Bacterial Biofilm: Its Composition, Formation and Role in Human Infections

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TACE plays an important role in the regulation of inflammation by its ability to cleave and release the extracellular portion of TNFR1 from the surface of airway epithelial cells and macrophages.

Therefore, SpA is involved in the S. The discovery of the new SpA- TNFR1 signaling axis highlights additional molecular targets to modulate the host immune response and to treat S. Thus, these toxins have both direct and indirect means to cause a lung damage [ 73 , 90 — 92 ]. However, little is known about the significance of these toxins in S. Pore formation on susceptible host cell membranes triggers alterations in ion gradients, loss of membrane integrity, activation of stress-signaling pathways, and cell death [ 93 , 94 ].

Interestingly, the dosage of the toxin can result in two different modes of activity. Low concentrations bind to specific cell surface receptors and form a heptameric pore.

This pore allows the exchange of monovalent ions, resulting in DNA fragmentation and, eventually, in apoptosis [ 96 ]. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor. Based on literature data, S. Depending on the chain length of their fatty acids or the mode of metabolism, these ceramides may have a number of effects in eukaryotic cells, including stimulation of second messenger systems, activation of MAPKs, changes in cell shape, and even apoptosis [ , ].

Study of Hayashida et al. Ectodomain shedding is a proteolytic mechanism of releasing the extracellular domains of cell surface proteins as soluble ectodomains that can regulate many pathophysiological processes, such as microbial pathogenesis, inflammation, and tissue repair [ , ]. Syndecan-1 is the major heparan sulfate proteoglycan of epithelial cells, which binds and regulates a wide variety of biological molecules through its heparan sulfate chains [ ].

The mechanism of syndecan-1 shedding was well characterized in a mouse model. In bleomycin-induced acute inflammation and lung injury, shedding of syndecan-1 by metalloproteinase-7 generates a chemokine gradient that attracts PMN into the alveolar compartment [ ]. Panton-Valentine leukocidin is one of several extracellular cytotoxins produced by S.

The toxin was first described by Van de Velde , but only in Panton and Valentine associated the leukotoxin with skin and soft-tissue infection.

Clinical studies propose the exotoxin PVL being a virulence factor in necrotizing diseases [ 24 , ]. Previous studies revealed that human and rabbit neutrophils are highly sensitive to the pore-forming properties of PVL and rapidly undergo cell death [ ].

Furthermore, it is generally accepted that myeloid cells are the prime target of PVL and that low concentrations of the toxin cause apoptosis, whereas higher amounts induce lysis of neutrophils [ ]. More than 20 years ago, it was suggested that this lytic toxin functions as a virulence factor in cutaneous infection [ , ]. Necrotizing pneumonia has long been recognized, but the association with PVL was made by Gillet et al.

Patients with PVL-positive S. However, several studies that used a diversity of animal models have created conflicting results concerning the role of PVL in pneumonia. In one study applying a mouse acute pneumonia model, Labandeira-Rey et al.

Using purified toxin or a laboratory strain of S. The mice showed symptoms of severe illness. It is of interest that when comparing isogenic S. They showed that the expression of PVL induces global changes in transcriptional levels of genes encoding secreted and cell-wall-anchored staphylococcal proteins, including SpA [ 73 ].

It should be mentioned that this statement is controversial: Diep and Otto [ ] explained that misinterpretation of the data due to the apparent lack of confirmatory experiments might have led to the model in which PVL plays a role in global gene regulation. Also, other groups fail to detect any pathogenic function of PVL in murine model of pneumonia.

Moreover, it was suggested that Hla, but not PVL, was essential for the pathogenesis of staphylococcal pneumonia [ 75 ]. Passive immunization with anti-PVL immune sera also failed to protect mice against challenge with USA in the murine pneumonia model [ 95 ], indicating that PVL is not necessary for the pathogenesis of pulmonary disease. Despite the role of PVL as a virulence factor in the lungs is controversial, the pulmonary immune response to PVL, especially responsiveness of alveolar macrophages to this toxin, is known [ ].

The recent study of Zivkovic et al. The alveolar macrophages are considered to represent the first line of defense against pathogens and express receptors, including TLRs, which recognize pathogen-associated molecular patterns [ ]. These pathways further modulate proinflammatory gene expression, which is crucial in shaping the innate immune response within the respiratory tract [ ].

The idea that TLRs could play an important role in bacterial toxin recognition is not uncommon. However, in contrast to data showing that LukF from S. The ability of PVL to induce inflammatory gene expression is independent of pore formation [ ].

These data are in line with previous observations, showing that both subunits of PVL are required to perform a pore [ ]. Furthermore, single submit LukS, but not LukF, is able to induce an inflammatory response, suggesting that inflammatory gene expression relies on cellular pathways independent of pore formation [ ].

