Combating bacteremia… with our dirty little secret

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Bacteremia is the presence of bacteria in the blood. Under normal circumstances, blood should be sterile, but with the insertion of a catheter into the body or through an open wound, bacteria can gain entry and take hold. Even small numbers of bacteria can be a real problem and make people very sick. Untreated bacteremia has been associated with serious health consequences ranging from sepsis, systemic inflammatory response syndrome (SIRS), septic shock and multiple organ dysfunction syndrome (MODS). Hospitals need to have a low bacterial detection threshold, and to achieve this, the blood is first cultured, which can take 48 hours and is applicable only to culturable microbes. To complicate things further, some patients have already been dosed with antibiotics or other drugs which can interfere with the culturing process. Bacterial genotyping from cultured blood can be very difficult due to the presence of PCR inhibitors such as charcoal used for the removal of antibiotics from patient blood (e.g., BacT/ALERT tubes).

The journey of combating bacteremia starts with the isolation of bacterial DNA from infected blood. At QIAGEN, we have an innovative solution, the QIAamp BiOstic Bacteremia DNA Kit. When the QIAamp BiOstic Bacteremia DNA Kit was originally designed, it was optimized for the extraction of bacterial DNA from infected blood. However, it has turned out to be useful for so much more.

The basic premise of the QIAamp BiOstic Bacteremia DNA Kit is to isolate DNA from a small number of bacteria inside a messy mixture of blood and anticoagulants or growth media. Standard blood or microbial kits won’t work for this. Microbial cells are harder to lyse than blood cells and the heme and lipids in the blood can be potent PCR inhibitors which need to be removed using specialized chemistry.

The QIAamp BiOstic Bacteremia DNA Kit combines bead beating and detergent for complete bacterial cell lysis with QIAGEN’s Inhibitor Removal Technology, for the removal of heme molecules and other inhibitors from DNA. The first steps in the QIAamp BiOstic Bacteremia DNA Kit protocol are to remove a portion of cultured blood; centrifuge to pellet all the cells and then bead beat the cell pellet with a strong detergent-based lysis buffer.

The kit uses 2 ml bead tubes containing fine garnet sand (0.15 mm) and is ideal for lysing microbial cells. Bead beating is followed by QIAGEN’s Inhibitor Removal Technology. This is the same powerful chemistry that is used in our soil and fecal kits and will remove all PCR inhibitors including heme, lipids, polysaccharides, humic acid and heparin.

Isolating DNA from a wide variety of samples

Isolating microbial DNA from cultured blood is a specific application. Soon after the QIAamp BiOstic Bacteremia DNA Kit was released, we found other situations where it was useful to get DNA from a dirty cell pellet. Customers started trying it on all kinds of other samples and it turned out to work fabulously. We thought it time to let you in on our dirty little secret. Dirty swab samples which contain PCR inhibitors (fecal, vaginal, wound, intestinal/stomach and environmental surfaces) tend to contain a relatively small number of cells with many PCR inhibitors. The QIAamp BiOstic Bacteremia DNA Kit’s’s strong lysis buffer combined with its one step Inhibitor Removal Technology turns out to be ideal for maximizing the DNA yields.

For your convenience, we have listed the quick protocols for the extraction of DNA from different samples such as swabs, dirty microbial pellets and uncultured blood.

Swabs

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To use a swab with this kit, start with the following protocol:
1. Add 450 µl of Solution MBL directly to the bead tube.
2. Place and rotate the swab in the buffer and let it soak for a few minutes to release the cells into the solution.
3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible.
4. Proceed with the 70ºC heating step and the rest of the normal protocol.

Dirty microbial pellets (sputum, saliva, washes from dirty surfaces; nasopharyngeal, colorectal and mixed culture isolates)

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Any situation where bacteria can be pelleted from a dirty (containing PCR inhibitors) liquid can work well with the QIAamp BiOstic Bacteremia DNA Kit. Some examples include sputum, saliva, washes from dirty surfaces (nasopharyngeal and colorectal) and mixed culture isolates. To utilize these types of samples with this kit, follow this protocol:
1. Centrifuge the liquid at 13,000 x g for 2 minutes to pellet the cells. The volume of liquid will depend on the sample. Aim for a wet cell pellet weighing 25 mg or less.
2. Remove the supernatant.
3. Resuspend the cell pellet in 450 µl of Solution MBL and add to the bead tube.
4. Proceed with the 70⁰C heating step and the rest of the normal protocol.

