“SCREENING FOR ANTIBIOTIC PRODUCERS IN SOIL FROM THE BANKS OF SEWER CANALS, AND TESTING THE EFFICACY OF ANTIMICROBIAL COMPOUNDS OBTAINED, AGAINST COLIFORMS”

1. “SCREENING FOR ANTIBIOTIC PRODUCERS IN SOIL FROM THE
BANKS OF SEWER CANALS, AND TESTING THE EFFICACY OF
ANTIMICROBIALCOMPOUNDS OBTAINED, AGAINST COLIFORMS”
(DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR
THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY)
By
JAMMALA VAMSIKRISHNA
Regd. No: 27016062010
Submitted to
DEPARTMENT OF MICROBIOLOGY
SRI VENKATESWARA UNIVERSITY, TIRUPATHI.
Under the supervision of
Sri. P.VIJAYA KUMAR, Msc , (Ph.D).
Department of Microbiology,
Government Degree College, Naidupet.
(Affiliated to Vikrama Simhapuri University)
MAY 2017

2. v
CERTIFICATE
This is to certify that the dissertation entitled “screening for antibiotic
producers in soil from the banks of sewer canals, and testing the efficacy of
antimicrobial compounds obtained, against coliforms”which is being
submitted by Mr. Jammala Vamsi Krishna, Roll No. 27016062010,for
the award of the degree of Master of Science in microbiology from Sri
Venkateswara University, Tirupathi, is a record of bona-fide research
work, carried out by under my supervision. The results embodied in this
thesis are new and have not been submitted to any other university or
institution for the award of any degree or diploma.
Place:
Date:
(P. Vijaya Kumar)
Lecturer (Assistant Professor Grade),
Department of Microbiology,
Govt. Degree College, Naidupet-524126,
S.P.S.R. Nellore, A.P.

3. ii

4. iii
SRI VENKATESWARA UNIVERSITY
TIRUPATI
CERTIFICATE
This is to certify that project report entitled, “screening for antibiotic
producers in soil from the banks of sewer canals, and testing the efficacy of
antimicrobial compounds obtained, against coliforms”is a bonafied work done
and submitted J.VAMSI KRISHNA , M.Sc., General Microbiology Course, Regd
No. 27016062010, Department of Microbiology and that it had not been previously
submitted for any degree, diploma or price of this or any Universities.
HEAD OF THE DEPARTMENT
INTERNAL EXAMINER EXTERNAL EXAMINER

5. ii
DECLARATION
I, Jammala Vamsi Krishna, M.Sc., Life Science, Department of
Microbiology, S.V.U, Tirupathi, hereby declare that my research work
incorporated in the dissertation entitled “Screening for antibiotic
producers in soil from the banks of sewer canals, and testing the
efficacy of antimicrobial compounds obtained, against coliforms” is
an authentic research work carried out at Department of Microbiology,
Govt. Degree College, Naidupet under the direct guidance and
supervision of Sri. P. Vijaya Kumar, Lecturer (Assistant Professor Grade),
Department of Microbiology, Naidupet. The project work is original and
no part of this work has been submitted for any other degree or diploma.
All the given information is true to the best of my knowledge.
Place:
Date: (J. Vamsi Krishna)

6. ix
ACKNOWLEDGEMENT
I would like to express my sincere gratitude to
Prof. CH PARAMAGEETHAM, Head of the Department of
Microbiology, S.V.U. for giving me a great opportunityto do this project
work and also for the support and motivation that she has provided. She
suggested different ways to approach a research problem and the need to
be persistent to accomplish any goal and for providing me an ample
environment to work.
My sincere thanks to my guide Sri. P.VIJAYA KUMAR for his
immense support and motivation. Humble thanks to him for suggesting
me to work on this research problem and teaching the research methods
to address the same. I also thank him for providing me suitable working
environment in his laboratory. I also thank him for his supportin learning
the basic techniques, with possible advancement, which will be of good
help for my future endeavors.
I thank the Principal, Govt. Degree College, Naidupet, for his kind
permission to work at his college.
I express my sincere thanksto jay kumar, charan teja,veena swarna
kumari research Scholers in department of microbiology who helped me
in successfully completing my project. Thanks to my parents for
supporting me throughout.Last but not least, I thank the god almighty for
helping me to complete this work outfacing obstacles occurred.
J. Vamsi Krishna

7. ii

8. xi
CONTENTS
List of tables...............................................................................................................................xiii
List of figures...............................................................................................................................xv
Abstract.........................................................................................................................................01
Introduction..................................................................................................................................03
Review of literature......................................................................................................................06
Objectives..................................................................................................................................... 09
Materials and methods................................................................................................................16
Results.......................................................................................................................................... 24
.
Discussion......................................................................................................................................25
Conclusion ……………………………………………………….………………………………………………….………………………..26
Bibliography ................................................................................................................................30

9. xiii
List of Tables
Table 1 List of a few commonly known microbes that produce antibiotics..............................2
Table 2 Composition of Eosin methylene blue agar (EMB; pH-7.2)........................................10
Table 3 Composition of MacConkey agar medium (pH-7.1)....................................................11
Table 4 Composition of Nutrient agar medium (pH-7.0)..........................................................11
Table 5 Composition of Czapek-Dox broth medium (pH-7.0)..................................................14
Table 6 Effect of antimicrobial compounds on growth of coliforms........................................20
Table 7 Effect of antimicrobial compounds on growth of coliforms-qualitative expression..21

