Introduction

A biofilm is defined as a collection of organisms which are attached to a surface and

surrounded by a matrix. This matrix is produced by the bacteria and is commonly

referred to as extracellular polymeric substances (EPS) or ‘‘slime’’. The matrix combines

with other substances in the surrounding tissues to form a structure known as a

glycocalyx. Confocal and electron microscopy have shown that biofilms have a well

defined structure, commonly mushroom shaped in the planktonic form [1]. They have

open channels which are believed to be related to nutrition and the removal of waste [1–

3]. Recent studies of the ultrastructure of the biofilm matrix show a characteristic

honeycomb appearance and a Nano wiring structural component which is believed

related to energy sharing [4]. The function of a biofilm is unknown but the organisms

which are associated within it become highly resistant to antibiotic therapy and to the

immune system [4–7].

The reasons for this are complex and are related to the inability of antibiotics to

diffuse through the biofilm, changes in the organisms due the increasing hypoxia within

the biofilm and the development of persistent or resistant cells which are thought to be a

mechanism of survival by the microorganisms. The composition of biofilm matrix is

highly variable and contains various proteins, polymers, complex carbohydrates and

water. The organisms in biofilms are commonly mixed and combined together to form

well defined structures by a mechanism called ‘‘Quorum Sensing’’, during which cell to

cell communication occurs through the interchange of small diffusible molecules

including peptides and chalones [1,2,8,9]. Within the biofilm the organisms undergo

metabolic changes in response to the hypoxia, and exchange genetic material. Many
bacteria including those commonly isolated from the burn wound are able to produce

biofilms. These include Staphylococcus aureus, Staphylococcus epidermidis,

Pseudomonas aeruginosa and Escherichia coli [1,2]. Biofilm related sepsis is well

documented in medical practice and is commonly associated with implantable medical

devices such as orthopaedic implants, cardiac pacemakers and centrally placed venous

catheters [10–13]. Biofilms additionally constitute a major problem in the sterilization of

medical instruments, particularly endoscopes. It has been estimated by the Center for

Disease Control in the United States that 60% of all chronic infections are related to

biofilm and include a wide range of illnesses such as cystic fibrosis, chronic obstructive

pulmonary disease, otitis media and prostatitis [1]. The Dental literature is prolific on

biofilms and dental plaque, with the major cause of periodontal disease and tooth loss

clearly identified as due to biofilms [1]. The clinical relevance of biofilms relates to a

wide range of medical disciplines, including neuro-surgery, orthopaedics, cardiology and

vascular surgery. Surgical devices or implants used in these disciplines which become

infected develop biofilms on their surface resulting in recalcitrant septic complications

which commonly require removal of the implants thus resulting in significant morbidity

and mortality. The chronicity of bone infections is believed to be associated with biofilms

[14,15]. Most biofilm research literature is related to in vitro studies and the majority of

clinical studies are related to complications involving medical implants. In recent years

there have been a number of publications showing the presence of biofilm on the mucosal

surfaces of both tonsils and adenoids, biofilm associated with helicobacter infections of

the upper gastro-intestinal tract and in ophthalmology [16–20]. Biofilm has also recently

been demonstrated in chronic wounds, particularly vascular or diabetic ulcers [21], and
has also been noted in the wounds of thermally injured mice [22]. Following a burn the

normal protective defence mechanisms of the skin, including defensins derived from the

keratinocytes and acidic secretions such as lactic and fatty acids from the sweat and

sebaceous glands, are either lost or impaired. As a result the wound may become

colonized and invaded by micro-organisms and systemic septic complications may ensue.

In addition, a generalized depression of the immune system may occur [23].

Biofilms impact significantly on human activity. The safe disposal of organic

waste relies largely upon biofilms found within sewage farms. Similarly, industrial

effluents are degraded by microbial communities existing as biofilms in effluent

treatment plants. Some of the undesirable effects of biofilms are the corrosion of

submerged metal surfaces in ships hulls, oil rigs and piers. Also, biofilms formed on the

inner surfaces of pipes reduce lumen capacity and impact on flow rates. The role of

biofilms in nature and disease has been reviewed [24]. Within the human body many

members of the natural flora have the potential to form biofilms, yet adverse effects are

rarely seen and protection against infection may result. One of the most extensively
researched human biofilm is dental plaque. Biofilms have been implicated in persistent

human diseases, such as respiratory infections in cystic fibrosis patients, dental caries,

gingivitis, periodontal disease, osteomyelitis, chronic prostatitis, otitis media,

endocarditis, infectious kidney stones and Legionnaire’s disease [25 ]. Biofilms most

commonly linked to human disease involve indwelling medical devices, particularly

catheters, prosthetic joints, stents, intrauterine implants, heart valves and contact lenses.

