Staphylococcus aureus. Left—culture smear showing gram-positive cocci in pairs, short chains, and clusters (S. aureus, Gram stain, ×1000); right—blood agar showing heavy growth of opaque, golden yellow colonies of S. aureus
Laboratory diagnosis is usually simple as staphylococci grow abundantly on media such as blood agar, chocolate agar, nutrient agar, and many other media. Characteristic colonies coupled with a few biochemical reactions are adequate to differentiate S. aureus from coagulase-negative staphylococci. Accurate identification of coagulase-negative staphylococci is aided by currently available automated methods such as VITEK 2 compact system from bioMérieux, France.
Drug resistance is common among staphylococci, the first resistance developing against penicillin in 1940s. Methicillin was the first compound introduced to combat resistance to penicillin; however, soon methicillin-resistant Staphylococcus aureus (MRSA) emerged that exhibited resistance to many other antibiotics. Currently, vancomycin is the most effective antibiotic.
Streptococci are gram-positive cocci that occur in pairs and short or long chains, catalase negative, and nutritionally fastidious; they require blood-enriched media for growth. There are several systems of classification of streptococci, and one of them is based on hemolysis (beta-complete hemolysis, alpha-partial hemolysis with greenish discoloration, gamma-no hemolysis) on sheep or horse blood agar. One of the most important ophthalmic pathogens includes Streptococcus pneumoniae, also known as Diplococcus pneumoniae (Pneumococcus), which is typically lanceolate shaped, capsulated occurring in pairs (Fig. 26.2 left), alpha-hemolytic on blood agar, tiny, transparent or translucent on chocolate agar with greenish discoloration (Fig. 26.2 right), and susceptible to optochin (ethyl hydrocuprein). Other alpha-hemolytic streptococci (optochin resistant) are collectively known as “viridans streptococci” that constitute several species, which are normal commensal in the throat but are potentially opportunistic pathogens. Differentiation of the species and antibiotic susceptibility testing is important as alpha-hemolytic streptococci have been reported to be resistant to aminoglycosides, penicillins, and fluoroquinolones .
Streptococcus. Left—culture smear of Streptococcus pneumoniae showing gram-positive elongated (lanceolate shaped) cocci in pairs with mild capsule (Gram stain, ×1000); right—chocolate agar showing tiny translucent confluent colonies of Streptococcus pneumoniae grown in a vitreous drop (37 °C, 2 days)
The genus Bacillus includes several species of gram-positive, large, thick, often beaded, spore-forming bacilli that grow on ordinary media producing rapidly growing large colonies (Fig. 26.3 left). They are ubiquitous and most common laboratory contaminants. Bacillus cereus is the most common pathogen. Species identification requires VITEK system or other automated methods. They are known to be associated with severe posttraumatic endophthalmitis . They are susceptible to vancomycin, clindamycin, and several aminoglycosides.
Bacillus. Sheep blood agar (left) and chocolate agar (right) showing rough, dirty yellow, translucent, flat colonies of Bacillus cereus grown in vitreous drops (37 °C, 2 days)
Corynebacterium and Propionibacterium species
Corynebacterium and Propionibacterium are two genera that are similar in many respects except that the former is aerobic and the latter is anaerobic. Several species have been reported as normal commensals in the conjunctival sac and lid margins. They are low virulent organisms associated with chronic endophthalmitis. Joseph et al. have reported a series of 16 cases of Corynebacterium endophthalmitis, and the clinical setting included trauma, cataract surgery, and penetrating keratoplasty surgery . Corynebacterium and Propionibacterium are small gram-positive bacilli that are arranged in Chinese letter pattern (palisades, L and V shapes—Fig. 26.4 left), non-motile, catalase positive, and ferment carbohydrates. Corynebacterium produce lactic acid and Propionibacterium produce propionic acid. Both organisms grow slowly and require enriched media for growth and special biochemical tests to identify species. They are usually susceptible to various groups of antibiotics. Tiny, raised, translucent, nonhemolytic colonies of Corynebacterium species grown in vitreous sample are seen in Fig. 26.4 (right).
