**2. Immune response**

Regarding bacterial keratitis there are several potential risk factors such as contact lenses, trauma, aqueous tear deficiencies, neurotrophic keratopathy, eyelid alterations or malposi‐ tion, decreased immunologic defenses, use of topical corticoid medications and surgery [4]. Trauma is a major risk factor for corneal infection in developing countries. In Paraguay, the percentage of cases with preceding trauma was 48%, in Madurai, South India, 65% and 83% in Eastern India [5, 6, 7]. By far the most common cause of trauma to the corneal epithelium and the main risk factor for bacterial keratitis in developed countries is the use of contact lenses, particularly extended-wear contact lenses. Patients with bacterial keratitis, 19-42% are contact lens wearers; incidence of bacterial keratitis secondary to use of extended-wear contact lenses is about 8,000 cases per year. The annual incidence of bacterial keratitis with

Traditionally the more common groups responsible for bacterial keratitis are: *Streptococcus sp.*, *Pseudomonas sp.*, *Enterobacteriaceae* (including *Klebsiella*, *Enterobacter*, *Serratia*, and *Proteus*), and *Staphylococcus sp.* Although there is also a wide variation depending on the setting of the series reported. A high percentage of *Staphylococcus sp.* (79%) was recorded in a study from Para‐ guay, although the reason for this is not clear. Another study found the highest proportion of *Streptococcus sp.* (46.8%), the authors noted that this figure was only 18.5% in 1986 and suggest that the trend might represent a genuine change in the bacterial flora owing to changes in the climate and environment [9]. A study from Bangkok [10] had the highest proportion of *Pseudo‐ monas* infections (55%). Interestingly, this study did not have the highest proportion of con‐ tact-lens wearers. Other studies reported far higher proportions of contact-lens wearers—for example, 44% in a study from Taiwan [11] and 50% in a study from Paris [12]. When compared the percentage of contact-lens wearers with the percentage of pseudomonal infections, the Spearman correlation coefficient was not statistically significant. Cohen et al. at Wills Eye Hos‐ pital reported a decline in contact lens-related ulcers: during 1991 to 1998, contact-lens wear accounted for 44% of all ulcers, but during 1992 to 1995, it accounted for only 30%. Liesegang reports the following risk factors for development of bacterial keratitis among contact lenses wearers: overnight wear, smoking, male sex, and socioeconomic status. The risk with thera‐

peutic contact lenses is much higher: approximately 52/10,000 per year [13].

organisms accounted for 29.1% of all Gram(+) cultures.

Jeng [14] commented on the emerging resistance of bacterial infections to fluoroquinolones. In addition to changes in resistance patterns, studies have also demonstrated changing pat‐ terns of causative organisms over time in a given geographical location. Varaprasathan et al [15] reported that the proportion of S*treptococcus pneumoniae* and *Pesudomonas aeruginosa* ul‐ cers in Northern California had decreased over a 50-year period, while that of *Serratia mar‐ cescens* had increased over the same period. Sun et al [16] reported a rise in the percentage of Gram positive (+) cocci in North China from 25% in 1991 to 70.8% in 1997, as well as a de‐ crease in Gram negative (-) bacilli from 69% to 23.4% over a similar period. Hsiao et al [17] reported on a 10 year follow up that there was a significant decrease in the percentage of Gram(+) microorganisms over time. The sensitivity of Gram(-) isolates to tested antimicrobi‐ als was >97% response for all the reported antibiotics; this was not the case for Gram(+) iso‐ lates, in which resistance to the antibiotics was more common, methicillin-resistant

daily-wear lenses is 3 cases per 10,000 [8].

86 Common Eye Infections

We have a lot of mechanisms to evade a bacterial infection: physical, chemical, microbiologi‐ cal, and immunological mechanisms. But not all of the mechanisms are described in a bacte‐ rial keratitis infection.

