakterien-Desoxyribonucleinsäure in vivo.
Naturwissenschaften. 1960;47(20):477. DOI:
7. Inoue S, Naoki T, Kinoshita T, Obata T, Yanagi H. Light extraction
enhancement of 265 nm deep-ultraviolet light-emitting diodes
with over 90 mW output power via an AlN hybrid nanostructure.
Appl Phys Lett. 2015; 106(13):131104. DOI:
8. Antopol SC, Ellner PD. Susceptibility of Legionella pneumophila
to ultraviolet radiation. Appl Environ Microbiol. 1979
9. Cervero-Aragó S, Sommer R, Araujo RM. Effect of UV irradiation
(253.7 nm) on free Legionella and Legionella associated with
its amoebae hosts. Water Res. 2014 Dec;67:299-309. DOI:
10. Oguma K, Katayama H, Ohgaki S. Photoreactivation of Legionella
pneumophila after inactivation by low- or medium-pressure
ultraviolet lamp. Water Res. 2004 Jun;38(11):2757-63. DOI:
11. Wilson BR, Roessler PF, van Dellen E, Abbaszadegan M, Gerba
CP. Coliphage MS-2 as a UV water disinfection efficacy test
surrogate for bacterial and viral pathogens. Proceedings, 1992
Water Quality Technology Conference; Toronto, Ontario;
November 15-19, 1992. p. 219-35.
12. Matsui M, Fujii S, Shiroiwa R, Amemura-Maekawa J, Chang B,
Kura F, Yamauchi K. Isolation of Legionella rubrilucens from a
pneumonia patient co-infected with Legionella pneumophila. J
Med Microbiol. 2010 Oct;59(Pt 10):1242-6. DOI:
13. DIN Deutsches Institut für Normung eV. Optical radiation physics
and illuminating engineering – Part 10: Photobiologically effective
radiation, quantities, symbols and action spectra. DIN 5031-10.
Berlin: Beuth Verlag; 2013.
14. Buchrieser C, Hilbi H, editors. Legionella: Methods and Protocols.
Totowa, NJ: Humana Press; 2013. DOI: 10.1007/978-1-62703-
15. Tang WZ, Sillanpää M. Bacteria sensitivity index of UV disinfection
of bacteria with shoulder effect. J Environ Chem Eng.
2015;3(4):2588-96. DOI: 10.1016/j.jece.2015.09.010
16. Hussong D, Colwell RR, O'Brien M, Weiss E, Pearson AD, Weiner
RM, Burge WD. Viable Legionella pneumophila Not Detectable
by Culture on Agar Media. Nat Biotechnol. 1987; 5(9):947–50.
DOI: 10.1038/nbt0987-947
17. Quek PH, Hu J. Indicators for photoreactivation and dark repair
studies following ultraviolet disinfection. J Ind Microbiol
Biotechnol. 2008 Jun;35(6):533-41. DOI: 10.1007/s10295-008-
18. DIN Deutsches Institut für Normung eV. Water quality –
Enumeration of Legionella (Draft version). Berlin: Beuth Verlag;
19. Australian Government. Guidelines for Legionella Control: In the
operation and maintenance of water distribution systems in
health and aged care facilities. Canberra: Australian Government;
2015. Available from:
20. Steinert M, Emödy L, Amann R, Hacker J. Resuscitation of viable
but nonculturable Legionella pneumophila Philadelphia JR32 by
Acanthamoeba castellanii. Appl Environ Microbiol. 1997
21. Alleron L, Merlet N, Lacombe C, Frère J. Long-term survival of
Legionella pneumophila in the viable but nonculturable state
after monochloramine treatment. Curr Microbiol. 2008
Nov;57(5):497-502. DOI: 10.1007/s00284-008-9275-9
Corresponding author:
Prof. Dr. Martin Hessling
Ulm University of Applied Sciences, Albert-Einstein-Allee
55, 89081 Ulm, Germany, Phone: 0731/50-28602
Please cite as
Schmid J, Hoenes K, Rath M, Vatter P, Hessling M. UV-C inactivation of
Legionella rubrilucens. GMS Hyg Infect Control. 2017;12:Doc06.
DOI: 10.3205/dgkh000291, URN: urn:nbn:de:0183-dgkh0002911
This article is freely available from
©2017 Schmid et al. This is an Open Access article distributed under
the terms of the Creative Commons Attribution 4.0 License. See license
information at
6/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
... 6 Its mechanism is based on the destruction of the DNA of undesired microorganisms. 7 In Schmid et al study, 8 bacterial solutions containing Legionella rubrilucens were spread on buffered charcoal yeast extract agar and exposed to 254 nm UV-C radiation from a mercury vapor lamp. Successful disinfection was verified by counting Legionella colonies after incubation and comparing the number of colonies on irradiated agar plates with those on unirradiated reference plates. ...
