Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum.

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔkstD, that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A, MFKDΔhsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔhsd4AΔfadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

Native valve endocarditis caused by Mycobacterium fortuitum in a patient of carcinoma breast.

We report an unusual case of native mitral valve endocarditis in a patient with carcinoma breast in remission. She presented with intermittent fever for 4 weeks. The patient had a chemo port in situ. Blood cultures flagged positive on the 3rd day of incubation. Staining revealed branching acid-fast bacilli, which were subsequently identified as Mycobacterium fortuitum using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The patient responded well to medical management alone. Only two such cases have been reported from India previously.

Proteomic analysis of nontuberculous bacteria protein spectra as the element of subtyping of strains.

Background: For the present, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry is the fastest and the most correct method for species identification of microorganisms. Apart from species-level identification, it allows to use a variety of approaches for the analysis and comparison of protein spectra of microorganisms of the same species, which are isolated from a patient at various disease states, that can be used in routine microbiological practice in laboratories fitted with mass analyzers. Methods: Two strains of Mycobacterium fortuitum and two strains of Mycobacterium peregrinum were isolated from sputum samples, which were obtained from patients with different clinical aspects of mycobacteriosis, whereat were reinoculated on the universal chromogenic culture medium "UriSelect 4." Further, the MALDI-ToF mass spectrometry method was used, aiming to obtain protein profiles, which were analyzed using the FlexAnalysis 3.0 software package. Results of the statistical proteomic comparison of mass spectra were visualized using MALDI Biotyper 3.0 Offline Classification software. Results: Presented clinical examples demonstrate that strains of the same species, which are isolated from the same patient at different times of infection, change their cultural properties. Dynamic changes in cultural properties are reflected in changes in protein profiles by comparison spectra of isolates at different stages of colonization, which is reflected in the correlation with the clinical condition of the patient. Conclusion: Thus, the mentioned examples of proteomic analysis, using MALDI-ToF mass spectrometry, demonstrate the possibility of subtyping of strains, that are isolated on a universal chromogenic culture medium, in case of detection in the culture signs of population's heterogeneity, based on cultural properties.

Historical evolution of the diseases caused by non-pigmented rapidly growing mycobacteria in a University Hospital / Evolución histórica de las enfermedades causadas por micobacterias no pigmentadas de crecimiento rápido en un Hospital Universitario

INTRODUCTION: Non-pigmented rapidly growing mycobacteria (NPRGM) are a group of organisms of increasing interest due to the growing number of potential patients and the difficulties for a proper treatment in many of them. However, the evolution of these diseases in a long period of time and its evolutionary changes has been described only in a scanty number of reports. MATERIAL AND METHODS: We performed a retrospective study between January 1st 2004 and December 31st 2017 in order to evaluate the clinical significance and types of diseases caused by NPRGM. Patients with isolates of NPRGM during this period were selected for the study, and clinical charts were reviewed using a predefined protocol. RESULTS: During this period we identified 59 patients (76 clinical samples) with isolates of NPRGM, with 12 cases of clinical disease and one patient with doubtful significance (including 6 respiratory tract infections, 2 catheter infections, 1 skin and soft tissue infection, 1 disseminated infection, 1 conjunctivitis, 1 prosthetic joint infection and 1 mastitis). Fifty percent of M. chelonae isolates, 37.5% of M. abscessus isolates and 23.33% of M. fortuitum isolates were clinically significant. None of the isolates of other species were significant. CONCLUSIONS: Most isolates in respiratory samples were contaminants/colonizations. M. abscessus was the main etiological agent in respiratory syndromes, whereas M. chelonae and M. fortuitum were more frequently associated with other infections, especially clinical devices and skin and soft tissue infections

INTRODUCCIÓN: Las micobacterias no pigmentadas de crecimiento rápido (MNPCR) son un grupo de organismos de interés creciente debido al número cada vez mayor de pacientes potenciales y a las dificultades en el tratamiento. Sin embargo, el número de estudios que analizan la evolución de estos casos a lo largo de un periodo de tiempo largo es escaso. MATERIAL Y MÉTODOS: Se realizó un estudio retrospectivo entre el 1 de enero de 2004 y el 31 de diciembre de 2017 para evaluar el significado clínico y los tipos de enfermedades causados por MNPCR. Se seleccionaron para ello aquellos pacientes con aislamientos de MNPCR, y se revisaron las historias clínicas mediante un protocolo predefinido. RESULTADOS: Se identificaron 59 pacientes (76 muestras) con aislamientos de MNPCR, de los cuales 12 presentaron enfermedad y uno tuvo un significado dudoso (incluyendo 6 infecciones respiratorias, 2 infecciones asociadas a catéter, 1 infección de piel y partes blandas, 1 infección diseminada, 1 conjuntivitis, 1 infección de prótesis osteoarticular y 1 mastitis). El 50 % de los aislamientos de Mycobacterium chelonae, el 37,5 % de Mycobacterium abscessus y el 23,33 % de Mycobacterium fortuitum fueron clínicamente significativos. Ninguno de los aislamientos de otras especies fue significativo. CONCLUSIONES: La mayoría de los aislamientos de muestras respiratorias resultaron ser contaminantes/colonizaciones. M. abscessus fue el principal agente etiológico en las infecciones respiratorias, mientras que M. chelonae y M. fortuitum fueron asociados con mayor frecuencia a otras infecciones, especialmente infecciones de piel y partes blandas e infecciones asociadas a dispositivos biomédicos

