High-throughput microbial gene detection seems like the future? - and technology used to identify dysbiosis
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TOPIC: High-throughput microbial gene detection seems like the future? - and technology used to identify dysbiosis

High-throughput microbial gene detection seems like the future? - and technology used to identify dysbiosis 07 May 2014 20:57 #8517

  • KeithBell
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Note by moderator: The first 7 posts in this thread were originally in this thread on "a better way to test pathogen inactivation":
forum.susana.org/forum/categories/26-hea...dsource-a-better-way
But they fit better into this separate thread.

++++++++++


High-throughput microbial gene detection seems like the future; here's food for thought, 2013:
Detecting Human Bacterial Pathogens in Wastewater Treatment Plants by a High-Throughput Shotgun Sequencing Technique

pubs.acs.org/doi/abs/10.1021/es400275r (full paper attached below)

Given mycobacterium tuberculosis found in "high abundance" in sludge and effluent, I'm not sure why TB isn't considered a waterborne disease. Experts still believe it's all about airborne transmission.
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Last Edit: 23 Jul 2014 22:16 by muench.

Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 08 May 2014 08:24 #8523

  • PatrickBBB
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Very interesting Keith, all though I do not think it is what the original poster (OP) was looking for.

The article states:
However, it is worth noting that such a molecular technique is difficult to exactly quantify pathogens in terms of cell number in influent, activated sludge, and effluent because of the high complexity to convert gene copy number to cell number.

Unfortunately it does not say how accurate it can be, but as Joe pointed out earlier in this thread; what's interesting is the pathogen concentration. (In order to conduct a QMRA) Could you explain what the article means by pathogenic bacteria relative abundance?

I also identified some other constraints to the method.

- It requires very expensive equipment (the sequencer used in this experiment was prized at 690000 USD when it was released in 2010)
- It requires a lot of competence.
- All though it does not specifically say it, I assume it requires a lot of time.

Also, what I want to see are real-time sensors that can monitor the microbial effluent (and influent in the case of a DWTP) quality. Light scattering seems more promising in this regard in my opinion.
Happy learning.
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Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 23 May 2014 16:37 #8713

  • KeithBell
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Patrick, I'm still learning about how this technology can and will be applied to sanitation. Pricing of the technology has become much more affordable and testing is very fast.

Modern day hero, environmental microbiologist, Dr. Rita Colwell uses molecular techniques. Here's a recent presentation with focus on cholera:


Here's Dr. Colwell's most recent paper published a few days ago about microbial communities in saliva:
Microbial Community Profiling of Human Saliva Using Shotgun Metagenomic Sequencing
www.plosone.org/article/info%3Adoi%2F10....journal.pone.0097699

General papers; I believe this kind of work can now be done on a laptop:
www.plosone.org/article/info%3Adoi%2F10....journal.pone.0060811
www.ncbi.nlm.nih.gov/pubmed/20566210
www.plosone.org/article/info%3Adoi%2F10....journal.pone.0079490
www.plosone.org/article/info%3Adoi%2F10....journal.pone.0076185
link.springer.com/article/10.1007%2Fs00253-012-4082-4

Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 20 Jul 2014 17:58 #9416

  • KeithBell
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Here are two very recent papers sanitation professionals should be interested in as they may help to determine indicator species and also talk about technology used to identify dysbiosis:

Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition
genomebiology.com/2014/15/6/R76

MALDI-TOF Identification of the Human Gut Microbiome
in People with and without Diarrhea in Senegal

www.plosone.org/article/info%3Adoi%2F10....journal.pone.0087419
In individuals with diarrhea, major commensal bacterial species such as E. coli were significantly decreased (85% versus 64%), as were several Enterococcus spp. (E. faecium and E. casseliflavus) and anaerobes, such as Bacteroides spp. (B. uniformis and B. vulgatus) and Clostridium spp. (C. bifermentans, C. orbiscindens, C. perfringens, and C. symbosium). Conversely, several Bacillus spp. (B. licheniformis, B. mojavensis, and B. pumilus) were significantly more frequent among patients with diarrhea.


Here's some press about the recent study posted above:
Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition

"Scientists Identify New Microbes Associated with Severe Diarrhea"
cmns.umd.edu/news-events/features/2305

"Previously Unheralded Microbes Cause And Prevent Severe Diarrhea"
www.science20.com/news_articles/previous...vere_diarrhea-139500

Note: what causes diarrhea in one population may be different from another as flora balance varies considerably, i.e., European vs. African, the underlying beauty of diversity.
Last Edit: 24 Jul 2014 15:55 by KeithBell.

Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 22 Jul 2014 09:10 #9430

  • joeturner
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Interesting, but irrelevant Keith.