... The obtained result is presented in Figure 1. 8 On average, a UV-C dose of 1.1 mJ/ cm 2 reduces the bacterial count by 90%, which is consistent with the results of two previous studies on other species Abstract Legionella infections caused by contaminated water are a widespread problem worldwide. Discharge lamps like mercury vapor lamps are widely known for the disinfection properties of their radiation, but they suffer technical disadvantages, like high voltages and toxic content, and are, therefore, not suitable for some infection control applications. ...
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Legionella infections caused by contaminated water are a widespread problem worldwide. Discharge lamps like mercury vapor lamps are widely known for the disinfection properties of their radiation, but they suffer technical disadvantages, like high voltages and toxic content, and are, therefore, not suitable for some infection control applications. New high-intensity ultraviolet (UV) and violet LEDs offer new approaches for Legionella control, because these bacteria are significantly light sensitive compared to other pathogens. One of the most important infection pathways is the inhalation of Legionella-containing aerosols during showering. This problem could be reduced by a single strong UV LED within the shower head, which irradiates the passing water for some milliseconds. This practice can be especially beneficial in hospitals and care facilities. UV light offers only a limited penetration depth, however, even in pure water. To disinfect larger water volumes, e.g., in water dispensers, visible violet LEDs are more appropriate. Unfortunately, up to now, neither approach has been given much attention by potential users.
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Green algae produce valuable lipids as carbon-recycling resources. Collecting whole cells with the intracellular lipids could be efficient without cell burst; however, direct use of the cells causes microbial contamination in environments. Then, UV-C irradiation was selected to satisfy the requirements of avoiding the cell burst and sterilizing cells with Chlamydomonas reinhardtii. UV-C irradiation with 1.209 mW·cm−2 showed enough sterilization activity for 1.6 × 107 cells·mL−1 of C. reinhardtii in a depth of 5 mm for 10 min. The irradiation showed no effects to composition and contents of the intracellular lipids. From the viewpoint of transcriptomic analysis, the irradiation displayed possibilities of (i) inhibition of the synthesis of lipids due to decrement of the transcription of related genes, such as diacylglycerol acyl transferase and cyclopropane fatty acid synthase, and (ii) activation of lipid degradation and the production of NADH2+ and FADH2 due to increment of the transcription of related genes, such as isocitrate dehydrogenase, dihydrolipoamide dehydrogenase and malate dehydrogenase. Irradiation until cell death could be insufficient to shift the metabolic flows even though the transcriptions were already shifted to lipid degradation and energy production. This paper is the first report of the response of C. reinhardtii to UV-C irradiation on the transcription level.
... Mercury-based UV lamps are commonly used in drinking water and wastewater treatment plants [18]. Disinfection efficacy using low-pressure and mediumpressure mercury UV lamps has previously been reported for several Legionella species (e.g., L. bozemanii, L. dumoffii, L. longbeacheae, L. micdadei, and L. pneumophila (Lp)) as well as Lp sg1, 2, 7, and 8 strains [22][23][24][25][26][27][28][29][30]. However, there are major disadvantages of mercurybased UV lamps compared to UV light-emitting diodes (LEDs). ...
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... In regard to the relationship between OPPPs and free-living amoebae, Cervero-Aragó et al. [170] determined that higher fluence was required for a 4-log (99.99%) reduction in Legionella species when co-cultured with amoeba. UV disinfection has been successfully applied to control Legionella in building water systems [167,171]. ...
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... Specificity of the measurement tool to UV-C light is important because many UV-C sources (e.g., medium-pressure mercury or xenon arc lamps) emit wavelengths outside of the germicidal UV-C range [7]. Even near-monochromatic UV-C sources such as low-pressure mercury and amalgam lamps emit wavelengths >300 nm ( Fig 1E) [34]. We asked two questions: (1) how much of the PCI color change from low-pressure amalgam bulbs is due to non-germicidal wavelengths, and (2) how susceptible are PCIs to perturbation by spurious solar exposure during transport or storage. ...
