Okafor N. Modern Industrial Microbiology and Biotechnology

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"" Modern Industrial Microbiology and Biotechnology

24.3.1.2 The development of previously non-pathogenic

microorganisms into pathogens

As clinical practice now use more invasive methods and people live longer, more and

more people now depend on adequate antimicrobial coverage. Especially in patients

who are immunocompromised, microorganisms which were previously non-pathogenic

or ordinary commensals have become pathogens due to the widespread use of

antibiotics. Thus, Proteus sp., Acinetobacter sp. and yeasts have all gained new status as

pathogens especially in intensive care units.

24.3.1.3 Need to develop anti-fungal antibiotics

Currently there are few satisfactory systemic anti-fungal and anti-viral antibiotics. In the

case of anti-fungal agents, currently few satisfactory systemic antifungal antibiotics

outside Amphothericin B seem to exist; even Amphothericin B is not always efficacious.

There is a similar dearth in the anti-viral antibiotics.

24.3.1.4 Need to develop antibiotics specifically for

agricultural purposes

The growing needs for antibiotics used in agriculture for combating plant diseases, in

animal feeds and in veterinary practice dictate that antibiotics be found specially for food

production, but not for human medicine because of the problem of resistance.

24.3.1.5 Need for anti-tumor and anti-parasitic drugs

Although microbial metabolites for combating tumors, helminthes and parasites cannot

be strictly described as antibiotics in the conventional sense, their production by the

fermentation of microorganisms may allow the loose use of the term anti-tumor

antibiotics and anti-helminthes antibiotics. Antibiotics need to be produced from

microorganisms for these purposes.

24.3.2 The classical method for searching for antibiotics:

random search in the soil

The classical method for the search for new antibiotics is by random search in the soil.

This method will be described briefly below as a setting for more recent methods.

Although the first important commercially produced antibiotic was discovered by

chance, most present day antibiotics were discovered by systematic search. The soil is a

vast repository of microorganisms and it is to the soil that search is turned when

antibiotics are being sought. The stages to be discussed below are not necessarily rigidly

followed; they are merely meant to indicate in a general manner some of the activities

involved in the development of antibiotics. The most important are:

(i) The primary screening: Several methods have been employed in primary screening.

(a) The crowded plate: This method is used to isolate soil organisms able to produce

antibiotics against other soil organisms. A heavy aqueous suspension (1:10; 1:100)

of soil is plated on agar in such a way as to ensure as much as possible the

development of confluent growth. Organisms showing clear zones around

Production of Antibiotics and Anti-Tumor Agents ""

themselves are isolated for further study. Different groups of organisms could be

encouraged to develop by altering the media used.

This method has the disadvantage that slow-growing antibiotic-producing

organisms such as actinomycetes are usually over grown and are therefore hardly

isolated. Furthermore, the test organisms used in this method are soil organisms.

The susceptibility of soil organisms to the antibiotics produced in the test, may

therefore be unrelated to the susceptibility of clinically important organisms.

(b) The direct-soil-inoculation method: This method is used when the aim is to isolate

antibiotics against a known organism or organisms. Pour plates containing the

test organisms are prepared. Soil crumbs or soil dilutions are then placed on the

plates. Antibiotic producing organisms develop which then inhibit the growth of

the organisms in the plate. They are recognized by the cleared zone which they

produce around themselves and they may then be picked out.

especially actinomycetes which may be obtained from soil without any previous

knowledge of their antibiotic-producing potential. The organism may come from

one of the two methods already indicated above.

The purified isolate is streaked across the upper third of plate containing a

medium which supports its growth as well as that of the test organisms. A variety

of media may be used for streaking the antibiotic producer. It is allowed to grow for

up to seven days, in which time any antibiotic produced would have diffused a

considerable distance from the streak. Test organisms are streaked at right angles

to the original isolates and the extent of the inhibition of the various test organisms

observed (Fig. 24.4).

