Introduction reports in the discovery of natural

Introduction

Bacterial
and fungal infections are very common problem in human population. The chemical
synthetic drugs which are used to remove this medical problem may have several
side effects to us1. Thus the lead
molecule of the effective drug can be synthesised from the natural product with
a therapeutic effect. It is expected that drug from the natural compound will
be renewable, naturally eco-friendly and easily obtainable2. Plants are the
potential source of novel bioactive compounds. The micro-organisms associated
within the plants, i.e , endophytes may also produce biologically active
compounds similar to their host plant3 in direct or
indirect manner.

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Endophytes, by definition, are -“microbes that colonize living, internal
tissues of plants without causing any immediate overt negative effects”4. Studies have shown that, more or less every
plant species examined have atleast one endohyte species5. There is a mutual relationship between the
host plant and their endophytes. Endophytes may produce secondary metabolites
which prevent growth of pathogen or may kill the pathogen and in return plant
gives shelter and nutrition to these endophytes. In support of this idea many
antimicrobial, antifungal compounds are isolated from the endophytes6. There are many reports in the discovery of
natural compounds with potential antimicrobial activity from fungal endophytes7.

Therefore the aim of this review is to take a look
on recent research on antimicrobial metabolites and antibiotics produced from
endophytic bacteria.

Ecology
and Diversity of Endophytic Bacteria

Endophytes are found within most of plant species5. Endophytes may
present herbaceous to woody plant. Endophytes enter into the host tissue
through root zone or aerial zone8. It has been
reported that they may enter through stomata, lenticels9 or through
wounds10.  Within the host plant endophytes reside
either intercellularly or intracellularly. They may also reside within the
vascular tissues of their host plant11.

From a single plant species different
types of endophytes can be isolated. It may also possible from various plant
species a wide variety of endophytes are reported9. The diversity
of endophytes include Gram positive as well as Gram negative bacteria like Achromobacter,
Acinetobacter, Agrobacterium, Bacillus, Brevibacterium,
Burkholderia,Chromobacterium, Curtobacterium, Enterobacter, Kocuria,
Lysinibacillus, Methylobacterium, Microbacterium,Paneibacillus, Pantoea,
Phyllobacterium, Pseudomonas, Rahnella, Rhodanobacter, Stenotrophomonas,
Streptomyces, Xanthomonas etc12,13.

 

 

Screening
of Antimicrobial Metabolites

Three basic and major
steps are involved in the screening of antimicrobial metabolites from
endophytes. 1st one is selection of plant material. It is very
important steps because selection of plants provides the opportunity to isolate
endophytes with ability to produce novel anti- microbial metabolites. So,
plants can be selected from special ecological environment. For example,
mangrove plants can be selected and endophytes can be isolated from them14. Other criteria
may be selection of traditional medicinal plants and isolation of endophytes
from them15. The second
step is preliminary screening of antimicrobial
activity . Crude extract of endophytes are tested for their antimicrobial
activity by agar cup method or paper disc diffusion method16.Then active subtances of crude extract can be separated by Thin
Layer Chromatography technique and other chromatographic techniques17. The third step is development of antibiotics from the
potent metabolites.The general procedure of separation of antimicrobial
compounds is given below:

Selrction
of plant material

 

Isolation of endophytic
bacteria through surface sterilization

 

Preliminary screening
for antimicrobial activity from the crude extracts of endophytes

 

Separation of active
compounds through chromatographic techniques

 

Development of antibiotics from the potent metabolites

Table 1 – List of some potent bacterial endophytes
with their antimicrobial property are given below:

Sl No

Host Plant

Potent
Endophyte

Activity Shown

Test Organism

Reference No

1

Panax ginseng

Paenibacillus
polymyxa
GS01,
Bacillus
sp. GS07,
and
Pseudomonas
poae
JA01
 

Antifungal

Phytopathogenic fungi

12

2

T. grandiflora
Polyalthia sp.
Mapania
sp.

Streptomyces
fulvoviolaceus,
Streptomyces
coelicolor,
Streptomyces
caelestis

Antifungal

Phytopathogenic fungi

18

3

Scutellaria
baicalensis
Georgi
 
 

Bacillus
amyloliquefaciens

Antibacterial,
Antifungal
 

Phytopathogenic,
food-borne
pathogenic and
spoilage
bacteria and fungi

19

4

Panax
notoginseng
 
 

Bacillus
amyloliquefaciens
subsp.
plantarum ,
Bacillus
methylotrophicus

Antifungal

Phytopathogenic
fungi and
nematode

20

5

Azadirachta
indica
A. Juss.
 
