Screening for microbes and strain

improvement

Maulik P. Suthar

© 2009, Maulik P. Suthar

 Microbial products

• Primary metabolites : Made during the organism’s growth phase.

Essential to an organism’s metabolism and can be intermediate
metabolites or end
• Secondary metabolites: Not essential to cell function or growth and
are usually made late in the organism’s growth cycle. Usually derived
from primary metabolites or intermediates of primary metabolites.
Most likely give the organism an advantage over competitors

Primary Secondary

© 2009, Maulik P. Suthar

 Methods for screening

• Microbial
• Genotypic
• Phenotypic
• Chemical

© 2009, Maulik P. Suthar

 Strain improvement

• Stable production strain : with a high titre and ideal production

profile, which makes a simple purification process possible. To
obtain such strains we use a combination of traditional methods
such as random mutagenisation and genetic engineering.

• Mutagenisation and selection very effectively increase the titres of

desired compounds. Gene technology is used to obtain an ideal
production profile, e.g. to inactivate genes leading to side products
and to improve the self-resistance of the production strain, which
increases both stability and titre.

© 2009, Maulik P. Suthar

 What are Antibiotics?

• Antibiotics = “against life”

• Antibiotics = molecules that stop microbes, both bacteria and fungi,
from growing or kill them outright.
• Antibiotics can be either natural products or man-made synthetic
chemicals.
• Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism, within a
particular environment, to affect neighbouring microbes.
• Can cause microbe death or regulate the growth of the neighbouring
microbes.
• These antibiotic agents are produced by both bacteria and fungi.

© 2009, Maulik P. Suthar

 Antibiotic Production

• Antibiotics are produced industrially by a process of fermentation,

where the source microorganism is grown in large amounts.

• As the antibiotics are produced by the microorganisms they have to

be removed to ensure they do not effect the bacterial population.

• Certain bacterial species are selectively mutated to produce the

maximum amount of the antibiotic agent.

© 2009, Maulik P. Suthar

© 2009, Maulik P. Suthar
© 2009, Maulik P. Suthar
© 2009, Maulik P. Suthar
 Mutations and Genetic Instability

• Spontaneous mutations include a variety of molecular insults,

including deletions, duplications, transpositions, insertions, base
pair substitutions and reading frame shifts.

© 2009, Maulik P. Suthar

 Induced Mutations

UV radiation
Chemical Mutations
1. 4-Nitroquinoline-1-oxide
2. Hydroxylamine
3. Methyl Methanesulfonate
4. Ethyl Methanesulfonate
5. N-methyl-N'-nitro-N-nytrosoguanidine

© 2009, Maulik P. Suthar

Mutagenesis and Strain Development

Mutation induction in Streptomyces is a complex process. Not all

mutagens are capable of inducing a high level of mutation in all
streptomycetes.
1. Treat spore suspension with chemical or UV mutagen.
2. Isolate improved mutants using screening techniques.

Shake Flask Screens are the most popular method for isolation of
improved mutants and is still used frequently today.
1. Treat with mutagen
2. Plate out on growth medium at low density
3. Use agar plugs to isolate single colonies
4. Innoculate liquid cultures with test colony.
5. Screen for increased productivity.

© 2009, Maulik P. Suthar

 Plate Based Techniques

• As before, treat spore suspension and plate at low density.

• Culture the spores into colonies on the plate.
• Overlay the colonies with membrane innoculated with test organism.
• Zones of exclusion are measured using image analysis.
• Largest zone of exclusion, highest concentration of antibiotic
diffused into the surrounding agar.

© 2009, Maulik P. Suthar

 Antibiotic Production

• Despite the wide variety of known antibiotics, less than 1% of

antimicrobial agents have any medical or commercial value.

Why screening ?
• In order to identify the useful antibiotics, a process of screening is
often employed.
• Using this method, isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products
which inhibit the growth of test organisms.

© 2009, Maulik P. Suthar

 Production with microbes

Fermentation
• Ability of microbes to produce ‘useful’ product
• Usually a naturally produce substances for growth, and
maintenance.
• Metabolites
• Primary metabolite – produce during growth
• Secondary metabolite – produce after growth stage

© 2009, Maulik P. Suthar

 Steps in Industrial fermentation

Isolation of microbes that produce product of interest

• Screen for the best producing strain: naturally or mutation.
• Optimize production condition (growth – basically) in lab
• Scale up from lab scale (up to 10 L) to industrial scale (>10,000 L)
(raw material?)