Prevalence and Species Spectrum of Pulmonary Nontuberculous Mycobacteria Isolates at a Tertiary Care Center / 대한임상미생물학회지

BACKGROUND: Pulmonary infection with nontuberculous mycobacteria (NTM) is increasing in South Korea. Since treatment strategy differs by NTM species, accurate identification is necessary. In this study, using Mycobacterium pulmonary isolates recently recovered from a general hospital in Seoul, the prevalence of NTM isolates was investigated. METHODS: A total of 483 Mycobacterium pulmonary strains isolated between May and November 2018 from an 814-bed general hospital in South Korea were analyzed. Bacterial species were identified based on nucleotide sequences of the 16S–23S rDNA internal transcribed spacer and the rpoB gene. RESULTS: From a total of 1,209 pulmonary specimens from patients suspected to be infected with mycobacteria, 324 deduplicate strains were isolated, comprising 90 Mycobacterium tuberculosis and 229 NTM strains. Among the NTM isolates, 61.5% (n=144) were Mycobacterium avium complex (MAC), including 92 M. avium and 52 Mycobacterium intracellulare, while 8.1% (n=19) represented Mycobacterium abscessus, including 10 M. abscessus subsp. abscessus and 9 M. abscessus subsp. massiliense. In addition, 12 (5.1%) Mycobacterium lentiflavum, 12 (5.1%) Mycobacterium gordonae, 6 (2.6%) Mycobacterium kansasii, and 5 (2.1%) Mycobacterium fortuitum were identified. In addition, Mycobacterium mucogenicum (n=2), Mycobacterium septicum (n=1), Mycobacterium colombiens (n=1), Mycobacterium asiaticum (n=1), and Mycobacterium celatum (n=1) were identified. CONCLUSION: Among the recently recovered Mycobacterium pulmonary strains, more than half were identified as NTM, and MAC was the most prevalent NTM, followed by M. abcessuss.

Coinfección por Mycobacterium fortuitum y Mycobacterium tuberculosis en abscesos esplénicos en un paciente con VIH / Co-infection by Mycobacterium fortuitum and Mycobacterium tuberculosis in splenic abscesses in a patient with HIV

RESUMEN Los pacientes con VIH son susceptibles a la infección por micobacterias. En el caso de las micobacterias de crecimiento rápido, grupo al que pertenece el Mycobacterium fortuitum (M. fortuitum), se han descrito infecciones en la piel, pulmones, ganglios linfáticos y enfermedad diseminada. Presentamos el caso de un paciente varón de 43 años, con diagnóstico previo de VIH en tratamiento antirretroviral, que acude por fiebre, astenia, pérdida de peso y diarrea crónica. Se realiza tomografía abdominal y se evidencia hepatoesplenomegalia con lesiones nodulares en el bazo. El cultivo esplénico, finalmente, fue positivo para M. fortuitum, con PCR positivo a Mycobacterium tuberculosis. Los protocolos actuales de tratamiento para este tipo de infecciones se basan en la susceptibilidad mostrada en los cultivos realizados. En lo que respecta a coinfecciones entre M. fortuitum y Mycobacterium tuberculosis, en pacientes VIH positivos, la información es aun menor.

ABSTRACT Patients with HIV are susceptible to mycobacterium infection. In the case of fast-growing mycobacteria, the group to which Mycobacterium fortuitum (M. fortuitum) belongs, infections have been described in the skin, lungs, lymph nodes and disseminated disease. We present the case of a 43-year-old male patient with pre-diagnosis of HIV in antiretroviral therapy, which comes as a fever, asthenia, weight loss and chronic diarrhea. Abdominal tomography is performed and hepatosplenomegaly is evidenced with nodular lesions in the spleen. The splenic culture was finally positive for M. Fortuitum, with positive PCR to Mycobacterium tuberculosis. The current treatment protocols for this type of infection are based on the susceptibility shown in the cultures performed. With regard to coinfections between M. Fortuitum and Mycobacterium tuberculosis, in HIV positive patients, there are even less information.