#### **2.1. Exterior defense**

The eye has several mechanisms to prevent colonization by bacteria, among which are three main types: the mechanics, such as blinking, or that the Tight Junctions present in the cor‐ neal epithelial cells, preventing the entry of bacteria into the corneal stroma or other intraoc‐ ular structures, it is important to mention that certain bacteria are able to penetrate the intact corneal epithelium such as *Corynebacterium diphtheriae*, *Haemophilus aegyptus*, *Neisseria sp.*, *Listeria sp.* and *Shigella sp.* [18, 19]; Chemicals, which are the presence of soluble molecules involved in controlling the growth of bacteria, such mechanisms are presence of lactoferrin, lysozyme, antimicrobial peptides, antibodies, etc. [20-21] and finally microbiologic mecha‐ nisms, these mechanisms refer the normal microbiota of the ocular surface (*S. epidermidis, S. aureus and Propionibacterium sp.*) [22-26], the microbiota generates substances called bacterio‐ cins, which will be mentioned later in item 3 of this chapter.

#### **2.2. Complement**

It was reported in murine models, that anaphylatoxins (C3a, C4a, and C5a) could be gener‐ ated when the cornea was injured with lipopolysaccharides (LPS), immune complexes, acid, or alkali. Interestingly membrane attack complex (MAC) could only be generated when the cornea was exposed to LPS or immune complexes. Cornea failed to generate MAC when af‐ fronted with acid or alkali. The immune response mounted to LPS or immune complex is similar to that generated against infectious agents like Gram(-) bacteria. Indeed the comple‐ ment system has been shown to play a critical role in protection against *Pseudomonas aerugi‐ nosa* infection that causes keratitis [27,28]. Additionally, complement activation is believed to play an important role in ulceration of human cornea induced by Gram(-) bacteria [29].

#### **2.3. Receptors**

There are different receptors that recognizes bacteria molecules, these receptors in general are called pattern recognition receptors and exists several types of receptors (TLR, CLR, NLR and RLR). In bacterial keratitis infections are studied in murine and *in vitro* models, the presence, activation and function of TLR. The functions described in TLR activation are cy‐ tokine secretion, chemokines secretion, and antimicrobian peptides secretion, recruitment of cells to inflammation site. For example corneal TLR4 expression is increased in *P. aeruginosa* infection and deficiency of this receptor in BALB/c mice resulted in a susceptible rather than resistant phenotype [30], these observations suggest that TLR4 is critical for resistance to *P. aeruginosa* keratitis.

#### **2.4. Effects of receptors activation**

#### *2.4.1. Chemokines*

In other studies, UV killed *S. aureus* and Pam3Cys (TLR2 synthetic ligand) stimulated the phosphorylation of MAP kinases, JNK, p38 MAPK and ERK, and the blockade of JNK, but not that of p38 or ERK phosphorylation, had an inhibitory effect on IκBα degradation and CXC chemokine production [31]. Furthermore they also found that corneal inflammation was significantly impaired in mice deficient in JNK1 mice compared with control mice, sug‐ gesting that JNK has an essential role in TLR2-induced corneal inflammation.

#### *2.4.2. Antimicrobian peptides*

Activation with pathogens and TLR agonists of ocular surface epithelial cells by also leads to the production of antimicrobial peptides such as hBD-2 and the cathelicidin LL-37 [32-34]. In an interesting *in vitro* study [35], Maltseva et al., reported that a MyD88 dependent in‐ crease in corneal epithelial hBD-2 expression caused by exposure to *P. aeruginosa* superna‐ tant was abrogated by the presence of a contact lens, thus giving new insight into the mechanism by which contact lens wear predisposes to *P. aeruginosa* keratitis.

Additional *in vivo* studies have shown that defensins and LL-37 play an important role in protecting the ocular surface from *P. aeruginosa* infections. In particular, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of LL-37, are more susceptible to *P. aeruginosa* keratitis, had significantly delayed bacterial clearance and an increased number of infiltrating neutrophils in the cornea [36]. A similar finding was re‐ ported in BALB/c mice following knock down of mBD-2 or mBD-3, but not of mBD-1 or mBD-4, by siRNA [37-38]. Furthermore Wu et al. also found that silencing mBD2, mBD3 or both defensins resulted in a significant upregulation of TLR2, TLR4 and MyD88 but not TLR5 or TLR9 [39].