(d) The agar plug method: This method is particularly useful when the test organism

grows poorly in the medium of the growth of the isolate such as fungi. Plugs about

0.5 cm in diameter are made with a sterile cork borer at progressive distances from

the fungus. These plugs are then placed on plates with pure cultures of different

organisms. The diameters of zones of clearing are used as a measure of antibiotic

production of the isolate. The method may be used with actinomycetes.

Fig. 24.4 The Cross Streak Method for the Primary Search of Antibiotic Producing Organisms

"" Modern Industrial Microbiology and Biotechnology

(e) The replica plating method: If a large number of organisms are to undergo primary

screening, one rapid method is the use of replica plating. This is a well-known

method used in microbial genetics. It was discussed in Chapter 5 dealing with the

production of mutants. The method consists of placing a sterile velvet pad on the

colonies formed in the crowded plate or soil inoculation plate, or on series of

discrete colonies to be tested for antibiotic properties. The pad is thereafter

carefully touched on four or five plates seeded with the test organisms. As a

landmark is placed on the pad as well as on the plates it is possible to tell which

colonies are causing the cleared zones on the tested plates (Fig. 24.5).

(ii) Secondary screening: Organisms showing suitably wide zones of clearing against

selected target organisms are cultivated in broth culture in shake flasks using

components of the solid medium in which the isolate grew best. Crude methods of

isolating the active antibiotic are developed by extracting the broth using a wide range of

extractive methods. With each extraction the resultant material is assessed for activity

against the target organisms at various dilutions. The extract is either spotted on filter

paper discs placed on agar seeded with the test organism or introduced into wells dug

out from the seeded agar with sterile cork borers. In this manner the most efficient

extractive methods and the spectrum of activity of the organisms are determined.

Colonies 1 - 7 are transferred by a velvet pad to plates seeded with

E. coli

Bacillus

sp., and

Proteus

sp.

respectively. Note that colonies 1,5,7 produce dear zones in

E. coli,

Bacillus

sp., and

Proteus

sp.

respectively.

Fig. 24.5 Replica Plating Method of Testing Antibiotic Producing Colonies

Production of Antibiotics and Anti-Tumor Agents ""!

Secondary screening is aimed at eliminating at an early stage any antibiotic which

does not appear promising either by virtue of low activity, other undesirable properties or

because it has been discovered previously.

Antibiotic spectrum: The minimal inhibitory concentration (MIC) is a means of

determining the activity of the isolated antibiotic and comparing this activity with those

of existing antibiotics. Tests involving agar diffusion such as filter paper discs or agar

wells described above are rapid and very useful for initial screening. However, they

involve not only the intrinsic anti-microbial potency of the antibiotic produced but also

its ability to diffuse through agar. The MIC has the advantage that it is performed in broth

thereby eliminating the disadvantage of large-molecule slower-diffusing antibiotics.

(iii) Other properties: The other qualities of the antibiotic outside anti-microbial activity

depend on its intended use. For antibiotics meant for clinical use, information on a

number of the following may be sought at this stage:

(a) Toxicity to mammals, determined by intra peritoneal injection into animals;

(b) Haemolysis is tested by observing the effect on blood agar;

susceptible organisms;

(d) The inactivation of the antibiotic by several enzymes from various organs is tested

by exposing the antibiotic to them;

(e) Acid stability is tested if the antibiotic is meant for oral fermentation

(f) Tetragonicity tests, which determine the effect on the unborn are carried out on

laboratory animals;

(g) For plant antibiotics, phytotoxicity as shown by damage to leaves in the laboratory

and in the green house, is determined;

(h) For feed antibiotics, low absorbability and low toxicity are desirable and are tested.

Several other tests designed to certify the safety of administering the antibiotic may be

carried out at this stage or performed later.

As part of the secondary screening, initial studies on the chemical nature of the crude

antibiotics is determined using paper chromatography, ultraviolet absorption, solubility

in acid and alkali, optical rotation, infra-red absorption, nmr, etc. The aim of this study is

to see if it is an already known antibiotic. If it is, then further work on it may be

abandoned. It is important to make this decision early. As has been shown previously,

the same antibiotic is often produced by a large number of different organisms. This

method known as ‘finger printing’ is more fully discussed when anti-tumor antibiotics

are examined.