 

Streptomyces
sp.,
Nocardia
sp.
 

Antibacterial,
Antifungal

Phytopathogenic
fungi,
Human
pathogenic bacteria
and
fungus

21

6

Plectranthus
tenuiflorus
 

Bacillus sp.
Pseudomonas
sp.
 

Antibacterial,
Antifungal
 

Human
pathogenic bacteria
and fungus

22

7

Wheat

Bacillus
subtilis

Antifungal

Phytopathogenic
fungi

23

8

Anthurium

B.
amyloliquefaciens

Antibacterial

Phytopatogenic
bacteria

9

9

Platycodon
grandiflorum
 
 

Bacillus
licheniformis,
Bacillus
pumilus,
Bacillus sp.

Antibacterial,
Antifungal
 

Phytopathogenic
fungi and
anti-human
food-borne
pathogenic
organisms

24

10

Artemisia
annua
 

Streptomyces

Antibacterial,
Antifungal
 

pathogenic
bacteria, yeast and
fungal
phytopathogens

25

11

Centella
asiatica

Bacillus
subtilis,
Pseudomonas
fluorescens
 

Antifungal

Phytopathogenic
fungi

26

12

Panicum
virgatum L.
 
 

Bacillus
subtilis, C.
flaccumfaciens,
Ps.
Fluorescens,
P.
ananatis

Antifungal

Phytopathogenic
fungi

27

13

Raphanus
sativus L
 

Enterobacter sp., B.
subtilis
 

Antibacterial,
Antifungal
 

Phytopathogenic
fungi, Human
pathogenic
bacteria

28

14

Memecylon
edule ,
Tinospora
cordifolia,
Phyllodium
pulchellum and
Dipterocarpus
tuberculatus
 
 

Bacillus
amyloliquefaciens

 
Antibacterial,
Antifungal
 

Human
pathogenic bacteria
and fungus

29

15

S.
lavandulifolia,
H. scabrum, R.
pulcher
 

Bacillus sp.

Antibacterial,
Antifungal
 

Human
pathogenic bacteria
and
saprophytic fungi

30

16

Aloe chinensis

Paenibacillus species

Antibacterial,
Antifungal
 

Pathogenic
bacteria and fungi

31

17

Epimedium
brevicornu Maxim.
 

Phyllobacterium
myrsinacearum
 

Antibacterial,
Antifungal
 

Phytopathogenic
fungi and
phytopathogenic
bacterium

32

18

11 mangrove
halophytic
plants
 

Bacillus
Thuringiensis
and
Bacillus
pumilus
 

Antibacterial

Shrimp
pathogens

9

19

Kandelia
candel

Streptomyces
sp.
 

Antibacterial

Several
pathogenic bacteria

33

20

Codonopsis
lanceolata
 

Bacillus
pumilus
B. subtilis
B.
licheniformis
 

Antifungal
 

Phytopathogenic
fungi

34

21

Polygonum
cuspidatum
 

Streptomyces
sp.

Antifungal
 

Pathogenic
fungi

35

22

Manihot
esculenta

Paenibacillus
sp.

Antifungal
 

Phytopathogenic
fungus

36

23

Bruguiera
gymnorrhiza
Rhizophora
stylosa
Kandelia
candel
 
 

Bacillus
amyloliquefaciens
 

Antibacterial,
Antifungal

Phytopathogenic
fungi and
phytopathogenic
bacteria

37

24

Monstera sp.
 

Streptomyces
sp.

Antifungal,
Antimalarial
 

Pythiaceous
fungi
and the human
fungal
pathogen,
malarial
parasite

38

25

Piper nigrum L
 

P. aeruginosa,
P.
putida and B.
megaterium
 

Antifungal
 

Phytopathogenic
fungus

39

26

Huperzia
serrata

Burkholderia
sp.