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With COVID-19 N95 shortages, frontline medical personnel are forced to reuse this disposable–but sophisticated–multilayer respirator. Widely used to decontaminate nonporous surfaces, UV-C light has demonstrated germicidal efficacy on porous, non-planar N95 respirators when all surfaces receive ≥1.0 J/cm ² dose. Of utmost importance across disciplines, translation of empirical evidence to implementation relies upon UV-C measurements frequently confounded by radiometer complexities. To enable rigorous on-respirator measurements, we introduce a photochromic indicator dose quantification technique for: (1) UV-C treatment design and (2) in-process UV-C dose validation. While addressing outstanding indicator limitations of qualitative readout and insufficient dynamic range, our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%) required for UV-C dose measurements. In a measurement infeasible with radiometers, we observe a striking ~20× dose variation over N95s within one decontamination system. Furthermore, we adapt consumer electronics for accessible quantitative readout and use optical attenuators to extend indicator dynamic range >10× to quantify doses relevant for N95 decontamination. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical considerations for both photochromic indicators themselves and UV-C decontamination processes.
... (e) Robust UV-C measurements must meet key specifications, including dynamic range of quantification (before the indicator saturates), relative measurement uncertainty (determined from error propagation from the confidence intervals on the calibration curve fit), accuracy of the measurement compared to a calibrated standard sensor, and specificity of the PCI response to the germicidal wavelength range (in order to accurately report germicidal activity). Specificity curves adapted from Kowalski 7 , Schmid et al. 26 , and Blickenstorfer and Aufmuth 27 . SARS-CoV-2 diagrams adapted from an image by Maya Peters Kostman for the Innovative Genomics Institute. ...
With COVID-19 N95 respirator shortages, frontline medical personnel are forced to reuse this disposable − but sophisticated − multilayer textile respirator. Widely used for decontamination of nonporous surfaces, UV-C light has germicidal efficacy on porous, non-planar N95 respirators when ≥1.0 J/cm^2 dose is applied across all surfaces. Here, we address outstanding limitations of photochromic indicators (qualitative readout and insufficient dynamic range) and introduce a photochromic UV-C dose quantification technique for: (1) design of UV-C treatments and (2) in-process UV-C dose validation. Our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%). Furthermore, we adapt consumer electronics for accessible quantitative readout and extend the dynamic range >10× using optical attenuators. In a measurement infeasible with radiometers, we observe striking 20× dose variation over 3D N95 facepieces. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical design considerations for both photochromic indicators and UV-C decontamination.
Polyoxometalates (POMs) are metal-oxo clusters formed by early transition metals in their highest oxidation state. More particularly, they exhibit adjustable redox properties, i.e. they can be reduced successively and reversibly to one or several electrons, such that they find their applications as redox mediators or electron reservoirs for electrocatalysis, solar energy conversion, molecular batteries or information storage. Previous results of POMs deposition onto surface characterized by electrical transport measurements were encouraging to envision the integration of active layers of POMs into nanodevices for molecular electronics. The electrical properties of the resulting device will depend on the assembly quality. The mastering of POMs immobilization onto substrates and the control of the POM/substrate interface is still required. In this context, NH2/NH3+-terminated organic monolayers grafted on oxide-free silicon substrates were prepared by hydrosilylation and post-modifications. After the electrostatic deposition of photoreducible (nBu4N)3[PMo12O40] POMs, the photoreduction of the immobilized POMs was studied by means of several characterization tools (XPS, UV-Vis-NIR spectroscopy, KPFM). Preliminary electrical characterization of a POM-based pseudo MOSFET prototype device was carried out to study the influence of the POM redox state on the device conductance and to study the possible photoswitching property. Concurrently, the covalent grafting of POM hybrids onto functionalized, hydrogenated or oxidized Si surfaces was explored during the project, with the prospects of a more stable, controlled and tunable POM/substrate interaction.
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Ultraviolet Germicidal Irradiation (UVGI) has grown significantly as an optimistic alternative to chemical disinfection for surface and air disinfection in the healthcare sector. The application of UVGI in robotic technology has led to new opportunities in developing the disinfection robot which allows us to prevent pathogen transmission, reduce human participation and cross infection, then achieve efficient, quick and environmentally friendly sterilization. The aim of this paper is to review the outstanding achievements in UV-C disinfection robot sector. This review is based on the reports on 35 types of UV-C disinfection robots in both academic research and commercial sector. The current and future trends in the UV-C lamp technologies and the technical capacities of the robot are also discussed.