© 2009, Maulik P. Suthar

Industrial organism

© 2009, Maulik P. Suthar

 AN INDUSTRIAL MICROORGANISM
MUST
1. Produce the product of interest in high yield
2. Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor, whey) [ALL NON-animal cf. Mad Cow Disease –
BSE]
– A major production cost of commodity chemicals is the
substrate.
– COMMODITY chemicals are inexpensive chemicals produced in
bulk, including ethanol, citric acid, and many others - DEE
antibiotics)
3. Be amenable to genetic manipulation – mutation, genetically
engineered (NYT Feb.?) - yet stable!
4. Be non-pathogenic

© 2009, Maulik P. Suthar

 Industrial micro-organisms
• Often fungi or streptomycetes (bacteria)
• Ideally produce spores or some other reproductive cell form that can be
easily inoculated into large fermenters
• Capable of growth & product formation in large scale culture
• Grow rapidly & produce product in relatively short period
• Organisms can be manipulated in large-scale culture to produce one or
more products in high concentration
• Organisms are often genetically altered by mutation or recombination
• Industrial strains can be very different from “wild type” of the organism
found in nature
• Able to grow in inexpensive liquid culture media – waste from other
industries- eg
– Corn steep liquor from corn milling (rich in N & growth factors)
– Whey from cheese production (rich in lactose & minerals)
• Should not be pathogenic (humans, animals, plants)
• Amenable to genetic manipulation

© 2009, Maulik P. Suthar

Antibiotic producing organisms

© 2009, Maulik P. Suthar

 Level of antimicrobial activity

• Effectiveness expressed in two ways

1. Minimal Inhibitory Concentration (MIC) : lowest concentration of
drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc. That inhibits is MIC

2. Minimal Lethal Concentration (MLC): lowest concentration of drug

that kills pathogen
- Subculturing into fresh medium can reveal MLC

© 2009, Maulik P. Suthar

 Antimicrobial tests

• Dilution Susceptibility Tests : involves inoculating media

containing different concentrations of drug
• Disk Diffusion Tests : disks impregnated with specific drugs are
placed on agar plates inoculated with test microbe 􀂄 drug diffuses
from disk into agar, establishing concentration gradient . observe
clear zones (no growth) around disks
– Standardized bacterial culture on spread on agar
– Filter paper disk impregnated with antimicrobial compound is placed on
agar
– Compound diffuses into agar creating a conc. Gradient
– Diameter of zone of Inhibition reflects the sensitivity of organism to
compound

© 2009, Maulik P. Suthar

The Etest- strip test

© 2009, Maulik P. Suthar

 How do the microbes produce these
products?
• Genetically modified microorganisms
• Clone genes that encode protein of interest into vectors and express
this genes in microbes used in production.
• Able to produce high amount of compounds than in original
organisms with ease of manipulation.
• Microbes able to utilize waste from other process as a raw material
for fermentation.

© 2009, Maulik P. Suthar

 Search for new antibiotics

Two approaches:
1. Bioprospecting
2. Use of molecular microbiology, computational chemistry ...

© 2009, Maulik P. Suthar

 Bioprospecting

Investigating naturally occurring compounds for antimicrobial activity

– Soil organisms
– streptomycetes, actinomycetes, fungi,
– Marine actinomycetes
– Plants
– Corals and other marine organisms
– Products from insects and animals eg frog skin secretions

© 2009, Maulik P. Suthar

Bioprospecting - Step 1

© 2009, Maulik P. Suthar

Bioprospecting - step 2

© 2009, Maulik P. Suthar

 Molecular approaches

• Molecular approaches – enables target identification and expression

→ facilitates in-vitro screening & identification of lead molecules
• Limited success with antimicrobials – many leads unable to cross
the bacterial cell wall/membrane eg t-RNA synthetase inhibitors,
FabH carbapenamase FabH,
• More successful examples – benzimidazole & indazole gyrase
inhibitors

© 2009, Maulik P. Suthar

 DNA microarrays

• High-density DNA microarrays allow researchers to monitor the

relative expression levels of thousands of genes (an entire microbial
genome)
• Enables studies of coordinated gene expression that occurs under
various microbial growth conditions
• Expose organism to various sub-lethal concentrations of antibiotics
• Enables identification of genes that are more active or less active in
the presence of antibiotics
• Could provide targets for further study –eg mechanism of action
(MOA) or identify targets with unknown MOA

© 2009, Maulik P. Suthar

The Design Bicycle

© 2009, Maulik P. Suthar

 Novel approaches

• Virtual screening
– Enzyme purification, crystallization and 3-D structural
determination of a drug target (X-ray crystallography, NMR)
– Once target site identified – molecules with potential to target can
be identified using computer modelling
• Approach only works for targets amenable to 3-D structure
determination
– many targets are too complex
– Various known or unknown structures docked to target site and
those with best fit are selected for “wet screening”
– Rational approach has been used for lead optimization for a
number of targets e.g. gyrase inhibitors, HIV protease
– not many significant leads for antibacterial targets.