The bacteria most frequently isolated form such infections are coagulase negative

staphylococci, but Gram-negative bacilli and Candida have also been associated with

implant infections. Often these infections manifest weeks or months after insertion of the

device, in patients who seem to have successfully overcome surgery. It is possible that

their device was unwittingly contaminated during the occasion when it was inserted, and

that slow growth of adherent contaminants may have preceded microbial dissemination

and acute infection. Removal of the offending device is normally indicated in such

infections

Control of Biofilm

The link between wound chronicity and biofilms has provided some valuable insight into

the reasons why some wounds fail to heal within predicted times, but it has created a

need to devise effective strategies to control biofilms. In established biofilms, microbial

species grow at reduced rates and often exhibit decreased susceptibility to antimicrobial

agents by factors up to 500 times [24]. The involvement of antibiotic-resistant strains in

biofilms contributes further to treatment difficulties [26]. Anti-biofilm measures can

broadly be divided into those that aim to remove or disrupt biofilms, and those that aim to

prevent them forming. Neither seems to be particularly effective and there is little clinical
evidence to confirm the efficacy of either of these approaches. The first clinical study in

which an anti-biofilm strategy was adopted in patients with critical limb ischaemia

utilised sharp debridement, coupled with lactoferrin and xylitol, to restrict the availability

of iron and to interfere with EPS formation, respectively [27]. Biological debridement of

wounds with maggots [28], or disruption of EPS by enzymes [29] have also been

suggested as potential remedies for biofilms. Antimicrobial agents such as chlorhexidine,

cadexomer iodine, hydrogen peroxide, octenidine, polyhexanide, povidone iodine and

silver have also been evaluated in vitro with variable results [30,31,32,33,34,35].

Honey has been proposed as a further anti-biofilm intervention because its most common

sugar molecule (fructose) interferes with adherence of P. aeruginosa to host cells [36].

Also, the inhibition of biofilms by honey in vitro has been reported [37,38,39]. The role

of quorum sensing molecules in influencing some of the genes that contribute to

enhanced virulence and biofilm formation has given rise to the idea that interventions that

limit quorum sensing might be used to control biofilms. Assays for screening natural

products for quorum sensing inhibitors have been developed [40,41], and indicate that

garlic inhibits quorum sensing in P. aeruginosa. A role in treating lung infections in

cystic fibrosis patients has been suggested [42]. It is conceivable that chronic wounds

might also be suitable targets for garlic extracts. The development of biofilm models is

providing a means to evaluate potential biofilm interventions [43,44,45,46,47], but

clinical evidence will clearly be needed before effective anti-biofilm interventions will be

accepted. The burden that chronic wounds currently represent on medical resources and

the prospect of increased numbers of chronic wounds in ageing populations is a major

concern. The reasons why wounds fail to heal are varied and complex. Establishing an
association between wound chronicity and the presence of biofilm has been an important

advance in knowledge that has generated much interest and speculation within the wound

healing arena. Not only are wound care specialists interested in learning about the nature

of biofilms and the means to diagnose biofilm infections, but they need effective

treatment strategies. At this time it is important to understand that routine biofilm

detection methods are not yet available and that the development of reliable biofilm

interventions is ongoing. Biofilms should neither be over-estimated nor underestimated.

Not all chronic wounds can be expected to be attributed to biofilms, so the development

of appropriate anti biofilm interventions will never eradicate all chronic wounds.

Undoubtedly, effective treatments will be developed in due course, yet still wounds in

some patients will continue to fail to heal. Until routine biofilm detection methods

become readily available, knowing when to adopt an anti-biofilm strategy will be

speculative.

Quorum Sensing

Quorum sensing is a process done by the groups which are decentralized to make

a decision to coordinate their behaviour. Most of the bacteria use quorum sensing to

interact with each other and to coordinate their gene expression according to their

population density. Individual components should have the following factors like

1) Assessing the number of other components they are going to interact

 2) Standard response once a threshold number is detected of the

components. Quorum sensing can occur in single bacterial species and also in diverse

species. In order to communicate they produce different molecules as signals. Most

common classes of the molecules include are Oligo peptides in N-acetyl Homoserine and

Lactones (AHL). Bacteria will have a receptor which uses quorum sensing to produce

and secrete molecules. The signalling molecule is inducer which activates the

transcription of certain genes after binding to the receptor [48].

For the activation of the gene transcription the cell should encounter the signalling

molecules in the environment which are secreted by the other cells. As the number of

the bacteria reduce the diffusion reduce the concentration of the inducer in the

surrounding medium to almost zero, so due to this reason bacteria will produce little

inducer. However due to the increase in the population the concentration of the inducer

passes threshold causing more synthesise of the inducer. This leads to a positive

feedback loop, by which receptor becomes activated. As there is an activation of the

receptor it induces the activation of other specific genes causing all cells to start

transcription at a same time. This coordinate behaviour of the bacterial cells can use in

variety of situations.