Biofilms can harbor a wide range of microorganisms, including opportunistic respiratory pathogens, and their establishment on engineered surfaces poses a risk to public health and industry. The emergence of compact germicidal ultraviolet light-emitting diodes (UV LEDs) may enable their incorporation into confined spaces to inhibit bacterial surface colonization on inaccessible surfaces, such as those in premise plumbing. Such applications necessitate knowledge of the quantitative response of biofilm growth rates to UV exposure on continuously irradiated surfaces. Herein, we performed experiments at varying flow cell temperatures in order to control baseline biofilm growth rates in the absence of UV; then, biofilm growth was compared under the same conditions but with simultaneous UVC irradiation. The inhibiting effect of UV irradiation on biofilm growth kinetics was diminished by more favorable growth conditions (higher temperature). Increasing the temperature by 10°C resulted in an increase in biovolume by 193% under a UVC (254 nm) intensity of ∼60 µW/cm². We further fitted an existing intensity response model to the biofilm growth data and analyzed the effects of temperature on model parameters, which were consistent with a hypothesized shielding effect arising from the deposition of extracellular colloidal materials. The shielding effect was found to result in breakthrough behavior of irradiated biofilms after 48 h, wherein accumulation of shielding substances eventually enabled biofilm establishment at even relatively high irradiation intensities (102.3 µW/cm²). With respect to applications of UVC irradiation for biofilm prevention, these results imply that surfaces more prone to bacterial colonization require disproportionately higher-intensity UVC irradiation for prevention of biofilm establishment, and continuous surface irradiation may be inadequate as a sole intervention for biofilm prevention in many scenarios.
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Introduction During pandemics, such as the SARS-CoV-2, filtering facepiece respirators plays an essential role in protecting healthcare personnel. The recycling of respirators is possible in case of critical shortage, but it raises the question of the effectiveness of decontamination as well as the performance of the reused respirators. Method Disposable respirators were subjected to ultraviolet germicidal irradiation (UVGI) treatment at single or successive doses of 60 mJ/cm2 after a short drying cycle (30 min, 70°C). The germicidal efficacy of this treatment was tested by spiking respirators with two staphylococcal bacteriophages (vB_HSa_2002 and P66 phages). The respirator performance was investigated by the following parameters: particle penetration (NaCl aerosol, 10–300 nm), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry and mechanical tensile tests. Results No viable phage particles were recovered from any of the respirators after decontamination (log reduction in virus titre >3), and no reduction in chemical or physical properties (SEM, particle penetrations <5%–6%) were observed. Increasing the UVGI dose 10-fold led to chemical alterations of the respirator filtration media (FTIR) but did not affect the physical properties (particle penetration), which was unaltered even at 3000 mJ/cm2 (50 cycles). When respirators had been used by healthcare workers and undergone decontamination, they had particle penetration significantly greater than never donned respirators. Conclusion This decontamination procedure is an attractive method for respirators in case of shortages during a SARS pandemic. A successful implementation requires a careful design and particle penetration performance control tests over the successive reuse cycles.
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Detection and monitoring of genetically engineered microorganisms released to the environment, as well as pathogens, are primary factors in risk assessment. Culture methods have been proposed for both detection and monitoring. However, microorganisms in natural systems may not always be culturable. We surveyed environmental samples collected from sources implicated in an epidemic of Legionnaires' disease and, although no cultures were recovered from environmental samples, numerous cells were observed by fluorescent microscopy when anti-L. pneumophila group 1 antibody was used. Similar observations have often been made by others. To study this loss of culturability, L. pneumophila strains were maintained in a microcosm (vessels containing sterilized environmental water) and assayed at intervals for growth an appropriate media, and lethality for chick embryos. At 4 C, the decimal rate of decline of colony forming cells was approximately 29 days; at 37 C it was 13 days. When microcosm water samples were injected into embryonated eggs, far greater chick embryo mortality was observed than could be accounted for by the number of culturable cells in the injections. Thus, previously non-culturable Legionella had multiplied once again and become culturable. These results indicate that samples that do not contain culturable cells, may contain cells that are viable, as demonstrated by their pathogenicity for chick embryos. The fluorescent antibody assay may provide a valuable indication of the presence of such viable but non-culturable cells. Keywords: Bioassay, Reprints, Cultures(Biology).
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We report what we believe to be the first clinical isolation of Legionella rubrilucens from a pneumonia patient co-infected with Legionella pneumophila. L. rubrilucens strains were found in both a patient's sputum and the water of a hot spring in which the patient bathed, and DNA analysis by PFGE showed that they were indistinguishable.