Kumar et al. [40] observed that pre-treatment with the TLR5 agonist flagellin markedly reduced the severity of subsequent *P. aeruginosa* infection in C57BL/6 mice. This was in part due to induction of corneal expression of the antimicrobial molecules, nitric oxide and CRAMP. They also observed similar results *in vitro*, as flagellin pre-treatment en‐ hanced *P. aeruginosa* induced expression of hBD-2 and LL-37 in human corneal epithelial cells [39]. These observations raise the possibility of utilizing TLR activation as a prophy‐ lactic means of preventing an overwhelming inflammatory response and corneal destruc‐ tion in *P. aeruginosa* keratitis.

#### **2.5. Recruited cells**

presence, activation and function of TLR. The functions described in TLR activation are cy‐ tokine secretion, chemokines secretion, and antimicrobian peptides secretion, recruitment of cells to inflammation site. For example corneal TLR4 expression is increased in *P. aeruginosa* infection and deficiency of this receptor in BALB/c mice resulted in a susceptible rather than resistant phenotype [30], these observations suggest that TLR4 is critical for resistance to *P.*

In other studies, UV killed *S. aureus* and Pam3Cys (TLR2 synthetic ligand) stimulated the phosphorylation of MAP kinases, JNK, p38 MAPK and ERK, and the blockade of JNK, but not that of p38 or ERK phosphorylation, had an inhibitory effect on IκBα degradation and CXC chemokine production [31]. Furthermore they also found that corneal inflammation was significantly impaired in mice deficient in JNK1 mice compared with control mice, sug‐

Activation with pathogens and TLR agonists of ocular surface epithelial cells by also leads to the production of antimicrobial peptides such as hBD-2 and the cathelicidin LL-37 [32-34]. In an interesting *in vitro* study [35], Maltseva et al., reported that a MyD88 dependent in‐ crease in corneal epithelial hBD-2 expression caused by exposure to *P. aeruginosa* superna‐ tant was abrogated by the presence of a contact lens, thus giving new insight into the

Additional *in vivo* studies have shown that defensins and LL-37 play an important role in protecting the ocular surface from *P. aeruginosa* infections. In particular, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of LL-37, are more susceptible to *P. aeruginosa* keratitis, had significantly delayed bacterial clearance and an increased number of infiltrating neutrophils in the cornea [36]. A similar finding was re‐ ported in BALB/c mice following knock down of mBD-2 or mBD-3, but not of mBD-1 or mBD-4, by siRNA [37-38]. Furthermore Wu et al. also found that silencing mBD2, mBD3 or both defensins resulted in a significant upregulation of TLR2, TLR4 and MyD88 but not

Kumar et al. [40] observed that pre-treatment with the TLR5 agonist flagellin markedly reduced the severity of subsequent *P. aeruginosa* infection in C57BL/6 mice. This was in part due to induction of corneal expression of the antimicrobial molecules, nitric oxide and CRAMP. They also observed similar results *in vitro*, as flagellin pre-treatment en‐ hanced *P. aeruginosa* induced expression of hBD-2 and LL-37 in human corneal epithelial cells [39]. These observations raise the possibility of utilizing TLR activation as a prophy‐ lactic means of preventing an overwhelming inflammatory response and corneal destruc‐

gesting that JNK has an essential role in TLR2-induced corneal inflammation.

mechanism by which contact lens wear predisposes to *P. aeruginosa* keratitis.

*aeruginosa* keratitis.

88 Common Eye Infections

*2.4.1. Chemokines*

**2.4. Effects of receptors activation**

*2.4.2. Antimicrobian peptides*

TLR5 or TLR9 [39].

tion in *P. aeruginosa* keratitis.

