Detection of amoeba-associated Legionella pneumophila in hospital water networks of Johannesburg

Abstract

The prevalence of free-living amoeba and associated Legionella spp. in hospital water systems may pose a risk of Legionnaires’ disease to immuno-compromised patients. This study investigated the occurrence of amoeba-associated Legionella pneumophila in three South African hospital water systems. A total of 98 water and/or biofilm samples were collected from the sterilisation unit, theatres, neonatal ward and intensive care units. Amoebae were isolated from 71 (72.4%) samples. Isolated amoebae were analysed using qPCR and culture methods to test for the presence of Legionella. L. pneumophila did not grow on selective media in any of the samples. A total of 7 out of the 71 (9.9%) amoeba-positive samples showed a positive reaction for L. pneumophila using qPCR. Although relatively few samples were positive for Legionella in this preliminary study, the association with amoeba still presents a potential public health risk to immuno-compromised patients when exposed to contaminated water.

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Southern African Journal of Infectious Diseases
ISSN: 2312-0053 (Print) 2313-1810 (Online) Journal homepage: http://www.tandfonline.com/loi/ojid20
Detection of amoeba-associated Legionella
pneumophila in hospital water networks of
Johannesburg
P Muchesa, M Leifels, L Jurzik, TG Barnard & C Bartie
To cite this article: P Muchesa, M Leifels, L Jurzik, TG Barnard & C Bartie (2018): Detection of
amoeba-associated Legionella pneumophila in hospital water networks of Johannesburg, Southern
African Journal of Infectious Diseases, DOI: 10.1080/23120053.2018.1434060
To link to this article: https://doi.org/10.1080/23120053.2018.1434060
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South Afr J Infect Dis
ISSN 2312-0053 EISSN 2313-1810
© 2018 The Author(s)
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Southern African Journal of Infectious Diseases 2018; 1(1):1–4
https://doi.org/10.1080/23120053.2018.1434060
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Detection of amoeba-associated Legionella pneumophila in hospital water
networks of Johannesburg
P Muchesa
a
*, M Leifels
b
, L Jurzik
b
, TG Barnard
a
and C Bartie
a
a
Water and Health Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, South Africa
b
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Bochum, Germany
*Corresponding author. emails: pmuchesa@uj.ac.za; chesap@gmail.com
The prevalence of free-living amoeba and associated Legionella spp. in hospital water systems may pose a risk of Legionnaires’
disease to immuno-compromised patients. This study investigated the occurrence of amoeba-associated Legionella pneumophila
in three South African hospital water systems. A total of 98 water and/or biolm samples were collected from the sterilisation
unit, theatres, neonatal ward and intensive care units. Amoebae were isolated from 71 (72.4%) samples. Isolated amoebae were
analysed using qPCR and culture methods to test for the presence of Legionella. L. pneumophila did not grow on selective media
in any of the samples. A total of 7 out of the 71 (9.9%) amoeba-positive samples showed a positive reaction for L. pneumophila
using qPCR. Although relatively few samples were positive for Legionella in this preliminary study, the association with amoeba
still presents a potential public health risk to immuno-compromised patients when exposed to contaminated water.
Keywords: amoeba, Legionella pneumophila, Legionnaires disease
Introduction
Legionella species are gram-negative, non-spore-forming, rod-
shaped or lamentous fastidious aerobic bacteria. They have
been isolated from man-made aquatic environments such as
cooling towers, hot tubs, air-conditioning systems and potable
water systems where they can proliferate at temperatures
between 20° and 50°C.1,2. Among the 58 described Legionella
spp., Legionella pneumophila serogroup 1 is the most common
serotype responsible for at least 84% of infections in humans.
Inhalation of aerosols containing Legionella spp. may result in
two kinds of infections, the mild, non-fatal, inuenza-like illness
Pontiac fever and the severe form of pneumonia and potentially
fatal Legionnaires’ disease (LD) in both community and health-
care settings. Aspiration of contaminated water or direct contact
with surgical wounds are the other less common modes of
transmission.3 Worldwide, there are few LD cases where the
environmental source of Legionella infection is determined
successfully. Studies have shown that contaminated potable
water supplies within hospitals could be responsible for hospital-
acquired LD cases.4,5
The discovery by Rowbotham6 that L. pneumophila in aquatic
environments can exist as an intracellular parasite of amoebae
has provided a link between bacterial interactions in the
environment and human disease. Legionella spp. are known to
naturally infect and survive within the amoebae genera that
include Acanthamoeba, Vermamoeba and Naegleria in the
environment.7–10 Internalised Legionella spp. are protected inside
amoeba-resistant cysts and can survive adverse aquatic
environment conditions like the presence of chlorine, commonly
used to treat water in engineered water systems. In addition to
survival, Legionella spp. can multiply inside the amoeba before
being released into the environment in vesicles or by lysis.11,12
Legionella spp. released from amoebae have been reported to
increase in virulence, biocide and antibiotic resistance as a result
of horizontal gene transfer between several intracellular bacteria
and their amoebae hosts.13–15 This can have remarkable public
health implications if immunocompromised patients are
exposed to water systems in hospitals contaminated with
amoeba and Legionella. Our previous studies have shown a high
amoeba prevalence and coexistence of other clinically important
gram-negative bacteria in public hospitals of Johannesburg.16
The current study is preliminary work to establish potential
sources of hospital-acquired LD by screening hospital waters
and/or biolm for the presence of amoeba-associated L.
pneumophila.
