Nocardia Identification at the Genus Level　– Reply –

We appreciate Dr. Fatahi-Bafghi’s comments on our case report published in the Circulation Journal.¹ Dr. Fatahi-Bafghi’s insightful comments included 2 important points. First, the method that we used to isolate Nocardia was unclear. Second, Dr. Fatahi-Bafghi questioned how Nocardia could be identified at the genus level, because other genera in the Actinomycetaceae (i.e., non-Mycobacterium tuberculosis, Gordonia, etc.) are similar to Nocardia in some phenotypic characteristics. Our answers to these comments are given below.

With regard to the bacterial isolation method, we had obtained the sample from pus in the autopsied heart. The bacteria were first cultured on sheep blood agar (Eiken Chemical Co., Ltd, Japan) for 24 h under a capneic environment (5% CO₂, 35℃), and then cultured for a further 24 h at room temperature. Fine, white colonies (<1 mm) were observed at 48 h, and dry colonies emitting a soil odor were observed at 72 h, indicating they were nocardial. Although anaerobic culturing was performed, no bacteria were observed.

With regard to the genus identification, Nocardia species are gram-positive, weakly acid-fast, and filamentous aerobic actinomycetes. In this case, the Gram stain revealed gram-positive branching filamentous rods. The Kinyoun acid-fast stain, using weak acid (0.5% sulfuric acid) for decolorization, showed partial acid-fast staining of the bacterial cells, whereas the Ziehl-Neelsen stain showed non-acid-fast staining. Microscopic findings of the colonies, which were cultured on Mueller-Hinton agar (Eiken Chemical Co., Ltd, Japan) under a capneic environment (5% CO₂) for 24 h, revealed branching hyphae. Although Actinomadura, Actinomyces, and Streptomyces also have branching filaments and hyphae, only Nocardia has the acid-fast property. Non-mycobacterium tuberculosis is different from Nocardia in terms of the acid-fast staining by both Kinyoun acid-fast and Ziehl-Neelsen stains. Gordonia does not have branching filaments and its colonies do not have a soil odor. These findings can be helpful to identify Nocardia at the genus level.

In our hospital, the diagnosis of Nocardia spp. is decided by colony morphology and smell, the presence of branching hyphae by microscopic analysis, its absence in anaerobic culture, and the results of several staining assays (i.e., gram-positive with the Gram stain, acid-fast with the Kinyoun stain, and non-acid-fast with the Ziehl-Neelsen stain). A limitation of our study was that the biochemical characteristics or phylogenetic analysis of 16S rDNA could not be obtained, so the specific species of Nocardia was not identified in this case. However, antibiotic susceptibility testing showed the Nocardia spp. to be susceptible to imipenem, tobramycin, amikacin, and minocycline, and resistant to ampicillin, indicating that it might be N. asteroides complex or N. farcinica.² Needless to say, because treatment of nocardiosis is dependent on the species, it is important to identify Nocardia at the species level.

Acknowledgments

The authors thank Masami Sato (Division of Medical Technology, Tokushima University Hospital) for her technical comments about the method and discussion.

Disclosures

The authors have no conflicts of interest to disclose.

• Akira Takashima, MD, PhD
• Shusuke Yagi, MD, PhD
• Hiroyuki Ito, MD
• Masataka Sata, MD, PhD
• Department of Cardiovascular Medicine, Tokushima University Hospital, Tokushima, Japan

References

• 1.    Takashima  A,  Yagi  S,  Yamaguchi  K,  Watanabe  S,  Yamamoto  N,  Ito  H, et al. Purulent pericarditis accompanying pericardial abscess induced by Nocardia in an immunocompromised patient. Circ J 2016; 80: 2409–2411.
• 2.    Brown-Elliott  BA,  Brown  JM,  Conville  PS,  Wallace  RJ Jr. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 2006; 19: 259–282.