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UV-C inactivation of Legionella rubrilucens
UV-C-Inaktivierung von Legionella rubrilucens
Abstract
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
Zusammenfassung
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-
weisen.
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
OPEN ACCESS
Figure 1: Absorption of a UV-C photon by DNA and formation of a thymine dimer
Introduction
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
plate.
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
Legionella
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-
ation.
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.
Notes
Acknowledgement
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-
terests.
5/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
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Corresponding author:
Prof. Dr. Martin Hessling
Ulm University of Applied Sciences, Albert-Einstein-Allee
55, 89081 Ulm, Germany, Phone: 0731/50-28602
hessling@hs-ulm.de
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
http://www.egms.de/en/journals/dgkh/2017-12/dgkh000291.shtml
Published:
2017-04-10
Copyright
©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 http://creativecommons.org/licenses/by/4.0/.
6/6GMS Hygiene and Infection Control 2017, Vol. 12, ISSN 2196-5226
Schmid et al.: UV-C inactivation of Legionella rubrilucens