The knowledge about host factors, which facilitate eradication of S. Surfactant protein A SP-A is the major protein component of pulmonary surfactant. It is involved in organization of large aggregates of surfactant phospholipids lining the alveolar surface and acts as an opsonin for pathogens [ ].

Previous studies established that SP-A modulates macrophage phagocytosis and a host pro- and anti-inflammatory responses that help in eradication of infection [ — ]. Recent study of Sever-Chroneos et al. Macrophage receptor SP-R is implicated in the ability of SP-A to coordinate the clearance of pathogens and apoptotic cells, and to participate in temporal control of inflammation in the lungs [ ].

Phagocytosis of SP-A-opsonized S. Finally, Sever-Chroneos et al. Binding of SP-A to SP-RS induces phagocytosis and release of anti-inflammatory mediators via association with SR-A, leading to an enhanced bacterial killing and resolution of the infection.

Importantly, it is proposed that temporal control of inflammatory responses via SP-RS and SR-A contributes to the proper recruitment and activation of neutrophils, facilitating eradication of S. The innate defense of the airway epithelial cells against S. Induction of the airway inflammation can be mediated by several staphylococcal determinants and corresponding receptors and is not necessarily dependent on the expression of a particular virulence factor that is crucial for the pathogenesis of S.

Among many virulence factors produced by S. The role of PVL in lung infection is debated due to conflicting data. The shedding of the plasma membrane proteins represents an important mechanism underlying S.

In addition, the airway epithelial cells regulate their own signaling capabilities by shedding some epithelial receptors e. Considerable progress has been made in our understanding of known virulence factors and their implication in pneumonia in the last few years. Several new properties of S.

A detailed analysis of function and mechanisms of action of each virulence factor could open the way to control the proinflammatory response in the lung by using specific inhibitors and may be helpful for the development of novel therapies for S. Indexed in Web of Science.

Subscribe to Table of Contents Alerts. Table of Contents Alerts. Abstract Airway epithelial cells play a major role in initiating inflammation in response to bacterial pathogens. Introduction Although a relatively unspectacular, nonmotile coccoid bacterium, Staphylococcus aureus is a dangerous human pathogen in both community-acquired and nosocomial infections. Virulence Factors of S. Adherence Factors Adhesins The attachment of S.

Regulation of Virulence Factors in S. Known Virulence Factors of S. SpA SpA is a good example of one of known and well-characterized S. Aires De Sousa and H. Also the ability of Klebsiella pneumonia to form biofilm takes place more successfully in a mix strains than individual strain [ 50 , 51 ]. Mechanisms of antibiotics and biocides resistance of biofilms are categorized into four classes which include a active molecule inactivation directly b altering body's sensitivity to target of action, c reduction of the drug concentration before reaching to the target site and d efflux systems Figure 3.

Biofilm antibiotic resistance level may vary among different sittings and the key factors responsible for this resistance may also differ. Regarding resistance, the primary evidence shows that conventional mechanisms are unable to explain the high resistance to antibacterial agents associated with biofilms, although this evidence cannot be ignored in resistance in the growth of adherent cells.

So it is suggested that the resistance posed by the adhered bacteria or biofilms may have some intrinsic mechanisms and are responsible for conventional antibiotic resistance [ 52 ]. Several mechanisms have been explored that are considered to be key factors in high resistance nature of biofilms. These mechanisms are a limited diffusion, b enzyme causing neutralizations, c heterogeneous functions, d slow growth rate, e presence of persistent non-dividing cells and f biofilm phenotype such adaptive mechanisms e.

Antibiotic resistance associate to biofilm. Description of the key mechanisms involved in antibiotic resistance such as enzyme causing neutralizations, presence of persistent non- dividing cells and biofilm phenotype. Diffusion of antibiotics can take place through the matrix of the biofilm. Diffusion or penetration of antibiotics to deeper layers of biofilm is affected by exopolysaccharide acting as a physical barrier.

When molecules direct interact with this matrix, their movement to the interior of the biofilm is slow down, resulting antibiotic resistance. This may also acts as a hindrance for high molecular weight molecules such as complement system proteins and lysozyme, and in liquid culture bacterial cells are readily exposed to antibiotics as compare to compact structure biofilm. Bacteria escape from biofilm that do not produce polysaccharide and are easily attack by immune system cells.

Inactivation of antibiotic takes place when bind to biofilm matrix. Presence of this matrix explains slow penetration of fluoroquinolones and aminoglycosides [ 39 , 55 , 56 ]. Low penetration of antibiotic is not sufficient to explain the biofilm resistance, other mechanisms have been assumed that must be involved. This is also suggested recently that slow diffusion of antibiotics permit plenty of time to establish a protective response to stress [ 39 ].

Antibiotics resistance in biofilm may be due to the presence of neutralizing enzymes which degrade or inactivate antibiotics. These enzymes are proteins which confer resistance by mechanisms such as hydrolysis, modification of antimicrobials by different biochemical reactions.

