2.1 Enterobacteriaceae:-
One of the extremely large family of
Gram-negative bacteria is Enterobacteriaceae
that includes many of the more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia
pestis, Klebsiella and Shigella, Proteus, Enterobacter, Serratia, and
Citrobacter.
Members of this family are
1) Rod
shaped
2) 1-5μm in length
3) Facultative
anaerobes
4) Non
spore forming
5) Few
of them are non-motile but some have peritrichious flagella for movement.
These bacteria are able to reduce
nitrate to nitrite and capable to ferment sugar to lactic acid and in various
other end products. Catalase reaction varies among the bacteria of this family.
Most of them are the part of normal gut flora mostly in humans and other
animal’s intestine. (Tahoun, 2011)
Most members of Enterobacteriaceae have peritrichious
type I fimbriae involved in the adhesion of the bacterial cells to
their hosts. Some of them produce endotoxins. Endotoxins reside in the
cell cytoplasm and are released when the cell dies and the cell wall
disintegrates. Some members of the Enterobacteriaceae produce endotoxins that
when released into the bloodstream following cell lysis, cause an inflammatory
and vasodilatory response. The most severe form of this is known as endotoxic
shock which can be rapidly fatal (Williams, et.al,
2010)
2.2 Escherichia coli:-
Escherichia coli was also named as
E.coli the German pediatrician Theodore Escherichia was first time identified
it from the fecalmaterials of healthy persons and described it in 1885.
Many strains or serotypes of E. coli are
found as commensal gut flora that colonizes the gastrointestinal tract of
newborns within a few hours of life. The majority of them do not cause any
disease unless the host is malnourished, immuno-suppressed or there has been a
disturbance in the intestinal barrier. Under these circumstances even commensal
E. coli can cause disease (James& Kaper, 1998). Serologically each E. coliisolate can be grouped by its
Lipopolysaccharide O-antigen, flagellar H-antigen, capsuleand fimbriae or pilli.
The pathogenic strains include seven
distinct categories of E. coli called
as virotypethat cause diarrhea.
These
can also be referred as pathotypes:
(1)
Enterotoxigenic E. coli(ETEC)
(2)
Enteroinvasive E.coli(EIEC)
(3)
Enteropathogenic E. coli(EPEC)
(4)
Enteroaggregative E. coli (EAEC)
(5)
Diffuse Adherent E. coli(DAEC)
(6) Enterohemorrhagic E. coli (EHEC)/ Shiga toxin
producing E.coli (STEC)
(7)
Opportunistic E.coli
(James& Kaper, 1998)
2.2.1 Enterotoxigenic E. coli (ETEC):-
Enterotoxigenic E. coli is the causative agent of diarrhea in humans, pigs,
sheep, goats, cattle, dogs, and horses. ETEC
uses fimbrial adhesions (projections from the bacterial cell surface)
to bind enterocyte cells in the small intestine. ETEC can
produce two proteinaceous enterotoxins:
- The larger of the two
proteins, LT enterotoxin, is similar to cholera
toxin in structure and function.
- The smaller protein, ST
enterotoxin causes cGMP accumulation in the target
cells and a subsequent secretion of fluid and electrolytes into the
intestinal lumen.
ETEC
strains are noninvasive, and they do not leave the intestinal lumen. ETEC is
the leading bacterial cause of diarrhea in children in the developing world as
well as the most common cause of traveler's diarrhea.
2.2.2
Enteroinvasive E.coli(EIEC):-
EPEC
also causes diarrhea like ETEC but the molecular mechanisms of colonization and
etiology are different. EPEC lack fimbriae ST and LT toxins, but they use
an adhesion known as intimin to bind host intestinal cells.
This virotype has an array of virulence factors that are similar to those found
in Shigella, and may possess a shiga toxin. Adherence to the
intestinal mucosa causes a rearrangement of actin in the host cell
causing significant deformation. EPEC cells are moderately invasive and elicit
an inflammatory response.
2.2.3 Enteropathogenic E. coli
(EPEC):-
EIEC
infection causes a syndrome that is identical to shigellosiswith profuse diarrhea
and high fever found only in
humans. The first stage in EPEC pathogenesis involves the initial adherence of
bacteria to epithelial cell. EPEC strains typically infect intestine via
“attaching and effacing”
2.2.4 Enterohemorrhagic E. coli (EHEC):-
The
most notorious member of this virotype is strain O157:H7, which causes
bloody diarrhea and no fever. EHEC can cause hemolytic-uremic
syndrome and sudden kidney failure. It uses bacterial fimbriae for attachment
is moderately invasive and possesses a phage-encoded shiga toxin that can
elicit an intense inflammatory response found in humans, cattle, and goats.
