Problemas de valor inicial

GRAM-POSITIVE BACTERIA

Gram-positive bacteria have a high content of peptido- glycan and a low content of lipid compared to gram- negative bacteria. When the lipid layer is dissolved, crystal violet and iodine form a complex on the cell wall that appears blue under the microscope, depicting gram- positive bacteria.

The most common organisms which infect the eye are the Staphylococcus aureus, Coagulase negative Staphy- lococci, Streptococcus pneumoniae and Streptococcus

viridans.

Staphylococcus aureus

Staphylococcus aureus are gram-positive cocci (0.5 to

1.5 μm), which are seen under the microscope in pairs or grape like clusters and grow as routine culture media within 18 to 24 hours. On blood agar, they appear as golden hemolysis (clear area). They are non-motile, non- spore forming, facultative anaerobes and are usually encapsulated. They are coagulase and catalase positive. Staphylococcus species resistant to oxacillin agents constitutes the methicillin resistant Staphylococcus aureus (MRSA).

Antibiotic susceptibility: The widespread use of

aminoglycosides, penicillins, cephalosporins and fluoroquinolones (third generation) is responsible for the increasing resistance of these organisms against these

drugs. They are susceptible to, bacitracin, chlorampheni- col, cefazolin and fluoroquinolones.

Coagulase Negative Staphylococci

The genus Staphylococcus consists of 32 species out of which 3 are coagulase positive and the rest are coagulase negative. Coagulase negative Staphylococci are normal inhabitants of human body especially the skin, mucous membrane and the eyelid margins.

Coagulase negative Staphylococci are microscopi- cally similar to Staphylococcus aureus and appear in pairs or grape like clusters, but on blood agar isolation, they appear as white to grayish colonies within 24 to 48 hours of incubation. These bacteria are non-motile, non-spore forming, facultative anaerobes, usually non-encapsula- ted, catalase positive. The most common bacteria implicated in bacterial keratitis is negative coagulase

Staphylococcus.

Antibiotic susceptibility: Just like Staphylococcus aureus the

resistance rate of 55 percent has been noted to fluoro- quinolones in coagulase negative Staphylococcus. They are susceptible to vancomycin, bacitracin and chloram- phenicol.

MICROCOCCUS SPECIES

They are gram-positive cocci, which may be present as saprophytes and inhabit the eyelid margins. They appear on blood agar as yellow distinct colonies.

Streptococcus and Related Bacteria

Streptococci appear microscopically as gram-positive cocci (Fig. 3.9), usually coccoid or coccobacilli and appear in chains of cocci on broth medium. They may be present as normal inhabitants especially in children. Streptococci are distinguished from Staphylococcus and Micrococcus species by negative catalase reaction S.pneumoniae is less virulent as compared to S.viridans. They are responsible for corneal ulcers, recalcitrant graft infections and infectious crystalline keratopathy.

Streptococcus species are differentiated by their

hemolysis patterns on blood supplemented agar media into the following:

1. Alpha-hemolytic streptococci which partially lyse the red blood cells and a greenish halo appears around a colony. Examples of alpha-hemolytic streptococci include Streptococcus pneumoniae and Streptococcus

viridans.

Figure 3.9: Gram-positive pneumococci (Courtesy: Dr H Sheorey,

2. Beta-hemolytic streptococci lyse red blood cells completely and a clear halo appears around a colony. An example of beta-hemolytic streptococci is Strepto-

coccus pyogenes which when identified from cornea

is pathogenic.

3. Gamma-hemolytic or non-hemolytic streptococci which have no hemolysis pattern around a colony. Antibiotic Susceptibility

They exhibit high resistance to polymyxin. They are susceptible to cefazolin, bacitracin, chloramphenicol and sulphacetamide. The in vitro susceptibility of Streptococ- cus species to fluoroquinolones is low.

Enterococcus

Enterococcus species are gram-positive ovoid cocci or

coccobacilli and are isolated or broth media with or without red blood supplementation. Enterococcus fecalis is the most common species isolated from the cornea which can cause corneal ulcer.

Diphtheroids

Diphtheroids appear microscopically as gram-positive pleomorphic rods. They are aerobic non-spore formers. The most common diphtheroid isolated from the compromised corneas include Corynebacterium and

Propionibacterium. They grow well on blood-supplemen-

ted media, in a CO2 atmosphere but it may require 24 to 48 hours extra for the colonies to appear. Enriched thioglycollate, liquid broth is a good medium for the colonies to appear. Diphtheroids are known to occur as saprophytes, although in compromised conditions they may also cause infectious keratitis.

