Magnetic Separation Technology
Speed detection of
Legionella pneumophila
in every water sample
Dynabeads® MAX Legionella
Magnetic Separation Technology
Dynabeads® MAX Legionella
Immunomagnetic separation (IMS) for fast, robust Legionella isolation
→→ Eliminate filtration- or centrifugation-based sample concentration prior to plating
→→ Perform high-throughput, rapid, and effective Legionella pneumophila sample preparation
→→ Obtain selective, high-purity Legionella capture that significantly reduces background flora
and eliminates PCR inhibitors
Dynabeads® MAX Legionella provides a rapid and highly reliable sample preparation process for the detection
and quantification of Legionella in environmental water samples. Through the use of specific antibody-coated,
small superparamagnetic particles, Dynabeads® MAX Legionella offers an improved, high-throughput,
semiautomated sample preparation methodology. Known as immunomagnetic separation (IMS), this
technology delivers comparable analytical results with significant improvements in process time,
labor, and material costs.
2
Legionella—sources of pathogen
Environmental testing practices
Legionella are gram-negative bacteria, ubiquitous in natural aquatic
Sporadic cases of legionellosis have been reported throughout
environments, and the causative agent of human legionellosis,
the world over the past 30 years, leading to the development of
also known as Legionnaires’ disease. Legionellosis is contracted
regulations and surveillance programs to address health and safety
primarily through the inhalation or aspiration of contaminated
considerations. The frequency of testing varies by country; some
potable water distributed through man-made systems such as
countries establish testing only after a clinical occurrence has
showers, taps, pools, spas, and fountains.1,2,3 In several Legionnaires’
been confirmed, while other countries require routine detection
disease outbreaks, infection has been associated with contami-
programs for the identification and quantification of Legionella
nated water aerosols generated by cooling towers, industrial
at suspected sources. The recognized presence of Legionella in
equipment, and room-air humidifiers.
water systems and the increased required testing suggest the
While the presence of Legionella in potable water systems
importance of improved standard detection procedures.
can often be relatively low, special environmental conditions such
as elevated temperatures, stagnation, and the presence of
of these systems has resulted in significantly increased testing of
Basic Legionella detection method
proven useful but time consuming
environmental samples in many countries.
Protocols utilizing culture on selective agar remain the gold
biofilms can cause enhanced colonization and amplification.4,5
The risk of human infection caused by Legionella contamination
standard for Legionella testing in most laboratories. Water
Legionella pneumophila
samples are concentrated through centrifugation or filtration.
The Legionella genus comprises over 50 species and 70 serogroups.
Concentrated organisms are then resuspended and inoculated
Legionella pneumophila serogroups 1–14 account for about 90% of
onto agar plates containing antibiotics to suppress growth of
reported cases of legionellosis. About 85–90% of such outbreaks are
other flora in the sample. Such protocols for the detection,
attributed to serogroup 1. The low prevalence of other Legionella
enumeration, and identification of Legionella are referred to in
species among clinical isolates relative to their environmental
standards such as ISO 11731, NF T90-431, NF T90-461, and others.
abundance suggests that these species are less pathogenic.
After incubation for 10–14 days, suspected Legionella colonies
As a result, testing methodologies capable of identifying the
are confirmed by standard microbiology techniques to establish
presence of Legionella pneumophila serogroups 1–14 have the
species and serogroups of Legionella present in the sample. A
greatest clinical significance.
variety of other methods such as PCR and immunoassays are
3
emerging as new approaches to speed the detection of Legionella
in environmental water sources.
3
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Magnetic Separation Technology
Current protocol limitations—the need for change
The standard method used to detect Legionella provides the basic means to achieve
quantification from environmental water samples, but has several limitations. The variety
of water sample types (i.e., potable water, cooling tower water, swabs) often requires
multiple steps and increased time to prepare different samples for analysis, resulting in
sample-to-sample inconsistencies that make results difficult to interpret clearly.
Concentration (filtration and/or centrifugation)
Depending upon sample type, the time to complete the concentration of 100 ml–1 L
samples can vary widely. The presence of suspended solids can require splitting of samples
or the introduction of crude filtration methods; vortexing or stomaching, to free the
bacteria and organic material from the filter, is required. To achieve the necessary detection sensitivity, additional centrifugation is often used to further concentrate the sample.