#### *2.5.1. Polymorphonuclear cells*

In animal models as characterized by bacterial invasion of the underlying stroma and in‐ tense neutrophil infiltration which results in corneal opacification and potentially loss of vi‐ sion [41-45]. In an murine model of *S. aureus* keratitis, exposure of corneal epithelium to *S. aureus* increased neutrophil recruitment to the corneal stroma, corneal thickness and corneal haze in normal C57Bl/6 mice, mice deficient TLR4 or TLR9, but not in mice deficient in TLR2 or MyD88, suggesting that *S. aureus*-induced corneal inflammation is mediated by TLR2 and MyD88 [46].

In 2005 Huang et al., reported that silencing TLR9 by siRNA in C57BL/6 mice resulted in less severe inflammation, reduced polymorphonuclear infiltration but consequently increased bacterial load [47]. These data suggested that TLR9 activation is required to adequately elim‐ inate bacteria but that it also contributes to corneal destruction.

#### *2.5.2. T cell populations*

Extensive study of the underlying mechanism of the pathogenesis of *P. aeruginosa* keratitis in experimental models has revealed that mice can be divided in two groups based upon their immune response to the pathogen [48]. BALB/c mice are resistant to *P. aeruginosa* infec‐ tion as they mount a Th2 based response that facilitates recovery and corneal healing. While C57BL/6 mice are susceptible to *P. aeruginosa* infection as they mount a Th1 based immune response leading to corneal perforation. Comparison among these mouse strains provides a unique opportunity to understand the immune response to *P. aeruginosa*.

Exists other type of efectors in the immune response not characterized yet, like the presence of other receptors like NLR or CLR. It is important to mention that the immune response previous described are in animal models or *in vitro* models; a few studies are in patients and we need to study in the future to explain the immunopathogenesis and found new treat‐ ments for patients.

### **3. Virulence factors and mechanisms of bacterial resistance**

To understand why bacterial keratitis is often of difficult treatment is necessary to first re‐ view the virulence factors and mechanisms of bacterial resistance, this will help us to make decisions about treatment, patient management and contribute to prevent the emergence and development resistant strains.

The treatment for bacterial keratitis consist mainly in antibiotics, so it is necessary to know: bacterial structure, biochemical action, identified important immunogens, and virulence fac‐ tors. Molecular biology also has had a great participation and that made possible the devel‐ opment of molecular techniques with applications to research to learn more about the bacterial virulence factors and in the diagnosis of pathogens to give a prompt and timely treatment [49].

#### **3.1. Virulence factors**

Virulence is a term that comes from the Latin virulent (virus = poison and virulent = poison‐ ous) and that is a property to allows pathogenic bacteria to colonize the host and thus obtain their nutritional requirements, for this it is necessary to evade the defense mechanisms, mul‐ tiply, establish and cause harm. All this is achieved through the expression of bacterial viru‐ lence factors (bacteriocins) that allow microbial adherence, invasion, or both, the harmfulness and pathogenic microorganism determines its virulence o their ability to do harm. Within the virulence factors we can mention the following:


#### **3.2. Bacterial resistance**

The principal objective about the study of bacterial virulence factors is the quest from new preventive and therapeutic tools against many infectious diseases. However, there is anoth‐ er condition called bacterial resistance[50].

Antibiotic resistance in bacteria has become a health problem worldwide. The developments of new antibacterial drugs, the indiscriminate and irrational use, besides the evolutionary pressure exerted by therapeutic use have gone masking the increase of the resistance. It ap‐ pears that the design or discovery of new antibiotics solve the problem, however, also new mechanisms of resistance are difficult to control

Infections caused by multiresistant bacteria, causing extensive morbidity and mortality and the cost per hospitalization and complications is high. The selective pressure plays an im‐ portant role in the occurrence of resistant strains and is favored by free prescription and for‐ mal therapeutic use, the widespread use of antimicrobials in immunocompromised patients, in the intensive care unit, the use of inadequate dose or insufficient duration of antimicrobi‐ al therapy and indiscriminate use without establishing a profile sensitivity of isolates. The selective pressure is a process of adaptation and this is not an attribute of individual organ‐ isms or nature or life, but it is attributes of a species. In Darwinian terms, the response to the selective pressure is not the individual, not life or nature as a whole but the population itself [51], this means that when a treatment is handled improperly, only susceptible organisms will be destroyed and reduce the bacterial load and hence the infection symptoms, however resistant microorganisms remain in small amounts and gives rise to a new generation of re‐ sistant strains (figure 1).

