Assays for compounds that make existing antibiotics effective against antibiotic resistant bacteria.

The figure above shows structures of the beta-lactam semi-synthetic antibiotic amoxycillin (top) and the different compound clavulanate (lower). These two compounds are used together as a powerful synergistic antibacterial combination therapy in current medical treatments. Although amoxycillin is a semi-synthetic penicillin, clavulanate is not even an antibiotic, but an inhibitor of the bacterial enzyme beta-lactamase, which is enzyme which makes many bacteria resistant to penicillins and the related cephalosporins. Clavulanate is thus augments or extends the effectiveness of beta-lactam antibiotics, and it is produced naturally by a soil Streptomycete. Note that amoxycillin has a side chain (with a phenol group) and the beta-lactam bicyclic ring system which includes N and S atoms. (Image from Sandoz.Com.)

The useful beta lactamase inhibitor clavulanate was discovered around 1970 using a clever but simple assay strategy.

It required a simple way of measuring inhibition of beta lactamase enzyme activity.

Thus the biological activity being sought was inhibition of enzyme catalysis.

For this another beta-lactam antibiotic, Nitrocefin, was most useful. This antibiotic is pretty useless in treating infection as it may cause cancer and many bacteria are resistant to it. But it is superb for detecting chemicals that inhibit beta-lactamases, such as clavulanate.

This is because it changes from yellow to red colour when exposed to beta-lactamase.

The diagram below illustrated how on overlay of soft agar containing a freshly made mixture of Nitrocefin plus beta-lactamase enzyme can reveal beta-lactamase inhibitors diffusing from a bacterial colony. the diagram shows schematically the cross section of petric dish culture on which an unknown type of soil bacteria have developed as a colony.

The most practically useful part of this strategy is that it can be cheaply and conveniently adapted to screen hundreds of thousands of novel bacterial isolates. It does this without demanding much expensive labour.

It is thus a cheap high through-put assay or screen.

This approach enabled a drug company to find soil microbes that produce then unknown biological activities of beta-lactamase inhibition in the 1970s. These activities included clavulanate. Late work by chemists led to other antibiotic extenders such as sulbactam and tazobactam being synthesised.

Further reading:
Link on Nitrocefin colour changes

More ambitious microtitre dish assays based on this idea.

Using microbes to mine copper.

The Escondida copper mine in Chile- the worlds largest copper mine. Photo BHP Billiton Group from here.

Thomas Barton has recently written a great book - The Australian Miracle: An Innovative Nation Revisited (Picador 2006) - which has many interesting things to say about commercial and technological innovation in Australia.

It has an interesting discussion of the use of bacteria to extract copper in the following passage (pages 223-4):

For many years, people in Australia have talked about the need to shift from an economy based on the production of physical goods to an economy based on the production of knowledge. This idea is so commonplace that it is difficult to imagine anyone interested in Australian technological and economic competitiveness not taking it up.

What does it mean, though? When most people hear this recommendation, they usually imagine it means that Australia needs to shift out of mining, agriculture and other traditional sources of wealth, and into design, computer programming, biotechnology and the arts.

In actuality, though, what the shift might imply is quite the opposite.

One of Australia's biggest biotechnology companies is now BHP Billiton, the mining giant. Little known to the outside world, the company has a biotech laboratory with around eighty employees whose main job is to develop the use of bacteria for minerals leaching. The group specialises in identifying bugs in hot springs and other extreme environments, and then breeds them up so that they become efficient at breaking down intractable ores. Furthermore, unlike a good number of other biotech companies, BHP Billiton is actually putting its products to work.

As part of a one-billion-dollar exercise at Escondida in Chile - the world's largest copper mine - the company is currently bioleaching a heap of ore that stretches across nine square kilometres. Biomining, for the first time in human history, is a reality.

How to find microbes that make Gold.

The production of microbial products is a huge global industry with many billions of dollars of products sold world wide that are originally derived from microbes. There is much scope to add new bioproducts to that list.

It requires innovative thinking, a good bioproduct discovery strategy, and alertness to opportunities that you might come across while you are reading about biology, science, and business.

Remember, during your searches for bioproduct candidates, the success of French microbiologist Louis Pasteur, who is famous for observing that fortune favours the prepared mind.

Some general approaches and ideas for discovery of novel bioactive compounds from microbes are:

1. Tap into microbial biodiversity
   
2. Search for novel organisms
3. Be alert to microbial culture and enrichment concepts. These include using indicator agar plates, for example containing enzyme substrates that change colour during a reaction.
4. Search in novel or extreme microbial environments.
5. Find and grow previously unculturable organisms.
6. Find compounds through gene cloning (metagenomics) from organisms that cannot be grown. see eg How to Find New Antibiotics, Jo Handelsman, The Scientist Volume 19 | Issue 19 | Page 20 | Oct. 10, 2005
   
7. Exploit suitable assays or screens to find novel biological activities. eg Coloured compounds produced by microbial colonies are easier to find than colourless compounds. Similarly substrates that produce colour (chromogenic compounds) or fluorescence (fluorogenic compounds) by a chemical reaction are extremly useful if a relevant or practically useful way of exploiting a reaction can be devised.
   