Methods
Sample collection
A walk-through assessment of the water systems of three
Johannesburg hospital facilities was conducted to identify areas
of high-risk exposure to waterborne pathogens. A systematic
sampling strategy was followed to collect and analyse water
and/or swab samples of every second water tap. Water samples
were collected from the cold-water system. A total of 98 samples
(51 water and 47 swab) were collected from Hospital A: the
sterilisation unit (n = 8) and theatres (n = 42); Hospital B: neonatal
ward (n = 13); and Hospital C: intensive care units (n = 35). The
samples were analysed within 24 h of collection. Swab samples
were collected by swabbing the inside surfaces of the taps prior
to opening them. Water samples (500 ml) were collected after
running the taps for 1– 2min in 1 litre of sterile sampling bottles
containing 5 mg/l sodium thiosulfate (Merk, Modderfontein,
South Africa). At each sampling site, water temperature was
measured with a portable COMBO TESTER® (Hanna Instruments,
Bedfordview, South Africa) according to the manufacturer’s
instructions. Residual chlorine was measured using a chlorine
photometer (Hanna Instruments, Bedfordview, South Africa)
according to the manufacturer’s instructions.
Sample analysis
As the rationale for this work is the detection and quantication
of amoeba-associated Legionella, an enrichment technique used
to detect amoeba was adapted from a previous study.16 Briey,
500ml of water sample and 10ml of Page’s amoebal saline buer
(PAS) swab suspension was concentrated by ltration using a

2 Southern African Journal of Infectious Diseases 2018; 1(1):1–4
0.45μm nitrocellulose membrane (Merk, Modderfontein, South
Africa). The membrane was placed upside down onto a non-
nutrient agar (NNA) plate overlaid with heat-killed E. coli (NNA-
HKEC plates). The plates were incubated at 32°C and checked
daily under light or inverted microscope for the appearance of
amoebal trophozoites and cysts for up to 21days. Plates with
amoebal growth were puried by aseptically cutting small agar
plugs, placing them upside down onto fresh NNA-HKEC plates,
and incubating as before. Once puried, amoeba were re-
suspended in 1 ml sterile PAS, inoculated into a sterile 24-well
at-bottomed microtiter plate (Life Technologies, Randburg,
South Africa), and again incubated at 32°C. The plates were then
observed for the morphological appearance of amoebae
trophozoites and/or cysts under an inverted microscope using a
40-x objective (SMM Instruments, Johannesburg, South Africa).
Amoeba from the amoeba-positive samples were lysed by
passing amoeba cells suspended in 300 μl Page amoebal saline
through a 27-gauge syringe lter with a pore size of 0.45 μm
three times to release any potential Legionella species. One
hundred microlitres of the resulting suspension were inoculated
on non-selective buered charcoal yeast extract (BCYE) and
selective Glycine-Vancomycin-Polymyxin-Cycloheximide(GVCP)
Legionella agar (Quantum Biotechnologies, Randburg, South
Africa) and incubated aerobically at 37°C for up to 10days to
culture Legionella. DNA was extracted from cultures from
remaining 100 μl amoeba suspension for PCR identication and/
or screening for L. pneumophila using the QIAmp DNA Blood
Mini Kit (Qiagen, Hilden, Germany) as per the manufacturer’s
instructions and amplied according to Myamoto et al.,17 with
minor adaptions to increase the specicity and sensitivity of the
assay according to Omiccioli et al.18 The DNA Blood Mini
extraction kit was chosen due to its high DNA recovery rate and
low susceptibility to the presumed co-concentration of organic
and inorganic polymerase inhibitory substances in the samples
as demonstrated by a previous study.19 Both quantitative and
qualitative detection of L. pneumophila was performed using
PCR with primers LpneuF (5′-CCGATGCCACATCATTAGC-3′) and
LpneuR (5′-CCAATTGAGCGCCACTCATAG-3′) and for the
quantication of L. pneumophila after the samples were shown
to contain Legionella DNA by gel electrophoresis, a TaqMan
probe, LpneuP (5′-6-carboxyuorescein [FAM]-
TGCCTTTAGCCATTGCTTCCG-BHQ1–3′). As stated above,
adapted amplication cycle conditions are listed in Miyamoto
et al. and Omiccioli et al. 17, 18 The amplication mixture consisted
of 25 μl of iQ supermix (Life Science, Veenendaal, the Netherlands),
0.4mg/ml of bovine serum albumin (Roche Diagnostics, Almere,
The Netherlands), 0.2μM each primer and probe, 0.2μM each
primer, and 10 μl of DNA template in a total reaction volume of
50 μl. To detect Legionella spp., the polymerase in the reaction
tubes was initially activated at 95°C for 90 s, followed by 43 cycles
of amplication using denaturation at 95°C for 3min followed by
annealing at 55°C for 30 s and extension at 72°C for 1min.