2.2.5 Enteroaggregative E.coli (EAEC):-
So
named because they have fimbriae which aggregate tissue culture cells,
EAEC bind to the intestinal mucosa to cause watery diarrhea without fever. EAEC
are noninvasive. They produce a hemolysin and an ST enterotoxin
similar to that of ETEC andfound only in humans (Maria, et.al, 2007). Nataro and Kaper first describe the EAEC in culture. Virulence
factor associated with EAEC are a heat stable toxin and several aggregative
adherence fimbriae (Rosane, et.al,
2013).
2.2.6 Diffuse adherent E.
coli (DAEC):-
Diffusely
adherent Escherichia coli have been consideredas adiarrhea
genic group of E. coli (DEC). They are characterized by the diffuse
adherence pattern on cultured epithelial cells HeLa or HEp-2.
It cause diarrhea in children and adults both.A key virulence factor in DAEC is
the production of adhesions. Three adhesions Afa,Drand F1845 are fimbrial
adhesions for the diffuse cells adherence to epithelial tissue. Afa and Dr
commonly express together and associated with the urinary tract infection (Rosane,
et.al, 2013).
2.2.7 Opportunistic E.coli:-
Are
non-pathogenic unless an environmental with in the gastrointestinal tract
promotes over population and growth of bacteria. Opportunistic behavior is
typical in postsurgical wounds on surgical implants and urinary tract
infection.
2.3 Staphylococcus aureus:-
S.
aureus is a facultative
anaerobicGram-positive coccal bacterium, also known as "golden
staph" and Oro staphira. In medical literature the bacteria is often
referred to as S. aureus or Staph aureus. Staphylococcus should
not be confused with the similarly named and medically relevant genusStreptococcus. S.
aureus appears as grape-like clusters when viewed through a
microscope, and has large, round, golden-yellow colonies, often
with hemolysis, when grown on blood agar plates. S. aureus reproduces asexually by binary
fission. The two daughter cells do not fully separate and remain attached to
one another. This is why the cells are observed in clusters (Chambers, 2001).
S.
aureus is catalase-positive. Catalase
converts hydrogen peroxide to water and oxygen. Catalase-activity
tests are sometimes used to distinguish staphylococcifrom enterococci and streptococci.
Previously, S. aureus was differentiated from other
staphylococci by the coagulase test. However it is now known that not
all S. aureus are coagulase-positive and that incorrect
species identification can impact effective treatment and control measures
Staphylococcus
aureus
Staphylococcus aureus
is a bacterium which is the member of the Firmicutes, and is frequently present
in the human respiratory tract and also on the skin. Although S. aureus is not
always pathogenic, it is a common cause of skin infections (e.g. boils),
respiratory disease (e.g. sinusitis), and food poisoning. Disease-associated
strains often promote infections by producing potent protein toxins, and
expressing cell-surface proteins that bind and inactivate antibodies. The
emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA) is
a worldwide problem in clinical medicine.
Each year, some 500,000 patients in American hospitals contract a
staphylococcal infection (Cole, et.al,
2001).
Staphylococcus was first identified in 1880
in Aberdeen, United Kingdom, by the surgeon Sir Alexander
Ogston in pus from a surgical abscess in a knee joint.This name
was later appended to Staphylococcus
aureus by Rosenbach who was credited by the official system of
nomenclature at the time. It is estimated that 20% of the human population are
long-term carriers of S. aureus which can be found as part of
the normal skin flora and in anterior nares of the nasal
passages. S. aureus is the most common species of
staphylococcus to cause Staph infections and is a
successful pathogen due to a combination of nasal carriage and bacterial
immuno-evasive strategies.
S. aureus can cause a range of illnesses, from minor skin infections,
such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded
skin syndrome, and abscesses,to life-threatening diseases such as, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome (TSS), bacteremia, pneumonia and sepsis.
Its incidence ranges from skin, soft tissue, respiratory, bone, joint,
endovascular to wound infections. It is still one of the five most common
causes of nosocomial infections and is often the cause of
postsurgical wound infections (Boost, et.al, 2008)
2.4Worldwide
discovery of bacteriocins from E.coli:-
Michael Feld garden and Margaret A. Riley in 1997 in
their study In this study attempt to characterize more completely the levels of
naturally occurring colicin resistance. They present a survey of colicin resistance
in 158 E. coli isolates from a variety of hosts and geographic regions of
United Kingdom. In addition, they have screened for resistance in 137 isolates
of non-E.coli enteric, which are close relatives of E. coli. Their data suggest
that resistance levels are high. Similarly high levels of interspecific
resistance to colicins are also observed. The frequency of colicin resistance
to eighteen colicins is exceptionally high in all four E. coli collections surveyed.
