Biofilm Theory for Lid Margin and Dry Eye Disease

*Maria Vincent, Jose Quintero, Henry D. Perry and James M. Rynerson*

### **Abstract**

Blepharitis and dry eye disease have long been viewed as two distinct diseases with overlapping presentations and separate etiologies. Evaporative dry eye, although frequently associated with aqueous deficiency, is also considered a separate entity. We propose viewing dry eye, both evaporative and insufficiency, as the natural sequelae of chronic blepharitis induced by biofilm. We suggest describing this one chronic disease as dry eye blepharitis syndrome (DEBS). The disease process begins when normal flora bacteria colonize the lid margin beginning shortly after birth. This colonization accompanies the development of a biofilm on the lid margin. As years pass, the biofilm matures, and the increased bacterial population initiates the production of inflammatory virulence factors, such as exotoxins, cytolytic toxins, and super-antigens, which persist on the lid margin for the rest of the patient's life. These virulence factors cause early follicular inflammation and later, meibomian gland dysfunction followed by aqueous insufficiency, and finally, after many decades, loss of the dense collagen in the tarsal plate. We proposed four stages of DEBS, which correlate with the clinical manifestations of folliculitis (anterior blepharitis), meibomitis (meibomian gland dysfunction), lacrimalitis (aqueous deficiency), and lid structure damage evidenced by increased lid laxity resulting in entropion, ectropion, and floppy eyelid syndrome.

**Keywords:** biofilm, blepharitis, demodex, dry eye disease, eyelids, meibomian glands, quorum-sensing gene activation, tear film

#### **1. Introduction**

Blepharitis was first described by ancient Egyptian physicians in the Ebers Papyrus, which prescribed potions such as "Cream with the Milk-of-a-Womanwho-has-borne-a-Son" [1, 2]. Despite centuries of study, little progress has been made in understanding or treating this disease. The long standing dogma of multifactorial, overlapping manifestations of blepharitis and dry eye have led to the use of inaccurate terminology that creates misunderstanding among both patients and providers [3, 4]. In order to develop our understanding of DEBS, we must first establish the correct use of the word blepharitis as suggested by the origin of the word (blepharon = lid, −itis = inflammation).

The next step in understanding dry eye disease and blepharitis as a single disease process is to identify the cause of eyelid inflammation. In 1954, Thygeson first recognized that blepharitis was associated with "abnormal Staphylococcus

#### **Figure 1.**

*Biofilm theory of dry eye disease: Schematic of six steps of bacterial biofilm development leading to the stages of DEBS.*

colonization" of the eyelid margin [5]. Thygeson was describing the process by which our normal lid margin flora bacteria, primarily *Staphylococcus aureus* and *S. epidermidis*, gradually over-colonize the patient's lid margin, and over time, become pathogenic [6]. This is made possible by the bacterial biofilm, which allows the bacteria to thrive despite antimicrobials and the immune system [7]. Understanding biofilm progression links the shared underlying pathology between dry eye and blepharitis.

Biofilms are defined as groups of microbial cells enclosed in a matrix made primarily of polysaccharide material that are intimately associated with a surface. Antonie van Leeuwenhoek is credited for the first observations of biofilm, when he described the biofilm on teeth in 1684. However, further study of biofilms was limited until the development of the electron microscope in the mid-1900s. Furthermore, it was not until 1982 that the term "biofilm" was introduced, after Costerton's observation of a *S. aureus* biofilm on a cardiac pacemaker lead [8]. More recent studies have shown that cell-to-cell interactions ("quorum sensing") within the biofilm upregulate certain gene products. Further studies have implicated biofilm in many disease processes including periodontitis, endocarditis, chronic prostatitis, and medical device associated infections, as the one described by Costerton [8–10].

This chapter will explain the six steps by which our normal margin lid flora become pathogenic and cause eyelid inflammation. This inflammation, in turn, leads to the four stages of DEBS: folliculitis, meibomitis, lacrimalitis, and lid structure damage (**Figure 1**). This understanding will allow us to encompass dry eye disease, blepharitis, and meibomian gland dysfunction (MGD) in one disease process, namely, dry eye blepharitis syndrome (DEBS).

#### **2. Biofilm**

Bacteria were among the first forms of life on Earth and have survived billions of years in a myriad of different environments. While they are unicellular microorganisms, and can live in a free-floating form, the development of a biofilm provides

**21**

*Biofilm Theory for Lid Margin and Dry Eye Disease DOI: http://dx.doi.org/10.5772/intechopen.89969*

remain in a desirable environment.