**Figure 1.** Selective pressure after a treatment with dosage, time or inadequate concentration of the antibiotic. (A) mixture of sensitive and resistant bacteria to an antibiotic (B) Resistant bacteria (C) Proliferation of bacteria resistant proliferation.

The phenotypic expression of bacterial resistance has intrinsic or acquired genetic basis and is mainly expressed by biochemical mechanisms [52]. Briefly describe the two mechanisms of bacterial resistance, the naturally occurring and acquired by the same bacteria.

#### *3.2.1. Natural resistance*

bacterial virulence factors and in the diagnosis of pathogens to give a prompt and timely

Virulence is a term that comes from the Latin virulent (virus = poison and virulent = poison‐ ous) and that is a property to allows pathogenic bacteria to colonize the host and thus obtain their nutritional requirements, for this it is necessary to evade the defense mechanisms, mul‐ tiply, establish and cause harm. All this is achieved through the expression of bacterial viru‐ lence factors (bacteriocins) that allow microbial adherence, invasion, or both, the harmfulness and pathogenic microorganism determines its virulence o their ability to do

**•** Adhesins. These substances are membrane receptors involved not only in the cell-cell in‐ teractions but also cell-extracellular matrix and cell-trafficking cell. Among the adhesins

**•** Invasins. Surface proteins which are responsible for reorganization of actin filaments near the cytoskeleton, thus, when a bacterium comes into contact with the host cell occurs a change in its structure similar to a drop of liquid on a solid surface falls due the reorgani‐ zation of the cytoskeleton so that it can be incorporated into the cell, once inside the bacte‐

**•** Impedins. Molecules that help the bacteria evade the host immune response to perpetuate and maintain their infectivity, as examples we can mention the mucinases that using me‐ chanical effects generated by the movement of flagella prevent skidding and disposal, al‐ so we can mention proteases that are found mainly in the mucous membranes and destroy the IgA antibodies. In addition exist molecules that help evasion of phagocytosis as coagulase, DNAse, phosphatases, LPS that interfere with complement and finally the

**•** Aggressins. Hypothetical substance held to contribute to the virulence of pathogenic bac‐ teria by paralyzing the host defensive mechanisms which, by their chemical nature, can lead to tissue damage, inflammation and shock. Some examples we can cite alpha and be‐

**•** Modulins are bacterial components that promote the production of cytokines among which we can find the lipopolysaccharide of Gram(-), superantigens and murein frag‐

The principal objective about the study of bacterial virulence factors is the quest from new preventive and therapeutic tools against many infectious diseases. However, there is anoth‐

Antibiotic resistance in bacteria has become a health problem worldwide. The developments of new antibacterial drugs, the indiscriminate and irrational use, besides the evolutionary

ta toxins, lytic enzymes, DNases, lipases, hyaluronidases, kinases, teichoic acid.

harm. Within the virulence factors we can mention the following:

production of toxic metabolites to overcome the normal flora.

ria uses actin to move from one cell to another.

find bacterial pili, fimbrial proteins, lipoteichoic acids and glycocalyx.

treatment [49].

90 Common Eye Infections

ments.

**3.2. Bacterial resistance**

er condition called bacterial resistance[50].

**3.1. Virulence factors**

Natural resistance is a constant feature of strains of the same bacterial species and is a per‐ manent mechanism determined genetically and furthermore correlated with dose of antibi‐ otic. Some examples of this, we can mention the resistance presented by *Proteus mirabilis* to the tetracyclines and colistin, *P. aeruginosa* to the Benzylpenicillins and trimethoprim-sulfa‐ methoxazole, aerobic Gram(-) bacilli to the clindamycin, *Klebsiella pneumoniae* to the penicil‐ lins (ampicillin and amoxicillin), [53].