On average, 75% of the strains surveyed were resistant to a particular colicin.
The average resistance to a particular colicin was the highest for the ECOR
collection with a frequency of 91.1% and was the lowest for the Indian
collection with a frequency of 67.5%.
Similarly Riley and Gordon in 1996
do the surveys of over 1000 isolates reveal that, on average, 30% of E. coli
produce colicin. Even higher levels of resistance to these same toxins were
observed in one survey of E. coli (the ECOR collection; Riley and Gordon 1992).
This study suggested that most E. coli are resistant to most colicins. On
average, 93% of the isolates were resistant to any one colicin, and 33% were multiple
resistant to all the colicins tested (Riley and Gordon 1992)
David M. Gordon and Claire L.
O’Brien in 2005 collect the 266 faecal isolates of Escherichia coli from humans
in Australia was assayed for the production bacteriocins and screened using a
PCR-based method for the presence of eleven colicins and seven microcins. Eight
different colicins were detected and all seven microcins. Of the strains
examined, 38 % produced a bacteriocin, 24 % produced a colicin and 20 %
produced a microcin. Of the 102 bacteriocin-producing strains, 42 % produced
one type of bacteriocin, 41 % produced two, 16 % produced three and one strain
was found to produce four different bacteriocins.
2.5 Bacteriocin:-
The method to control the pathogenic bacteria
is the production of bacteriocin from bacteria.
Both Gram positive and Gram negative bacteria produce bacteriocin (Rowaida,et.al, 2009).
Historically the term “Bacteriocin” was
applied to antibiotic like compounds with specificityprimarily restricted to
bacterial strains. This can be thought of as microbial “murder” of
onesrelative, but their specificity and chemical composition served to
distinguish them from so-called classical antibiotics. (Reeves, 1979).
To go back to the first bacteriocin
descriptions amounts to studying the first worksconcerning bacterial
antagonism. Such bacterial antagonism was described by the pioneersof
microbiology during the last decades of the 19thcentury. At that
time, the molecular basisof bacterial inhibition was abstruse, so it was
difficult to distinguish antagonism due tobacteriocin from that provoked by
other compounds such as antibiotics, organic acids, orhydrogen peroxide, except
on the basis of their spectrum of activity, usually narrower thanthat of other
ones. (Desriac, 2010).
The
bacteriocins are the proteinaceous substance act as the bactericidal agent that
inhibit and often kill the some closely related species of the bacteria. Bacteriocins inhibit the spoilage of food and
also pathogenic bacteria. (Veeranan,et.al, 2009).
Bacteria express produce and utilize these
proteins for survival and proliferation of an organism in a mixed population.
The production of these ribosomally synthesized antimicrobial
substance was first described in Escherichia coli which is the Gram negative
bacteria (Aylin, et.al, 2001 ) .One
of the best known bacteriocin from Gram negative bacteria is colicin produce by
the strains of E.coli are large peptide 29-90 kDa , in size. Characteristics of
colicins are it involved in cell attachment, translocation and bactericidal
activity and attach with the specific receptor of target cells on the outer
membrane and in result their activity range would be narrow (Teather, et.al, 1999).
One common event found in Gram negative
Bacteriocin production is lethality effect of the producing cells. The
bacteriocin is generally produced and released which cause cell death these
three events are all directed by the Bacteriocin lysis protein also expressed
and produced by the host cell. Gram positive bacteriocins are differing from
gram negative in two ways. First Bacteriocin production is not necessarily the
lethal event it is for gram negative bacteria also gram positive bacteriocins
have evolved Bacteriocin specific regulation.
Bacteriocin also produced by the Gram positive
bacteria which are small peptide 3-6 kDa in size in most of cases. They are
divided in to two large classes,
Class
1: Lantibiotics bacteriocins.
Class2:Non-lantibiotic bacteriocins.
The post-translational modifications occur in
both but in case of lantibiotics additional post translational
modifications are also occur which giving rise to a variety of unusual amino
acid that include unsaturated amino acid
and also a number of other modified amino acid residues. Both classes of
peptide antibiotics show the great variety of heterogeneity with respect to
amino acid sequence and their effect on the target species. Most of the gram
positive bacteria increase the membrane
permeability of cytoplasmic membrane because they are membrane active compound
and also demonstrate the broad spectrum of bactericidal activity than colicin
due to their activity they do not require the specific cell surface receptor
and the absence of outer membrane restrict their access to the cytoplasmic
membrane (Teather, et.al, 1999).