(iv) Further laboratory evaluation: If and after all the above, the antibiotics is promising,

then further experimentation is done in shake flasks as a preparation for pilot

production. The optimal conditions of growth are determined; the most suitable medium,

optimal pH, temperature, length of fermentation etc. are all determined.

(v) Pilot plant production: The results obtained in previous experimentation are fed into

the pilot plant. The material produced is subjected to further safety tests and chemical

analysis. Enough materials are made available for the structure to be determined.

""" Modern Industrial Microbiology and Biotechnology

Methods for the isolation are improved and perfected. Clinical tests on a limited scale

may be carried out at this stage. The Journal of Antibiotics regularly carries several articles

describing the procedures for the chemical analysis of many new antibiotics. Unless the

organism is already known, it is also described and may be named. The mode of action,

the route of biosynthesis and strain improvement are undertaken, but the production of a

good antibiotic does not await their successful completion.

(vi) Plant production: The production plant utilizes all the information obtained in the

pilot experimentation.

(vii) Certification: A government agency must approve the antibiotic before it becomes

available for general use. In the US, it is the Food and Drug Administration. In the UK and

EU countries, it is the European Medicines Agency (EMEA) which was established in

1993 and is based in London. The process for the certification of drugs by the FDA is

discussed in greater detail later in Chapter 28.

(viii) Marketing and financing: The marketing and financing of the business are of

paramount importance since the aim of the producing firm is profit maximization.

24.4 COMBATING RESISTANCE AND EXPANDING THE

EFFECTIVENESS OF EXISTING ANTIBIOTICS

Many ways have been devised to combat microbial resistance to existing antibiotics or

expand the effectiveness and to discover new ones. These include modifying the

procedures for the classical search in soil, searching for antibiotics in novel

environments, and chemically manipulating the antibiotics directly or through using

mutant microorganisms.

24.4.1 Refinements in the Procedures for the Random

Search for New Antibiotics in the Soil

To increase the chances of finding new antibiotic - producing organisms, some

refinements of the classical methods of searching for antibiotic producers were

introduced as shown below.

24.4.1.1 The use of super-sensitive mutants

By using super-sensitivity strains of test organisms, organisms producing only small

amounts of an antibiotic may be detected. Antibiotics so produced are useful because

they may have a wider spectrum than those of the same class already in existence.

Furthermore they may provide better substrates for semi or muta-synthesis. This method

has led to the discovery of novel Beta-lactam antibiotics, thienamycin, olivanic acid,

nocardicins, clavulanic acid etc. It is remarkable that several natural clavulanic acid type

compounds (e.g. 2-hydroxymethylclavam) have significant antifungal properties. The

use of super-sensitive mutants has shown that Beta-lactam antibiotics are produced by a

wider spectrum of organisms – ascomycetes, fungi imperfecti and actinomycetes – than

was previously thought.

Production of Antibiotics and Anti-Tumor Agents ""#

24.4.1.2 The application of criteria other than death or inhibition

Reactions such as irregular growth of the fungal mycelium, inhibition of sporulation, or

some readily ascertainable deficiency rather than death may be used to follow the

antibiotic effect. When anti-fungal antibiotics are sought, the clear zone principle is

employed using yeasts or fungal spore in the test plate. With this method the existing

antifungal antibiotics, namely polyenes as well as cycloheximide and actimycins were

found. If criteria less drastic than death e.g. abnormal growth of hyphae, or inhibition of

zygosphore formation, then a wide range of antibiotics may be found. Thus some new

antibiotics have been found in actinomycetes including boromycin, venturicidin and

mikkomycin, with this method.

24.4.1.3 Search for antibiotics effective in

conjunction with other antibiotics

Some antibiotics while being effective are not permeable through the wall of the test

organism or the pathogen. Such antibiotics should be sought in nature by using test-

organisms with deficient cell walls, protoplasts, or by the incorporation of detergents or

EDTA which enable the permeation of antibiotics into the organism. When they are

discovered they can be made permeable by using them in conjunction with wall-

inhibiting antibiotics or compounds, or coupling them to compounds which bacteria

ingest by active transport.