50% compared to 14% in controls [20].

them a strong, virtually impenetrable defense structure [8]. Furthermore, Absalon et al. and Pickering et al. suggest that free-floating bacteria are the minority in nature by describing the biofilm as "the prevailing microbial lifestyle" [11, 12]. The biofilm helps bacteria by acting as armor against host defense responses and desiccation. It enhances survival across species by allowing bacteria to produce virulence factors, concentrate nutrients, and communicate with other bacterial species [13]. Biofilms are involved in many infections and are present in almost any environment – they form plaque on the teeth and can lead to corrosion of metal pipes. They are involved in recurrent infections from medical devices – from sutures to prostheses. Although they can also be found as floating mats submerged in or on top of liquids, they are usually sticky and adhere easily to any surface [14]. For example, *S. epidermidis* and *S. aureus* produce a protein called "adhesin", which functions as a glue, ensuring a strong adhesion between the biofilm and its host surface [15]. Once adhered, they are hard to dislodge, allowing the bacteria to

Biofilms are likely to grow wherever there is moisture, nutrients, and a surface [16]. These are all present at the lid margin, which has the added benefit of its inherent warmth. It is well known that the lid margin is home to normal flora bacteria consisting of mainly coagulase-negative species such as *S. epidermidis* [6]. It is also well known that species of Staphylococcus, especially *S. epidermidis*, produce biofilms [17]. In addition, a recent study by Kivanç demonstrated that 32 out of 34 isolates cultured from eyes immediately after cataract surgery were positive for being biofilm-forming species [18]. Taking all this information into account, it should come as no surprise that biofilms easily develop on the lid margin.

Furthermore, to avoid irritating our eyes with soap when we wash our face, we instinctively keep our eyes tightly shut, lid margin against lid margin, effectively blocking access to an area that needs cleaning as much as or more than any other area of the body. Therefore, the biofilm accumulates microscopically year after year, layer upon layer, without any removal. Even if home scrubs are attempted, the adhesin "glue" can prevent biofilm elimination. As patients age, the biofilm continues to accrue, leading to each of the stages of DEBS over time. This process starts much earlier in contact lens wearers, since the contact lens is itself an inert foreign body, producing a very early biofilm that allows protection for bacteria. Biofilm formation on contact lens and contact lens cases has been well documented [19]. This also helps explain why dry eye disease is more common in contact lens wearers,

The biofilm forms a multi-laminar substrate that provides more surface area for bacterial replication, which in turn leads to vast over-colonization of the surface. The over-colonization within the biofilm and increase in bacterial population density is what leads to quorum-sensing gene activation [21]. The discovery of quorumsensing gene activation by Hastings in 1999, was a groundbreaking study that lead to increased understanding of bacterial virulence [22]. Hastings demonstrated that populations of bacteria can sense when their densities achieve a certain quorum, and once that number or density is reached, dormant genes are activated [23]. The bacteria signal to each other using chemical messengers called homoserine lactones (HSLs) as well as through electric currents produced by potassium ions [24]. When enough bacteria are in close proximity to each other, the signals from these the surrounding bacteria sum to indicate a quorum [25]. These newly activated genes produce a wide array of virulence factors, many of which are extremely inflammatory. The bacteria wait to produce these factors until they have the protective biofilm in place to shield from the host immune response [26]. The inflammation from the host response to these virulence factors is the real destructive force in inflammatory lid disease, causing low-grade, chronic inflammation, beginning on

#### *Biofilm Theory for Lid Margin and Dry Eye Disease DOI: http://dx.doi.org/10.5772/intechopen.89969*

*Ocular Surface Diseases - Some Current Date on Tear Film Problem and Keratoconic Diagnosis*

colonization" of the eyelid margin [5]. Thygeson was describing the process by which our normal lid margin flora bacteria, primarily *Staphylococcus aureus* and *S. epidermidis*, gradually over-colonize the patient's lid margin, and over time, become pathogenic [6]. This is made possible by the bacterial biofilm, which allows the bacteria to thrive despite antimicrobials and the immune system [7]. Understanding biofilm progression links the shared underlying pathology between

*Biofilm theory of dry eye disease: Schematic of six steps of bacterial biofilm development leading to the stages of* 

Biofilms are defined as groups of microbial cells enclosed in a matrix made primarily of polysaccharide material that are intimately associated with a surface. Antonie van Leeuwenhoek is credited for the first observations of biofilm, when he described the biofilm on teeth in 1684. However, further study of biofilms was limited until the development of the electron microscope in the mid-1900s. Furthermore, it was not until 1982 that the term "biofilm" was introduced, after Costerton's observation of a *S. aureus* biofilm on a cardiac pacemaker lead [8]. More recent studies have shown that cell-to-cell interactions ("quorum sensing") within the biofilm upregulate certain gene products. Further studies have implicated biofilm in many disease processes including periodontitis, endocarditis, chronic prostatitis, and medical device associated infections, as the one described by

This chapter will explain the six steps by which our normal margin lid flora become pathogenic and cause eyelid inflammation. This inflammation, in turn, leads to the four stages of DEBS: folliculitis, meibomitis, lacrimalitis, and lid structure damage (**Figure 1**). This understanding will allow us to encompass dry eye disease, blepharitis, and meibomian gland dysfunction (MGD) in one disease

Bacteria were among the first forms of life on Earth and have survived billions of years in a myriad of different environments. While they are unicellular microorganisms, and can live in a free-floating form, the development of a biofilm provides

process, namely, dry eye blepharitis syndrome (DEBS).

**20**

**2. Biofilm**

dry eye and blepharitis.