Antifungal
 

Phytopathogenic
fungi

9

27

300
plants from
upper
Amazonian
Rainforests
 
 
 

Streptomyces
sp.
Micromonospora
sp.
Amycolatopsis
sp.

Antibacterial,
Antifungal

Range
of potential fungal and
bacterial
pathogens

40

28

Lycopersicon
esculentum
 

Streptomyces
sp.,
Microbispora
sp.,
Micromonospora
sp.
and Nocardia
sp.
 

Antibacterial,
Antifungal
 

Phytopathogenic
fungi and
phytopathogenic
bacteria

41

 

 

 

 

 

 

Antibiotic
production by Endophytic Bacteria

 

In 1957, the production of classical
phytohormone gibberellins from the endophytic fungus (Gibberella fujikuroi) of Rice plant had increased the importance
and significance of endophytes42. Later, in
1993, the discovery of Taxol, a novel anticancer drug, from Taxomyces andreanae , an endophytic
fungus of Taxus brevifolia43  motivated the endophytic research in a new
way. Furthermore, researchers are working on endophytic fungi and as well as
bacteria to explore their novel and unique natural bioactive compounds for
their commercial value. The natural products from endophytes have been proven
to produce antibiotics. Ecomycins, Pseudomycins,
Munumbicins, Kakadumycins are some examples of the novel antibiotics produced
by endophytic bacteria.

      

     Ecomycins

 

The discovery of antibiotics from
endophytic bacteria include the ecomycins from the known grass endophytic
bacterium,  Pseudomonas viridiflava44.
This endophyte exists in the tissues of many grass species. The identified and partially
characterized three antifungal lipopeptides produced by P. viridiflava strain
EB273 are called as Ecomycin A, B and C.
The
Ecomycins represent a family of novel lipopeptides and are made up of some
unusual amino acids including homoserine and ??hydroxy aspartic acid.
Out of these three molecules, the Ecomycin A is similar to (amino acid composition)
an already reported antibiotic syringotoxin45. However,
based on the molecular weight and amino acid compositional data, ecomycins B
and C represent related set of  lipopeptides which do not possess
phenylalanine, lysine, arginine, ornithine or diaminobutyric acid. These are constituents
of such compounds similar to the pseudomycins, syringomycins, syringostatins
and syringotoxin45,46,47.
Each ecomycin contained ? -hydroxy aspartic acid, threonine, serine,
homoserine, glycine, alanine and an unknown amino acid and with one exception
(syringotoxin) distinguishes them from all other antifungal lipopeptides.
However, ecomycins A and B yielded significant level of alanine. Ecomycin A was
the only member of the family containing 2,4-diaminobutyric acid and ornithine.
Ecomycin C had a level of alanine similar to that of ecomycin A44.

   

 

 

     Pseudomycins

 

The Pseudomycins is a group of peptide
antifungal compounds. It is isolated from liquid cultures of Pseudomonas
syringae, which is  a plant?associated
bacterium. The P. syringae is a member of the Pseudomonadaceae family of
Proteobacteria phylum. These antifungal peptide are mainly lipopeptides
containing aminoacids like L?chlorothreonine, L?hydroxy
aspartic acid and both D?and L?diaminobutyric acid. There
are four types of pseudomycins, pseudomycins A?D, have effective
activity against the human pathogen, Candida albicans, C. neoformans.
Pseudomycins A?C
contain hydroxyaspartic acid, serine, arginine, lysine and diaminobutyric acid.
Pseudomycin D has a molecular mass of 2401Da and is more complex than
pseudomycins A?C48.

