Gram-Negative Bacilli in Ocular Disease



Gram-Negative Bacilli in Ocular Disease


Nada S. Jabbur

Terrence P. O’Brien



Bacteria can cause disease in host tissue by various mechanisms. Gram-negative bacteria have a unique outer membrane containing lipopolysaccharides (LPS) that include a molecular organization pathogenic to the host. Host interaction and colonization are facilitated by complex outwardly radiating organelles, flagella (arising from basal membrane structures and used for locomotion), and pili (used for adherence to the target cell). When present, capsules made of slime-like polysaccharides make phagocytosis difficult. The bacteria also produce a myriad of proteolytic enzymes aimed at destroying the host cell. The host cell surface carries glycoproteins and glycolipids that act as receptors for microbial attachment. Some bacteria have invasive properties that allow them to invade host defenses. Biofilm formation (when bacteria adhere to prosthetic devices), hypoxia, and the host’s immune status are significant enhancers of pathogenesis. Finally, bacteria have developed different strategies for antibiotic resistance.

Gram-negative rods have been identified as major pathogens in major human organs, including the eye where infection can result in rapid destruction of ocular tissues. Species of Pseudomonas,1,2,3,4,5,6 Moraxella,7,8,9,10,11,12,13 Acinetobacter,14,15,16 Haemophilus,17,18,19,20 Brucella,21 Francisella,22 Pasteurella,23 Capnocytophaga,24,25 Aeromonas,26,27 Alcaligenes28 and the Enterobacteriaceae, including Escherichia,29 Shigella,30 Klebsiella,31 Proteus,32,33,34 Serratia,35,36,37 Yersinia38,39 and Enterobacter,40,41 Helicobacter,42 Salmonella,43,44,45 and Achromobacter xylosoxidans46 have all been clinically identified as human ocular pathogens.

Pseudomonas aeruginosa keratitis, the most common ocular infection caused by a gram-negative rod, can lead to blindness. The pathogenesis of Pseudomonas keratitis is multifactorial and has been studied in murine and rabbit models. Not many experimental models, however, have focused on other gram-negative rod ocular infections. Recently, Hazlett and her coworkers showed that the contact lens-wearing rat model allows a unique analysis of the early effects of bacterial challenge in extended-wear contact lenses in the absence of corneal scarring, used in most rodent models. The rat model should be valuable to delineate further the effects of contact lens wear, including the testing of additional contact lens-related complications.47 To understand the pathogenesis of disease caused by gram-negative bacilli, researchers have indirectly gained knowledge from nonocular animal models such as those of Escherichia coli pyelonephritis48,49 and Klebsiella pneumoniae pneumonia.50


PATHOGENESIS AND ANIMAL MODELS OF INFECTION


Microorganism Virulence


Endotoxin

The unique outer membrane of gram-negative bacteria, containing phospholipids, LPS, and proteins, has been implicated as a major player in pathogenesis. The LPS portion of the envelope is both a major cell surface antigen and a principal molecule triggering the host response to bacterial infection. It is made up of three regions: the O-specific region (region I), responsible for the O or somatic antigenicity of the species and subspecies; the core polysaccharide (region II); and lipid A (region III), which is closest to the cell wall. The term “endotoxin” was used before discovering that it is the lipid A portion of the LPS. Different sugar sequences, linkage groups, and substituents cause different antigenic (serologic) specificities. The presence of O-antigen in Serratia marcescens enhances its adherence to inert and biologic surfaces.51 Region II is less variable than region I. Region III is highly conserved among eukaryotes, with little microheterogeneity among genera and species. Several members of the family Enterobacteriaceae express additional antigenic polysaccharides in the form of capsular or K antigens and flagellar H proteins.

Lipid A is crucial in that it is responsible for the pathophysiologic effects associated with gram-negative bacterial infections and bacteremia, including pyrogenesis as well as hematologic, immune, and endocrinologic effects. Systemic exposures to endotoxin may result in hypotension, disseminated intravascular coagulation, and death. Most of these effects are mediated by release of cytokines and some by clotting and activation of complement.


Capsule

Encapsulated strains of bacteria have capsules that consist of high-molecular-weight polysaccharides that form gels and allow bacteria to adhere to the host target cell surface. The capsule acts as a protective “shield” because it is hydrophilic and poorly immunogenic. First, the polysaccharide-rich composition of the capsule strongly inhibits phagocytosis by the hydrophobic surface of the host cell. Second, many capsules are poor immunogens52 and complement activators.53 If phagocytosis occurs, it requires specific opsonizing antibodies against the capsule. The thickness of the capsule in Francisella has been correlated with virulence, and in Brucella, encapsulated smooth colonies are ingested less readily than rough colonies.54 The virulence of Klebsiella50 and Yersinia55 species has also been correlated with capsular presence and protection. Immunologic diversity of the capsule within the same species explains the basis for stereotyping. Although most ocular isolates of Haemophilus influenzae are non-encapsulated, the encapsulated form of type b is more virulent than the encapsulated type d.56


Flagella and Fimbriae (Pili)

When seen with transmission electron microscopy (TEM), the outer membrane of a gram-negative rod can take on a peritrichous appearance because of the flagella and more numerous pili that surround it. Flagella are larger than pili (they measure 16 to 18 nm in diameter) and allow for motility of the organism toward and within the host tissue. Ninety-five percent of clinical isolates of Pseudomonas are flagellated,57 and the burned-mouse model of P. aeruginosa has shown that flagella-deficient mutants are significantly less virulent.58 Flagellar proteins have been studied as vaccines. Rudner and associates59 showed that systemic or topical immunization of mice with strain-specific flagellar proteins or antiflagellar antibody homologous to the specific strain of bacteria protected them from pseudomonal keratitis.