#### *3.2.2. Acquired resistance*

Bacterial species, which by nature is sensitive to an antibiotic, can be genetically modified either by mutation or by acquisition of resistance genes (plasmids, transposons and inte‐ grons), these are evolutionary and their frequency depends on the use of antibiotics. An ex‐ ample of mutation of a gene involved in the mechanism of action of an antibiotic is the DNA gyrase involved in DNA replication process of enterobacterias and that a mutation in these genes can confer resistance to quinolones; can also be mutations generated in genes encod‐ ing the porins which results in blocking the entrance of the antibiotic into the microorgan‐ ism. The acquisition of resistance genes can be obtained by transfer from a strain of a species identical or different, mechanisms responsible for these are the plasmids, transposons and integrons [53-54].

The plasmids and transposons are mobile genetic elements which carry resistance genes. The plasmids are fragments of bacterial DNA with variable length; some have the ability to replicate independently of the genetic machinery available to the cell. Other hand transpo‐ sons are sequences of DNA (double stranded) which can be translocated from chromosome to chromosome or a plasmid to plasmids, thanks to a proper recombination system, this adds to the ability of plasmids to move from one cell to another during conjugation, this al‐ lows the acquisition of resistance genes from bacteria of the same species or different species which facilitates the expansion of the resistance strains. Some plasmids and transposons have elements called integrons gene that allows them to capture more exogenous genes de‐ termining the development of resistance to several antibiotics (multiple resistance). Antibi‐ otics particularly affected by this mechanism are the beta-lactams, aminoglycosides, tetracyclines, chloramphenicol, and sulfonamide, an example is the resistance presented by *Escherichia coli* and *P. mirabilis* to ampicillin [55].

#### **3.3. Resistance mechanisms**

Bacterial resistance both acquired and natural can be approached from the standpoint mo‐ lecular and biochemical and can be classified into three basic mechanisms of resistance ex‐ pressed according to the mechanism expressed and the antibiotics mechanism action and may occur simultaneously [55]. The figure 2 shows a schematic representation of the mecha‐ nisms of resistance.

**•** Inactivation of antibiotic by destruction or modification of chemical structure. Is a molec‐ ular process characterized by the production of enzymes that carry out this function. For example, enzymes that destroy the chemical structure of an antibiotic against beta-lacta‐ mases are characterized by hydrolyzing the beta-lactam nucleus through amide bond cleavage and erythromycin esterase which catalyses the hydrolysis of the lactone ring of the antibiotic, while the enzymes responsible to the modification of the structure we can mention the chloramphenicol acetyl transferase, enzymes that modify aminoglycosides, lincosamides and streptogramins, other enzymes belonging to this group are acetylases, adenilasas and phosphatases [56, 57].


#### **3.4. Biofilm production**

*3.2.2. Acquired resistance*

92 Common Eye Infections

integrons [53-54].

*Escherichia coli* and *P. mirabilis* to ampicillin [55].

adenilasas and phosphatases [56, 57].

**3.3. Resistance mechanisms**

nisms of resistance.

Bacterial species, which by nature is sensitive to an antibiotic, can be genetically modified either by mutation or by acquisition of resistance genes (plasmids, transposons and inte‐ grons), these are evolutionary and their frequency depends on the use of antibiotics. An ex‐ ample of mutation of a gene involved in the mechanism of action of an antibiotic is the DNA gyrase involved in DNA replication process of enterobacterias and that a mutation in these genes can confer resistance to quinolones; can also be mutations generated in genes encod‐ ing the porins which results in blocking the entrance of the antibiotic into the microorgan‐ ism. The acquisition of resistance genes can be obtained by transfer from a strain of a species identical or different, mechanisms responsible for these are the plasmids, transposons and

The plasmids and transposons are mobile genetic elements which carry resistance genes. The plasmids are fragments of bacterial DNA with variable length; some have the ability to replicate independently of the genetic machinery available to the cell. Other hand transpo‐ sons are sequences of DNA (double stranded) which can be translocated from chromosome to chromosome or a plasmid to plasmids, thanks to a proper recombination system, this adds to the ability of plasmids to move from one cell to another during conjugation, this al‐ lows the acquisition of resistance genes from bacteria of the same species or different species which facilitates the expansion of the resistance strains. Some plasmids and transposons have elements called integrons gene that allows them to capture more exogenous genes de‐ termining the development of resistance to several antibiotics (multiple resistance). Antibi‐ otics particularly affected by this mechanism are the beta-lactams, aminoglycosides, tetracyclines, chloramphenicol, and sulfonamide, an example is the resistance presented by