24.4.1.4 Use of organisms of recent clinical

importance as test organisms

The classical search used routine organisms such as E. coli and Bacillus. The result as has

been shown has been that fewer and fewer new antibiotics have been discovered. In

recent times new organisms previously of little importance in clinical practice have

emerged, due to the widespread use of antibiotics as important medical organisms. These

include anaerobes, Gram-negatives e.g. Proteus, Beta-lactamase producing gonococci,

facultatives, haemolysis, etc. These should be used as the test organisms in place of the

previous ones.

24.4.2 Newer Approaches to Searching for

Antibiotics

In spite of the above modifications and refinements in the classical methods for searching

for new antibiotics, antibiotics with new structures were not discovered and cross-

resistance among the available antibiotics continue to occur. In recent times some newer

approaches to discovering new antibiotics have been adopted.

24.4.2.1 Search in novel environments

Systematic search for antibiotics is usually from soil. Other natural bodies exist which

can provide novel organisms. Two of such habitats will be discussed in this section,

namely the sea and white blood cells.

""$ Modern Industrial Microbiology and Biotechnology

24.4.2.1.1 The sea as a habit for prospecting for micro-organisms

producing antibiotics (and other drugs)

The seas and oceans occupy 70% of the earth surface. Until recently they were not

exploited as sources of antibiotic-producing organisms. Although they would present

new difficulties such as the need for a boat, they are a unique habitat. They are not only

twice the land area of the earth, they contain large amounts of salt and other mineral

nutrients, have fairly constant temperature, and have a higher hydrostatic pressure and

less sunlight in the deeper regions. The coastal area is constantly changing with tides

and such areas should be expected to have a wide variety of organisms, peculiar to the

littoral environment.

Although deep ocean exploration is still in its infancy, many scientists now believe

that the deep sea harbors some of the most diverse ecosystems on Earth. This diversity

holds tremendous potential for human benefit. More than 15,000 natural products have

been discovered from marine microbes, algae, and invertebrates, and this number

continues to grow. The uses of marine-derived compounds are varied, but the most

exciting potential uses lie in the medical realm. More than 28 marine natural products are

currently being tested in human clinical trials, with many more in various stages of

preclinical development. To date, most marketed marine products have come from

shallow and often tropical marine organisms, due mainly to the ease of collecting them.

But increasing scientific interest is now being focused on the potential medical uses of

organisms found in the deep sea, much of which lies in international waters. These

organisms have developed unique adaptations that enable them to survive in dark, cold,

and highly pressurized environments. Their novel biology offers a wealth of

opportunities for pharmaceutical and medical research and a growing body of scientific

evidence (Table 24.2) suggests that deep sea biodiversity holds major promise. The

medicines in the Table do not include antibiotics, but it could be because search for them

was not conducted in this particular study. Nevertheless the search for antibiotics in the

sea has indeed led to the discovery of new and unique antibiotics. These include

antibiotic SS-228R from Chainia sp. effective against Gram-positive bacteria and tumors,

bromopyrrole from a marine Pseudomonas, and leptosphaenin from marine fungi.

24.4.2.1.2 Antibiotic sources other than microorganisms: bactericidal/

permeability increasing protein (BPI)

Antibiotics are produced also by higher organisms – plants and animals – and they also

should be screened by the regular method of antibiotic screening. However because of

established practice, antibiotics from such higher organisms have been usually screened

for anti-tumor and anti-viral activity. Nothing intrinsic in materials from higher

organisms should stop them from acting against microorganisms in suitable cases. In

this section Bactericidal/permeability increasing protein (BPI) will be discussed as an

example of a novel antimicrobial agent derived from a living thing higher than

microorganisms.