The proteins that play a main role in the quorum sensiting were LuxS and

LuxP[49,50,51]. Past few decades has a witnees that about 70% of the bacterial

population which cause infections are resistant to drugs which are commonly used for the

treatment. Some are resistant to all antibiotics and can treated only with experimentally.

Even pencillin resistant bacterial pneumonia 25% cases identified of which 25% of cases

were more resistant to more than one antibiotics. Present researchers were focused on the
disrupting the bacteria’s ability to communicate, making them non pathogenic. This

gives our body to eliminate the bacteria naturally through immune system function. One

of the advantage of the anti-quorum sensing approach is to control the infections .

Fig. 3. Targeted genes of Quorum sensing

The advantage of the anti-quorum sensing approach to controlling infection is that there

are few evolutionary forces that select for resistance — there is little in the process that

would create resistant strains. Since the compounds kill none of the bacteria, any resistant

mutations must compete with living, non-resistant individuals. In other words, there is no

survival advantage to the resistant mutations, and natural selection does not come into

play. Resistant

strains will be unlikely to occur.

Quorum sensing inhibitors

Application of quorum sensing antagonists

Antagonists for AHL-mediated quorum sensing

Delisea pulchra halogenated furanones as quorum sensing-mediated processes are often

involved in the interaction with plant and animal hosts; it might not be surprising that

these higher organisms have developed mechanisms to disrupt quorum sensing. One of

these mechanisms is the production of quorum sensing antagonists: molecules that can

bind to quorum sensing response regulators, but fail to activate them . The red marine

alga D. pulchra has developed such a defense mechanism to protect itself from extensive

bacterial colonisation [52]. The alga produces halogenated furanones as antagonists for

AHLmediated quorum sensing. Structural similarity between AHL and the D. pulchra

halogenated furanone (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5H)-furanone. The

R in the AHL molecule is usually an alkyl group consisting of between 3 and 13 carbons,

which can have an oxo or hydroxyl substitution at the second carbon halogenated
furanones most probably bind to LuxR type proteins without activating them [52,54,55,]

showed that swarming of the pathogen Serratia liquefaciens on agar plates could be

inhibited completely by adding 100 mg/l of (5Z)-4-bromo-5-(bromomethylene)-3-butyl-

2(5H)- furanone. This furanone could also suppress the expression of bioluminescence

genes, located on a reporter plasmid in S. liquefaciens, without affecting the growth rate

of the bacterium. Interestingly, [56] found that the furanone inhibited extracellular toxin

production in a pathogenic V. harveyi strain. Mortality in the shrimp Penaeus monodon

was reduced to 50% after intramuscular injection with diluted cell supernatant extracts

from V. harveyi cultures grown in the presence of the halogenated furanone, compared to

extracts from untreated cultures.

Fig. 1. Structural similarity between AHL and the D.pulchra halogenated furanone (5Z)-
4-bromo-5- (bromomethlene0-3-butyl-2(5H)-furanone. The R in the AHL molecule is
usually an alkyl group consisting of between 3 and 13 carbons, which can have an oxo or
hydroxyl substitution at the second carbon.
 auto inducer of V.harveyi

Antagonistic AHL molecules Apart from the D. pulchra furanones, it was shown

that some naturally occurring AHL molecules could stimulate quorum sensing-regulated

pigment production in the Gram-negative bacterium Chromobacterium violaceum, while

other AHLs completely inhibited this phenotype [57]. The stimulatory or inhibitory effect

was linked to the structure of the acyl side chain of the molecules: AHLs with an acyl

side chain containing up to eight carbons were stimulatory, acting as quorum sensing

agonists. AHLs with an acyl chain containing 10 carbons or more, on the other hand,

were inhibitory and acted as quorum sensing antagonists. Apart from that, research by

[58] indicated that heterologous AHLs are potent antagonists for AHLmediated quorum

sensing in bacteria that express the LuxR-type proteins at native levels. Overexpression

of these proteins abolished the repression. Interestingly, research by Swift [59,60 ]

indicated that heterologous AHLs are able to reduce virulence factor production by the

aquatic pathogens A. hydrophila and A. salmonicida. In the first report,[59] showed that

serine protease production by A. salmonicida was delayed and that the final concentration

was reduced to 50% by applying N-(3-oxododecanoyl)-l-homoserine lactone at a

concentration of 10 µM. In a second research, [60] demonstrated that N-(3-oxodecanoyl)-

l-homoserine lactone, at a concentration of 10 µM, inhibited quorum sensing-regulated

exoprotease production by the Gram-negative pathogen A. hydrophila as well. Two other

3-oxo substituted long-acyl AHLs, N-(3-oxododecanoyl)-l-homoserine lactone and N-(3-

oxotetradecanoyl)-l-homoserine lactone, had a similar effect.

