Staphylococcal infections are reported to cause very important problems in hospitalized and immunocompressed patients worldwide due to their tough and irresponsive treatment by antibiotics. Biofilm-embedded bacteria that gain resistance to immune defense and antibiotics by antibiotic degrading enzymes, efflux pumps, and certain gene products of which expression are changed by the quorum sensing cause chronic and recurrent infections such as indwelling device–associated infections. Biofilm-embedded sessile community has heterogeneous cells that have wide range of different responds to each antimicrobials. Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) that are mostly known pathogenic strains can induce gene expression of biofilm that has an important role in the pathogenesis of staphylococcal infections and causes bacterial attachment and colonization on biotic such as tissues or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion when microorganisms exposed to stress conditions. This expressed and matured biofilm causes bacterial spread to whole body, consequently, spread of infection in to whole body. It is hard to treat biofilm infections, and new agents are being researched to prevent formation and dissemination of biofilm. Defining the virulence and the role of biofilm of S. epidermidis and S. aureus in chronic and recurrent infections such as indwelling device–associated infections, the mechanism and the global regulation of biofilm production by quorum-sensing system, inactivation of biofilm formation, and the resistance patterns of biofilm-embedded microorganism against antimicrobials are important.
- staphylococcal biofilm
- mechanism and regulation of biofilm formation
- quorum-sensing system
- antimicrobial resistance of biofilm
- Staphylococcus aureus
- Staphylococcus epidermidis
Staphylococci that construct the human skin flora can contaminate indwelling devices. By this way, they are inserted to human by contaminated indwelling devices. When microorganisms exposed to stress conditions, gene expression of biofilm is induced as a stress response. The biofilm that is a slime-like glycocalyx causes bacteria to survive in the stress conditions. Staphylococci adhere, colonize, and infect biotic surfaces such as tissue or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion and causes bacterial spread to whole body by forming biofilm that is a slime-like glycocalyx [1, 4, 5]. The virulence and the role of biofilm of
2. The biofilm, virulence, and Staphylococcus
2.1. The pathogenesis of Staphylococcus biofilm
The biofilm has an important role in the pathogenesis of staphylococcal infections. The biofilm causes bacteria to survive in the stress conditions such as UV damage, metal toxicity, anaerobic conditions, acid exposure, salinity, pH gradients, desiccation, bacteriophages, and amoebae and to resist antibiotics, antimicrobials, and host immune defense [5–8]. The main pathogen of implant infections is staphylococci that cause 80% of all prosthetic infections . The biofilm of bacteria causes chronic infections such as indwelling device–related infections, chronic wound infections, chronic urinary tract infections (UTI), cystic fibrosis pneumonia, chronic otitis media (OM), chronic rhinosinusitis, periodontitis, and recurrent tonsillitis . The biofilm infections are the main important problems in hospitalized and immunocompressed patients in worldwide due to their tough and irresponsive treatment by antibiotics. In biofilm, bacteria are not distrupted completely by antibiotics even high doses of antibiotics used
2.2. Staphylococcal biofilms as a virulence factor
The biofilm that anchored to abiotic or biotic surfaces is a slime-like glycocalyx in which sessile community of microorganisms embedded. This extracellular polymeric substance that is constituted by matrix of polysaccharide, teichoic acids, extracellular DNA (eDNA), and staphylococcal proteins is produced by biofilm producing microorganisms [4, 17, 18]. Polysaccharide intracellular adhesin (PIA) is a specific polysaccharide in glycocalyx composed of β-1,6–linked N-acetylglucosamine residues (80–85%) and non-N-acetylated D-glucosaminyl residues that are an anionic fraction and contain phosphate and ester-linked succinate (15–20%) . Although PIA is a main mechanism of biofilm formation in
2.3. Mechanisms of biofilm formation
Bacterial biofilm formation is a complex and multifactorial process. The biofilm formation process consists of adherence/adhesion/attachment, aggregation/maturation/accumulation, and detachment/dispersal phase. The last step is the dispersal of mature biofilm-embedded bacteria out of the biofilm  (Figure 1).
2.3.1. Attachment (adhesion or adherence) phase
When conditions favor biofilm formation, biofilm formation that begins with the adherence of the bacteria to a surface that act as a substrate for microbial adhesion continues with the aggregation formed by cell–cell adhesion  (Figure 1).