Bacteriocins are characterized due to their
high level of molecular diversity. Bacteriocin production is a complex high
energy demanding process, requiring not only the synthesis of the bacteriocin prepeptide
butalso involved in the production of a set of auxiliary proteins that ensure
bacteriocin maturation, secretion, regulation and immunity (Ruth,et. al, 2007).
2.5.1 Methods of classification:-
The
alternative methods of classification include method of killing, genetics
(large plasmids, small plasmids, chromosomal), molecular weight and chemistry
their (large protein or polypeptide) and method of production
(ribosomal, post ribosomal modifications, non-ribosomal).
The bacteriocins are classified into Class I,
Class IIa, and Class III.
Class I bacteriocins:-
The class I bacteriocins are small
peptide inhibitors and include nisin and other lantibiotics.
Class II bacteriocins:-
The class II bacteriocins are small
(<10 kDa) heat-stable proteins. This class is subdivided into five sub
classes. The class IIa bacteriocins (pediocin-like bacteriocins) are the
largest subgroup. The class IIa bacteriocins are used in the food preservation
as well as in medical applications because they have broad range of activity.
One example of Class IIa bacteriocin is pediocin.
The class IIb bacteriocins require two
different peptides for their activity. One example of this class is lactococcin
G which permeabilizes cell membranes for monovalent ions such as Na and K, but
not for divalents ones.
Class IIc cover the cyclic peptide
bacteriocins possesses the N-terminal and C-terminal regions which are covalently
linked.
Class
IId cover single-peptide bacteriocins which are not post-translated modified.
The one of the best example of this group is the aureocin A53which is highly
stable. The bacteriocins of this class are stable in highly stable environment.
The most recently proposed subclass is the
Class IIe which encompasses those bacteriocins composed by three or four
non-pediocin like peptides. The best example is aureocin A70 a four-peptides
Bacteriocin and which is highly active against L. monocytogenes
with a tremendous biotechnological application (Franz et.al, 2007).
Class III bacteriocins:-
Class III bacteriocins are the large (>10
kDa) protein and heat-labile bacteriocins. This class is subdivided in two
subclasses: subclass IIIa or bacteriolysins and subclass IIIb.
Subclass IIIa contain those peptides that kill
bacterial cells by degradationand cause cell lysis. The best studied
bacteriolysin is lysostaphin, a 27 kDa peptide that hydrolysis several Staphylococcus spp.
cell walls especially S. aureus. Subclass IIIb consist of
those peptides that do not cause cell lysis and kill the target cells by
disrupting the membrane potential.
Class IV bacteriocins:-
Class IV bacteriocins are known as complex
bacteriocins containing lipid or carbohydrate moieties. (Franz et.al, 2007)
2.5.2
Medical importance:-
The most of the bacteria used as medicines
because they are made by non-pathogenic bacteria which normally colonize
the human body. Bacteriocins also serve as cancer treatment.
Bacteriocins were tested as AIDS drugs
around 1990 but did not progress beyond in-vitro tests on cell lines.Bacteriocins can target individual
bacterial species kill many microbes to the broad spectrum.
2.6 Bacteriocins in human health:-
In addition to bacteriocinogenic probiotics,
purified or partially purified bacteriocins also hold great promise with
respect to the treatment of target pathogenic bacteria and may ultimately be employed
as pharmabiotics and novel alternatives to existing antibiotics. Mersacidin,
produced by Bacillus sp. strain HIL
Y85, was also active against methicillin-resistant Staphylococcus
aureus (MRSA ). This
bacteriocin was able to completely eradicate MRSA
from the nasal epithelium of the mouse, independent of the colonization time
and number of inoculations. It has also been shown that a single dose of
mutacin B-Ny266, produced by Streptococcus mutans, was 100%
protective when administered intraperitoneally to mice previously infected with
methicillin-susceptible S. aureus (Mota, et.al, 2005). Finally, it is noteworthy that both lacticin 3147 and
thuricin CD produced by Lactococcus lactis DPC3147 andBacillus
thuringiensis DPC6431, respectively, exhibited inhibitory activity
againstC. difficile.