TEM also reveals fimbriae, also known as pili, that measure 4 to 10 nanometers in diameter. These microfibrils can vary in number (2 to 12 in Pseudomonas) and can be classified according to their function as adhesins, lectins, evasins, aggressins, and sex pili. Fimbriated cells adhere to surfaces with specificity and thus allow colonization of a specific host tissue cell. Attachment to host cells is a first step in Pseudomonas keratitis. Stern and colleagues60 demonstrated that murine corneal trauma predisposes to ulceration, not by increasing the exposed area of deepithelialized stroma for entry of the organisms, but by providing an injured epithelial edge to which P. aeruginosa can adhere. Purified Pseudomonas pili were found to compete with whole bacteria in saturating the binding receptors of the host tissue.61 Knutton and coworkers,62 in their experiments with enterotoxigenic E. coli, showed that before invasion, these microorganisms adhere to the mucosal surface of intestinal cells with pili that allow them to hold on to specific mucosal receptors.


Proteolytic Enzymes

Exotoxin A,63 alkaline protease,64 exoenzyme S,65 phospholipase C,66 hemolysin,67 and elastase68 have all been implicated as players in the pathogenesis of Pseudomonas infections. Whereas endotoxins are part of the cellular wall, exotoxins (also known as cytolysins) are extracellular enzymes and are easily separated from the envelope. Most of the cytolysins that have been isolated and purified are made up of two components. One part binds to the target cell and allows the other part, the enzyme, to pass through the cell membrane. After preparing a specific combination of amino acids and a metal-chelating moiety as an inhibitor to Pseudomonas elastase (a zinc metal-loendopeptidase), Kessler and associates68 demonstrated that intrastromal injection of the inhibitor first, followed by elastase, prevented corneal melting. Furthermore, subconjunctival injections of the elastase and inhibitor only delayed corneal melting, suggesting that the reversal of corneal melt by the elastase is better carried out intrastromally. Purified P. aeruginosa hemolysin injected into the corneas of rabbits was found to induce an extensive leukocytic invasion of the corneal stroma.67 In Moraxella angular conjunctivitis, the pathogenesis for lid maceration is caused by proteases from inflammatory cells rather than proteases elaborated by Moraxella.69 Both encapsulated and untypable isolates of H. influenzae produce an IgA protease that degrades the protective secretory IgA (sIgA) elaborated by host mucosal surfaces.70,71


Bacterial Receptors and Host Ocular Surface


Host Surface Glycoproteins and Glycolipids

Adherence of bacteria to the corneal epithelium is a prerequisite for keratitis. Both P. aeruginosa and Staphylococcus aureus were found to bind to rabbit corneal epithelial cells in vitro. P. aeruginosa bound, in multiple layers, to the periphery of cells grown on glass slides, whereas S. aureus bound more randomly to the cell surface. E. coli did not bind significantly to those cells. The peripheral location of Pseudomonas binding is probably caused by its affinity to macromolecules of the cell surface involved in cell-cell interaction.72 Panjwani and coworkers73 also demonstrated that P. aeruginosa binds to rabbit corneal neutral glycosphingolipids. They later demonstrated that P. aeruginosa also binds to specific phospholipids (phosphatidylinositol and phosphatidylserine) extracted from rabbit corneal epithelium.74 These two molecules in ocular mucus or at the corneal surface may function as bacterial receptors and allow specific host-bacterium interaction and initial colonization.

Pseudomonas pili also bind to corneal epithelial receptors, which are glycoproteins.75 More specifically, such carbohydrate receptors have been studied and include sialic acid,76,77 N-acetylmannosamine,78 mannose,79 galactose,80 N-acetylglucosamine,81 and L-fucose.82 Although rabbit and human corneal epithelial cell models were not able to show detectable levels of the glycolipid asialo GM1,83 the incidence of murine P. aeruginosa keratitis was significantly reduced after mouse corneas infected with Pseudomonas were treated with a serum containing antibodies specific to asialo GM1, a glycolipid to which bacterial pili and LPS usually bind. This experiment, performed by Hazlett and associates,84 provides evidence that antibodies against host corneal receptors can significantly inhibit bacterial binding in vitro. In addition, when applied topically in vivo, the antibodies did confer some immunity and decreased the severity of the disease. Also Hazlett et al. have shown that vasoactive intestinal peptide (VIP), which is an antiinflammatory neuropeptide, downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis85 and that testican 1, also known as SPOCK which is a highly conserved proteoglycan with protease activity, promotes resistance against P. aeruginosa-induced keratitis through regulation of MMP-2 expression and activation.86 Earlier on, Hazlett et al. had shown that the neuropeptide substance P (SP), among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the antiinflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.87

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Jul 11, 2016 | Posted by in OPHTHALMOLOGY | Comments Off on Gram-Negative Bacilli in Ocular Disease

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