Accumulations of these enzymes occur in the glycocalyx from the biofilm surface by the action of antibiotics. Neutralization by enzymes is enhanced by slow penetration of antibiotics and also antibiotics degradation in the biofilm.

In cystic fibrosis which is caused by P. During a study when filters impregnated with antibiotics was applied on K. Studies performed on determination of microbial growth in biofilms by using a microelectrode with probes to direct measure oxygen concentration in different areas of the biofilms [ 39 ].

The biofilms are heterogeneous nature both metabolically and structurally and both processes such as aerobic and anaerobic occur at the same time. So response against antibiotics may be different in different areas of the biofilms. On surface of biofilm there is a high level of activity of antibiotics while inside the biofilms, slow or absent growth reduces the sensitivity of the cells to antimicrobials [ 59 ].

In the various sub layers of biofilm, aerobic or facultative anaerobic microbial populations help us to know the differential susceptibility to various antibiotic therapies. Antibiotics response to the planktonic forms is different from the adhered cells. Action of aminoglycosides is affected by limitation of oxygen and anaerobic growth of microorganisms, which is affected by the presence of oxygen and pH gradients.

Slow growth of microorganisms occurs due to limited availability of nutrients which confer resistance to antibiotics. In case of biofilm a gradient of nutrients resulting in metabolically active cell periphery or surface layer and inactive cells within its interior [ 60 ].

Bacterial cells are attack by both penicillin and ampicillin only when they are growing. So due to slow growth resistance has been determined in different bacterial strains such as resistance to cetrimide on E. It has been shown that this resistance was due to slow growth [ 13 , 61 ]. There are some natural peptides produce during host innate immune response act as antibacterial providing protection to the body [ 62 ].

In Cystic fibrosis patients, using ciprofloxacin and tetracycline can clear active growing cells and it is suggested that a combination of antibiotic colistin with other two antibiotics ciprofloxacin and tetracycline will be very effective in clearing P. After a purling antibiotics treatment of biofilm, a very small number of bacterial cells remain viable, called persistent cells. These cells may or may not give this resistance to their progeny and return to their normal state after the release of the applied stress or pressure.

The persistent cells stop their replication for small duration for the survival of the community. Their adaptive mechanism is not related to the mechanism followed by the cells during stress environmental damage.

Persistent cells can bear multiple antibiotic doses and work for survival. When density of bacterial cells number in stationary phase raised to maximum, persistent cells increase in number indicting their main role in survival [ 66 ]. There are certain evidences for the presence of persistent cells in biofilm: During biofilm formation, bacteria produce some products called secondary metabolites. These products are not required by the cell for their growth.

These metabolites function as signaling molecules thus enhancing formation process of biofilms [ 68 ]. Biofilm phenotype is regarded as community cells that confer no response to antibiotics treatment. These characteristics have proposed the presence of specific genes.

However at present time, this differential gene expression has not been proven fruitful for describing this mechanism [ 28 ]. Efflux pumps are protein structures, either express constitutively or intermittently. These pumps may have substrate specificity. Similar compounds can be transported by these pumps that may be involved in multidrug resistance [ 69 , 70 ].

Efflux pumps, inside the bacteria in the periplasmic area, are involved in antagonized accumulation of antibiotics. Five families of efflux transporters have been identified in prokaryotes. Over expression of the efflux pumps have been considered to be responsible for antibiotic resistance in P. Permeability of outer membrane is very important for the antibiotic diffusion through different routes.

A key role is played in transportation of hydrophilic molecules by outer membrane channel proteins porins present in Gram-negative bacteria from the outer environment to the periplasmic space. Mutation in porins encoding genes can result in production of non-functional or altered proteins. These mutant porins have low permeability for the passage of hydrophobic molecules [ 73 ].

OprD is a specific porin present in P. Loss of OprD in P. The differential expression of porins coding genes, occur in biofilm, leading to antibiotic resistance. When the expression of ompC and three other genes osmotically regulated is increased then the bacterial cells grow as biofilms [ 39 ].

Diverse phenotype within biofilms plays a significant role and is responsible for the resistant infections. Authors have reported this phenomenon for many genera and species which are exemplified by Pseudomonas and Staphylococcus genus, and also some species of Enterobacteriaceae [ 2 ].

Biofilms have the capability to develop bacterial subpopulation to switch to the quiescent state as small-colony known as small colony variants SCVs. These variants cells have very less susceptibility to growth phase dependent killing of antibiotics. Also have a defective catalase activity which interferes with oxidative metabolism [ 74 , 75 ].