10. xv
List of figures
Figure 1 Green metallic sheen formed by colonies of E. coli on EMB agar medium...........17
Figure 2 Fish-eye-like colonies of E. coli and colorless colonies of Salmonella spp. on
MacConkey agar medium...........................................................................................................17
Figure 3 Sub culture of E. coli on a nutrient agar medium slant............................................18
Figure 4 A representative image of the crowded plate technique performed........................18
Figure 5 A representative image of the scale-up culture of unknown bacterial culture (Left)
and the unknown fungal culture................................................................................................19
Figure 6 A representative picture of solvent extraction. Upper layer was the antibiotic extract
in n-hexane. Bottom layer was the aqueous production medium...........................................20
Figure 7 Assay of antimicrobial activity by disc diffusion test against E. coli culture. E1 -
extract-1; E2 - extract-2; B – blank disc; K – kanamycin disc. The test was performed in
triplicates and average results were reported..........................................................................21
Figure 8 Graphical representation of the assay of the antimicrobial activity of the antibiotic
extract on E. coli..........................................................................................................................21
Figure 9 Assayof antimicrobial activity by disc diffusion testagainst Salmonellaspp. culture.
E1 - extract-1; E2 - extract-2; the other unlabeled disc was blank. The test was performed in
triplicates and average results were reported...........................................................................22
Figure 10 Graphical representation of the assayof the antimicrobial activity of the antibiotic
extract on Salmonella spp...........................................................................................................23

11. 1
ABSTRACT
Soil microbiota produce a number of antibiotics known to date. Screening for new
antibiotics has become one of the thrust research areas in microbiology. Particularly, with the
emergence of antibiotic resistance, better and broad spectrum antibiotics are needed to be
identified. Antagonism among the microbial flora of same soil-niche is responsible for production
of antimicrobial compounds as well as for the development of microbial drug-resistance towards
those compounds. Eventually, this phenomenon leads to the production of new or modified
antibiotics. In the present study, screening for antibiotic producing microbes was done in soil
samples from the banks of sewer canals, where antagonism between sewage-contaminant
coliforms and other microbes would routinely occur. After primary screening by crowded plate
technique, one bacterial strain (unidentified) and one fungal strain (unidentified) were isolated.
Solvent extraction of the antimicrobial compounds was followed by disc diffusion test to determine
the effect of the extracts on coliforms. These strains produced certain antimicrobial compounds
(unknown) which had antibacterial activity against E. coli, and Salmonella spp., which were
isolated from the same niche. Simultaneous comparison of this antimicrobial activity with that of
standard commercially available antibiotics like Kanamycin helped in assessing the efficacy of the
antimicrobial compounds extracted. Identification of the isolated strains and the extracted
antimicrobial compounds shall be a future perspective.
Key words: Antibiotics, Sewage, Coliforms, E. coli, Salmonella spp., Primary Screening,
Crowded Plate Technique, Disc Diffusion Test, Solvent Extraction, Kanamycin, Chloramphenicol.

12. 2
INTRODUCTION
Among microbial flora, Antagonism is a very common phenomenon. Microbes exhibit
antagonism at every niche. They show this property by secreting antimicrobial compounds. These
antimicrobial compounds inhibit the growth of other microbes in the same microenvironment.
Those microbes that secrete antimicrobial compounds gain upper hand in occupying space and in
nutrient procurement, and thus, they prevail in that particular niche. Many microorganisms are
known today for their antibiotic production (Table 1)
Table 1 List of a few commonly known microbes that produce antibiotics.
Antibiotic producer Antibiotic produced Spectrum of activity
Pencillium notatam Penicillin Gram positive bacteria
Pencillium chrysogenum Penicillin Gram positive bacteria
Pencillium griseo fulvum Griseo fulvin Dermatophytic fungi
Cephalosporium acremonium Cephalosporin Broad spectrum
Bacillus subtilis Bacitracin Gram positive bacteria
Streptomyces erythreus Erythromycin Gram positive bacteria
Streptomyces fradiae Neomycin Broad spectrum
Streptomyces griseus Streptomycin Gram negative bacteria
Streptomyces orientalis Vancomycin Gram positive
Micromonospora perpurea Gentamycin Broad spectrum
Antibiotics are substances produced by microbes to limit the growth of other microbes in their
vicinity. They either inhibit the growth or destroy microbial cells (Duerden et al., 1993).
Antibiotics are produced as secondary metabolites by several microbes like actinomycetes, and
fungal molds (Demain, 2000) in order to reduce the consumption of nutrients, water, space etc.,
by other microbes. This gives the antibiotic producing microbe a clear advantage over the niche,
where they are dwelling. Limited groups of microorganisms produce antibiotics that are clinically
used. According to Cooke and Gibson (1983), antibiotics that affect pathogen but not the host cells
are considered as useful antibiotics. Many antibiotics, which would work against bacteria, have
been identified. There are a few antifungal antibiotics that can be used to treat fungal infections
(Rusell, 1977). However, the emergence of new diseases and development of multiple-antibiotic
resistance pose a challenge to the use of antibiotics. Therefore, the need for screening for new
antibiotic producers is inevitable (Roberts, 1998). Soil is the major source for antibiotic producers,
as it harbours actinomycetes, the group of bacteria that produce most of the antibiotics. Soil

