DNA Analysis of Chobani Yogurt: Guest Post by Nick Conley

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chobani 1Identification-by-DNA has had eclectic uses: outing fake sushi, detecting horsemeat in Ikea meatballs, and capturing a murderer thanks to a telltale hair from Snowball the cat, for example. Nick Conley decided to take on Chobani yogurt, a delicious product accused a few weeks ago of harboring a contaminant. I love Chobani yogurt and their company headquarters is in a tiny town about an hour from me in upstate New York.

Nick sent me his blog post from his company, Epibiome, and has graciously offered to share it. He’s joined at Epibiome by physicist Aaron Tynes Hammack and physical chemist Hsiao-lu Lee. Nick’s PhD is in chemistry and he did a post-doc in developmental biology, both at Stanford, generating an impressive publication record and a cosmetics company. But most important to me is that he has basset hounds, my favorite dog.

Many thanks Nick!

Sept 9      We had just extracted bacterial DNA from a set of facial microbiome samples when I decided to reward myself with a cup of lime-flavored Chobani Greek yogurt. Oddly, the foil on the cup was bulging, the yogurt was thin, and it was extremely carbonated. Note the comparison to a “normal” strawberry flavored cup in the photo.

A quick google search brought me to Chobani’s Facebook page, which showed that many people were reporting the same issue. Since lots of bacteria are capable of lactic acid fermentation, we decided to extract the bacterial DNA from this funky lime cup and a “normal” strawberry cup and do 16S rDNA sequencing to identify the bacteria present.
Since the time we commenced our study, Chobani announced a voluntary recall and has since reported that it believes the contamination is from a mold (Mucor circinelloides).

Our technique will only give us information about the bacteria present, not about molds, which are a type of fungus. Nevertheless, we are very excited to see if there are any unexpected bacteria in the culture and to understand how the mold contamination changes the composition of the original bacteria in the yogurt culture. We will report our results within a few days.

And yes, I ate the lime yogurt and I was fine. The carbonation was exciting, generating a kefir-like (mojito-like?) yogurt. While Greek yogurt purists might take issue (would they really be eating lime-flavored yogurt anyway?), I think Chobani should try to achieve the carbonation with a lactic acid fermenting bacterium and launch it as a new product.

Sept 10     We are happy to announce the bacterial 16S rDNA sequencing results from our two Chobani Greek yogurt samples: a strawberry-flavored cup of normal appearance, and a recalled lime cup.

We have determined unambiguously that the lime cup used in our study was involved in the recall because the IMS number matches exactly that posted on the FDA recall site (and the yogurt showed all of the characteristics). No such IMS number could be located on the strawberry cup, whose contents appeared normal, so we used it to compare with the recalled lime yogurt.

We do not contest anything Chobani has reported about the cause of the recall, including the mold contaminant and its non-hazardous nature. Although the FDA is investigating complaints of illness related to the recalled yogurt, no determination has been made whether or not such a link exists. Importantly, the data we present below do not in any way show that the recalled Chobani yogurt was capable of causing illness or caused illness of any kind.

Our study was conducted purely to satisfy curiosity, and our experiment has several shortcomings, including: (a) a very small samples size (n = 2), (b) the comparison of 0% fat strawberry and 2% fat lime flavors, (c) inhomogeneity in the yogurt cup, which could lead to an unrepresentative sample being collected, (d) PCR bias PCR bias, (e) the possibility of misidentification of organisms sharing a common 16S rDNA sequence, (f) sequencing errors resulting in mischaracterization of bacterial taxonomy, (g) incorrect alignment databases, and (h) possibility of DNA contamination. There may be other shortcomings of which we are not aware.

