Microbiology and Infectious Diseases postgraduate teaching

Guest posting: Dr Syeda Navqi, Microbiology Registrar, Pathology North. 

Group: Aerobic actinomycetes

• Gram positive bacteria that are usually filamentous and branched, commonly producing a fungus like mycelium that fragments into rod shaped or short coccoid form.
• All grow better under aerobic than anaerobic conditions, a feature distinguishing them from most organisms in the genus Actinomyces.
• The organisms containing mycolic acids in their cell walls (included in the genera Nocardia, Rhodococcus, Gordonia, Tsukamurella, and Corynebacterium) are rather closely related on the basis of molecular genetic studies.  c.f. Streptomyces, Actinomadura, Dermatophilus have no mycolic acid.
• Nocardia is the most important genus as the most commonly isolated aerobic-actinomycete human pathogens.
• There are approximately 87 validly named species included in Nocardia genus.

Identification

 Microscopic Morphology

• Should be the initial step in organism identification
• On direct Gram smears, organisms generally appear as very long, obviously branching, thin, and finely beaded Gram-positive rods
• The modified acid-fast stain used on direct specimens as well as on colonial growth. Acid-fast cells will clearly be red; cells that stain purple or light pink may or may not be truly acid fast. The acid fast reaction has been reported to be most reliable when the test is performed with colonies after 1 to 4 weeks of growth.
• Growth requirement and Medium : Slow growing, require minimum 48-72 hrs before colonies appear, grow at room temp, incubate for 2-3 weeks.

Blood agar, chocolate agar, brain heart infusion agar, Sabouraud dextrose agar, and Lowenstein-Jensen medium support the growth of most aerobic actinomycetes; Buffered charcoal yeast extract agar (BCYE) is particularly useful for the recovery of Nocardia species. Specimens from sterile sites or concentrated sterile body fluids can be inoculated directly onto these media.

Specimens from respiratory sites, skin, and other potentially contaminated sites, such as mycetomas, should additionally be inoculated onto selective media, such as modified Thayer-Martin agar and selective BCYE (containing polymyxin B, anisomycin, and either vancomycin.)

Colonial morphology

• Variable
• Colony color may best be seen on the reverse when colonies are grown on translucent media (such as Sabouraud agar), as color may become obscured on the surface by the powdery aerial hyphae typically produced by members of this genu. Of the genera that are partially acid fast, only Nocardia species regularly produce aerial hyphae.
• Chalky, matte or velvety, powdery irregular, wrinkled, or smooth; generally apparent  pink, orange, red, purple, gray, yellow, peach, or white on the reverse; smooth or granular; soluble brown or yellow pigments may be produced.

• Nucleic acid amplification: molecular methods for the detection of Nocardia directly from clinical specimens are well developed
• MALDI-TOF: some limitations to date

Susceptibility Testing

• Basic biochemical testing may be paired with susceptibility testing to achieve preliminary identifications.
• Some species or species complexes have predictable susceptibility patterns that may assist in isolate characterization.
• These patterns should not be used exclusively as identification techniques, as many newly described species have not been tested for their antibiotic susceptibilities.

Epidemiology and clinical significance

• Normal inhabitants of soil and water that are responsible for decomposition of plant material.
• Facultative intracellular pathogen capable of growth in various human cells.
• Infections can occur in both immunocompetent and immunocompromised hosts.
• N. asteroides, N. brasiilensis and N. otitidiscavarium are the major cause of infections.
• Infections generally result either from trauma-related introduction of the organism or, particularly in immunocompromised patients, from inhalation and the resulting establishment of a pulmonary focus.
• Extrapulmonary disease  usually results from haematogenous spread from a pulmonary site; the brain is one of the most common secondary sites of infection.
• Various Nocardia species implicated as the causal agents of keratitis and other ocular infections.
• In 1988, a breakthrough in the clinically useful categorization of pathogenic nocardial isolates was provided by Wallace and his coworkers. They divided organisms phenotypically resembling N. asteroides into six different drug pattern types and one additional miscellaneous group. With more-recent molecular characterizations, numerous different species have been described within this set of organisms, which came to be known as the Nocardia asteroids complex. These include
• Nocardia abscessus (drug pattern I),
• Nocardia cyriacigeorgica (drug pattern VI),
• farcinica (drug pattern V),
• Nocardia nova (drugpattern III),
• Nocardia wallacei (drug pattern IV), and isolates of drug pattern II.
• Other new Nocardia species are continually being described, and undoubtedly,by current species definition criteria, many more will be described in the future.

Differentiation of Nocardia from other Aerobic actinomycetes