**Figure 1.**

*DEBS.*

Costerton [8–10].

them a strong, virtually impenetrable defense structure [8]. Furthermore, Absalon et al. and Pickering et al. suggest that free-floating bacteria are the minority in nature by describing the biofilm as "the prevailing microbial lifestyle" [11, 12].

The biofilm helps bacteria by acting as armor against host defense responses and desiccation. It enhances survival across species by allowing bacteria to produce virulence factors, concentrate nutrients, and communicate with other bacterial species [13]. Biofilms are involved in many infections and are present in almost any environment – they form plaque on the teeth and can lead to corrosion of metal pipes. They are involved in recurrent infections from medical devices – from sutures to prostheses. Although they can also be found as floating mats submerged in or on top of liquids, they are usually sticky and adhere easily to any surface [14]. For example, *S. epidermidis* and *S. aureus* produce a protein called "adhesin", which functions as a glue, ensuring a strong adhesion between the biofilm and its host surface [15]. Once adhered, they are hard to dislodge, allowing the bacteria to remain in a desirable environment.

Biofilms are likely to grow wherever there is moisture, nutrients, and a surface [16]. These are all present at the lid margin, which has the added benefit of its inherent warmth. It is well known that the lid margin is home to normal flora bacteria consisting of mainly coagulase-negative species such as *S. epidermidis* [6]. It is also well known that species of Staphylococcus, especially *S. epidermidis*, produce biofilms [17]. In addition, a recent study by Kivanç demonstrated that 32 out of 34 isolates cultured from eyes immediately after cataract surgery were positive for being biofilm-forming species [18]. Taking all this information into account, it should come as no surprise that biofilms easily develop on the lid margin.

Furthermore, to avoid irritating our eyes with soap when we wash our face, we instinctively keep our eyes tightly shut, lid margin against lid margin, effectively blocking access to an area that needs cleaning as much as or more than any other area of the body. Therefore, the biofilm accumulates microscopically year after year, layer upon layer, without any removal. Even if home scrubs are attempted, the adhesin "glue" can prevent biofilm elimination. As patients age, the biofilm continues to accrue, leading to each of the stages of DEBS over time. This process starts much earlier in contact lens wearers, since the contact lens is itself an inert foreign body, producing a very early biofilm that allows protection for bacteria. Biofilm formation on contact lens and contact lens cases has been well documented [19]. This also helps explain why dry eye disease is more common in contact lens wearers, 50% compared to 14% in controls [20].

The biofilm forms a multi-laminar substrate that provides more surface area for bacterial replication, which in turn leads to vast over-colonization of the surface. The over-colonization within the biofilm and increase in bacterial population density is what leads to quorum-sensing gene activation [21]. The discovery of quorumsensing gene activation by Hastings in 1999, was a groundbreaking study that lead to increased understanding of bacterial virulence [22]. Hastings demonstrated that populations of bacteria can sense when their densities achieve a certain quorum, and once that number or density is reached, dormant genes are activated [23]. The bacteria signal to each other using chemical messengers called homoserine lactones (HSLs) as well as through electric currents produced by potassium ions [24]. When enough bacteria are in close proximity to each other, the signals from these the surrounding bacteria sum to indicate a quorum [25]. These newly activated genes produce a wide array of virulence factors, many of which are extremely inflammatory. The bacteria wait to produce these factors until they have the protective biofilm in place to shield from the host immune response [26]. The inflammation from the host response to these virulence factors is the real destructive force in inflammatory lid disease, causing low-grade, chronic inflammation, beginning on

the lid surface, the structures of the lid margin such as lash follicles, meibomian glands and connective tissue, and eventually affecting the accessory lacrimal glands as it progresses.

*S. epidermidis* produces a small amount of a moderate cytolytic toxin, a phenolsoluble modulin, but *S. aureus* produces two groups of highly destructive and immunogenic exoproteins: exotoxins and enzymes [27–29]. Many exotoxins are super-antigens that signal T cells to secrete large amounts of cytokines, and thus, massive inflammation. Exotoxins are responsible for toxic shock syndrome, food poisoning and scalded skin syndrome (toxic shock syndrome toxin, staphylococcal enterotoxins A-E and G-I, and exfoliative toxins A and B respectively) [30, 31]. The enzymes produced consist of nucleases, proteases, lipases, hyaluronidase, and collagenase, all capable of destroying host tissue [32]. Cytolytic toxins, including hemolysins and leukocidins, further contribute to the inflammatory cascade by destroying or damaging cells [33].

These toxins and enzymes permeate the biofilm and its surroundings, creating the same massive inflammation that leads to acute, severe debilitating disease as in scalded skin syndrome, food poisoning, and even death, as in the case of toxic shock syndrome [34]. As the biofilm spreads, more areas reach the quorum needed to activate virulence factors. Thus, inflammation spreads from the lid margin, to within the lash follicles, meibomian glands, accessory lacrimal glands, possibly to the main lacrimal gland and eventually to nerve endings and even the connective tissue of the eyelid, which can affect the structural integrity of the eyelids [35]. Decades of this toxicity, and the resulting inflammation, leads to nonselective damage [36]. While the body manages to ward off some of the effects of this toxic environment until later in life, eventually no part of the lid is immune to this chronic, progressive inflammation [37].