The issue is this: we cannot measure directly all of the microbial pathogens in faecal waste hence the need for a reliable indicator that we can measure which models the breakdown of all the pathogens that we cannot measure.

What these papers seem to be showing is the gut flora of people with and without diarrhea in Senegal - and have found a measurable difference between groups.

This is essentially descriptive. I don't think they are saying that one could analyse faecal waste and use this suite of species to tell whether pathogens have been destroyed. But even if they are saying that, it still is not very helpful because you would have to be able to plate out lots of different microbes, which is expensive and not really the point of an indicator.
Previously trained and worked as a Soil Scientist and worked on projects composting sewage sludge.
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Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 22 Jul 2014 17:34 #9437

  • christoph
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Dear all,
the title of the threat is "Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way?"

This is a topic I am very interested in. I don´t find it polite nor good for the quality if there are posts about totally other subjects and I would like to ask if we could not put up a rule which enables the forum organization to open a new section where all "misleading" posts are put or ask the "misleading"poster if he/she would like to open a new threat.

What do you think? I know I am missleaing s well with this post, but my post is related to the posts above (but feel free to reorganize I you feel it should not be here).

Yours
Christoph
Last Edit: 22 Jul 2014 17:35 by christoph.

Re: Wanting a better way to test pathogen inactivation? Us too! Can you help me crowdsource a better way? 23 Jul 2014 18:36 #9457

  • KeithBell
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joeturner wrote:
But even if they are saying that, it still is not very helpful because you would have to be able to plate out lots of different microbes, which is expensive and not really the point of an indicator.


Hey Joe, based on your statement it appears you may not understand that 454 pyrosequencing based on amplification of microbial DNA is very fast and cheap. It's high throughput, meaning one pass of a probe reveals tons of data based on programming. I'm not an expert in this area, but believe this is the future. The equipment may be cost-prohibitive at present, but there are many institutions owning this equipment looking for research projects.

The same technology used to determine microbial populations in human intestines in the diarrhea studies I posted is also used in WWTP, but this is in its infancy. Functional medicine doctors order the same tests for their patients, called the future of medicine. Here's a 2012 WWTP study using pyrosequencing; this is opening up new worlds of information including previously unclassified bacteria dominant due to the wastewater treatment process:
Bacterial communities in different sections of a municipal wastewater treatment plant revealed by 16S rDNA 454 pyrosequencing
www.ncbi.nlm.nih.gov/pmc/articles/PMC3586070/

The original post states: "What pathogens should we test? Right now we are planning on a kit for helminth and E.coli."

My post is quite on topic as one of the studies I posted states E. coli is actually significantly decreased in diarrhea. This should be eye-opening to many who never considered E. coli as protective. The study implicates Bacilli as cause of diarrhea, so perhaps Bacilli would be a good indicator.

Thanks for your patience, folks, just trying to be helpful.
Last Edit: 24 Jul 2014 16:25 by KeithBell.

Re: High-throughput microbial gene detection seems like the future? - and technology used to identify dysbiosis 24 Jul 2014 16:23 #9479

  • KeithBell
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Here are a smattering of recent studies detailing flora shift in WWTP (wastewater treatment plants) as revealed by 16S rDNA 454 pyrosequencing. This is potentially very useful information which may help improve WWTP technology, but it also may give powerful justification for ending the practice of defecating and urinating in water.

In some cases, papers are not open access, but abstracts are also useful and it's good to know these new studies exist:

Exploring Variation of Antibiotic Resistance Genes in Activated Sludge over a Four-Year Period through a Metagenomic Approach
pubs.acs.org/doi/abs/10.1021/es4017365

Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach
www.sciencedirect.com/science/article/pii/S0043135414003728

Characterization of Bacterial Communities in Sediments Receiving Various Wastewater Effluents with High-Throughput Sequencing Analysis
link.springer.com/article/10.1007/s00248-014-0370-0

And a couple of scientific reviews likely including information culled from metagenomic studies:

Bacterial diversity and antibiotic resistance in water habitats: searching the links with the human microbiome
onlinelibrary.wiley.com/doi/10.1111/1574...sAuthenticated=false

Human health implications of clinically relevant bacteria in wastewater habitats
link.springer.com/article/10.1007/s11356-013-1594-0

This recent piece in The Guardian speaks toward the end of mixing human waste with water:

Why the modern bathroom is a wasteful, unhealthy design
Piped water may be the greatest convenience ever known but our sewage systems and bathrooms are a disaster

www.theguardian.com/lifeandstyle/2014/ju...ful-unhealthy-design
Last Edit: 24 Jul 2014 20:56 by muench.
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