**2. Causes of antibiotic failure in biofilm**

Antibiotic resistance is the acquired ability of a microorganism to resist the effect of an antimicrobial agent and is associated with inheritable antibiotic resistance. On the other hand, antibiotic tolerance is a transient and nonheritable phenotype defined by the physiological state of biofilm cell populations. Also it can be provided by biofilm-specific characteristics that limit drug diffusion and activity [7]. For an antimicrobial agent to act on biofilm-forming microorganisms, it must overcome some factors, such as an increased number of resistant mutants, high cell density, molecular exchanges, substance delivery, efflux pump, and persistent cells.

### **2.1 Antibiotic penetration**

Antibiotic molecules ought to penetrate throughout the biofilm matrix to impact the covered cells. The extracellular polymeric matrix influences the amount of the molecule, which is transferred to the inner layer of biofilm and interacts with an antibiotic agent, so it provides an anti-spread barrier for an antimicrobial agent. Biofilm EPS confers a physical barrier containing numerous anionic and cationic molecules such as proteins, glycoproteins, and glycolipid that can bind charged antimicrobial agents and provide shelter for microorganisms [8]. For example in *Pseudomonas aeruginosa* biofilms, Pel exopolysaccharides, an EPS component is able to spread cationic antibiotics such as aminoglycosides and, thus, provides tolerance to these molecules [9].

The adsorption sites of the matrix also limit the transportation of antimicrobial substances. Glycocalyx layer, component of EPS, can accumulate antibacterial molecule up to 25% of its weight and serve as an adherent for exoenzymes [10].

It is commonly accepted that in written materials lowered antibiotic penetration toward the EPS layer does not adequately clarify the risen resistance of microorganisms forming biofilm against most antimicrobial agents. The act of lowered antibiotic penetration in developing biofilm is not clear due to the fact that even antibiotics, which quickly disperse the biofilm, do not lead to notable cell death. It is suggested that reduction of antibiotics penetration might provide time for an adaptive phenotypic response, which can probably reduce susceptibility [11].

**137**

*Antibiotic Resistance in Biofilm*

**2.3 DNA in biofilm matrix**

tance to certain antimicrobial agents [15].

against cationic peptide and aminoglycoside [17].

**2.4 Growth rate, stress response, and persistent cells**

cation restriction in *P. aeruginosa* [18].

forming biofilm [14].

*DOI: http://dx.doi.org/10.5772/intechopen.92388*

**2.2 Accumulation of antibiotic-degrading enzymes in the matrix**

β-lactamases in the biofilm matrix as a defense mechanism.

The microorganisms that form biofilm are able to collect high amounts of

When *P. aeruginosa* biofilm matrix accumulates β-lactamases, it can lead to increased hydrolysis of antibiotics, such as imipenem and ceftazidime. It is demonstrated that *P. aeruginosa* PAO1-J32 biofilms have shown high promoter (ampC β-lactamases) activity, which is determined by scanning confocal laser photomicrographs [12]. Also, while ampicillin cannot reach the deeper layers of *Klebsiella pneumoniae* biofilms associated with β-lactamase activity, deletion of β-lactamase increases the amount of ampicillin that reaches the deep layer [13].

Extracellular DNA (eDNA) is a significant and common component ingredient of the bacterial biofilm matrix. The eDNA can be obtained endogenously without quorum sensing-mediated release, from the outer membrane or from the cell integrity-degraded biofilm microorganisms [14]. DNA can increase biofilm resis-

One of the mechanisms by which the DNA increases biofilm resistance is that it causes changes in outer membrane because DNA is an anionic molecule; it is able to chelate cations, such as magnesium ions and cause a lowering Mg2+ concentration in membrane. Magnesium restriction in *P. aeruginosa* and *Salmonella enterica* serovar Typhimurium is an environmental signal that induces energizing of the two-component systems PhoPQ and PmrAB to provide antimicrobial resistance [16].