As it had to be expected that not all samples would be positive
for Legionella, a two-tier approach was chosen to conserve
resources: in a rst step, the eciency of PCR was conrmed
qualitatively by agarose gel electrophoresis with 8 μl of the PCR
product on 2% gel as shown in Figure 1. Quantication of
amplicons in GU/l (genomic units per litre) as well as data analysis
was performed with a real-time PCR Rotor-Gene 6000 Cycler
(Corbett Life Science, Mortlake, Australia) on samples positive for
Legionella according to ISO-TS 12869:2012 and the TaqMan
Probe. In brief, a calibration range that comprised four serial
dilutions of 25 to 25 000 GU of L. pneumophila (ATCC 33152) per
well using a working calibration solution (Corbett Life Science,
Mortlake, Australia).20 This was used to interpolate the
concentration of DNA amplicons of the samples under
investigation within the linear response range of the qPCR
method.
Statistical analysis
Statistical analysis was used to determine whether L. pneumophila
co-occurred with amoebae. The collected data were analysed
with SPSS®, version 20 (SPSS Inc., Chicago, IL, USA), using crossing
tables and a chi-square test (asymptotic signicance, 2-tailed).
Signicance was set at p < 0.05. Pearson’s chi-square test was
used to test for association between amoeba and L. pneumophila.
The interpretation was performed at 95% condence limit.
Results
The water temperature of the three hospitals at the time of
sampling ranged between 20.7° and 27.3°C (mean 22.5°C) at
hospital A, 20.7° and 27.3°C (mean 22.5°C) at hospital B and 20.0°
and 23.7°C (mean 21.6°C) for hospital C. The chlorine
concentrations of the individual hospitals were 0.01–0.17mg/l
(mean 0.09mg/l) for hospital A, 0.21–0.28mg/l (mean 0.23mg/l)
for hospital B and 0.01–0.32 mg/l (mean 0.23 mg/l). Amoebae
were isolated from 72.4% (n = 71) of the 98 water and biolm
Figure 1: Agarose gel (2%) electrophoresis of the rst-step PCR products of water samples from three hospitals performed with primers LEG 225 and
LEG 858, which amplies approximately 654bp (arrow) of the 16S rRNA gene and a 100bp ladder.

Detection of amoeba-associated Legionella pneumophila in hospital water networks of Johannesburg 3
samples, of which 69.4% (n = 68) were microscopically identied
as Vermamoeba vermiformis and 30.6% (n = 30) identied as
Acanthamoeba spp.
All samples cultured on BYCE and GVCP agar were negative for
Legionella species. However, a total of 7 (six water and one swab)
out of 71 (9.9%) amoeba-positive samples showed a positive
reaction for L. pneumophila using the q-PCR. The positive samples
were isolated from the central sterilisation service department
(CSSD) (sample 167); theatre tap (sample 402); neonatal ward
cubicle tap (sample 870); from the cardiothoracic intensive care
unit (ICU) (sample 1109) and trauma ICU (samples 1130; 1302;
1286) (see Figure 1). Legionella co-occurred with the amoeba V.
vermiformis in all samples and this coexistence between the two
species was statistically signicant (p < 0.05). Using qPCR, the
positive samples were quantied to determine the GU/l. Sample
1302 showed the highest concentration with 3.8 × 102 GU/l,
corresponding to the strongest band signal on the agarose gel
(see Figure 1). Sample 870 had the lowest concentration (2.7 × 100
GU/l), corresponding to the weakest band signal on the agarose
gel (Table 1).
Discussion
Generally, all the measured water temperature and residual
chlorine for all three hospitals were within the limits prescribed by
the South African National Standard for Drinking Water Systems.21
In this study, amoeba-associated Legionella spp. did not grow on
BCYE and GVCP agar. However, L. pneumophila were detected in
9.9% (7/71) of samples using conventional and quantitative PCR.