In both filtration and centrifugation steps, bacteria can be lost.
Background flora
The presence of background flora in many concentrated samples can interfere with
subsequent Legionella plate growth and lead to an underestimation of the real number of
Legionella present. This interference is typically addressed through the use of selective
media. This may result in prolonged incubation periods and a reduction in Legionella
growth. In addition, treatment of sample concentrates with heat or acid prior to plating
is often employed. The additional steps used to reduce background flora, can add significantly to both the time and cost of analysis.
Change has arrived
Dynabeads® MAX Legionella provides a simplified means to address the limitations of
the most commonly used sample preparation protocols. Dynabeads® MAX Legionella
provides a system that is:
→→ Fast—no lengthy filtration steps needed
→→ Specific—identify the targets that matter most
→→ Scalable—process small or large numbers of samples simultaneously
4
Dynabeads®—a proven track record
For more than 15 years, leading research organizations, government agencies, and analytical
and diagnostic companies have chosen Dynabeads® as their preferred tool for capture,
purification, and concentration of selected targets from biological samples. Based on the
use of magnetic separation technology (Figure 1), Dynabeads® provide an effective means
to achieve rapid target isolation essential for effective biological analytics. This rapid and
flexible technology makes even complicated protocols simple and has been successfully
used for a wide range of applications across many biological fields including molecular
biology, immunology, and microbiology. Dynabeads® technology is:
→→ Easy, flexible, and scalable
→→ Robust and highly reproducible
→→ Designed for both manual and automated protocols
Figure 1—Dynabeads® rapid and selective magnetic
isolation tools. Dynabeads® are superparamagnetic, monosized polymer particles. When added
to a sample, Dynabeads® bind to the desired target
(cells, nucleic acids, proteins, or other biomolecules). This interaction relies on the specific
affinity of the ligand on the surface of the beads.
The beads respond to a magnetic field, allowing
bound material to be rapidly and efficiently separated from the rest of the sample.
Dynabeads® immunomagnetic separation (IMS)
The attachment of specific, high-affinity antibodies to the surface of magnetic Dynabeads®
provides a valuable means to isolate specific biological entities from complex matrices.
The use of selective antibodies directed against surface antigens of intact organisms
provides a powerful, improved tool to existing methods for microbial analysis (Figure 2).
Dynabeads® IMS tools are available for the isolation of Salmonella, Listeria, Legionella
species, Cryptosporidium, Giardia, and the verocytotoxin-producing Escherichia coli (VTEC)
serotypes O157, O145, O111, O103, and O26. Dynabeads® IMS products have been designed
for both manual and automated separation of target organisms from food, water, and
environmental samples. In addition, their use greatly increases sensitivity of analysis.
Figure 2—Novel Dynabeads® IMS technology.
Dynabeads® are coated with affinity-purified
antibodies against specific surface markers
on microbial pathogens that are common
contaminants of food, water, and environmental
samples.
5
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Magnetic Separation Technology
Principle of Dynabeads® MAX Legionella
sample preparation
Dynabeads® MAX Legionella utilizes immunomagnetic separation (IMS) technology to
improve and speed the preparation of water samples for analysis (Figure 3). Dynabeads®
MAX Legionella uses a magnet and high-affinity antibody-coated Dynabeads® to
effectively capture and concentrate Legionella from environmental water samples. This
novel technology ensures a reproducible and scalable approach for multiple samples
simultaneously, reducing overall processing time and costs. In addition, the use of
antibody-based selective capture of Legionella significantly reduces background flora
that can lead to additional downstream processing steps and time.
Figure 3—Sample preparation is easy with Dynabeads®
magnetic bead–based separation. 1: Individual tubes
containing 45 ml of suspected Legionella-contam­
inated environmental water are prepared for
testing. 2–3: Dynabeads® MAX Legionella and
buffer are added to each sample, and the tubes
are incubated for 60 min using a sample mixer
(Figure 4). During incubation, Legionella present in
the samples are selectively captured on the highaffinity antibody-coated magnetic Dynabeads®.