Detectable colonial morphological changes caused by SCVs in biofilms, leads to increased adherence, auto-aggregation, increased hydrophobicity and low level motility. It can also withstand wide range of harsh environmental stress conditions so this is considered as survival mechanism for biofilms. Phase variation was considered as the process of cellular internal rearrangement but recently it is consider as phase variation that occur due to genetic elements interaction [ 76 , 77 ].

Various mechanisms involved in antibiotic resistance due to biofilm are shown in Figure 3. Microscopic evaluation of specimens from chronic wounds often indicates the presence of biofilms. Traditionally, three phases are use to describe wound microbiology. These three phases described as: Contamination refers to the presence of bacteria in the wound, whereas the term colonization is used for bacterial community which is multiflying within the wound but not causing tissue damage.

However, they adversely affect wound healing [ 78 ]. Some researchers have suggested that bacteria may play a vital role in normal wound healing [ 78 ]. However, the specific role of bacterial community in wound healing is still under debate. The wound communities have polymicrobial nature.

Bacteria can find its way to a wound from exogenous soil and water and endogenous skin, saliva, urine, and faeces sources.

This is particularly the case for Corynebacteria and coagulase-negative Staphylococcus which are considered as commensal skin organisms. However, the multiple microbial population interactions in chronic infections are not completely understood. Trengove and his co-workers studied 52 patients and concluded that bacterial diversity and wound chronicity is correlated [ 79 , 80 ].

In , it has been hypothesized that bacteria which colonize human chronic wounds may exist as biofilm communities. Later in , Akiyama and his co-workers collected patients specimen who were suffering from the skin diseases bullous impetigo, atopic dermatitis, and pemphigus foliaceus and used safranin, ConA, and immunofluorescent staining with CSLM to study and demonstrate the presence of S. Likewise Kirketerp-Moller et al. Microscopic evaluation of specimens from 50 chronic wounds and 16 acute wounds was done by James et al.

Biofilm formation in wounds has been investigated in-vivo using model organisms murine and porcine. Akiyama and co-workers investigated S. These mice were treated with cyclophosphamide to inhibit leukocytes. Normal mice rapidly clear the inoculated bacteria because of a strong PMN response. Bacterial biofilm interrupts the human immune system in several ways.

In initial stages of chronic wound, antibiotic treatment is considered to be the immediate step. But in case of mature and established biofilm, antibiotic therapy is least effective and has only short term effects on both inflammation and healing.

Clinicians have to depend on the results from a swab or biopsy, which barely represent all microorganisms present in the wound. The bacterial community residing in biofilm can be up to times resistant to antimicrobials [ 33 ]. Even silver treatment which is considered to be quite effective now a days and incorporated in wound dressings, is least effective on biofilm [ 86 , 87 ]. This result in the formation of complex biofilm composed of infecting bacteria and their exoproducts, and mineralized stone material.

Hellstrom in for the first time examined the stones that were collected from his patients and discovered the occurrence of bacteria deep inside them [ 88 ]. Microscopic analysis of stones, which were removed from infected patients, have revealed that bacteria present in the stones are organized to form microcolonies surrounded by an anionic matrix composed of complex polysaccharides and minerals [ 89 ].

Urine flow is obstructed by these infectious stones and thus causes severe inflammation and infection that can lead to kidney failure [ 90 ]. Bacteria and host components form complex biofilm that cause infection lesion in endocarditis. This biofilm is known as vegetation and can cause disease by three main mechanisms [ 61 ].

First, the vegetation disrupts the function of valve by creating leakage, turbulence and flow of blood. Secondly, the vegetation causes bloodstream infections, and may lead to recurrent fever, chronic systemic inflammation, and other severe complications.

Thirdly, sometimes vegetation breaks off into pieces and these pieces are then carried to extremities in the circulation system embolization. Brain and kidneys are particularly vulnerable areas of the body.

Vegetation is usually treated by prolonged administration of intravenous antibiotics or surgical excision of the infected valve [ 91 ]. Cystic Fibrosis patients most commonly afflicted with P. These infections are generally divided into two steps. First, intermittent respiratory infections are developed in CF patients [ 11 ].

In second stage, permanent infections with P. This persistant infection is clinically very important as it causes permanent failure of lungs [ 96 , 97 ]. Biofilms cause several diverse kinds of human infections. Biofilm is involved in otitis media with effusion.

Fluid accumulates in the middle ear cavity of the patient, thus affecting speech development and learning capability of the patient. However, the complete etiology of the problem is still not clealy understood [ 97 ]. In acute osteomyelitis, certain areas of bone necrose and produce favourable conditions for biofilm development [ 98 ].

Biofilms have also been identified in most indwelling medical device infections and also in biliary tract infections, periodontitis, ophthalmic infections [ 99 ].

In-vitro studies have indicated that human leukocytes have the ability to penetrate biofilms of S. They investigated the primary mechanism of antimicrobials in normal mice, and found that it was due to the penetration of PMNs into the biofilm.