Direct from (uncultured) blood

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Customers often want to avoid culturing blood because of the time and possible bias involved. The QIAamp BiOstic Bacteremia DNA Kit can also be used for the isolation of bacterial DNA directly from blood without culturing, but less starting sample should be used to avoid clogging of the spin column. To use the kit for DNA isolation from whole blood, follow this protocol:
1. Centrifuge 50–500 µl of whole blood at 13,000 x g for 2 minutes to pellet all the cells. If the blood is too viscous to pellet, premix the blood with an equal volume of sterile PBS before centrifuging.
2. Pipette off the supernatant and dispose of as hazardous waste.
3. Resuspend the cell pellet in 450 µl of Solution MBL and add to the bead tube.
4. Proceed with the 70ºC heating step and the rest of the normal protocol.
In addition to the sample types mentioned above, the QIAamp BiOstic Bacteremia DNA Kit has been used for filtered plant washes, feces and even raw milk. We’ve listed some recent references below. So, what started as a bacteremia kit is now much more!
Learn more about the QIAamp BiOstic Bacteremia DNA Kit and the QIAGEN innovative microbiome solutions for your sample preparation.

We are looking forward to hearing your exciting story about how you used different starting materials for extraction of DNA using the QIAamp BiOstic Bacteremia DNA Kit.

References

Direct from blood:
• Gutiérrez, R., Cohen, L., Morick, D., Mumcuoglu, K.Y., Harrus, S. and Gottlieb, Y. (2014) Identification of different Bartonella species in the cattle tail louse (Haematopinus quadripertusus) and in cattle blood. Appl. Envir. Microbiol. 80, 5477–5483.
From cultured infected tissue:
• Fritz, S.A. et al. (2013) Mupirocin and chlorhexidine resistance in Staphylococcus aureus in patients with community-onset skin and soft tissue infections. Antimicrob. Agents Chemother. 57, 559–568.
• Fritz, S.A. et al. (2013) A serologic correlate of protective immunity against community-onset Staphylococcus aureus infection. Clin. Infect. Dis. 56, 1554–1561.
Direct from swabs:
• Lerner, A., Romano, J., Chmelnitsky, I., Navon-Venezia, S., Edgar, R. and Carmeli, Y. (2013) Rectal swabs are suitable for quantifying the carriage load of KPC-producing carbapenem-resistant Enterobacteriaceae. Antimicrob. Agents Chemother., 57, 1474–1479.
• Marrazzo, J.M. et al (2012) Extravaginal reservoirs of vaginal bacteria as risk factors for incident bacterial vaginosis. J. Infect. Dis. 205, 1580–1588.
Filtered plant washes:
• Maignien, L., DeForce, E.A., Chafee, M.E., Eren, A.M. and Simmons, S.L. (2014) Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities. MBio. 5, doi: 10.1128/mBio.00682-13.
Direct from saliva:
• Francavilla, R. et al. (2014) Salivary microbiota and metabolome associated with celiac disease. Appl. Envir. Microbiol. 80, 3416–3425.
Direct from feces:
• Zaborin, A. (2014) Membership and behavior of ultra-low-diversity pathogen communities present in the gut of humans during prolonged critical illness. MBio. 5, doi: 10.1128/mBio.01361-14.
Direct from raw milk:
• Ercolini, D., De Filippis, F., La Storia, A. and Iacono, M. (2010) “Remake” by high-throughput sequencing of the microbiota involved in the production of water buffalo mozzarella cheese. Appl. Envir. Microbiol. 78, 8142–8145.
Blood culture:
• Ziga, E.D., Druley, T. and Burnham, C.A. (2010) Herbaspirillum species bacteremia in a pediatric oncology patient. J. Clin. Microbiol. 48, 4320–4321.

 

Authors: This post was written with contributions from both Heather Martinez and Vishwadeepak Tripathi.

This article was compiled from the contributions of multiple authors. Please see the end of the post for details.

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