4–5: The tubes are placed in a magnetic stand
and the Dynabeads® are pulled to the side of
each tube along with their target Legionella cells.
Decanting or aspiration removes non-Legionella
micro­organisms and other unbound contam­
inants. 6–8: A quick wash is carried out using
buffer provided, and the beads are resuspended.
9: Finally, concentrated samples are resuspended
in 1 ml buffer provided and are ready for culture
plating or PCR.
6
1. Place water sample
in tube.
2. Add Dynabeads®
and buffer.
3. Legionella binds to
Dynabeads®.
4. Place near magnet.
5. Aspirate or decant
supernatant.
6. Add wash buffer.
7. Place near magnet.
8. Aspirate or decant
supernatant.
9. Remove sample for
plating or PCR.
Dynabeads® sample processing—simplify your
capture process and reduce time
Dynabeads® sample mixers provide continuous gentle tilting and rotational motion for
optimal binding of Dynabeads® MAX Legionella with the target in a sample. The mixers
combine reliability and ease of use in a lightweight unit. Dynabeads® sample mixers come
in a variety of formats and attachments to accommodate a range of sample tube sizes and
numbers, as well as magnets (Figure 4).
The BeadRetriever™ system (Figure 4) is an automated instrument that provides the
ability to accelerate and simplify the testing of some water samples using Dynabeads®
MAX Legionella. Designed as a small, benchtop tool, the BeadRetriever™ system is pre­
programmed for the automatic isolation of Legionella, as well as other pathogenic
Dynabeads® MX2 Sample Mixer
microorganisms, using the Dynabeads® IMS technology.
Figure 4—Dynabeads® sample mixers and the
BeadRetriever™ system. The Dynabeads® MX2
Sample Mixer (above) is used to hold and mix
multiple sample tubes. The Dynabeads® Rotator
Sample Mixer (below) and mixing rods provide
end-over-end rotational mixing of multiple
sample tubes. With the BeadRetriever™ system
(left), all Dynabeads® MAX Legionella protocol
steps are automatically carried out in disposable
tubes with simple push-button operation. This
innovative instrument enables simultaneous
processing of up to 15 samples in ≤40 min, covering Dynabeads®-driven capture, purification, and
concentration of selected microorganisms.
BeadRetriever™ system
Dynabeads® Rotator Sample Mixer
7
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Magnetic Separation Technology
Rapid, high-throughput sample preparation
Filtration currently serves as the basic method for preparing environmental water samples
to be tested for the presence of Legionella (Figure 5). This initial filtration step is designed
to concentrate the sample and improve the sensitivity of the plating assay that follows.
However, due to the nature of the water samples, the filtration time is often inconsistent
and can significantly delay analysis.
Dynabeads® MAX Legionella is an effective alternative. Using Dynabeads® IMS
technology and semiautomated sample processing, only 45 ml of sample is required to
achieve the necessary assay sensitivity. The significantly lower sample volume and rapid
processing reduce labor time and cost. In addition, the selective power of IMS technology
improves the quality of sample preparation, significantly reducing background flora and
enabling easier identification, quantification, and confirmation of suspected colonies.
Conventional filtration method
Dynabeads® MAX Legionella method
Sample: 100 ml–1 L
Sample: 45 ml
Pre-filter
Filter
Whole
sample
filter
Split
sample
filter
Untreated
Filter elution
Centrifugation
Figure 5—Comparing IMS and filtration methods for
sample preparation. Standard filtration methods can
be complicated and require multiple processing
techniques. The Dynabeads® IMS method is the
same for any sample type. Filtration can take from
10 min to 2 hr depending on the sample type,
number of clogged filters, or centrifugation steps
needed. IMS only takes 1 hr 15 min from start to
finish every time.
8
Dynabeads® MAX Legionella
capture and concentration
Plating on selective and/or
nonselective agar
Total time required: 1 hr 15 min
Untreated
Heattreated
Acidtreated
Plating on selective and/or
nonselective agar
Total time required: up to 2 hr
Sensitive Legionella cultures
14
depend in part on the level of contamination present in the suspected water sources
as well as the number of distal outlets testing positive for the presence of Legionella.