Corynebacterium diphtheriae and Listeria monocytogenes

are corneal pathogens, which can breach the intact epithelium without prior trauma.

Antibiotic susceptibility: They are susceptible to vanco-

mycin, chloramphenicol, ofloxacin and partially to cefazolin. Diphtheroids demonstrate a good in vitro susceptibility to most antibiotics except to trimethoprim.

Mycobacterium

Mycobacteria are obligate parasites and opportunistic pathogens. They are gram-positive bacteria which are slightly curved or straight bacilli. They appear as “ghosts” or beaded gram-positive rods on Gram stain.

Mycobacterium chelonae and Mycobacterium fortuitum are

responsible for keratitis after refractive surgery. On blood agar and chocolate agar plates, the colonies may take a week to appear. Routine mycobacterial isolation medium such as Löwenstein-Jensen medium is more definitive for isolation or organisms.

When mycobacteria are suspected to cause keratitis, special acid-fast stains should be used to stain them as they have a cell wall with high content of lipid which resists Gram staining.

Antibiotic susceptibility: They are susceptible to clarithro-

mycin and amikacin.

Bacillus

Bacillus species are large, gram-positive rods but may

sometimes be gram-variable. They are spore bearing, catalase positive, motile and grow aerobically or as facultative anaerobes. The most important corneal pathogens are Bacillus cereus and Bacillus anthracis, which appear as large, grainy, dry beta-hemolytic colony on agar media. Most organisms are opportunistic pathogens.

Antibiotic susceptibility: They are susceptible to aminogly-

cosides, fluoroquinolones and sulphacetamides.

Nocardia

Nocardia species belong to the group of actinomycetes

and appear as gram-positive, branching, filamentous bacteria. They are aerobic, non-motile, partially acid-fast and appear as white, tiny, dry colonies.

They are associated with infections after trauma, contact lens wear and following laser in situ kerato- mileusis (LASIK) surgery. The pathogenic species are

Nocardia asteroids and Nocardia brasiliensis.

Antibiotic susceptibility: They are susceptible to sulpha-

cetamide, trimethoprim – sulfamethoxazole and amikacin.

GRAM-NEGATIVE BACTERIA

Gram-negative bacteria appear red because the lipid layer is not removed by the decolorizing step. A blue crystal violet complex cannot form in the cell wall and safranin counter stains the bacteria (Fig. 3.10).

The common gram-negative bacteria, which infect cornea include Pseudomonas aeruginosa, Serratia marce-

19 scens, Moraxella species and Haemophilus species.

Generally gram-negative infection occurs due to bacilli. Rarely, gram-negative cocci may cause infection such as in cases of Neisseria gonorrhoeae, Neisseria meningitides and Branhamella catarrhalis.

Pseudomonas aeruginosa

P. aeruginosa are aerobic bacteria, which appear as gram-

negative bacilli. It grows between 30-37°C and on trypticase broth supplemented with 5 percent sheep blood appear grayish or greenish, metallic-appearing as gelatinous colonies. These colonies have a grape-like odor. The colonies of P. aeruginosa are oxidase positive. The infections due to this organism are generally fulminant because of the presence of virulence factors (exotoxin A, proteolytic enzymes), presence of pili, which attach to the cells and production of alginate, which is a polysaccharide polymer, which inhibits phagocytosis.

Contact lens wearers, debilitated patients, patients with systemic diseases and in intensive care units are at risk for Pseudomonas keratitis.

SERRATIA MARCESCENS

Serratia marcescens is an opportunistic pathogen, which

along with corneal epithelial breakdown due to trauma or contact lenses can lead to corneal ulceration. On Gram stain they appear as coccobacilli and its colonies are reddish in color. They are a frequent contaminant of contact lenses and contact lens solutions.

Antibiotic susceptibility: They are susceptible to fluoroqui-

nolones and aminoglycosides and have intermediate susceptibility to chloramphenicol, trimethoprim and sulfasoxazole.

MORAXELLA

Microscopically, they appear as gram-negative brick shaped diplobacilli (Fig. 3.11). They cause chronic conjunctivitis and acute corneal ulceration. Moraxella

lacunata is the commonest species involved. The colonies

appear grayish in color and appear pitted. HAEMOPHILUS

Haemophilus appear as tiny gram-negative coccobacilli. These bacteria require factor V and factor X for their growth and hence grow better on chocolate agar than on blood agar. The colonies appear as grayish in color and have a musty smell.

GLUCOSE FERMENTERS AND NON-FERMENTERS Glucose fermenters include Stenotrophomonas and

Alcaligenes species and glucose non-fermenters include Escherichia coli, Enterobacter, Klebsiella and Proteus species.