Synthetic AHL-mediated quorum sensing antagonists Based on the findings mentioned

above, several research groups started to investigate the activities of different synthetic

AHL and furanone analogues with respect to quorum sensing [62,63,64]. As far as we

know, a synthetic derivative of the D. pulchra halogenated furanones, (5Z)-4-bromo-5-

(bromomethylene)-2(5H)-furanone, is the most active AHL antagonist mentioned in

literature thus far. This furanone, dosed in a concentration of 10 µM, could almost

completely reduce virulence factor expression in pure cultures of P. aeruginosa PAO1

[64]. Interestingly, the furanone was equally active on biofilm bacteria compared to

planktonic cells, making them susceptible to sodium dodecyl sulphate and antibiotics. In

the absence of the furanone, on the contrary, 100- to 1000-fold higher doses of antibiotics

are required to eradicate biofilm bacteria compared to their planktonic counterparts

[65,63] showed that the furanone was also active in vivo in mouse lungs after infection

with a P. aeruginosa strain harboring a fluorescent AHL reporter plasmid. The

fluorescence signal from the P. aeruginosa strain was significantly reduced after

subcutaneous injection of the furanone. After 8 h, however, the signal reappeared,

indicating that the furanone had cleared from the mouse blood 2. Antagonists for AI-2-

mediated quorum sensing The AI-2 structure has only recently been elucidated [67].

Therefore, not much effort has been done to disrupt AI-2-mediated quorum sensing so

far. In one report, however, [68] found that the halogenated D. pulchra furanone
Compound 2, previously described as an AHL antagonistic analogue, could completely

inhibit AI-2- regulated swarming of E. coli. Moreover, the furanone decreased thickness

of E. coli biofilms by 55% and the percentage of live cells in the biofilms by 87%.

Finally, the furanone also inhibited AHL-mediated as well as AI-2-mediated

luminescence in V. harveyi. The furanone might also attenuate virulence of this aquatic

pathogen since the expression of some virulence factors was also found to be regulated

by its quorum sensing system.

Chemical inactivation of quorum sensing molecules

It has been established for a long time that AHLs are chemically inactivated via alkaline

hydrolysis, yielding the cognate acyl-homo serine [69]. To our knowledge, the only other

chemical inactivation that has been studied so far is the reaction with oxidised halogen

antimicrobials. These antimicrobials, at a concentration of approximately 0.14 mM, were

found to decrease the concentration of 3-oxo-substituted AHLs to about one-fourth after

1 min incubation, but had no effect on un substituted ones [70]. Moreover, the

inactivation of 3-oxo AHLs was shown to proceed in the presence of polysaccharide

biofilm compounds despite the much higher concentration of the latter compared to the

AHL concentration. In a further study, [71] unravelled the inactivation mechanism by

liquid chromatography mass spectrometry. Apparently, the reaction kinetics is largely

influenced by the pH of the reaction mixture. At pH 6, a 3-oxo AHL molecule reacts

quickly with two molecules of hypo bromous or hypo chlorous acid, yielding a 2,2-

dihalo-3-oxo AHL molecule. Subsequently, the acyl chain is hydrolysed, yielding a fatty
acid and 2,2-dihalo-Nethanoyl-l-homoserine lactone. At pH 3, the reaction stops after the

halogenation steps, whereas at pH 8, the lactone ring of 2,2-dihalo-N-ethanoyl-l-

homoserine lactone is hydrolysed, yielding 2,2-dihalo-N-ethanoyl-l-homoserine. These

data indicate that treating culture water with low concentrations of strong oxidising

agents, such as ozone [72], might be useful as an anti-infective therapy in aquaculture by

removing the quorum sensing molecules of pathogens. Enzymatic inactivation and

biodegradation of quorum sensing molecules. The ability to degrade AHLs seems to be

widely distributed in the bacterial kingdom. Enzymes that are able to inactivate AHLs

have been discovered in species [53] belonging to the h-Proteobacteria [73,74,75,76].

These bacteria might block the quorum sensing systems of their bacterial competitors to

obtain a selective advantage over them. This could be the case, for instance, for those

microbes living in proximity of bacteria that regulate the production of antibiotics via

quorum sensing [77]. The actual inactivation of the signal compound can be mediated by

two types of enzymes: AHL lactonases and AHL acylases. Moreover, eukaryotic

acylases might be able to inactivate AHLs as well [78].