13. 3
microorganisms have been screened for their antagonistic metabolites such as antibiotics for a long
time (Dulmage and Rivas 1978).
The antibiotics listed in table 1 are naturally produced anti microbial compounds. They are
often conjugated with other compounds or chemically modified to show greater antimicrobial
potential, which are called semi-synthetic antibiotics. E.g. Penicillin - G is natural, while Penicillin
- V is synthesized by a chemical modification. The use of antibiotics has been effective and being
prescribed for many infectious diseases nowadays. However antimicrobial resistance poses a big
problem to the use of these antibiotics. The resistance mechanisms that microbes adapt to become
resistant are needed to study and it is essential to target such mechanisms in order to avail the
benefits of these antibiotics. At this juncture, discovery of new antibiotics that could prove
effective against resistant microbes is quite essential. Screening for antimicrobial compounds from
the niches that harbor resistance microbial population could be helpful in identifying more
potential antimicrobial compounds or producers of such new antimicrobial compounds.
In this study we aimed at screening for antibiotic producers from the soil samples obtained from
banks of sewer canals. Such soils harbor routine fecal flora like Escherichia coli, Salmonella,
Enterobacter etc. which would normally present in sewage. These sewage could invariably
contain drug resistant microbes. At the same time, development of antagonistic microbes against
such resistant population is also possible at the same soil-niche due to the competition between
the drug resistant microbes and the niche-residents. Based on this principle, we screened such
soil samples for antagonistic association among microbes, using crowded plate method.
Microbes, thus identified for production of antimicrobial compounds, were cultured for antibiotic
production in broth media. At the same time, E.coli and Salmonella species were isolated from
the sewage from the same location and grown as pure cultures. Upon extraction of the
antimicrobial compounds by solvent extraction method, their efficacy was tested on the isolated
cultures, using disc diffusion test; standard commercial antibiotic discs were used to compare the
efficacy. Therefore this study stands as a primary step in addressing the necessity of screening
for the new antibiotics at niches where both resistance microbes and possible their antagonistic
microbes well. Sewage contains coliforms as contaminants from human faces. When cultured on
EMB agar, a selective medium for the isolation of coliforms, colonies of E.coli (most strains)
produce a characteristic green sheen through fermenting lactose and producing strong acids.

14. 4
Rapid acidification of the EMB agar is critical in formation of the green metallic sheen. Colonies
of other coliforms that do not ferment lactose produce either colorless or lavender colonies. On
MacConkey agar medium, E. coli produces a characteristic fish-eye-colony with pink periphery,
where as Salmonella spp. produce a normal colorless colony. These principles aid in identifying
these two microbes at a preliminary level from a sample containing coliforms; consequent sub-
culturing aids in pure culturing and preservation of the identified colonies/strains.
Coliforms of the sewage often contaminate the soil on the banks of sewer canals, where a possible
antagonistic interaction would occur among the microflora of that particular niche. This may lead
to the production of antibiotics by the indigenous flora against contaminant coliforms. Sometimes,
it is possible that the coliforms constitute drug-resistant varieties; in such cases induction of new
antibiotics by the indigenous flora against those drug-resistant microbes may occur eventually. To
screen for such antibiotic producers, crowded plate technique is employed; in which, antibiotic
producers, which suppress the growth of other microbes around their colonies are easily identified.
When grown in liquid media, the antimicrobial substances produced by cells that have the capacity
to produce such compounds are diffused into the medium. Since, antibiotics are secondary
metabolites; the production of such antimicrobial substances can be achieved upon prolonged
incubation under optimal conditions. Inducer nutrients may be used for more production of the
compounds.
Most of the antibiotics being readily soluble in organic solvents like hexane, can be extracted into
a variety of polar and non polar organic solvents. Methanol denatures membranes and proteins and
thus let most of the compounds freely release into the medium. Besides, such compounds would
partition into methanol upon agitation of the aqueous-broth with equal volumes of methanol, even
though methanol is miscible with water. n-hexane, a universal solvent, is used to further extract
those compounds i.e. as a complete extraction medium. At the same time, it floats as an immiscible
layer upon aqueous-portion, which can be separated out, using a separating funnel.

15. 5
REVIEW OF THE LITERATURE
Pathogenic microorganisms
Around 2000 types of microbes that mostly belong to bacteria, fungi, protozoa, and viruses
cause diseases in humans; they are referred to as ‘pathogens’. Those microorganisms that can
infect and/or produce various toxins tend to be pathogens. Pathogenicity of different microbes is
different in some degrees (Gilbert, 1977). According to Duerden et al. (1993), pathogens which
infect human may come from either exogenous or endogenous sources. Bacterial infections usually
can be cured through antibiotics but the emergence of multi-drug resistance pathogens has created
obstacles in treating diseases.
Multiple-drug resistant pathogenic microorganisms
The presence of antibiotics is supposed to suppress the infection of pathogenic microorganisms
(Hamilton-Miller, 2004). However, misuse and wide-use of antibiotics for medication and animal
breeding in inappropriate dosage have been creating antibiotic resistance among pathogens (Lynch
et al., 2004; Theuretzbacher, 2009). This is either due to the mutations that occur in bacterial
chromosome or development of antibiotic resistant strains through the exchange of genetic
material (Cooke& Gibson, 1983; Roberts, 1998; Cirz et al., 2005). Besides, according to Chung et
al. (2008), the emergence of multi-drug resistant bacteria are also due to inheritance factors.
The discovery of new antimicrobial drugs and renewed derivatives of the previous antibiotics
may be useful for a limited period of time owing to the development of resistance (Roberts, 1998;
van der Waaij et al., 2000). Some of the common examples of these multiple-drug resistant
pathogenic microorganisms are the methicillin or multidrug resistant Staphylococcus aureus
(MRSA), multiple-drug resistant (MDR) Enterococci, and multi-drug resistant Streptococcus
pneumoniae (Hart, 1998; Huycke et al., 1998; Xu et al., 2009). These Multi-drug-resistant
pathogens can cause severe diseases especially in patients who are immuno-compromised and for
those who stays in intensive care unit (ICU) (Weber et al., 2007). Besides bacteria, microbes like