These signal molecules are responsible for the rearrangement of the PA3552-3559 operon. The operon encodes to protein having enzymatic activity that attaches aminoarabinose to Lipid A part of the lipopolysaccharide layer, so it provides resistance

A polyamine, spermidine, localized to the outer membrane contributes to saving the cell from aminoglycosides and cationic peptides that are antimicrobial agents by lowering outer membrane penetrability for these positively charged molecules. Spermidine synthesis is another resistance mechanism induced by eDNA-associated

Playing a physical role in defense against antibiotics, eDNA has also provided horizontal transfer of antibiotic resistance genes between microorganism cells

During growth in biofilm structures, physiological heterogeneity happens due to the occurrence of oxygen and other nutrients gradient in biofilms. This gradient is created because cells that are close to the surface of the biofilm consume obtainable nutrient sources and oxygen before the nutrients disperse into depth of the biofilm [19]. Nutrient and oxygen concentration gradients develop and cause bacterial populations that display different growth rates [20]. The effect of many antibiotics depends on growth. Because most antibiotics aim at some kind of produced macromolecule, it is unexpected that these agents will have much impact on the microorganisms in biofilm that limit macromolecular production, so conventional antibiotics

In biofilms, a small subpopulation of bacteria can be reversibly transformed into slowly growing cells. These cells are known as persistent or dormant cells. Persistent cells are generated stochastically or under endogenous stress (e.g., oxidative stress

are usually less affected against metabolically inactive or slow-growing cells.

*Bacterial Biofilms*

gene transcription [4].

persistent cells.

**2.1 Antibiotic penetration**

lular DNA (eDNA) and in physical interactions [4].

checkpoint during the development of biofilm [6].

**2. Causes of antibiotic failure in biofilm**

A biofilm can be described as a microbially derived sessile community characterized by cells. These cells are irreversibly attached to a surface or interface or to each other, are inserted in a matrix of extracellular polymeric substances (EPSs) that they have produced, and exhibit an altered phenotype in terms of growth rate and

EPSs consist of proteins, cellulose, alginates, extracellular teichoic acid, poly-Nacetyl, and other organic compounds [4, 5] and play a critical role in the formation of glucosamine, lipids, nucleic acids, phospholipids, polysaccharides, and extracel-

The stages that occur during the biofilm development are the initial attachment of the planktonic cell to the surface, followed by cell differentiation, EPS secretion, maturation, and dispersion of biofilm [6]. It can be summarized in three main stages: irreversible adhesion to the surface, being followed by bacterial division and production of the extracellular matrix, and, finally, disassembly of the matrix and dispersion of bacteria [2]. Quorum Sensing (QS) is one of the regulatory mechanisms that plays an important role in coordinating biofilm formation in many species but QS may not be the primary regulatory mechanism and serves as a

Antibiotic resistance is the acquired ability of a microorganism to resist the effect of an antimicrobial agent and is associated with inheritable antibiotic resistance. On the other hand, antibiotic tolerance is a transient and nonheritable phenotype defined by the physiological state of biofilm cell populations. Also it can be provided by biofilm-specific characteristics that limit drug diffusion and activity [7]. For an antimicrobial agent to act on biofilm-forming microorganisms, it must overcome some factors, such as an increased number of resistant mutants, high cell density, molecular exchanges, substance delivery, efflux pump, and

Antibiotic molecules ought to penetrate throughout the biofilm matrix to impact the covered cells. The extracellular polymeric matrix influences the amount of the molecule, which is transferred to the inner layer of biofilm and interacts with an antibiotic agent, so it provides an anti-spread barrier for an antimicrobial agent. Biofilm EPS confers a physical barrier containing numerous anionic and cationic molecules such as proteins, glycoproteins, and glycolipid that can bind charged antimicrobial agents and provide shelter for microorganisms [8]. For example in *Pseudomonas aeruginosa* biofilms, Pel exopolysaccharides, an EPS component is able to spread cationic antibiotics such as aminoglycosides and, thus, provides tolerance to these molecules [9].

The adsorption sites of the matrix also limit the transportation of antimicrobial

It is commonly accepted that in written materials lowered antibiotic penetration toward the EPS layer does not adequately clarify the risen resistance of microorganisms forming biofilm against most antimicrobial agents. The act of lowered antibiotic penetration in developing biofilm is not clear due to the fact that even antibiotics, which quickly disperse the biofilm, do not lead to notable cell death. It is suggested that reduction of antibiotics penetration might provide time for an adaptive phenotypic response, which can probably reduce susceptibility [11].

substances. Glycocalyx layer, component of EPS, can accumulate antibacterial molecule up to 25% of its weight and serve as an adherent for exoenzymes [10].

**136**