 

      Munumbicins

 

The munumbicins were isolated from a Streptomyces
NRRL 30562 that colonizes snakevine (Kennedia nigriscans), a plant
species that was used to prevent wound sepsis. There are 4 types of Munumbicins
namely Munumbicins A, B, C and D. These novel bioactive substances are active
against plant pathogenic fungi and bacteria, and a Plasmodium species.
The munumbicins act against Gram?positive bacteria such
as Bacillus anthracis, Streptococcus pneumoniae, Enterococcus faecalis
and Staphylococcus aureus.49.The
interesting fact is that, the methicillin?resistant strain of S.aureus
(MRSA, ATCC 33591) and a vancomycin?resistant strain of E.
faecalis (VREF, ATCC 51299) are two of the Gram?positive
munumbicin?sensitive
bacterial strains that are commonly drug?resistant. The drug
resistance strain of Mycobacterium tuberculosis is sensitive to munumbin
B but the drug susceptible strain of this organism is less sensitive to this
antibiotic.  The munumbicins C and D are effective
against Gram positive and negative bacteria, along with that they are also effective
against the malarial parasite  Plasmodium
falciparum. It is reported that munumbicin D is more powerful than
chloroquine, the gold?standard antimalarial drug. Munumbicins
B, C and D compounds are not effective against human pathogenic fungi50. Each
of the four munumbicins compounds consists with the presence of Glx (glutamic
acid or glutamine), Pro, Thr and Val, except for munumbicin C, which had an
extra proline49.

 

     Kakadumycins

 

Kakadumycins produced (in culture)
by endophytic bacterium Streptomyces (NRRL30566) from a fern?leaved
Grevillea tree (Grevillea pteridifolia, Synonym: Grevillea
chrysodendron R.Br.) native to the northern territory of Australia.
Kakadumycin A, has antibacterial activity similar to Munumbicins; and it also
shows activity against P. falciparum. Kakadumycin A is chemically
related to echinomycin, which is a Streptomyces derived quinoxaline
antibiotic, that acts as potential anticancer drug51.

 

    Xiamycins

 

The Xiamycins is one of the
indolosesquiterpenes. There are 2 types of xiamycins, one is Xiamycin a which
is a pentacyclicindolosesquiterpene and another one is xiamycin b that is a
indolosesquiterpenes. Along with this two type of xiamycins other two new type
of indolosesquiterpenes isolated from the culture broth of Streptomyces sp.
strain HKI0595, a bacterial endophyte of the mangrove tree, Kandelia candel.
Their research findings suggest that these Xiamycins do have moderate to strong
antimicrobial activities against several bacteria, including methicillin?resistant
Staphylococcus aureus and vancomycin?resistant Enterococcus
faecalis33.
Interestingly, Xiamycin?A also shows  anti?HIV activity52.

 

    Oocydin

 

Another interesting discovery was
oocydin A, a chlorinated macrocyclic lactone produced by Serratia marcescens
living inside the aquatic plant species Rhyncholacis penicillata53.
Strobel et al. speculated that oocydin A may contribute to the natural
protection of R. penicillata against oomycete pathogens that are
prevalent in the aquatic environment1.

 

    Other
Antibiotics

 

Antifungal activity is also shown by the
endophytic bacteria, Paenibacillus
polymyxa associated with Wheat plant by producing Fusaricidin A–D active
compound54. Coronamycin
is
produced by a verticillate Streptomyces
sp. isolated as an endophyte from an epiphytic vine, Monstera sp., found in the Manu region of the upper Amazon of Peru
shows antimalarial, antifungal activity against pythiaceous fungi and the human
fungal pathogen Cryptococcus neoformans38.

 

Conclusion

 

The objective of this
paper was to review the diversity of secondary metabolites with anti-microbial
activities produced by endophytic bacteria. This review covered 8 antibiotics
with diverse activities against plant pathogens, produced from different kinds
of endophyteic bacteria inhabiting from a range of plant species.This
anti-microbial substances can be isolate and purified from endophytic bacteria.
If any of the metabolites show potent activity then those can be processed for
future drug development. Those may also be used as antibiotics. With the help
of microbial fermentation process this bioactive natural compounds can be
produced from the endophytic bacteria inexhaustible manner. Finally this
research provides better bioactive antimicrobial substances
without any side effect to human, plant and environment. So, this review suggests that as bacterial endophytes are potentially
vital sources for antimicrobial metabolites and antibiotics thus significant
numbers of antibiotics and antimicrobial metabolites are remain to be
discovered from less explored or unexplored endophytic bacteria.