8. It's possible to find a novel microbe to make or provide a useful biological activity previously unknown in microbes using a well chosen assay.
9. Exploit specific or sensitive assay approaches.
10. Possibly identify a novel "drug" target to help develop an assay.
11. Use a high through-put assay or screening approach ( eg screens of millions of colonies on plates, screens in microtitre dishes).
12. Screen pools of different compounds or microbes in the one assay to find which pool has the posive microbe.
13. Use of chemical analysis techniques such as Mass spectroscopy, in innovative ways to allow high through-put detection on novel molecules
14. Encapsulate individual cells or samples in polmeric microscopic beads to facilitate screening
    

Pundit is going to add useful hyper links to this list to expand this information.

But why not help the Pundit and put your questions and comments about them below so we can discover good ideas by dialog.

For example, answer this question I'm putting forward to start you thinking about useful approaches:

How are sensitive and specific assays helpful to the bioproduct discovery process, and can you think of particular opportunities they open up?

There is plenty of helpful general reading that can help you prepare for the task of exploiting microbial novelty:

• Pages 26- 32 of The textbook Microbial Biotechnology by A.N. Glazer and H. Nikado, Freeman, 1995 are a good short introduction to some previously discovered microbial bioproducts such as plastics and drugs.
• Chapter 15. of Microbe, M. Schaechter et al ASM Press 2006 is very good background reading about bacterial diversity.
• Several previous posts at this site deal with compounds, such as siderophores, that are produced by soil streptomycete bacteria (these posts are readily accessible via the index hyper-link in the side-bar on the right.)
  

Process improvement in the antibiotic industry.

Year

Process improvement also occurs in the antibiotic industry. This example shows how microbiologists improve the process levels (concentrations) in production of an antibiotic over time. The slide is from an old talk by Arnold Demain of MIT.

The point it that many different experiments, such as genetic selections for improved microbe strains, and changes to growth media based on physiology experiments, allow process costs to be driven down over time.

Can you suggest examples of different biotechnology process improvements from previous published scientific work and document and explain how they were achieved?

There are so many different products to chose from, and so many different ways of reducing costs of production, so let me have your ideas in the comments. Let's work them up together for a series of Microbe Pundit Posts to share around.

For example how have ethanol costs been reduced?

Technological Learning Curves: Renewable Energy Redux.

Renewable Energy Redux. Data showing learning curve demonstrating reduction in cost for Brazilian ethanol (blue) undercuts price of petroleum based liquid fuel (red) by 2005. From a talk by S. Coelho April 2006.

Welcome back to students of the Microbe. This semester, The Pundit is going to concentrate on Microbial Biotechnology.

He's especially emphasising technological innovation. It's this innovation that has delivered most economic growth this last century, and that's what solves problems in this world - prosperity and resources that better management other problems in societies feasible.

The message is clear: intellectual assets and imagination saves lives, rain-forest, and pristine wilderness.

To start the innovation ball rolling, The Pundit is posting some examples of how innovation over time allows biotechnology products to be made more cheaply and efficiently.

This is the slow burn of incremental improvement year after year, only a few percent per year, but eliminating much poverty over decades.

A super example is ethanol fermentation used in Brazil to make liquid fuels. That is, sugar cane based biofuel.

This started out being relatively energy, land, and cost inefficient in the early 1980s, and bioethanol fuel nearly collapsed as an industry in Brazil when oil prices fell because it was very costly. It dragged subsidies from other areas of agriculture where investment was sorely needed. Not so today.

In this case many technological innovations have allowed ethanol from sugar cane to become energy efficient (with fuel energy output being 3-4 times the fuel energy input EOIR) .

Brazilian ethanol is now cost competitive with petroleum liquid fuels without any subsidy. These innovations, including biotechnology-based method changes in fermentation and strain improvement, are ongoing, and there is ample room for further efficiency improvement and more innovation.

A detailed economic documentation of this case is given by Goldemberg et al 2004.

Biomass and Bioenergy 26 (2004) 301 – 304
Short communication
Ethanol learning curve—the Brazilian experience
Jose Goldemberg, Suani Teixeir Coelhob, Plinio Mario Nastaric, Oswaldo Lucond

Abstract

Economic competitiveness is a very frequent argument against renewable energy (RE). This paper demonstrates, through the Brazilian experience with ethanol, that economies of scale and technological advances lead to increased competitiveness of this renewable alternative, reducing the gap with conventional fossil fuels.