Staphylococcal adherence to an abiotic surface of indwelling prosthetic device depends on physico-chemical structure of medical device and surface components of Staphylococci such as wall teichoic acid (WTA) , lipoteichoic acid (LTA) , accumulation-associated protein (Aap) , autolysins AtlA  and AtlE . The staphylococcal adherence to a biotic surfaces such as host cells and plasma protein-coated prosthetic surface is mediated by cell wall-anchored (CWA) proteins such as the fibrinogen-binding protein SdrG/Fbe of
Several microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that are able to bind to human matrix proteins such as fibronectin and fibrinogen and colonize are expressed in
2.3.2. Accumulation (aggregation or maturation) phase
After adherence of staphylococcus to biotic and abiotic surfaces, exopolysaccharide (EPS) such as PIA or PNAG that are produced by
In the initial cell-surface interaction of motile bacteria, adherence of motile cell to surface is facilitated by flagella of motile cell. After adherence motile species that undergo cellular differentiation in biofilm lose their motility by paralyzing their flagella and become nonmotile . Klausen et al.  revealed that wild-type strain and isogenic flagellar mutant of
2.3.3. The detachment (or dispersal) phase
In the detachment stage, sessile cells turn into planktonic state that can spread and colonize other surfaces and form biofilm on these infected regions  (Figure 1). Detachment of microorganisms from biofilm can be caused by bacteria themselves, such as enzymatic degradation of the biofilm matrix such as dissolution of adhesins by proteases, nucleases, and a group of small amphiphilic α-helical peptides, known as phenol-soluble modulins (PSMs) functioning as surfactants , and quorum sensing or by external forces such as fluid shear forces, corrosion, and human intervention  (Figure 2). During detachment of motile microorganism rather than staphylococcus, cells express genes that are for motility such as transcription of pilus and ribosomal proteins and are almost seen in planktonic cells .
2.4. Types of biofilm formation
2.4.1. PIA-dependent biofilm formation
Positively charged PIA provides intercellular attachment via binding to bacteria of which surface is negatively charged . All
2.4.2. PIA-independent biofilm formation
Biofilms not only can be constructed by
PIA-independent biofilms were constructed by accumulation-associated proteins (Aap) of
Scientists determined that medical MRSA isolates produce protein-dependent biofilm such as FnBP- and Aap-dependent biofilms in animal models that have indwelling device–associated infection. O’Neill et al.  and McCourt et al.  revealed that biofilms of certain isolates of HA-MRSA from CC8 and CC22 and CA-MRSA from USA300 lineage (CC8) were FnBPs-dependent.
Autolysin Atl that is a wall-anchored protein of
In biofilm production of
2.5. The global regulation of biofilm formation
2.5.1. The regulation of Staphylococcal biofilm by
Biofilm production is provided by the equilibrium between the productions of amyloid fibrils and phenol soluble modulins (PSMs) that are extracellular polymeric substances and their catabolism by enzymes such as nucleases and proteases that are expressed by agr-QS regulator system that use two-component system signal transduction system (TCS). The control of planktonic and sessile bacteria and the biofilm expression is regulated by coordinated mechanisms  (Figure 2).
The biofilm formation of staphylococci is fully expressed
Staphylococcus use quorum-sensing systems (QS) for intercellular communication and biofilm formation. Accessory gene regulator (Agr) system regulates cell density-dependent gene expression using two-component signal transduction system . Agr and LuxS systems that are required for autoinducer peptide (AIP) production as a pheromone are quorum-sensing systems in staphylococci . Bacteria sense pheromones as stimuli that are released by the density of bacteria belonging to the same group and express biofilm formation . AIP production starts in exponential phase of bacterial growth . There are four proteins that are sensor histidine protein kinase AgrC, DNA-binding response regulator AgrA, AgrD that is a prepheromone, and AgrB that exports and modifies AgrD, present in this system. The signal is transported to bacteria by binding of AIP to AgrC. When AIP binds to AgrC, DNA-binding regulator AgrA is activated by His-dependent phosphorylation of AgrC . By the binding of activated DNA-binding regulator AgrA to P2 and P3 promoters in
The regulation mechanisms of RNAIII for target genes can be at transcriptional and translational level, and its regulation can be direct or indirect. Fourteen stem-loop and two long helices construct structure of RNAIII. Each domain regulates the expression of each target gene. Translation of α-hemolysin (
Staphylococcal virulence factors are expressed with accessory gene regulator (agr) system in response to cell density . During the beginning of the biofilm-related staphylococcal infection, adhesion factors (surface proteins) such as MSCRAMMs are upregulated. After initial attachment and colonization had been happened, during early stationary growth phase of bacteria, toxins and other acute virulence factors such as degradative exoenzymes (such as δ-hemolysin, lipases and proteases that disperse bacteria) are upregulated and non-aggressive colonization surface proteins such as MSCRAMMs are downregulated by
Phenol-soluble modulins (PSMs) are surfactant-like staphylococcal peptides and are controlled by
Agr (AIPs) of each strain belongs to different
To control biofilm-associated staphylococcal infections, production of virulence factors and antibiotic resistance, QS can be disrupted by inhibition of signal production, degrading signals, and suppressing synthase and receptors .