16. 6
fungus such as Candida albicans can also confer resistance towards different antibiotics as stated
by Gulshan and Rowley (2007), C. albicans has been involved in broad studies by researchers due
to its property that can resist different antimicrobial drugs. The number of multi-drug resistant
pathogenic microbes has increased over time and there are only limited therapeutic drugs that are
applicable to combat these pathogens (Roberts, 1998; Demain & Sanchez, 2009). Therefore, there
is a need for continuous discovery of new antibiotics in order to make treatments under antibiotics
remain effective (Roberts, 1998; van der Waaij et al., 2000).
Antibiotics
Antibiotics are substances produced by microbes which can be used to inhibit the growth of
other microorganisms at low concentration (Russell, 1977). In their naturalhabitats, bacteria utilise
the antibiotics they produce as protective substances bypreventing the invasion of other bacterial
species. Protection is not the only function ofantibiotics. Hence, according to Linares et al. (2006),
antibiotics also act as signalling molecules that bacteria use as a means of communication between
cells.Antibiotics can be classified according to their mode of actions. Antibiotics areclassified as
broad-spectrum antibiotics when they have the ability to affect a wide rangeof Gram-positive and
Gram-negative bacteria while antibiotics that only effective towards certain group of bacteria are
known as narrow-spectrum antibiotics. Several mechanismsof actions of antibiotics have been
discovered by scientists. These actions include the inhibition of cell wall, protein and nucleic acids
synthesis (Lambert, 1977; Brooks et al., 2001; Tortora et al., 2007). There are three important
groups of microorganisms which are responsible for the production of antibiotics. These are the
Gram-positive rod shape bacteria such as Bacillus, actinomycetes, and fungi such as
Cephalosporium and Penicillium (Tortora et al., 2007). Actinomycetes are the Gram-positive
bacteria that contribute most of the clinically use antibiotics and as stated by Oskay et al., (2004),
the discovery of new biological metabolites particularly useful antibiotics from actinomycetes
need a vast amount ofisolates. Majority of the antibiotics that have been identified and presently
in use are isolated from the bacteria under the genus of Streptomycetes. Examples of these
antibiotics are tetracycline and streptomycin. As compared to antibacterial agents, the development
of antifungal agents is not achieved high advancement. This is because the lethal targets for
infectious fungal species are hard to be identified due to the similarity of metabolic pathways
possessed by them and their host, mammals since both are classified as eukaryotes (Imada & Hotta,

17. 7
1992). Griseofulvin and nystatin are examples of antifungal antibiotics that can be used to cure
infections caused by Trichophyton and C. albicans, respectively (Russell, 1977).
Sources of natural occurring antibiotics
Antibiotics-producing microbes can be isolated from different sources such as soil and marine
microbes, endophytes, lichens, and even animals. According to von Bubnoff (2006), some
scientists are continually searching for novel antibiotics producing microorganisms from
extraordinary places such as deep sea mud and seaweeds. In addition, endophytes which inhabit
in higher plants also become one of the important sources of antibiotics which are effective against
different types of pathogens (Strobel &Daisy, 2003). An early study conducted by Burkholder et
al. (1944) had shown that lichens have high potential to produce useful antibiotics. In their study,
of the 42 lichens species, 64.29% of the species did show active antimicrobial activity against S.
aureus and B. subtilis. Besides, animals also become target for scientists to search for novel
antibiotics. According to Margavey et al. (2004), a broad spectrum of antibiotics, squalamine has
been successfully isolated from the stomach tissues of dogfish shark Squalus acanthias. As
discussed above, there are many sources where antibiotics can be discovered but soil still remains
the most important target for most researchers in their efforts to discover novel antibiotics that
have pharmaceutical values. This is because many microbes especially bacteria that reside in soil
have the ability to produce biologically Active secondary metabolites such as useful antibiotics.
Antibiotic producing soil microbes
Soil is a reservoir where most antibiotics producing microbes and their secondary metabolites
can be found. Actinomycetes are Gram-positive bacteria which form spore and filamentous and
they are the most important group of antibiotic producing soil microorganisms since they
contribute to 75% of the identified products which are widely. Streptomycetes can be widely found
in both terrestrial and aquatics environments especially where nutrients are highly abundant such
as in the soil, hay, and composts (Locci, 1989). Besides, there are several factors which can
influence the distribution of streptomycetes which including the temperature, moisture, pH, and