2.7Colicin:-
Colicins are the macromolecules which are
produced by some strains of Escherichia coli and related Enterobacteriaceae
family. They occur in protein structures and specified by Col plasmid (Aylin, et. al, 2001). More than 20 colicins
have been described in E. coli distinguished by the absence of
cross-immunity between the producing strains (Nomura 1963). Colicins are
encoded on a diverse group of colicin plasmids (Col plasmids), which usually
encode at least two additional colicin-related proteins: an immunity protein,
conferring specific immunity to the host and against that colicin, and a lysis
protein, involved in lysing the host cell and in releasing colicin into the
environment. This group of genes is called a “colicin cluster.” Colicins are
abundant in natural populations of E. coli.
The first colicin was identified by Gratia in
1925 as a heat-labile product present in cultures of E.coli. After
this variety of colicins produced by different strains of the entire group of
bacteria (E. coli, Shigella, and Citrobacter)
were characterized. The name colicin was
coined by Gratia and Fredericq in 1946, who demonstrated their protein nature
and the specificity of their activity spectra. Colicin kills sensitive cells
according to single-hit kinetics and colicin is not active against the
producing bacteria due to the presence of a specific antagonist protein called
the immunity protein. (Eric, et.al,
2007)
Colicins are the best characterized
bacteriocins. Some colicins inhibits
(1) Peptidoglycan synthesis
(2) Lipopolysaccharides O- antigen synthesis,
but mostly act as the
(3) Membrane permeabilization which is
followed by nuclease activity (Zgur,et
.al, 2013)
Colicins are divided into two groups according
totheir cross-resistance patterns.
Group A: A, E1, E2, E3, E9, K, L, N, S4, and X
Group B: B, D, G, H, I, Ia, Ib, M,Q, S and V.
It was later shown that the A and B groups are
also distinguished by their mechanism of release from the producing cell. In
general, group A colicins are encoded by small plasmids and are released into
the medium, whereas group B colicins are encoded by large plasmids and are not
secreted. However, some colicins might belong to one group and share homologies
with colicins of the other group. (Pilsl,et.al,
1995)
Colicin itself is translocated through the
cell envelope rather than any putative constituents that play an intermediate
role during its action. The demonstration in 1971 that colicin E3 is a specific
RNasethat makes one cut in the 16S rRNA gene significantly changed the model of
colicin action. Further on, the endonuclease activity of numerous colicins was
demonstrated, with each one specifically cleaving a particular nucleic acid at
a precise site. Colicins E2, E7, E8, and E9 cleave DNA (108, 574, 628), and colicins
E3, E4, and E6 hydrolyze rRNA while colicins D and E5 cleave tRNA (Eric, et.al, 2007)
In 1963, Nomura demonstrated that the various
colicins have different modes of action: colicins E1 and K inhibit all
macromolecular synthesis, colicin E2 causes DNA breakdown, and colicin E3 stops
protein synthesis (Nourma, 1963).
Although the ecological role of colicins is
not clear, several studies reveal that they play a role in bacterial
competition and invasion. Under conditions of stress, a small fraction of
colicinogenic cells are induced to produce colicin and lysis proteins, an
action mediated by the SOS response. The production of lysis protein results in
the lysis of the host cell and in the release of colicin. The colicin protein
attaches to specific cell surface receptors on nearby E. coli cells and
kills the invaded cell by one of four mechanisms, which include DNA and rRNA
degradation. If the invaded cells possess the same Co1 plasmid, they will be
immune to the effects of the colicin, through a specific interaction between
the immunity and colicin proteins (Pugsley, 1984). Colicins of both group A and
B target the E.coliby interacting
with the specific outer membrane proteins. In group A colicins A, E1 and E9
target the vitamin B12 transporter BtuB (Eric, et.al, 2007).
2.8Microcins:-
The
second classes of bacteriocins produced by E. coli, the microcins, are less
well understood.Thegene cluster may be chromosomally or plasmid encoded
andcomprises two genes: the microcin gene, which encodes thebactericidal
protein, and the immunity gene. Cells areinduced to produce the microcin
protein under specificconditions, such as iron limitation. The release of
microcinsis not a consequence of cell lysis; rather, the microcin isactively
secreted from the cell. The export of microcins canbe complex and often
involves microcin-specific proteins aswell as basic ‘housekeeping’ proteins (David
M. Gordon and Claire L. O’Brien, 2006). Most microcins arethought to bind to
surface receptors on target cells involved in iron uptake. The manner in which
microcins kill cells isnot generally known, but some disrupt the target
cell’smembrane potential. Fourteen microcins have been reported, of which only
seven have been isolated and fully characterized(Severinov,et.al, 2007).
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