Below are pie charts showing the taxonomic order associated with the most abundant 16S rDNA reads from the normal-appearing strawberry cup and the recalled lime cup.

chobani 2Chobani indicates that they have three probiotic cultures in their yogurt: Lactobacillus acidophilus, Lactobacillus casei, and Bifidus. The first two belong to taxonomic orders Lactobacillales and the latter belongs to Bifidobacteriales, which is consistent with what we found by sequencing bacterial DNA in the normal strawberry cup.

chobani 3However, the recalled lime yogurt shows a considerably different distribution. Out of a total of 1,950,616 reads for this sample, 190,331 (9.8%) belonged to the order Oceanospirillales. Fortunately, the sequence data contain more information than the order; they also give insight into the taxonomic family of the bacterium (Oceanrickettsiaceae) and the genus (Oceanrickettsia). This is where things get extremely obscure, however, because aside from a couple of sequence entries in Genbank in Genbank  on an organism sharing the same genus (Oceanrickettsia ariakensis), we know almost nothing about these bacteria. So little, in fact, that the taxonomic family and genus have not been formally accepted and the organism has been relegated to a list of “unclassified Gammaproteobacteria.”

800px-Oyster_Ushimado03sFrom what little we can glean, two Chinese scientists affiliated with the South China Sea Institute of Oceanology, Drs. Xiangyun Wu and Yang Zhang, deposited the sequence data for Oceanrickettsia ariakensis into the database in 2005 and indicated that they isolated the bacteria from an oyster (Crassostrea ariakensis). They believed that the bacteria infected the oyster and contributed to its massive die-off.

It’s very important to note that there is no evidence that this bacterium would be pathogenic in humans. Furthermore, the work of Wu and Zhang doesn’t appear to have been independently verified or subjected to peer review (to the best of our knowledge).

Does this mean that the Chobani yogurt recall had anything to do with oyster contamination? Absolutely not! It’s possible that the bacteria whose DNA we detected may have already been present in tiny quantities in one of the yogurt’s ingredients, and when the mold took over the yogurt culture, it created conditions that allowed it to thrive. In support of this theory, we observed 198 reads out of 1,713,939 total that corresponded to order Oceanospirillales (0.012%) in the strawberry yogurt. Or perhaps our findings do provide some insight into the contamination source. It’s too early to say.

But what we can say is that we have a lot more to learn about bacteria. We can only culture (and therefore, thoroughly study) about 1% of the bacterial species on the planet. With the latest generation of sequencing technologies, we can sequence everything. Wonder what else is out there? So do we.

398px-Basset_hound_0003I haven’t stopped eating Chobani yogurt and am still a big fan! Many trusted brands have faced a recall at some point during their history. Microbial contamination is a constant concern among food manufacturers and is not uncommon. The overwhelming majority of the time, the extra passengers we call bacteria simply go along for the ride, completely unnoticed.

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7 Responses to DNA Analysis of Chobani Yogurt: Guest Post by Nick Conley

  1. r says:

    the Oceanospirillales calls might be the mold mitochondrial signal.

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    • Nick Conley says:

      Fungal (mold) taxonomists generally use the 18S ITS2 amplicon for identification. To the best of my knowledge, it is very unlikely that the bacterial 16S primers we employed would amplify any DNA from the mold.

      We have preserved samples of the yogurt by deep freezing, and may commence a mold sequencing study in the next few weeks. In addition, we will publish a more detailed species-level analysis of our data shortly.

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  2. Beth Skwarecki says:

    The first pie chart doesn’t seem to show any green/Bifidobacteriales … is the color coding wrong or am I misunderstanding the explanation?

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    • Nick Conley says:

      There is a small green sliver at exactly 12 o’clock. I can see it quite clearly when I view it on a high-res monitor, but my Galaxy S3 doesn’t display it properly.

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  3. gillt says:

    errors (e) and (f) are kinda the same. I’ve attempted Sanger sequencing of bacterial genes before and it’s extremely difficult due to high sequence similarities among bacteria. Lets see some raw data!

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    • Nick Conley says:

      Thanks for your comment gillt. The subtle distinction between (e) and (f) is that (e) can occur even if the sequence data are accurate due to 16S homology between different species and (f) results from improper base calling. Note that we are using an Illumina MiSeq platform, which is considerably different than Sanger (chain-termination) sequencing. We are also targeting the 16S hypervariable region. Checkout this publication:
      http://res.illumina.com/documents/products/appnotes/appnote_miseq_16s.pdf

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  4. Joe Q says:

    Hopefully The Food and Drug Administration Recalled the products. Products derivated from milk are delicate because of the Bacterias it might contain.

    Lactobacillales are positives bacterias used for the fermentation of yogurs and other products derivated from Milk.

    Joe Q

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