Legionnaires’ disease is a severe form of pneumonia that can occur in epidemics of several hundred cases. This atypical pneumonia is characterized by a high mortality rate and affects primarily elderly and immuno-compromised individuals. An in-depth understanding of the ecology and virulence of Legionella spp. will contribute to an efficient and sustainable elimination of the bacteria from anthropogenic water systems and might lead to the development of novel therapeutics. The many different methods described in Legionella: Methods and Protocols are all specifically adapted and developed for the study of distinct features of L. pneumophila, and have already led to exciting discoveries. Most of these techniques can also be applied to the study of other bacterial pathogens, in particular intracellular pathogens like Shigella, Salmonella or Listeria. . Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Legionella: Methods and Protocols will not only be useful for research groups studying Legionella, but also for a broader scientific community studying the epidemiology, typing, physiology, pathogenesis, immunity, genetics and evolution of other bacterial pathogens.
In research, design, and validation of UV disinfection systems of a given bacteria, shoulder effect makes prediction of fluence required difficult. This study theoretically analyzes the fluence required to inactivate any bacteria vs. E. coli as the reference bacteria, at the same Log I. Bacteria sensitivity index (BSI) is defined as the ratio between the inactivation rate constant ki of any bacteria for the linear portion of the dose response curve to that kir of E. coli. Among three sets of fluence requirement reported by the US EPA [4], Sommer et al. [5,20], and Chang et al. [6], the fluence required from the EPA is selected as the most suitable reference according to its predictive power as well as regulatory purpose. In the present study, shoulder broadness (SB), H0, is approximated with the intercept of the linear portion at the Log I = 0. A universal equation which can be used to predict the fluence required of any bacteria with a shoulder: H = (0.914 ± 0.055)∗(BSI)E∗(Log I) + H0. Furthermore, shoulder broadness index (SBI) is defined as the ratio between the fluence difference of any bacteria and the reference bacteria such as E. coli divided by the SB of E. coli. To validate the equation, an independent set of fluence data during UV disinfection of four different ARBs reported by McKinney and Pruden [8] was used. When the predicted fluence by using BSI of the ARBs is compared with the reported fluence at different Log I levels with less than 10% error with Log I up to 5.
Deep-ultraviolet (DUV) aluminum gallium nitride-based light-emitting diodes (LEDs) on transparent aluminum nitride (AlN) substrates with high light extraction efficiency and high power are proposed and demonstrated. The AlN bottom side surface configuration, which is composed of a hybrid structure of photonic crystals and subwavelength nanostructures, has been designed using finite-difference time-domain calculations to enhance light extraction. We have experimentally demonstrated an output power improvement of up to 196% as a result of the use of the embedded high-light-extraction hybrid nanophotonic structure. The DUV-LEDs produced have demonstrated output power as high as 90 mW in DC operation at a peak emission wavelength of 265 nm.
Water systems are the primary reservoir for Legionella spp., where the bacteria live in association with other microorganisms, such as free-living amoebae. A wide range of disinfection treatments have been studied to control and prevent Legionella colonization but few of them were performed considering its relation with protozoa. In this study, the effectiveness of UV irradiation (253.7 nm) using low-pressure lamps was investigated as a disinfection method for Legionella and amoebae under controlled laboratory conditions. UV treatments were applied to 5 strains of Legionella spp., 4 strains of free-living amoeba of the genera Acanthamoeba and Vermamoeba, treating separately trophozoites and cysts, and to two different co-cultures of Legionella pneumophila with the Acanthamoeba strains. No significant differences in the UV inactivation behavior were observed among Legionella strains tested which were 3 logs reduced for fluences around 45 J/m(2). UV irradiation was less effective against free-living amoebae; which in some cases required up to 990 J/m(2) to obtain the same population reduction. UV treatment was more effective against trophozoites compared to cysts; moreover, inactivation patterns were clearly different between the genus Acanthamoeba and Vermamoeba. For the first time data about Vermamoeba vermiformis UV inactivation has been reported in a study. Finally, the results showed that the association of L. pneumophila with free-living amoebae decreases the effectiveness of UV irradiation against the bacteria in a range of 1.5-2 fold. That fact demonstrates that the relations established between different microorganisms in the water systems can modify the effectiveness of the UV treatments applied.
Legionella pneumophila, a facultative intracellular human pathogen, can persist for long periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing systems is often difficult. We tested L. pneumophila survival after monochloramine treatment. Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal-yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed after >4 months after treatment. These results demonstrate for the first time that L. pneumophila could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L. pneumophila in water networks is generally done by enumeration on standard solid medium. This method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long periods and should be resuscitated by amoeba. These cells constitute potential sources of contamination and should be taken into account in monitoring water networks.