BPI, is a protein and has been studied for several decades. It is derived from the white

blood cells, polymorphonuclear leucocytes, the primary phagocytic white blood cells

responsible for part of the body’s innate immune response. BPI has great affinity for the

lipopolysaccharide layer (LPS) of Gram-negative bacteria. It immediately arrests the

Production of Antibiotics and Anti-Tumor Agents ""%

growth of Gram-negative bacteria, increases the permeability of the outer and inner

membranes of the Gram-negative bacterial wall and eventually kills the organism. It is

attractive as a possible antibiotic for several reasons. First, it is highly potent and specific

against Gram-negative bacteria, which include many important human pathogens; it is

at least 10 times more potent than any known mammalian anti-microbial protein or

peptide. Second, it is non-toxic to mammalian cells. Third, it maintains its anti-microbial

Table 24.2 Deep sea compounds in development for medical use (July, 2005)

Name Application Source Depth/Location Status Comments

E7389 Cancer: Sponge: 330 ft (100 m) Phase I

non-small Lissodendoryx New Zealand clinical trials

cell lung sp.

and other

types

Discode- Cancer: Sponge: 460 ft (140 m) Phase l trials Toxicity

morlide solid tumors Discondermia Bahamas (completed) similar to

Dissolute Taxol ®;

works on

multi-drug

resistant

tumors

Diclyos- Cancer Sponge: Order 1,460 ft (442 m) Preclinical Toxicity

tatin -1 Lithistida, Jamaica Development similar to

Family Taxol ®

Corallistadae

Sarcodictyin/ Cancer Coral: 330 ft (100 m) Preclinical Toxicity

Eleutherobin Sarcodictyon Mediterranean Development similar to

(related roseum Taxol ®

compounds)

Salinospor- Concer: Microbe: More than Preclinical Will enter

amide A melanoma, Selinospora 3,300 ft (1,000 Development clinical trails

colon, breast, m) North in 2005;

non-small Pacific Ocean potency 35x

cell lung omuralide

Topsentin Anti- Sponge: 1980 ft Preclinical

inflammatory: Spongosporites (1,000 m) Development

arthritis, skin ruetzleri Bahamas

irritations

Cancer: colon

(preventive)

Alzheimer’s

Orthopedic Bone grafting Coral: Family More than Preclinical Resources

implants Isididae 3,300 ft Development risk of

(1,000 m) mammalian

North Pacific disease

Ocean

""& Modern Industrial Microbiology and Biotechnology

activity in the complex environment of body fluids, unlike many mammalian anti-

microbial agents. Finally it is not only a potent antimicrobial agent, but it also reduces the

effect of the inflammatory response known as ‘septic shock’ which the

lipopolysaccharide of the Gram-negative cell wall induces in patients. None of the other

proteins able to bind to the Gram-negative lipopolysaccharide is able to neutralize the

effect of toxic shock. It is currently at the stage of clinical trials.

24.4.3 Chemically Modifying Existing Antibiotic:

The Production of Semi-synthetic Antibiotics

The production of a semi-synthetic antibiotic involves the use of a fermentation-derived

antibiotic, which is then modified by the addition of side chain to give rise to an antibiotic

with new properties. As has been discussed, a well-known example is the modification of

penicillin G to 6-APA and the subsequent use of chemical reaction to produce semi-

synthetic penicillins. Another example achieved in a different manner is the chemical

alteration of specific sites in an antibiotic in order to render the antibiotic immune to an

enzyme which destroys it, as is done with streptomycin.

24.4.4 Modifying an Existing Antibiotic Through

Synthesis by Mutant Organisms: Mutasynthesis

This method is one in which a mutant of an antibiotic producing organism is fed different

precursors leading to the production of new antibiotics. Since the original production of

hybridicins from the neomycin synthesizing Strep. fridiae, this method has been used in

the production of paromomycin by Strep. rimogus forma paromonycins, ribostamycin by

Strep. ribosidificus and butrosin by Bacillus circulans.