6
Different countries and states have established various standards for testing frequency,
water sources to be tested, and, if detected, the levels of Legionella that trigger various
No. of samples in
correct cfu/ L category
The contraction of Legionnaires’ disease following exposure to Legionella is believed to
12
10
8
6
4
2
0
remedial action plans.
<100
Filter method
Dynabeads® MAX Legionella provides the sample preparation necessary to reach the
level of detection most often required. While IMS technology only requires 45 ml of sample
(compared to 100 ml–1 L when sample is prepared by filtration), studies have shown that
Dynabeads® MAX Legionella is capable of achieving detection level results comparable to
standard filtration methods (Figure 6).
100–1,000
>1,000
cfu/L categories
IMS
Figure 6—When detecting Legionella from regularly tested potable water samples, the Dynabeads®
IMS and standard filtration methods result in similar
outcomes. Filtration was performed using the
standard, ISO 11731 method. Out of 50 samples
tested by both filtration (starting with 1 L) and
IMS (starting with 45 ml) and analyzed by plategrown cultures, all 50 produced similar detection
level results (i.e., <100 cfu/L, 100–1,000 cfu/L, or
>1,000 cfu/L).
Greater sensitivity with Dynabeads® MAX Legionella
Environmental water assessments are routinely performed over a range of starting sample
450
volumes (100 ml–1 L) depending on the levels of Legionella detection established in
350
resulting in recovery loss. In addition, recovery from the membrane requires large
reconstitution volumes (as much as 20 ml), reducing the benefits of concentration.
Dynabeads® MAX Legionella provides several advantages over standard filtration
methods. The use of IMS technology introduces a gentle mechanism for concentrating
Legionella that significantly improves recovery of intact Legionella organisms. In addition,
the final recovery of Legionella from IMS beads is performed in just 1 ml of buffer. The
higher concentration of final product can result in detection levels that exceed those
achieved with filtration-prepared samples (Figure 7).
300
cfu/plate
regional standards. Typical filtration methods can “trap” Legionella on the membranes,
400
250
200
150
100
50
0
Sample 1
Filter method
Sample 2
Sample 3
IMS
Figure 7—Colony counts per plate for IMS method
compared to filtration method. Dynabeads® IMS
technology can be more efficient than standard
filtration methods when detecting Legionella
from known positive hospital potable water
samples. Various samples containing 100 ml for
processing via filtration, or 45 ml for processing
via IMS, were analyzed by plate-grown cultures.
Despite using <50% of the original starting
volume, in all cases the IMS method provided
higher colony counts per plate than the filtration
method.
9
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10
Magnetic Separation Technology
Selectively capture and concentrate a broad range
of Legionella species
Legionella pneumophila, serogroups 1–14 account for about 90% of reported cases of
legionellosis, with most outbreaks attributable to serogroup 1. Other Legionella species
are rare among clinical isolates, suggesting that these species are less pathogenic. As a
result, testing methodologies capable of identifying serogroups 1–14 have the greatest
clinical significance.
Dynabeads® MAX Legionella is capable of isolating most Legionella species present
in environmental waters (Table 1). This high-affinity antibody bead has shown broad reactivity for the most commonly encountered Legionella species and has proven to be an
effective means of capturing and concentrating Legionella from environmental water
samples.
Table 1—The Dynabeads® IMS and standard filtration methods yield identical species identification. As confirmed
by plate-grown cultures using agglutination testing and molecular typing methods, the IMS method has
been shown to detect Legionella pneumophila serogroup 1, serogroups 2–14, and Legionella spp. Both IMS
and filtration methods can detect low numbers of organisms per sample, but only the IMS method can
produce these results while lowering background contamination.
Legionella pneumophila
serogroup 1
Legionella pneumophila
serogroups 2–14
Legionella spp.
Filtration methods
•
•
•
Dynabeads® MAX
Legionella (IMS)
•
•
•
Sample preparation
method
Significant reduction in background flora
The presence of nonpathogenic Legionella and other microorganisms significantly affect the
detection of Legionella species in environmental samples. These organisms can contribute
to the clogging of filters and can carry over to the final concentrates. Culture media
designed to suppress background can help, but can also inhibit Legionella growth, leading
to an underestimation of the real number of Legionella cells present in the original sample.