© 2009, Maulik P. Suthar

 Novel approaches

2. Genome-wide antibacterial targets - sequencing of bacterial genomes

– Genomes of around 80 bacteria now completely sequenced - > 100 others underway Eg S
aureus
• Using microarray methods - estimated that genome contained 265-359 essential
genes
• 60% broadly conserved in clinically relevant bacteria
• Currently marketed drugs already target about 15 of the essential gene products
• Identification of targets
– Targets whose genes are essential for in-vitro growth
• All currently marketed antibiotics inhibit or kill bacteria grown in vitro
– Targets that only expressed in vivo or are essential for growth and survival in the infected
host, eg P pilus gene in UTI E coli
• Biofilm production by Pseudomonas aeruginosa
• Bioinformatic analysis of genomic data can assist
– Identify genes conserved in multiple bacterial species
• Determining if a gene is essential
– Random mutagenesis
– Targeted mutagenesis

© 2009, Maulik P. Suthar

 Identification of targets

• Target gene then cloned and sequenced

• Corresponding protein expressed in a standard expression system
(eg Pichia pastoris, Baculovirus or E. coli)
• Target protein is purified
• Biochemical assay developed for screening large number of diverse
low molecular wt. compounds to identify target inhibitors (“hits”)
• “Hits” characterized in terms of potency, mechanism of inhibition
and enzyme spectrum and selectivity

© 2009, Maulik P. Suthar

 Genomics and target based
identification of new antimicrobials
• Once whole cell activity is established need to check if the
antimicrobial activity is linked to the intended target or via an
unintended mechanism
– Use defined mechanism of action (MOA) assays
– These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency, spectrum and selectivity.

• As lead compound series are identified and optimized, DMPK (drug

metabolism and pharmacokinetic) studies are undertaken and
animal models of infection are applied.

© 2009, Maulik P. Suthar

 Search for new antibiotics

• Stage 1 – primary screen

Elementary tests to determine activity in-vitro against small number of
key organisms
• Stage 2 - secondary screen
Selected compounds tested more extensively – aim to determine if
compound could be useful in clinical medicine
• Stage 3 – toxicity testing
Compounds thoroughly tested in animals

© 2009, Maulik P. Suthar

 Search for new antibiotics

• Stage 4 – human pharmacokinetics

Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administration
• Stage 5 clinical trials
Effectiveness & acceptability of the compound tested in patients

© 2009, Maulik P. Suthar

 Parameters which determine the
usefulness of an antibiotic
• Demonstrable activity against target pathogen(s)
• Breadth of antibacterial spectrum & degree of activity of the
compound
• Bacteriostatic or bactericidal?
• Any cross-resistance with existing antimicrobials?
• How readily resistance emerges in important pathogens?
• How resistant it is to destruction by bacterial enzymes?

© 2009, Maulik P. Suthar

 Parameters which determine the
usefulness of an antibiotic
• Stability to degradation by animal tissues
• Extent to which it is bound to serum proteins
• Pharmacokinetics in experimental animals following administration
by various routes
• Ability to protect animals from experimental infections
• Freedom from toxic effects in animals
• Human pharmacokinetics in volunteers – after administration via
various routes
• Side effects in volunteers – pain on injection, nausea, headache or
other symptoms not predicted by toxicity tests in animals

© 2009, Maulik P. Suthar

 Antibiotic Targets

• The major classes of antibiotics affect 1 of 3 targets in bacteria cells:

(1) Cell wall biosynthesis
– penicillins
– cephalosporins
– vancomycin(non-ribosomal peptide)
(2) Protein synthesis
– erythromycin (macrolidepolyketides)
– tetracycline (aromatic polyketides)
– streptomycin, kanamycin(aminoglycosides)
(3) DNA replication
– quinolones(Cipro)

© 2009, Maulik P. Suthar

 Antibiotic Targets

Antibiotics work by exploiting biochemical differences between our

eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis
-block synthesis of peptidoglycan, the covalently cross-linked
peptide/glycannetwork, which imparts osmotic resistanceto cell
(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase
center of bacterial ribosome
-stop elongation of growing peptide chains
(3) DNA replication
-inhibit gyrase, essential enzyme that uncoilsintertwined circles of
DNA after replication of the circular bacterial chromosome

© 2009, Maulik P. Suthar

 Questions

• Importance of industrial microorganisms

• Properties of useful industrial organisms
• Differentiate primary and secondary metabolites
• Importance of control of microbial growth processes
• Outline the key steps in the production of penicillin and
semisynthetic penicillins
• Outline the steps in the production of tetracyclines
• Describe the key approaches in the search for new antibiotics
• Describe the steps involved in isolation of new antibiotics from soil
organisms
• Describe the importance of molecular advances in the search for
new antibiotics
• Outline the steps in the development of a new antibiotic
• Describe the parameters that determine the usefulness of a new
antibiotic

© 2009, Maulik P. Suthar