18. 8
climate (Williams etal., 1972b; Williams, 1978 in Locci, 1989). Used in clinical applications
(Oskay et al., 2004; Ceylan et al., 2008).
According to Demain (2000), there will be about 500 antibiotics from actinomycetes
continually being discovered each year. As reported by Oskay et al. (2004), of the 50 isolates that
they obtained from actinomycetes, 34% of the isolates did produce antibiotics. Besides, from the
recent research work conducted by Ceylan et al. (2008), they also reported that 15 isolates obtained
from the genus of Streptomycetes showed the capability of producing antibacterial substances
towards both Gram-positive and Gram-negative bacteria which resistance to different antibiotics.
Streptomyces’s are categorized in the family of Streptomycetaceae (Anderson & Wellington,
2001) and different species of bacteria classified under this genus contribute mostly to the useful
biologically active substances such as antibiotics that have been authorized. Besides, the ability of
Streptomyces to act as useful biological control agents in retarding the growth of pathogenic fungi
which infected plants also has been reported by many researchers. These pathogenic fungi may
either arise from soil or air (Oskay, 2009). In addition, the current research conducted by Oskay
(2009) has discovered that there was a novel strain of Streptomyces assigned as Streptomyces sp.
KEH23 that has a high potential to produce useful antibiotics which can actively against pathogens
that infected plants and human being.
Besides Streptomycetes, other Gram positive soil bacterium such as Rhodococcus has also
been identified to have high potential of producing useful antibiotics when theyare in stress
condition. As reported by Robson (2008), a research team led by KazuhikoKurosawa has isolated
the amino glyceride antibiotic, rhodostreptomycin produced byRhodococcus. This antibiotic is
effective against many types of test microorganism whichincluding the hardy strain
Streptomycetes.

19. 9
OBJECTIVES
To address the research problem, the work is divided into four major objectives as
described below.
This objective helps in obtaining the sewage fecal flora, which could contain drug resistant
species. Determination of antibiotic sensitivity of them gives an idea about their
susceptibility/resistance against standard commercially available antibiotics. At the same time,
these cultures may be used as test organisms to test the efficacy of newly isolated antimicrobial
compounds, obtained during this study.
This objective helps in identifying antibiotic producers among the soil samples that were
contaminated with soil sample. This objective focuses on the prime concept of this study.
Working on this objective is essential to grow the isolated cultures that produce antimicrobial
compounds at optimal conditions, favoring them to produce antibiotics. Solvent extraction helps
in harvesting the antimicrobial compounds.
Working on this objective concludes the study as this gives an idea on the drug resistance of
the microbial isolates, if any, efficacy of the newly isolated antimicrobial compounds. At the
same time, a comparison made with the commercially available antibiotics helps in
understanding the novelty of the newly isolated compounds.
 “To isolate, identify and grow E. coli and Salmonella species as pure cultures”
 “To screenfor antibiotic producers from soil samples at the banks of sewer canals”
 “To produce and extract antibiotics from the isolated antimicrobial cultures”
 “To test for antimicrobial activity of the antimicrobial compounds obtained against
the isolated cultures of E. coli and Salmonella spp.”

20. 10
Materials and Methods
 Isolation and identification of E. coli and Salmonella spp.
Materials:
Sewage sample, EMB agar medium, MacConkey Agar medium, Nutrient-Agar medium,
sterilized laminar air-flow chamber, incubator, hot-air oven, autoclave, weighing balance,
refrigerator, sterile petri-plates, sterile test tubes for serial dilution, autoclaved distilled water, L-
shaped bent-glass rod for spreading the inoculum, sterile non-absorbent cotton, sterile pipettes,
inoculation loop, spatula, Antibiotic extract, nutrient agar medium, test cultures (E. coli and
Salmonella spp.), filter paper-discs, commercially available antibiotic discs (kanamycin),
sterilized laminar air-flow chamber, shaker-incubator, hot-air oven, autoclave, weighing balance,
sterile petri-plates, autoclaved distilled water, sterile non-absorbent cotton, sterile glass pipettes,
L-shaped bent-glass rod for spreading the inoculum, sterile forceps, spatula, punching machine,
centimetre scale, etc.,
Composition of EMB agar medium:
Table 2 Composition of Eosin methylene blue agar (EMB; pH-7.2)
Ingredients g/1000 mL g/100 mL
Peptone 10 1
K2HPO4 2 0.2
Lactose 5 0.5
Sucrose 5 0.5
Eosin 0.4 0.04
Methylene blue 0.065 0.0065
Agar 13.5 2.8
Composition of MacConkey agar medium:
Table 3 Composition of MacConkey agar medium (pH-7.1)
Ingredients g/1000 mL g/100 mL
Peptone (meat and casein) 3 0.3
Pancreatic digest of gelatine 17 1.7

21. 11
Lactose monohydrate 10 1
Bile salts 1.500 0.15
Neutral red 0.030 0.003
Agar 13.500 1.35
Crystal violet 0.001 0.0001
Sodium chloride 5 0.5
Composition of Nutrient agar medium:
Table 4 Composition of Nutrient agar medium (pH-7.0)
Ingredients g/1000 mL g/100 mL
Peptone 5 0.5
Beef extract 3 0.3
NaCl 5 0.5
Agar 20 2
 Sewage samples were collected from different sewer canals, using thread-tied screw-cap
tubes.
 These samples were pooled, and serial dilution of the sample was made up to 10-8 dilution
with autoclave-sterilized distilled water.
 1000 mL each of EMB agar, MacConkey agar media were prepared as per the composition
given in tables 2, and 3, respectively. The media were sterilized in an autoclave at 121ºC
temperature and 15 lbs pressure for 20 minutes. 20 mL aliquots of media were poured into
sterile petri-plates and the media were allowed to sit for solidification.
 100 mL of nutrient agar medium (NAM) was prepared as per the composition given in
table 4. 10 mL aliquots of the NAM were poured into sterile test tubes, using sterile glass
pipettes, the tubes were labeled and cotton-plugged. Cotton-plugged tubes with medium
were kept in a slant position, until solidification so as to obtain NAM-slants for sub-
culturing.
 0.1 mL of serially-diluted sewage samples from each dilution were spread on the EMB
agar medium-plates by using incineration-sterilized L-shaped bent-glass rod. The plates
were labeled appropriately.
 These plates were incubated at 37ºC for 24-48 h in an electric incubator which has a
thermostat.