The use of high-affinity, selective antibodies immobilized to Dynabeads® MAX
Legionella magnetic beads enables selective capture and concentration of Legionella
present in environmental water samples (Figure 8). Immunomagnetic separation reduces
background flora in plated samples by as much as 80–85% when compared to standard
filtration methods.
Figure 8—Dynabeads® IMS method reduces background growth. Plated samples were prepared from potable
water samples using either the IMS method (left) or the filtration method (right). Samples were untreated
and plated directly to BCYE (buffered charcoal yeast extract) medium. The results indicate that the IMS method
dramatically reduces nonspecific carryover.
11
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Invitrogen service and support
For fast, sensitive, and reliable detection of Legionella pneumophila in environmental and
potable water samples, get Dynabeads® MAX Legionella today. For more details, contact
[email protected].
Ordering information
Product
Quantity
Cat. no.
Dynabeads® MAX Legionella
15 ml
A10655
Dynabeads® MX2 Mixer (for 14 x 30–50 ml tubes)
1 unit
159-08
Dynabeads® Rotator Mixer (for 12 x 10–15 ml tubes or 14 x 1.5 ml tubes)
1 unit
947-01
Mixing Rod for Dynabeads® Rotator Mixer (for 14 x 30–50 ml tubes)
1 unit
947-02
Dynabeads® MPC®-6 x 50 ml (magnetic stand for 6 x 50 ml tubes)
1 unit
A10757
Dynal MPC®-1 (magnetic stand for 1 x 5–50 ml tubes)
1 unit
120-01D
Dynal MPC®-6 (magnetic stand for 6 x 5–15 ml tubes)
1 unit
120-02D
Other IMS Products
Quantity
Cat. no.
Dynabeads® anti-Cryptosporidium
1 ml
730-01
Dynabeads® anti-Cryptosporidium
5 ml
730-11
Dynabeads® MAX E. coli O157
1 ml
A10714
Dynabeads® MAX E. coli O157
5 ml
A10715
Dynabeads® anti-E. coli O157
1 ml
710-03
Dynabeads® anti-E. coli O157
5 ml
710-04
Dynabeads® EPEC/VTEC O103
2 ml
710-11
Dynabeads® EPEC/VTEC O111
2 ml
710-09
Dynabeads® EPEC/VTEC O145:H57
2 ml
710-07
Dynabeads® EPEC/VTEC O26
2 ml
710-13
Dynabeads® GC-Combo
1 ml
730-02
Dynabeads® GC-Combo
5 ml
730-12
Dynabeads® anti-Listeria
5 ml
710-06
Dynabeads® anti-Salmonella
5 ml
710-02
BeadRetriever™ System
1 unit
159-50
240 samples
159-51
1 unit
159-52
BeadRetriever™ Tubes & Tips
BeadRetriever™ Tube Rack
Visit our website at www.invitrogen.com/microbiology or contact us by email at [email protected] for more information.
References
1. Cianciotto, N.P. (2001) Pathogenicity of Legionella pneumophila. Int J Med Microbiol 291:331–343.
2. Fields, B.S. et al. (2002) Legionella and Legionnaires’ Disease: 25 years of investigation. Clin Microbiol Rev 15:506–526.
3. Steinert, M. et al. (2002) Legionella pneumophila: an aquatic microbe goes astray. FEMS Microbiol Rev 26:149–162.
4. Emtiazi, F. et al. (2004) Investigation of natural biofilms formed during the production of drinking water from surface
water embankment filtration. Water Res 38:1197–1206.
5. Walker, J.T. and Marsh, P.D. (2004) A review of biofilms and their role in microbial contamination of dental unit water
systems (DUWS). Int Biodeterior Biodegradation 54:87–98.
6. Stout, J. et al. (2007) Role of environmental surveillance in determining the risk of hospital-acquired legionellosis:
a national surveillance study with clinical correlations. Infect Control Hosp Epidemiol 28:818–824.
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