24.5 ANTI-TUMOR ANTIBIOTICS

24.5.1 Nature of Tumors

Each cell in the animal (and human) body has a definite function which it carries out in

cooperation with other cells. Thus the brain, the skin and the intestines are composed of

specialized cells which cooperate to carry the functions of these organs. Sometimes

however a cell in any part of the body may no longer cooperate with others with which it

normally functions in an organ. Such cells divide indiscriminately and independently of

the others to form a structure called a tumor or a neoplasm. Sometimes the body restricts the

growth of tumors by forming a capsule round them. Under these conditions they do not

spread: they are known as benign tumors. Other tumors however grow rapidly and are

not restricted by a capsule. Such tumors are malignant. The cells in malignant tumors

often break off and are carried via blood vessels and lymphatic vessels to other parts of

the body where they initiate new tumors. When such secondary growth occurs away

from the primary tumor the situation is known as metastasis.

Tumors are further classified according to the type of tissue they attack. Some of these

will be mentioned: a malignant tumor composed of epithelial cells is called a cancer or a

carcinoma. Adenocarcinomas are tumors formed around the mucous membranes such as in

the alimentary canals. Sarcomas are connective tissue tumors. The term ‘hard’ tumor is

Production of Antibiotics and Anti-Tumor Agents ""'

sometimes used to distinguish neoplasms formed in the solid parts of the body such as

the gut, bones, brain etc. from those of blood such as leukemia which is a neoplasm of the

white blood cells. Neoplasms are treated by one or more of three methods: (1) by surgery

to remove the cancer; (2) by radiation, which aims at selectively destroying the cancer

cells and (3) by chemotherapy or the use of chemicals which affect the tumor cells without

damaging the normal cells. When such chemicals are produced by microorganisms they

are called anti-tumor antibiotics. Chemotherapy is particularly useful when the disease

has metastasized to several sites in the body so that it becomes practically impossible to

achieve any success by surgery or by radiation. It is also used after treatment by surgery

or radiation to attack those cancerous cells missed by the other two treatments. Many of

the chemotherapeutic agents used in cancer treatment are secondary metabolites

produced by microorganisms, especially of the genus Streptomyces. This chapter is

concerned with these metabolites known as anti-tumor antibiotics.

24.5.2 Mode of Action of Anti-tumor Antibiotics

The anti-tumor antibiotics are heterogeneous in their chemical natures. Some of the best

known groups used in clinical practice include anthracyclines, actinomycins and

bleomycins (Fig. 24.7). In terms of their modes of action their common characteristic

seems to be interaction in some form with DNA. Daunomycin and adriamycin which are

anthracyclines link up base pairs and thus inhibit RNA and DNA synthesis.

Mithramycin and chromomycin A

which are actinomycins inhibit DNA – dependent

RNA synthesis. On the other hand bleomycins which are peptides react with DNA and

cause it to break. Other anti-tumor antibiotics operate through alkylation e.g.

streptonigrin, mitomycin C, and profiromycin. Still others interfere with membrane

functions or interact with the micro-tubules in the cell.

The basis of all chemotherapy whether with anti-bacterial or with anti-tumor drugs is

the ability of the drugs to selectively attack the pathogen or the errant tumor cell. In the

well-known case of penicillin for example, the absence of mucopeptides in animal cell

wall is the key to the operation of the drug. Several mechanisms have been suggested

which allow the selective attack of anti-tumor drugs on tumor cells. These include

inability of the tumor cell to repair damage by the anti-tumor drug, higher distribution of

the drug in tumor than in normal cells, greater ability to inactivate tumor cells. These are

based on structural and bio-chemical differences between normal and tumor cells.

Unfortunately anti-tumor antibiotics as well as other anti-tumor drugs do not always

discriminate successfully between tumor and normal cells. Varying degrees of toxicity

therefore usually accompany the use of anti-tumor antibiotics. The most severe of these is

damage to the bone marrow which is involved in synthesis of blood components a

damage that may be fatal. Toxicity is in many cases being successfully handled clinically

by various means including reduced dosage, change of route of administration, etc. Less

toxic antibiotics are also being produced by semi-synthesis or by the modification of the

antibiotic molecule.

24.5.3 Search for New Anti-tumor Antibiotics

The search for anti-tumor antibiotics is more difficult than that of anti-bacterial or anti-

fungal agents in terms of methodology and interpretation. In the search for the latter