Bacterial AHL lactonases [79] screened more than 500 field and laboratory bacterial

isolates for AHL-inactivating activity. Of these, 24 isolates showed different levels of

enzymatic activity in eliminating AHLs. The enzyme responsible for the AHL-

inactivating activity (AiiA) was isolated from the strain that showed the strongest

activity, Bacillus sp. Strain 240B1. The purified enzyme, at a concentration of 50 mg/l,

reduced the concentration of N-(3-oxohexanoyl)-l-homoserine lactone from 20 µM to

about 5 µM after 10 min. Reaction between a 3-oxo AHL and halogen antimicrobials

(HOX; hypobromous or hypochlorous acid) at pH 6. First, two a-halogenation reactions

occur, yielding 2,2-dihalo-3-oxo AHL. Subsequently, the acyl chain is hydrolysed,

yielding a fatty acid and 2,2-dihalo-N-ethanoyl-l-homoserine lactone. The R is an alkyl

group consisting of between 3 and 13 carbons, which can have an oxo or hydroxyl

substitution at the second carbon. Electrospray ionisation-mass spectrometry of the

hydrolysis product revealed that the AiiA enzyme opens the lactone ring to produce N-(3-

oxohexanoyl)-l-homoserine. Further research demonstrated that genes encoding AHL-

degrading lactonases are widespread in many Bacillus species. These AiiA homologues

showed about 90% sequence homology at the amino acid level. The first evidence

indicating that enzymatic AHL inactivation could be used as a biocontrol strategy was

provided in the study of [79]. Expression of the AiiA enzyme in transformed Erwinia

carotovora decreased the production of cell wall degrading enzymes by the pathogen to

about 10% and inhibited soft rot disease symptoms in susceptible plants almost

completely. In a further in vivo study,[84] tested the efficacy of using an AHL-degrading

Bacillus sp. strain for the biocontrol of plant diseases. The Bacillus sp. strain could

reduce potato tuber soft rot caused by E. carotovora to about 15% and crown gall in

tomato caused by Agrobacterium tumefaciens to about 10%. AHL degradation by the

Bacillus sp. Strain offered a protection as effective as or better than antibiotic production

by a Pseudomonas chlororaphis biocontrol strain. Moreover, degradation of AHLs had

not only a preventive, but also a curative biocontrol activity. Recently, similar results

were obtained with Bacillus thuringiensis [84].

Bacterial AHL acylases

AHL acylases in Variovorax paradoxus and Ralstonia

At the same time as [79] discovered an AHL-degrading Bacillus sp. strain, [85] isolated a

strain that could use AHLs as the sole source of carbon and nitrogen. This strain, V.

paradoxus VAI-C, was enriched on a medium with 500 mg/l N-(3-oxohexanoyl)-l-

homoserine lactone as the sole source of carbon and nitrogen. The researchers deduced

from experiments with radiolabeled AHL Enzymatic inactivation of AHLs. Cleavage of

the amide bond by an AHL acylase enzyme yields a fatty acid and homoserine lactone.

Cleavage of the lactone ring by an AHL lactonase enzyme yields the corresponding

acylated homoserine. The R is an alkyl group consisting of between 3 and 13 carbons,

which can have an oxo or hydroxyl substitution at the second carbon. V. paradoxus

cleaves the AHL by an AHL acylase enzyme, releasing homoserine lactone and a fatty

acid. Subsequently, the fatty acid is used as carbon source via the hoxidation pathway.

Further research is needed to elucidate how the bacterium obtains the nitrogen from the

homoserine lactone moiety. More recently, [86] isolated a bacterium, Arthrobacter sp.

strain VAI-A, capable of degrading and utilising the nitrogenous breakdown products of

AHL signal molecules. Interestingly, the AHLdependent growth rate and yield of a

coculture of the Arthrobacter sp. strain and V. paradoxus VAI-C were superior to those of

monocultures of these bacteria, indicating that consortia may have a synergistic effect in

quorum sensing signal turnover and mineralisation.

Recently, [75] isolated an AHL-inactivating bacterium, Ralstonia sp. Strain XJ12B, from

a mixed-species biofilm. The enzyme responsible for the AHL-inactivating activity

(AiiD) was purified and subsequently, N-(3-oxodecanoyl)-l-homoserine lactone was

incubated with the purified enzyme. Electrospray ionisation-mass spectrometry of the

hydrolysis product demonstrated that the AiiD enzyme hydrolyses the amide bond of
AHLs. Expression of the AiiD enzyme in transformed P. aeruginosa PAO1 inhibited

swarming of the pathogen and reduced mortality of the nematode Caenorhabditis elegans

to about 15%, compared to wildtype PAO1.

Specific AHL inactivation by AHL acylases produced by pseudomonads

Another recent research, [88], indicated that some substrate specificity of AHL-

inactivating enzymes can exist. The researchers isolated a soil pseudomonad, strain PAI-

A, that inactivated AHLs by means of an AHL acylase enzyme. In contrast to V.

paradoxus and Ralstonia sp. strain XJ12B, the pseudomonad could only utilise AHLs

with acyl side chains longer than eight carbons. Since the 16S rRNA gene from strain

PAI-A showed high similarity to the one from the pathogen P. aeruginosa PAO1, the

ability of the pathogen to degrade AHLs was investigated as well. In accordance with the

results obtained for the strain PAI-A, the pathogen started to grow on long-acyl AHLs but

not on short-acyl AHLs. The investigators presumed that degradation of its own long-

acyl AHL, N-(3-oxododecanoyl)-l-homoserine lactone, may play a role in the regulation

of the quorum sensing system of P. aeruginosa. Such a signal turnover control

mechanism had already been found in A. tumefaciens [73].