2.5.2. The regulation of Staphylococcal biofilm by other than Agr
Two-component regulator gene locus encoded by arlRS is regulated by
The biofilm formation of
2.5.3. Inactivation of ica by sequences
220.127.116.11. Tetranucleotide tandem repeat
2.6. Treatment of biofilm
To provide protection against
Nitric oxide (NO) that is a product of anaerobic respiration can cause dispersal of microorganism from mature biofilm by stimulation of c-di-GMP phosphodiesterases activity . c-di-GMP biosynthesis inhibitors can be an alternative treatment for preventing biofilm formation and mature biofilm dispersal. The combinations of dispersin B (EPS-degrading enzymes) and disinfectants such as triclosan with antibiotics that are used in the treatment of wound and skin infections provides synergistic removal of biofilms .
3. The mechanisms of antibiotic resistance in biofilm-embedded microorganism
Biofilm-embedded bacteria are more resistant to antimicrobial agents than planktonic bacteria. It is difficult to eradicate biofilm, and this causes serious clinical problem .
Antibiotic resistance (tolerance) that is caused by biofilm and permit bacteria to survive is a physiological state by which mutational changes not caused . Impermeability of peptidoglycan by efflux pumps, antibiotic-degrading enzymes, the charge of polymers , and certain gene products that are produced in biofilms  are the other antibiotic resistance mechanisms of bacteria rather than the biofilm . Biofilm can gain higher antibiotic tolerance by antibiotic degrading enzymes such as beta-lactamases, efflux pumps, and certain gene products of which expression are changed by the quorum sensing as a stress response [3, 74]. Biofilms resist to beta-lactam antibiotics by beta-lactamases. Beta-lactamases that are produced by bacteria play a key factor in the biofilm caused resistance to beta-lactam antibiotics .
3.1. The heterogeneous sessile community and the physiology of biofilm
Biofilm-embedded sessile community has heterogeneous cells that are in the different growth states. Bacterial growth rate is reduced by stress conditions such as nutrient and oxygen limitation at the lower parts of the biofilm, and low metabolic activity. Low metabolically active cells (slow growing cells) are seen at the deeper parts of the biofilm, whereas high metabolically active cells (rapid growing cells) are seen at the surfaces of the biofilm. These heterogeneous cells that consist of low and high metabolically active cells have wide range of different responds to each antimicrobial. Antibiotic penetration through the biofilm is reduced by reduced bacterial growth rate. The biofilm-related resistance mechanisms such as oxygen limitation and low metabolic activity, reduced antibiotic penetration through the biofilm, and gaining genetic adaptations such as increased changes in the genes of the DNA repair systems play a key factor in the biofilm tolerance to antibiotics . But some antibiotics such as colistin are just effective against slow-growing cells seen at the deeper parts of the biofilm not against rapid growing cells that acquired adaptive resistance by upregulation of the LPS-modification (arn) operon . Persister cell population that is present in the biofilms of
3.2. Nutrient limitation
Some researchers demonstrated that nutrient limitation-related antibiotic resistance is not due to the reduced growth rate of microorganism, but rather to the activation of regulated stress responses. Nutrient limitation-related antibiotic resistance is controlled by complex regulatory pathways . During starvation, the activation of the stringent response participates in antibiotic resistance such as fluoroquinolone resistance in
3.3. Biofilm matrix
Usually, the decreased antibiotic penetration through the biofilm is caused by antibiotics that may bind to the structural contents of biofilm matrix  rather than reduced diffusion of antibiotics through the biofilm matrix  (Figure 3).
3.4. Agr expression
Antibiotic susceptibility of biofilm-embedded bacteria decreases according to the planktonic state. The virulence of
Agr expression of biofilm producer staphylococcus has also been associated with the drug resistance of some antibiotics. It has been also revealed that the effect of rifampin against
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