The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements.

UNLABELLED: Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae. IMPORTANCE: Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium's growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae.

Cutaneous infection by Mycobacterium haemophilum and kansasii in an IgA-deficient man.

BACKGROUND: The prevalence of infections by nontuberculous mycobacteria (NTM) has steadily increased over the past decades, especially in immunocompromised patients. CASE PRESENTATION: We present a patient with IgA-deficiency and mixed cutaneous infection by two slowly growing mycobacteria, Mycobacterium (M.) haemophilum and M. kansasii. CONCLUSIONS: Cutaneous M. haemophilum infections most often result from HIV or transplantation-associated immunosuppression. Rarely, M. haemophilum may also infect healthy patients or iatrogenically immunosuppressed patients without transplantation. M. kansasii is one of the most frequent NTM and large awareness exists about its involvement in human diseases. Mycobacterial diagnosis of cutaneous infections should be considered in long-lasting skin lesions.

Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities.

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.

[Mycobacterium haemophilum as the cause of lymphadenitis in the neck in an otherwise healthy boy]. / Mycobacterium haemophilum als veroorzaker van lymphadenitis colli bij een niet-zieke jongen.

In an 8.5-year-old boy with severe recurring unilateral swelling of the neck, infection with Mycobacterium haemophilum was established. In addition to the more usual causative agents, cervical lymphadenopathy in children can, in rare cases, be caused by M. haemophilum. The skin tests did not differentiate between Mycobacterium avium and Mycobacterium tuberculosis infection. There was no evidence of specific immune deficiencies. The swelling did not respond to treatment with four tuberculostatic drugs (rifampicin, isoniazid, pyrazinamide and ethambutole), nor to repeated surgical excision. Finally, M. haemophilum grew on the culture despite sub-optimal culture conditions. Thereupon the patient was treated with co-trimoxazole and recovered in two weeks. This is the first patient with cervical lymphadenopathy caused by M. haemophilum to be described in the Netherlands. M. haemophilum grows on an iron-rich medium at a relatively low temperature (30-32 degrees C). In cases of cervical lymphadenopathy, it is advisable to consider M. haemophilum as a cause and to adjust the culture conditions accordingly.

Mycobacterium haemophilum infection in a Japanese patient with AIDS.

Mycobacterium haemophilum has been described as a pathogen that causes cutaneous lesions in immunocompromised patients. A specimen from a skin ulcer on the leg of a Japanese patient with acquired immunodeficiency syndrome yielded acid-fast bacilli on blood agar plates after 4 weeks of incubation at 30 degrees C, but the organism was not found on Ogawa egg slants. The organism was identified as M. haemophilum, on the basis of 16S rRNA gene sequence analysis. Prolonged culture in an optimal environment that includes an iron supplement, and growth temperatures at 28 degrees to 33 degrees C are necessary to grow M. haemophilum. Genotypic characterization of 16S rRNA is useful for a rapid diagnosis of this slowly growing mycobacterium.

[Mycobacterium haemophilum infection in a patient with AIDS]. / Mycobacterium-haemophilum-Infektion bei einem Patienten mit AIDS.

HISTORY AND FINDINGS: A 35-year-old HIV-infected man with a CD4 cell count of 100/microliter who had returned from a holiday in Spain presented with fever, chronic diarrhoea, cough, oral ulcers, subcutaneous nodules of about 1 cm in diameter and crusted skin ulcers of about 2 cm in diameter at his right arm, both wrists and buttocks. INVESTIGATIONS: Microscopic examination and culture of smears of a skin ulcer revealed acid-fast bacteria. Mycobacterial cultures of blood, sputum, urine and stool remained sterile. TREATMENT AND COURSE: Before the microorganisms were identified culturally, atypical mycobacteriosis was assumed and treatment with rifampicin, ethambutol, isoniazid and clarithromycin was started. Mycobacterium haemophilum was identified by using molecular biological techniques. Within 3 weeks the patient became afebrile and the skin ulcers healed completely. After a 7-week course, the treatment had to be stopped, and one month later painful subcutaneous nodules developed again at his arms and legs. A relapse of Mycobacterium haemophilum infection was confirmed by culture of a fine needle aspirate of a nodule. The same treatment was restarted and the nodules disappeared. CONCLUSIONS: Mycobacterium haemophilum, first identified in 1978, is an emerging pathogen in immunocompromised patients. Clinical manifestations usually are skin ulcers, subcutaneous nodules and subcutaneous abscesses, and less frequently, systemic infection. Treatment options of this life threatening disease have yet to be defined but therapeutic response to tuberculostatic combination therapy has been observed. Since Mycobacterium haemophilum is a fastidious organism, special laboratory methods are required for cultivation as well as for identification.

Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B).

We developed an in vitro model to study the temperature-regulated cytotoxicity and intracellular growth of Mycobacterium haemophilum in cultured human epithelial and endothelial cells. M. haemophilum associated with human epithelial and endothelial cells at similar rates when incubated at 33 and 37 degrees C, but only the epithelial cell line supported the multiplication of this organism. M. haemophilum grew equally well with epithelial cells at both temperatures. The aminoglycoside antibiotic amikacin was used to study the intracellular growth of M. haemophilum in the epithelial cells at 33 and 37 degrees C. Although an approximately equal number of bacteria were found within cells after 2 days of incubation at both temperatures, intracellular replication of M. haemophilum was 1,000-fold greater at 33 than at 37 degrees C. This intracellular multiplication was associated with destruction of the monolayers at 33 but not at 37 degrees C, and only culture filtrates from infected monolayers incubated at 33 degrees C were cytotoxic to fresh epithelial cell monolayers. This strain of M. haemophilum also produced contact-dependent hemolysis of sheep erythrocytes, demonstrating the possible presence of a cytolysin. These studies suggest that M. haemophilum has a preference for growth with cultured human epithelial cells. In addition, intracellular growth is best at 33 degrees C in epithelial cells, and this correlated with cytotoxicity at this temperature. This phenotype may be caused by induction of a soluble cytotoxic component, possibly a hemolytic cytolysin.