Eukaryotic acylases

Since several bacterial AHL acylases had been found to inactivate AHLs by deacylation,

[78] investigated the ability of an eukaryotic counterpart of these bacterial enzymes to

inactivate AHL molecules. Different AHLs were shown to be inactivated by the porcine

kidney acylase I enzyme. Since the inactivation was best at high pH, it could have been

due to simple alkaline hydrolysis of the lactone ring. However, the acylase could reduce a
model biofilm, made mainly by a Pseudomonas and a Microbacterium species, indicating

that the enzyme might be able to inactivate AHLs at environmentally relevant

concentrations. Nevertheless, further research is needed to investigate to what extent

eukaryotic acylases can indeed inactivate AHLs and whether these enzymes have any

significance in the in vivo inhibition of quorum sensing-mediated gene expression.

Enzyme activity and substrate threshold levels

Several bacteria were shown to be able to degrade AHLs if these molecules were present

at µM to mM concentrations [74,79]. Unfortunately, no enzyme activity studies have

been conducted so far. Moreover, previous research indicated that the biodegradation

abilities of micro-organisms can be limited to above certain threshold substrate levels

[89]. As transcription of quorum sensing-regulated genes can be activated at AHL

concentrations as low as 1 nM [65], it still has to be established whether substrate

specificity of the AHL-degrading enzymes is sufficiently high to decrease the AHL

concentration to below this threshold. However, mixed substrate growth experiments

indicate that threshold concentrations might be lower or even nonexistent if other carbon

sources are present (as in most natural ecosystems) than if there is only one single carbon

source . Indeed, several in vivo studies showed that the degradation of AHLs might

continue until below the level needed for activation of quorum sensing-regulated

virulence genes [75,80,84]

The opposite way: application of quorum sensing agonistic analogues

All techniques discussed so far aim to inactivate quorum sensing-regulated virulence

factor expression., however, tested an opposite strategy: they activated quorum sensing-
regulated virulence factor expression by using quorum sensing agonists. The idea behind

this strategy was that by adding the signal molecule of a pathogen, virulence factor

expression would be activated at low population density. Subsequently, the virulence

factors could trigger the activation of the host’s defense system allowing resistance to

develop. The, disease in tobacco plants caused by E. carotovora was reduced to 10% by

applying a 5 mM solution of the pathogen’s own AHL. Furthermore, the ability of E.

carotovora to cause disease after local inoculation with pathogens per plant was

decreased to about half in transgenic tobacco plants producing the pathogen’s AHL

compared to wildtype lines. If the infection was already established or if the inoculum

size was increased by a factor 4 or more, however, there was no significant difference

between AHL-producing and control plants. Apparently, the pathogen was present in

sufficiently large numbers to overwhelm the defense system of the plant in these cases.

Taken together, these results are in accordance with the original hypothesis that quorum

sensing is used to avoid premature virulence factor production and subsequent activation

of plant defense responses. As this research was conducted with plants, it would be very

interesting to conduct similar tests with (aquatic) animals to investigate whether their

immune systems—specific or nonspecific—could be activated successfully if a quorum

sensing pathogen is present by the application of the microbe’s signal molecule.

Limitations to disruption of bacterial quorum sensing

• Resistance development

A first limitation to the use of quorum sensing disrupting techniques as a new

antiinfective herapy might be resistance development, [58] indicates that bacteria could
simply circumvent quorum sensing blockade by overexpressing quorum sensing genes.

These researchers found that many synthetic AHL analogues were potent inhibitors of

quorum sensing responses in wildtype A. tumefaciens, whereas in a transformed strain

that overexpressed the A. tumefaciens LuxR homologue TraR, inhibition was not

detected for any of the analogues. However, as disruption of quorum sensing is less likely

to pose a selective pressure for development of resistance than conventional antibacterial

compounds do [64], the chance that a quorum sensing mutant will arise might be rather

small.