22. 12
 Upon incubation the plates were observed for the growth of desired colonies. EMB agar-
plates were screened for the colonies that produce green-metallic sheen around them, which
is a characteristic feature of E. coli which ferment lactose and produce acid rapidly which
is responsible for green metallic sheen. Colorless or light purple colonies were identified
to be lactose non-fermenters.
 These colonies were picked separately by using an inoculation loop and streaked on to
MacConkey agar medium-plates, by quadrant-streaking method of streak-plate culturing;
and the plates were labeled appropriately.
 These plates were incubated at 37ºC for 24-48 h in an electric incubator which has a
thermostat.
 Upon incubation the plates were observed for the growth of desired colonies. Fish-eye
colonies with pink periphery were identified as E. coli colonies, colorless colonies were
deemed to be Salmonella spp.
 Identified colonies were picked separately, using inoculation loop and streaked on NAM-
slants, which were prepared earlier. Labeling was done appropriately. Sub-cultures were
incubated at 37ºC for 24-48 h. Once growth occurred, the sub-cultures were transferred to
refrigerator (4ºC) for storage until further use.
 Primary screening for antibiotic producing microbes in soil samples obtained from
the banks of various sewer canals by crowded plate technique.
100 mL of nutrient agar medium was prepared as per the composition given in the table 4. The
media were sterilized in an autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes. 20
mL aliquots of media were poured into sterile petri-plates and the media were allowed to sit for
solidification.
 Upon solidification, a pinch of soil inoculum was sprinkled uniformly on sterile nutrient
agar plates. The plates were incubated at 37ºC for 72 h or more in an electric incubator
which has a thermostat.
 During incubation, the plates were observed at different intervals, for colonies which had
clear zone of inhibition surrounding them i.e. no growth would be seen in their vicinity. In

23. 13
other words, compounds secreted by these colonies do suppress the growth of other
microbes around those colonies.
 Such colonies were picked-up, sub-cultured as per the procedure given in section 3.1.

24. 14
 Production of antimicrobial substances
Composition of Czapek-Dox broth medium
Table 5 Composition of Czapek-Dox broth medium (pH-7.0)
Ingredients g/1000 mL g/100 mL
Sucrose 30 3
Sodium nitrate 2 0.2
K2HPO4 1 0.1
MgSo4 0.5 0.05
Potassium chloride 0.5 0.05
Ferrous sulphate 0.01 0.001
Scaling-up was done by the following procedure
 250 mL each of nutrient broth, and czpek-dox broth media were prepared as per the
composition given in table 4 (except agar) and table 5 respectively. The media were
sterilized in an autoclave at 121ºC temperature and 15 lbs pressure for 20 minutes.
 Upon cooling, the conical flasks with media were transferred to sterile laminar air-flow
chamber, where, plenty of the microbial cultures that produce antimicrobial substances
were added separately to the flasks, and labelled appropriately. Cultures that possess fungal
morphology were inoculated into czapek-dox broth.
 The flasks were incubated at 37ºC for 24-72 h in an electric shaker-incubator which has a
thermostat. Besides optimum temperature, continuous agitation was provided in shaker-
incubator.
Production of antimicrobial substances was achieved by the following procedure
 1000 mL each of nutrient broth, and czpek-dox broth media were prepared as per the
composition given in table 4 (except agar) and table 5 respectively, as production media.
The production-media were sterilized in an autoclave at 121ºC temperature and 15 lbs
pressure for 20 minutes.
 Upon cooling to room temperature, the conical flasks with media were transferred to sterile
laminar air-flow chamber, where, scaled-up cultures were added to the production media.

25. 15
 The flasks were then incubated at 37ºC for 72 h or more for mycelial cultures in an electric
shaker-incubator which has a thermostat. At the end of the incubation, antimicrobial
compounds were deemed to be produced and diffused into the broth media, as per the
principle.
 Extraction of antibiotics from broth media by solvent extraction method
 Equal volumes of extraction media and methanol were taken in a separating funnel and the
mixture was shaken vigorously for 5 min.
 An equal volume of n-hexane was added to the mixture followed by shaking the contents
vigorously for 5 min.
 The contents were then allowed to settle for 30 min. The lower aqueous layer is collected
into sterile conical flask and the upper hexane-extract is collected into another conical
flask.
 The first three steps were repeated twice, to extract any remaining compounds from the
aqueous phase, and the hexane-extract thus obtained was pooled.
 n-hexane extract was transferred to a wide beaker, covered with filter paper, and dried
gradually, by evaporating n-hexane.
 The extract from fungal sample was referred to as ‘extract-1’ and that from bacterial culture
was referred to as extract-2
 The dried extract was stored in a refrigerator until further use.
 Assay of the antimicrobial activity of the extracted antimicrobial substances - disc
diffusion method (Kirby-Bauer method)
 Filter paper discs were cut using a punching machine and 100 mL of nutrient agar
medium was prepared as per the composition given in the table 4. The paper-discs
and the media were sterilized in an autoclave at 121ºC temperature and 15 lbs