Since they were positive in the ISO certied qPCR, it islikely that
there were residues of amoeba-resistant L. pneumophila present,
which entered a viable but non-culturable (VBNC) state. Similar
studies conducted in Italy detected L. pneumophila in 33.3%
(22/66) of hospital water systems,10 while in Greece Legionella spp.
were detected from 16.9% (22/130) of hospital water samples.22
Another study of hospital water systems in Taiwan reported 62.5%
(10/16) of the samples to be positive for L. pneumophila.23 The
highest concentration obtained in this study of 3.8 × 102 GU/l for
L. pneumophila was quantied from trauma ICU. This is lower than
the concentration peak of 4.0 × 104 GU/l of L. pneumophila that
was quantied in the otorhinolaryngology, pathologic anatomy,
and paediatrics and surgery wards of an Italian hospital.10 Several
factors such as dierent geographical locations, dierent
molecular detection methods used as well as matrix features of
the water source might explain the variability in the prevalence
and concentration values.
Although relatively few samples were positive for Legionella in
the current study, which according to its rationale did not
account for the Legionella eventually present in the water phase,
the coexistence between Legionella and V. vermiformis was
statistically signicant (p < 0.05). This suggests that these FLA
may serve as reservoirs of Legionella, thus contributing to the
environmental survival of Legionella as well as transmission
vectors in hospital settings by releasing them into the water
stream. Therefore, their detection may still indicate a risk to
hospitalised patients as Legionella spp. are known to replicate
rapidly intracellularly within protozoan hosts for prolonged
periods of time forming amoebic vesicles that can contain
hundreds of Legionella cells.6,24 The relatively few positive
Legionella detected may be attributed to the exclusion of free-
living Legionella detection in the method.
Health-care-associated Legionnaires’ disease (LD) has been
reported worldwide.25,26 In South Africa, the rst investigation of
an outbreak of LD in a Johannesburg teaching hospital in 1985
reported 12 cases in hospitalised patients, with two patients
conrmed to have acquired the disease in the hospital.27 A more
recent surveillance study of two South African hospitals by
Wolter et al.28 reported Legionella in 21 (1.2%) cases of patients
diagnosed with HIV or tuberculosis infections. However, this
study did not aim to prove if the infections were acquired from
the hospital environment. Hospital-associated LD cases in South
Africa may be under-reported due to the lack of robust and
reliable surveillance mechanisms and lack of accurate diagnosis.
Routine sampling of hospital water supplies with the application
of amoebal enrichment and co-culture techniques to resuscitate
bacteria in the VBNC state can be eective strategies to prevent
and manage hospital-acquired LD.5,29 Therefore, future work will
focus on the detection of Legionella spp. from hospital water
supplies and hospital surfaces, and comparing them with clinical
samples. This will establish any link between occurrence of the
organisms in the hospital environment with LD infection in
patients using amoeba culture and molecular techniques.
Knowledge on the occurrence of amoebae-associated L.
pneumophila in the hospital water systems will also provide
baseline information to monitor potential outbreaks that may be
facilitated by their presence.
Conclusions
Free-living amoebae were detected in 71 (72.4%) of the 98 water
and biolm samples collected. From the amoebae-positive
samples, Legionella did not grow on BCYE and GVCP agar.
However, L. pneumophila were detected in 9.9% (7/71) of samples
using conventional and real-time PCR. The seven L. pneumophila
positive samples were from trauma ICU, cardiothoracic ICU, a
neonatal ward and a central service sterilisation unit. Patients
and health-care workers might be exposed to waterborne
amoeba-associated L. pneumophila
Compliance with ethical standards – The manuscript does not
contain clinical studies or patient data.
Disclosure statement – No potential conict of interest was
reported by the authors.
Funding – This work was supported by Water Research
Commission [grant number K5/2138].
ORCID
M Leifels http://orcid.org/0000-0002-1295-3410
L Jurzik http://orcid.org/0000-0002-8086-0427
Table 1: Quantitative and qualitative results of PCR for Legionella
pneumophila and free-living amoebae isolated from hospital water
Notes: S = swab; W = water; GU/l = genetic units per litre; CSSD = central service
sterilisation unit; ICU = intensive care unit.
Hospital amoebae Sample source L. pneumophila
(GU/l)
Hospital A V. vermiformis CSSD (S) 2.7x10
0
V. vermiformis Theatre (W ) 1.6x10
1
Hospital B V. vermiformis Neonatal ward
(W)
2.9x10
0
Hospital C V. vermiformis Cardiothoracic
ICU (W)
4.7x10
1
V. vermiformis Trauma ICU (W) 1.4x10
1
V. vermiformis Trauma ICU (W) 1.6x10
1
V. vermiformis Trauma ICU (W) 3.8x10
2

4 Southern African Journal of Infectious Diseases 2018; 1(1):1–4
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Received: 02-06-2017 Accepted: 26-01-2018
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