• Lack of specificity

Apart from resistance development, the lack of specificity could also confine the use of

quorum sensing disrupting techniques to control pathogens. Most quorum sensing

disrupting techniques developed so far are not specifically blocking the quorum sensing

system of one or more pathogens. Most AHL degrading bacteria, for instance, inactivate

a wide range of AHL molecules. However, not all bacteria found to contain a quorum

sensing system are pathogens. Quorum sensing was shown, for example, to be involved

in nodule formation by Rhizobium species and in antibiotic production by fluorescent

pseudomonads [77]. As could be the case for these growth promoting and biocontrol

activities of plant-associated bacteria, gross disruption of quorum sensing might

adversely affect yet unknown favourable quorum sensing-regulated processes in aquatic

ecosystems. On the other hand, it will probably be difficult to develop techniques that

only disrupt the quorum sensing system of a single pathogenic species. It is clear that

much more knowledge about the occurrence and function of quorum sensing in

aquaculture systems will be necessary in order to be able to develop a new anti-infective

strategy. The results obtained by using techniques that disrupt quorum sensing systems of

pathogenic bacteria indicate that it is a promising alternative for antibiotics in fighting

bacterial infections. This new approach might also have value in aquaculture since a link

between quorum sensing and virulence factor expression in several aquatic pathogens has

been demonstrated. Unfortunately, data about the impact of quorum sensing on virulence

(i.e. the net result of all virulence factors) of aquatic pathogens are still lacking.

Fundamental research in the quorum sensing domain will undoubtedly provide more

precise insights into the mechanism by which the expression of quorum sensing-regulated

genes is activated or inhibited. This research will make it possible, for example, to

conduct a more directed search for antagonists. So far, most of the research has been

done on the AHL-mediated quorum sensing systems of Gram-negative human and plant

pathogens. However, it can be expected that more techniques to disrupt quorum sensing

systems of other pathogens will be developed in the future. Before this new strategy can

be applied in aquaculture, there are some important topics that should be faced. First of

all, the impact of disrupting the quorum sensing system of several aquatic pathogens on

their virulence should be studied in full depth in order to elucidate whether it is a valid

anti-infective strategy in aquaculture. Secondly, the different quorum sensing disrupting

techniques should be investigated further in order to determine which technique(s) suits

best for the aquaculture system of interest. In this view, one should try to get an idea

about the impact of the techniques on the health of the final consumer of the aquaculture

product and also on the aquaculture system itself since gross disruption of quorum

sensing might adversely affect yet unknown favourable quorum sensing-regulated

processes. Moreover, some practical problems—such as the cost of a treatment and how
to deliver the quorum sensing disrupting compound or organism to the site of action—

should be considered. Finally, the problem of eventual resistance development should not

be neglected although disruption of quorum sensing is less likely to pose a selective

pressure for development of resistance than conventional antibacterial compounds do.

Honey is one of the sweet food eat by the humans. It is made by the bees using the

process regurgitation and store it in wax honey combs inside the beehive from flowers.

The genus apis is the most commonly referred one. Beekeepers collect the honey will

consumed by the humans. Honey production from other bees and insects has different

properties. Beekeeping practices encourage the over production of honey.

Monosaccharide’s fructose and glucose presence makes the honey sweeter5. Most of the

microorganisms do not grow in honey. Sometimes it may contains endospores of

bacteria which may harm humans. It has a long history of human consumption and used

in various food and beverages as flavouring agent. Flavours are based on nectar source

at present various grades of honey are available. China, Argentina, Turkey and U.S were

the top producers of natural honey

It has been used by the humans to a variety of ailments by topical applications and it

found that the honey has antiseptic and antibacterial properties. In Ayurveda a medicinal

book from India gave evidence that it has sweetness with added astringent as end taste. It

maintains blood pressure.

Recently it showed that it has the activity towards MRSA (methicillin resistant

staphylococcus aureus) as a antimicrobial agent honey may have the potential to treat the

variety of ailments. Main reason for its antibacterial activity is low water activity

causing osmosis, hydrogen peroxide effect, high acidity, and antibacterial activity of
methylglyoxal. It has effective activity on killing drug resistant biofilms which plays a

main role in chronic rhinosinusitis

Conclusion

Honey has been noted to have significant wound healing properties by the laboratory

evidence of antimicrobial action. There are different methods reported in in-vitro honey

sensitivity tests. The methodology in this study differs from some other studies, but

follows the same principle of exposure of micro-organisms to honey dilutions in a

reproducible manner. Its simplicity should ensure it can be reproduced by any tropical

laboratory involved in wound cultures. However adding honey to agar at 500C may have

led to some diminution in antimicrobial activity as honey is heat labile. Whilst in- vitro

evidence of 1-in -10 dilution of honey as an antimicrobial exists, in-vitro Nigerian

studies have cited 40% concentration as effective, and 30% ineffective against gut

bacteria. This study used 33% effectively even though the organisms used here are

different.

The isolates for this study came from chronic wounds, a number of which had become

resistant to several antimicrobials. The coliform organisms were not further characterised,

nor the specific strains of the isolates identified as the laboratory lacked facilities to do

this. The possibility of having different strains of the same organism accounting for the

results exists. The study seems to agree with other studies that honey is effective against

multi antibiotic resistant organisms. The antimicrobial effect of honey has been attributed

to several properties. Its hypertonicity leads to desiccation and subsequent destruction of

micro-organisms. Honey contains inhibines as hydrogen peroxide flavonoids and

phenolic acid. It also contains gluconic acid, and tetracyclines. However not all honey

samples contain all these substances, which may in part explain the differences in the

efficacy of different honey samples.