26. 16
pressure for 20 minutes. 20 mL aliquots of media were poured into sterile petri-
plates and the media were allowed to sit for solidification.
 Antibiotic extract was taken from refrigerator and cooled to room temperature. The
extract was re-dissolved in about 0.5 mL of n-hexane. Upon sterilization, the paper-
discs were placed in this antibiotic-solution and dried in laminar air-flow chamber.
 Separate inoculum of each test organism (E. coli and salmonella spp) was prepared
by dispersing the test cultures in a few mL of sterile distilled water taken in a test
tube.
 0.1 mL of this inoculum was added to the surface of the NAM plates and was spread
by using sterile L-shaped bent-glass rod. Immediately, antibiotic discs were placed
on the surface of the medium with the help of a sterile forceps, by positioning them
distant enough from each other and the rim of the plate. Each plate was also placed
with kanamycin-discs as control.
 The plates were incubated at 37ºC for 24-48 h in an electric incubator which has a
thermostat.
 Upon incubation, the plates were observed for zone of inhibition around the
antibiotic-discs. The area of the zone of inhibition was calculated using the formula
“Πr2”. In which, r denotes the radius of the zone of inhibition, and the results were
tabulated.

27. 17
RESULTS
Isolation and identification of E. coli and Salmonella spp.
 Colonies with green metallic-sheen periphery were observed on EMB agar. This is a
characteristic feature of E. coli. Therefore, the colonies were identified as that of E. coli.
(Figure 1)
 Colorless colonies without green metallic sheen were observed on EMB agar. Lactose non-
fermenters produce such colonies on EMB agar. Therefore, these colonies were assumed
to be Salmonella spp.
 ‘Fish-eye’ like colonies with pink periphery were observed on MacConkey agar. This is a
characteristic feature of E. coli. Therefore, the colonies were identified as that of E. coli.
(Figure 2)
 Colorless colonies were developed on MacConkey agar, which further confirmed the
identity of Salmonella spp. (Figure 2)
 Sub-culturing of these strains was successful in preserving the cultures. (Figure 3)
Figure 1 Green metallic sheen formed by
colonies of E. coli on EMB agar medium.
Figure 2 Fish-eye-like colonies of E. coli and
colorless colonies of Salmonella spp. on
MacConkey agar medium.

28. 18
Primary screening for antibiotic producing microbes in soil samples obtained from the
banks of various sewer canals.
 One microbial colony was found to be antagonistic, forming a clear zone of growth
inhibition around it. However, this colony has fungus-like mycelial morphology. Therefore
this organism was assumed to be a fungus.
 Another colony with a clear zone of growth inhibition around it was also observed in the
crowded plate, which was having the morphological characteristics of a bacterial colony.
Figure 3 Sub culture of E. coli on a
nutrient agar medium slant.
Figure 4 A representative image of the
crowded plate technique performed.

29. 19
Production of antimicrobial compounds
 The microbial strain that was assumed to be a fungus was well grown in, the fungal medium
that was used for scale-up process, confirming the assumption. Therefore this culture was
continued to grow in czapek-dox broth for production of antibiotics.
 Another microbial strain was assumed to be a bacterium; it has grown well in nutrient broth
medium, giving rise to enormous growth in nutrient broth. Hence, this culture was grown
in the same medium for production of antibiotics.
Extraction of antibiotics from broth media by solvent extraction method
 Two separate extracts one each from the unknown fungal culture and the unknown bacterial
culture were made. The final dried extract was highly viscous, indicating the positive
outcome of the solvent extraction procedure.
Figure 5 A representative image of the scale-up culture of
unknown bacterial culture (Left) and the unknown fungal culture.
.

30. 20
Assay of the antimicrobial activity of the extracted antimicrobial substances
 Extract-1 had showed highest antimicrobial activity against both the coliforms.
 When compared the antibacterial efficacy against the isolated strain of E. coli, extract-1
had 1.45 times higher efficacy than that exhibited by kanamycin, an antibiotic that is known
to be effective against gram negative bacteria like E. coli. (Tables 6 and 7)
 When compared the antibacterial efficacy against the isolated strain of Salmonella spp.,
the antimicrobial potential of extract-1 was approximately 16 times greater than that of the
kanamycin. (Tables 6 and 7)
Table 6 Effect of antimicrobial compounds on growth of coliforms.
Test organism
Area of the inhibitory zone (cm2)
Blank Extract-1 Extract-2 Kanamycin
E. Coli 0.0 18.1 3.8 12.5
Salmonella 0.0 16.6 2.0 1.1
Data are Mean values of duplicate assays, with negligible standard deviation. Formula
used to calculate the area of the circle was “Πr2”. Three to four values of diameter
were taken using cm scale and average diameter was divided by two to get the radius
(r)
 Extract-2 had a very lesser antimicrobial activity against E.coli when compared to that of
the extract-1 and kanamycin. (Tables 6 and 7).
Figure 6 A representative picture of
solvent extraction. Upper layer was the
antibiotic extract in n-hexane. Bottom
layer was the aqueous production
medium

31. 21
 In inhibiting the growth of salmonella spp. it was as effective as a two fold higher than that
of kanamycin. However, it was still 8 times lesser than that of the extracty-1. (Tables 6 and
7)
Table 7 Effect of antimicrobial compounds on growth of coliforms-qualitative expression
Test organism Extract-1 Extract-2 Kanamycin Blank
E. coli +++ + + -
Salmonella ++ + +++ -
++ = Very good inhibition; ++ = Good inhibition; + = Moderate inhibition; - = No inhibition.
Figure 7 Assay of antimicrobial activity by disc diffusion test against E. coli
culture. E1 - extract-1; E2 - extract-2; B – blank disc; K – kanamycin disc. The
test was performed in triplicates and average results were reported.

