Legionella pneumophila Detection in Water by rRNA (without PCR)

A rapid and innovative test system for Legionella, especially Legionella pneumophila, in water
Shyam Verma1, Jvo Siegrist2, Jens Boertz2, and Katja Vetter3
1Sigma-Aldrich/Supelco,

Bellefonte, PA, USA, 2Sigma-Aldrich/Fluka, Buchs (SG) Switzerland, 3Scanbec, Bitterfeld-Wolfen, Germany

Abstract
The current ISO method (ISO 11731:1998; 11731-2 2004) used to detect and identify the
Legionella species takes at least 10 days and requires several steps with different media.
Therefore, new rapid methods for the detection of Legionella are of great interest.
HybriScan Legionella kit and HybriScan Legionella pneumophila kit use ribosomal RNA as
a modern detection target. The rRNA is greater in number than DNA (no PCR is needed),
and is only present in living cells. Therefore, it perfectly suits specific and sensitive
detection. Within 2.5 hours, a direct quantitative detection of 10,000 colony forming units
(CFU) per liter is easily possible and 1-10 CFU/L can be detected with an enrichment step
in BCYE medium.

Table 2. Comparison of Different Technologies for Detection

of Beer Spoilage Bacteria
Cultivation
based method

PCR

VIT

HybriScan

Method

cultivation based
method with
optical microscopic read out

PCR/real-time
PCR

fluorescence
microscopy

sandwich
hybridization and
photometrical signal
read out

Detection
spectrum

detection and
identification of
all beer spoilage
microorganisms

identification of all
relevant Beer
spoilage
microorganisms
possible

Lactobacillus sp.
and Pediococcus
damnosus,
Lactobacillus sp. +
L.brevis, Pectinatus
+ Megasphaera
cerevisiae

identification of all
relevant Beer
spoilage
microorganisms
possible

Sample
preparation

selective preenrichment

enrichment and
lysis of bacteria,
if necessary preenrichment

selective preenrichment

enrichment and lysis

of bacteria, if
necessary preenrichment

Time

3 to 7 days

3 hours to 2 days

2 days

3 hours to 2 days

Costs per test

ca. $1

$13

$17

$3

Detection limit
(cfu)

1-5 x 103

1 x 103

1-5 x 103

Devices

None

PCR cycler

fluorescence
microscope

microplate reader

Advantages

high sensitivity,
relatively cheap

high sensitivity,
quantitative
analysis

simple detection
technology set up,
detects only living
cells (RNA)

rapid and sensitive,

qualitative and
quantitative
detection of living
cells, cost efficient
analysis

Disadvantages

time consuming,
no detection of
non-culturable
microbes, labor
expensive

expensive
devices needed,
no discrimination
between live and
dead cells, not
officially accepted

time consuming, low

sample throughput,
expensive, not
automatable,
difficult data
analysis, not
officially accepted

no differentiation of
serotypes or
subspecies, limited
probe design (rRNA
target), not officially
accepted

Introduction
Legionnaires disease is a dangerous and infectious pneumonia which can affect anybody,
but those at highest risk are the elderly, sick, smokers, or other people with weak immune
systems.
The causative organism is the Legionella pneumophila, which easily forms aerosols that
can be inhaled, causing infection. The natural source of Legionella pneumophila is all
types of water, but particularly water containing algae, rust, sludge, scale, and organic
compounds. It is known that Legionella are able to form biofilms inside water pipes where
they are protected against antimicrobial treatments. The infected water droplets can be
produced in such places as whirlpool spas or air conditioners since these organisms
prefer warm environments but can also occur in fresh water such as rivers and ponds.
A new sandwich hybridization test was developed in a microtiter plate format which takes
less than 2.5 hours and has a sensitivity of 10,000 CFU per liter and 1-10 CFU per liter
after an overnight enrichment in BCYE medium.
The quantification is possible with this kit using photometric methods. Compared to PCR,
this system does not count dead cells, as rRNA from dead cells is depleted within a few
hours. Also this technique is more robust (no contamination risk during assay), less
expensive, and unaffected by the sample matrix. The rapidity, sensitivity, reliability,
robustness, adaptability to sample matrix, and time savings meet todays analytical
microbiology demands.

Experimental
A new sandwich hybridization test was designed on a 96-well microplate and is done in ~ 2
to 2.5 hours. A positive result is visible to the naked eye but quantification is possible with
this kit using photometric methods and standards.
Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin-labeled
capture probe is used to immobilize the target sequence on a solid support plate
(streptavidin-coated microtiter plate). A digoxigenin labeled detection probe provides an
enzyme linked optical signal read-out. Detection results from application of anti-DIGhorseradish peroxidase Fab fragments. The bound complex is visualized by horseradish
peroxidase substrate TMB. Photometric data are measured at 450 nm and compared with
standard solutions.
As the detection of Legionella is quite tricky, two additional certified microorganism
standards were used as positive controls since they can be used as qualitative and
quantitative positive controls (see Table 1).

Results
By using two different probes for detection of microbial rRNA, false-positive results are
almost impossible. The quantitative analyis of 39 Legionella positive samples with HybriScan
were compared with ISO 11731 (GVPC and BCYE Agar) and no significant difference could
be found. HybriScanD Legionella even showed to be more sensitive on low concentrations..
Figure 2. Comparison of ISO 11731 (purple) versus HybriScanD Legionella (yellow)

Vitroids Test Strains

Origin

Strain No.

CFU

Cat. No.

Legionella bozemanll

NCTC

11368

50000

RQC02908

Legionella pneumophila

ATCC

12821

50000

RQC02008

CFU /mL

Table 1. Discs with Certified Reference Test Strains of Legionella

Inclusivity in terms of organism and serogroups detected:

HybriScanD Legionella: all of the genus Legionella including L. pneumophila
HybriScanD Legionella pneumophila:
all species of L. pneumophila
HybriScanI Legionella pneumophila:
all species of L. pneumophila
Figure 1. Principle of the Assay

Comparison of these different technologies is given in Table 2. By comparing HybriScan to

PCR, ELISA or FISH, the benefits of this rapid test system become obvious:
Fast, cost-efficient analysis
Inexpensive read-out technology
High sensitivity and specificity
Detection of organisms based on ribosomal RNA through HybriScan rapid test system is
faster and efficient. The HybriScan can detect much more than Legionella, the
development of an assay for a new organism is relatively easy and fast.

References
1. T. Leskel, A. Tilsala-Timisjrvib, J. Kusnetsovc, P. Neubauera, A. Breitenstein,
Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich
hybridization assay, J. Microbiol. Meth., 62, pp. 167179 (2005).
2. ISO 11731, Water quality - Detection and enumeration of Legionella (1998).

Besides the classical methods, a comparison with three different rapid methods was made.
PCR, ELISA and FISH are also methods used for Legionella detection. The differences are
shown in Table 2.

T414055

HybriScan is a trademark of ScanBec GmbH.

Vitroids is a trademark of Sigma-Aldrich Co. LLC.