This study clearly shows that whilst honey shows in- vitro antimicrobial action against

aerobic organisms, it did not inhibit all isolates of the same organism taken from different

wounds. This has been reported previously and indicate the need for routine sensitivity

studies (as with systemic antibiotics) before honey application. The findings of

occasional resistance in differing isolates of the same organism, and complete inhibition

for previously resistant organisms following prior inoculation with pure honey raise

important issues. This finding of occasional resistance in differing isolates of the same

organism followed 24 hour incubation. After 72 hours no organism was grown. In a

previous study following 48 hour incubation no tested organism grew. Perhaps those

organisms would not have grown in our series if they were all observed at 48 or 72 hours

after incubation. However as different sensitivity patterns exist for differing isolates of

the same organism even in antibiotics both in-vitro and in-vivo, this may not be a result

of the methodology. It may explain the occasional ineffectiveness of honey dressings in

our environment. This finding appears to support the decision of some authors who

combine honey with antibiotics. Synergy for pseudomonads has been reported. It may

also indicate the need for more frequent dressings in wounds infected by resistant

organisms. Twice daily dressing with honey has been reported as effective by some

workers, but in such wounds infected by resistant organisms, especially those highly

exuding wounds that quickly dilute the concentration of honey on the wound surface,
four to six hourly dressings may be necessary to get good results. More sensitive

organisms will still be inhibited by less concentrated honey though diluted by tissue fluid;

higher local concentrations of honey are important for less sensitive isolates as indicated

by the study. Routine laboratory sensitivity tests for honey will help the clinician decide

which wound infection will require such treatment. The rate of improvement in such

wounds may also be slower when treated with honey alone.

Contamination of honey samples by organisms has been attributed to a number of factors,

most especially recent contamination from handlers and containers. Gamma-irradiation

sterilizes honey without causing it to lose antimicrobial qualities and is recommended.

The purity of commercial honey samples obtained locally is questionable. Some

unscrupulous traders sell boiled sugars in its place. This not only strengthens the need for

routine laboratory sensitivity tests, but also raises the question of a simple reliable test for

pure honey. A lot more work needs to be done to determine the place of the flammability

test. The activity of honey extract against MRSA is of great importance since these

strains cause major problems in hospitals. Honey phenolics are partially responsible for

the variation in the antibacterial activity of the honeys tested. A better procedure for the

recovery of antibacterial phenolics was also developed. Using that procedure some of the

phenolic acids responsible for part of the antibacterial activity have been identified;

however, further work needs to be done to identify the rest of the components present.

Honey can be recommended as an alternative treatment for infected wounds or ulcers,

especially those that are caused by antibiotic- resistant bacteria. . A number of reasons for

this have been suggested: shrinkage disruption of the bacterial cell wall due to the

osmotic effect of the sugar content; induction of an unfavourable environment with low
water activity, thereby inhibiting bacterial growth; and a low pH of 3.6 and the

fermentation of honey, producing alcohol in. sitar. Honey acts as a highly viscous barrier

preventing bacterial penetration and colonization of the wound surface.

The bacterial quorum-sensing concept is revolutionary because it questions the

fundamental view that microbial cells live as single entities and that their response to the

environment is driven by primarily chemical and physical stimuli. The previously held

view that microbial cells respond as single entities is overly simplistic. Bacterial cells can

communicate with each other through autoinducer molecules which function as signaling

molecules. Thus, it is logical that if we are to control the proliferation and survival of

bacterial communities, we should be developing strategies to interrupt or modulate these

communication signals. Our knowledge about how different foods and their ingredients

influence the different spoilage organisms and bacterial pathogens is still very limited

from a microbial cell–signaling point of view. Even though there is a growing body of

literature on AIs and their inhibitors, significantly more detailed information is needed,

especially as it relates to food microbiology. We need to know whether processed and

natural foods contain AIs, AI-like “mimic” compounds, or quorum-sensing inhibitory

molecules. We need to have a clear understanding of how these foods and food

components influence microbial cell signaling. Our understanding of autoinducers and

quorum sensing is in its infancy, and we predict that in the next 5–10 years we will

identify a much larger repertoire of cell-signaling compounds. Some of these compounds

may hold the key to preservation of foods and preventing the growth and persistence of

pathogens in foods. Understanding the relationships that exist between the food

ingredients, food processing, handling, and food consumption methods, and the cell-
signalingbased microbial activity of pathogens and spoilage bacteria is critical. We must

attempt to understand these relationships because such understanding can be important in

formulating the next generation of foods that are microbiologically safe.

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