32. 22
Figure 8 Graphical
representation of the assay of
the antimicrobial activity of
the antibiotic extract on E.
coli.
Figure 9 Assay of antimicrobial activity by disc diffusion test against Salmonella spp. culture.
E1 - extract-1; E2 - extract-2; the other unlabeled disc was blank. The test was performed in
triplicates and average results were reported.

33. 23
Figure 10 Graphical representation of the assay of the antimicrobial activity of the antibiotic
extract on Salmonella spp.

34. 24
DISCUSSION
Primary screening for antibiotics has been an ever-fresh research problem owing to the
emerging need for new and effective antibiotics against emerging and drug resistant pathogens.
Soil, Aquatic eco-systems, forests, mountains, etc are being routinely screened for unidentified
and new species of microbes that have antimicrobial potential. However, there are countless
microbial niches across the planet; therefore, it has become practically impossible to screen all the
microenvironments for microbial antagonism. On the other hand, continuous adaptation of
microbes to changes in their microhabitat could result in emergence of better antimicrobial strains
than that were previously identified. Therefore, in this study, soil samples from the banks of sewer
canals are selected as a source for microbes that produce antimicrobial compounds. Contamination
of such soils by means of sewage adds coliforms to those soil niches. These microbes would
generally be human gut-derived and these microbes could also be drug resistant-pathogens, by
chance. At the same time, interaction of the sewage contaminants with the indigenous microflora
of soil at the sewer canal-banks, could invariably lead to antagonism, which may result in
production of new or modified antimicrobials against the contaminants.
Based on this, it is hypothesized that “the coliforms from sewage sample are susceptible to
antimicrobial compounds that are produced by the antagonistic indigenous flora of the soil at banks
of sewer canals”.
This study is planned to be executed in the following steps
1. Isolation, and identification and culturing of E. coli and Salmonella species as pure
cultures,
2. Screening for antibiotic producers from soil samples at the banks of sewer canals,
3. Production and extraction of antibiotics from isolated antimicrobial cultures,
4. Testing of the antimicrobial activity of the extracted antimicrobial compounds against the
isolated cultures of E. coli and Salmonella spp.
The first objective of the study was achieved by culturing sewage samples from different sewer
canals on EMB and MacConkey agar media. Since these media are selective as well as differential,

35. 25
they helped in identifying lactose fermenters and non-fermenters by colony morphology and the
biochemistry behind the colony appearance. On EMB agar, those bacterial colonies, with green
metallic sheen, were identified as E. coli and that were colourless were assumed to be Salmonella
spp. These findings were confirmed by re-growing the cultures on the same medium. When these
cultures were grown on MacConkey agar medium, the culture that was previously identified as E.
coli developed fish-eye like colonies with pink periphery. This further confirmed the identification
of the microbe. On the other hand, formation of colourless colonies on MacConkey agar by the
culture that was previously identified as Salmonella spp., assured on the assumption of the
cultures’ identity, but it could not have been absolutely confirmed.
Crowded plate technique employing NAM was useful to identify two antimicrobial-strains from
the soil samples from the banks of various sewer canals. In contrast to the fact that NAM is
intended to grow bacteria, one out of the two identified antimicrobial-strains was observed to have
fungal morphology. Enormous growth of it in czapek-dox broth confirmed that this is a fungal
strain. Thus, two unidentified antimicrobial-strains were obtained by the screening procedure that
included a fungal culture and a bacterial culture.
The process of antibiotic production involved scale-up and expansion, which helped in the
production of sufficient antimicrobial-compounds. The compound produced by culturing the
unidentified fungal strain (in czapek-dox broth) was referred to as extract-1 and the other
compound produced by culturing the unidentified bacterial culture (in nutrient broth) was referred
to as extract-2. Solvent extraction provided a means to obtain crude antimicrobial compound.
Methanol treatment of the broth culture followed by extraction of the organic compounds into n-
hexane facilitated the harvest of antimicrobial extracts, as crude organic compounds.
Since extract-1 was produced by a fungal strain,
1. Identification of this fungal strain, strain improvement, and optimization of the production
process are needed, to improve the antibiotic production.
2. Identification of the exact organic compound that is responsible for the observed
antimicrobial activity should be carried out by testing of each molecular-fraction (can be
obtained by chromatography) of the crude extract, against each of the test organisms
followed by identification of the compound with reference to its optical spectrum, using
spectroscopy.

36. 26
CONCLUSION:
Highest antimicrobial activity was shown by extract-1 against both the isolated coliform strains,
which is promising, as the compound inhibited the growth of the test organisms to far greater
extent than did kanamycin.
Although the extract-2 was twofold effective than kanamycin, against Salmonella spp. Further
research on the compound is lesser promising than the effect of extract-1. However, identification
of this bacterial strain, strain improvement, and optimization of the production process could be
of some research interest.
Altogether, the primary screening carried out in this study resulted in isolation of a fungal strain
and a bacterial strain, which produced antimicrobial compounds. Since, the efficacy of these
compounds is promising, it is concluded that “screening for microbes with antimicrobial activity
would be promising and of research importance, when carried out using the soil samples that
harbour pathogens as a contaminants.

37. 27
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