Left—culture smear of Corynebacterium species showing gram-positive bacilli arranged in Chinese letter pattern (Gram stain, ×1000); right—sheep blood agar showing tiny, yellowish raised, translucent, nonhemolytic colonies of Corynebacterium amycolatum grown in vitreous drops (37 °C, 3 days)
Nocardia and Mycobacterium species
Nocardia species are strict aerobic gram-positive, beaded, branching filamentous bacteria that may break into short bacilli (Fig. 26.5 left). They may appear as gram-negative filaments with gram-positive beads and are acid fast with weak acid (1% H2SO4) in modified Ziehl-Neelsen stain (Kinyoun stain) owing to mycolic acid in their cell wall. The colonies on blood and chocolate agar are often chalky white and dry (Fig. 26.5 right). They occur as saprophytes in nature and have been reported from posttraumatic as well as postoperative endophthalmitis . Amikacin is the drug of choice although the organism is susceptible to many other antibiotics. MALDI-ToF mass spectrometry is more reliable than biochemical reactions in species identification.
Left—gram-positive, thin, beaded, branching filaments of Nocardia asteroides (Gram stain, ×1000); right—tiny, chalky white, nonhemolytic colonies of Nocardia asteroides on sheep blood agar grown in vitreous drops (37 °C; 4 days)
Mycobacterium tuberculosis is a rare cause of endogenous endophthalmitis . Ocular infections are more commonly caused by atypical mycobacteria. M. chelonae and M. manitobense endophthalmitis are microbiologically challenging to diagnose [26, 27]. DNA sequencing was applied for the identification of M. manitobense cultured from vitreous of a patient with post-cataract surgery endophthalmitis .
Mycobacteria are gram-positive, slender, beaded bacilli that stain poorly with gram stain but are strongly acid fast in Ziehl-Neelsen stain. In contrast to M. tuberculosis, atypical mycobacteria are rapid growers and grow within 7 days on regular laboratory media such as blood agar and chocolate agar. Biochemical tests or molecular methods may help identify the species; VITEK cards are not available for Mycobacterium and Nocardia species identification. In recent times MALDI-ToF mass spectrometry has emerged as an useful tool for species identification.
Pseudomonas species and Burkholderia cepacia
Pseudomonads are saprophytic, ubiquitous, usually slender, gram-negative bacilli (Fig. 26.6 left). Unlike Enterobacteriaceae family members such as Klebsiella species and Escherichia coli, they are non-fermenters of sugars and oxidase positive. They are a common cause of endophthalmitis and are dreaded agent of cluster endophthalmitis. Most members possess potent virulence factors (enzymes and toxins) and can destroy tissues rapidly. P. aeruginosa is very virulent.
Left—slender gram-negative bacilli in culture smear of Pseudomonas aeruginosa (Gram stain, ×1000); right—large, flat, moist, greenish colonies of P. aeruginosa grown in the inoculum of vitreous on sheep blood agar (37 °C; 2 days)
Pseudomonads can utilize a variety of compounds for nutrition and grow even in distilled water. They produce non-lactose-fermenting colonies on MacConkey’s agar and utilize citrate, and most species are motile. VITEK 2 compact system is very reliable for species determination. P. aeruginosa often produces large, gray, moist colonies with greenish pigment and beta-hemolysis on blood agar (Fig. 26.6 right).
Antimicrobial resistance among P. aeruginosa is a global concern. Nosocomially acquired isolates tend to be more resistant than community-acquired strains. Several mechanisms such as mutations in genes encoding porins, efflux pumps, penicillin-binding proteins, and chromosomal beta-lactamase contribute to the resistance. In addition P. aeruginosa strains may contain extended spectrum beta-lactamases and metallo-beta-lactamases that can degrade imipenem. Rise in multidrug-resistant P. aeruginosa has resulted in revisiting the use of toxic drugs like colistin and polymyxin B.