Bacterial resistance both acquired and natural can be approached from the standpoint mo‐ lecular and biochemical and can be classified into three basic mechanisms of resistance ex‐ pressed according to the mechanism expressed and the antibiotics mechanism action and may occur simultaneously [55]. The figure 2 shows a schematic representation of the mecha‐

**•** Inactivation of antibiotic by destruction or modification of chemical structure. Is a molec‐ ular process characterized by the production of enzymes that carry out this function. For example, enzymes that destroy the chemical structure of an antibiotic against beta-lacta‐ mases are characterized by hydrolyzing the beta-lactam nucleus through amide bond cleavage and erythromycin esterase which catalyses the hydrolysis of the lactone ring of the antibiotic, while the enzymes responsible to the modification of the structure we can mention the chloramphenicol acetyl transferase, enzymes that modify aminoglycosides, lincosamides and streptogramins, other enzymes belonging to this group are acetylases, In nature, bacteria can grow like planktonic or free-floating, but can also grow colonies em‐ bedded in a matrix known as biofilm. Deserves special mention the formation of biofilms, since being a microbial ecosystem composed of one or more microorganisms associated with living or inert surface with functional features and complex structures can be considered a virulence factor and the same time a resistance mechanism. Biofilm formation enables the adhesion to the surface where the bacteria is present and can be one of many causes of chronic infections, for example the chronic infectious keratitis. The structural organization of the bacterial biofilm is composed of polysaccharides, nucleic acids and proteins and all this set is known as extracellular polymeric substances (EPS) and its production is affected by the nutritional quality of the environment in which bacteria develop when the environment is suitable to form biofilms with multiple microcolonies, so the structure that forms is so great that prevents phagocytosis and effects of the immune system against them, for this reason is considered a virulence factor. A very important advantage from the clinical point of view is that biofilms confer resistance to antibiotics such that the dose can be increased thousands of times without causing damage [59]. Two hypotheses to explain the resistance generated by the production of biofilms, the first indicating that occurs a limited penetration of the drug and the bulk is left on the surface such that the antibiotic never reaches its target. The second refers to the physiological limitation and proposes that some microorganisms within the biofilm can exist in a more recalcitrant phenotypic state. Anderl JN et al [60] in a study of *K. pneumoniae* found that the planktonic form was sensitive to ampicillin and re‐ ported minimum inhibitory concentration (MIC) of 22µg/mL while the same strain that grew as a biofilm presented a survival of 66% increasing the concentration of ampicillin to 5000µg/mL which corresponds to 2500 times the MIC.

**Figure 2.** Resistant mechanism (1) Altered target site of the antibiotic and altered permeability barriers (2) Inactivation of antibiotic by destruction or modification of chemical structure (3) Efflux pumps (4) acquisition of resistance genes by fagos (5) Plasmids (6) Transposons and Integrons (7) modification by mutation of topoisomerase.

Having recognized the role of biofilm as responsible of infectious diseases, it is necessary the search for new approaches in both the treatment and prevention. A proposal to counteract this resistance factor is the alteration of the surface to inhibit adhesion. In the area of oph‐ thalmology, for example, chelating agents could be used in contact lens solutions, mainly iron-trapping agent which is necessary for adhesion of the pili of *Pseudomonas sp.* [59, 60].

**Figure 3.** Biofilm production (1) Planktonic bacteria encounter a submerged surface. They begin to produce slimy ex‐ tracellular polymeric substances (EPS) and to colonize the surface. (2) EPS production allows the emerging biofilm community to develop a complex three-dimensional structure (3) Biofilms can propagate through detachment of small or large clumps of cells that releases individual cells.