Antimicrobial Susceptibility Testing and Molecular Characterization of Mycobacterium fortuitum Isolates in China / 生物医学与环境科学（英文）

We performed molecular identification of clinical isolates of Mycobacterium fortuitum (M. fortuitum) and conducted drug susceptibility testing to analyze the in vitro susceptibility of clinical M. fortuitum isolates and potential molecular mechanism conferring resistance to fluoroquinolone and macrolide drugs. The results showed that moxifloxacin had the highest in vitro activity against M. fortuitum, and most M. fortuitum isolates were resistant to clarithromycin and linezolid in China. The loss of genetic mutation in clarithromycin- and amikacin-resistant isolates indicates that some other intrinsic mechanism conferring clarithromycin and amikacin resistance plays an essential role in M. fortuitum infection.

Infección urinaria por Mycobacterium fortuitum en paciente con infección por el VIH / Urinary infection due to Mycobacterium fortuitum in a patient with HIV infection

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Infección de prótesis total de rodilla por Mycobacterium fortuitum / Knee prosthetic-joint infection due to Mycobacterium fortuitum

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Cutaneous Mixed Infection of Mycobacterium fortuitum and Mycobacterium intracellulare after a Fat Graft / 대한피부과학회지

Atypical mycobacteria, widely distributed in nature, are opportunistic infection strain, rare pathogen in immunocompetent patients. Recently, the increase of invasive cosmetic treatment and surgery has increased the infection of atypical mycobacteria. We report a case of cutaneous infection by Mycobacterium fortuitum and Mycobacterium intracellulare that occurred after a fat graft. A 62-year-old female patient presented painful, erythematous nodules and purulent discharge on her face after a fat graft. A skin biopsy and culture were performed. Polymerase chain reaction-hybridization to identify mycobacterium other than tuberculosis revealed M. fortuitum and M. intracellulare. The patient was treated with clarithromycin, rifampicin, and ethambutol for 1 month after excision and drainage of pus, after which the skin lesions improved. In cases of refractory cutaneous infection after an invasive cosmetic procedure, atypical mycobacterial infection should be suspected.

Mycobacterium fortuitum como causa de infección del orificio del catéter de diálisis peritoneal. Caso clínico y revisión de la literatura / Mycobacterium fortuitum as a cause of peritoneal dialysis catheter port infection. A clinical case and a review of the literature

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Diferenciación de especies del complejo Mycobacterium fortuitum mediante espectrometría de masas / Differentiation of Mycobacterium fortuitum complex species using mass spectrometry

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Spondylitis with an Epidural Abscess due to Mycobacterium fortuitum: A Case Report / 대한내과학회지

Mycobacterium fortuitum is a rapidly growing mycobacteria that causes skin and soft tissue infection, pulmonary infection, and occasionally disseminated disease in severely immunocompromised patients. While three cases of spondylitis caused by M. fortuitum have been reported, no case has been reported in Korea. We describe the first reported case of M. fortuitum spondylitis and an epidural abscess after lumbar acupuncture, and a surgical procedure to treat spinal stenosis.

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens / 대한임상미생물학회지

BACKGROUND: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. METHODS: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. RESULTS: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. CONCLUSION: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

Mycobacterium fortuitum-related lymphadenitis associated with the varicella-zoster virus

Lymphadenitis caused by non-tuberculous mycobacteria is an uncommon manifestation in immunocompetent individuals. Here, we report a case of Mycobacterium fortuitum infection in a previously healthy 9-year-old patient who developed cervical lymphadenitis evolving to a suppurative ulcer associated with a varicella-zoster virus infection. We discuss the relationship between the varicella-zoster virus and the immune response of the host as an explanation for the unusual progression of the case.

Isolation of Mycobacterium fortuitum in sputum specimens of a patient with chronic cough: Is it clinically significant?

Managing chronic cough is diagnostically challenging especially in primary care. This case report highlights the difficulties experienced in approaching a case of chronic cough from a primary care perspective. The discussion also involves the clinical significance and treatment dilemma of M. fortuitum chelonae complex that was isolated from the sputum cultures of an elderly woman who presented with chronic cough for more than a year.

Postsurgical Wound Infection Caused by Mycobacterium conceptionense Identified by Sequencing of 16S rRNA, hsp65, and rpoB Genes in an Immunocompetent Patient / 대한임상미생물학회지

Rapidly growing mycobacteria are ubiquitous in the environment and are increasingly being recognized as opportunistic pathogens. Recently, a new species, Mycobacteium conceptionense, has been validated from the Mycobacterium fortuitum third biovariant complex by molecular analysis. However, there are few reports, and postsurgical wound infection by this species is rare. We report a case of postsurgical wound infection caused by M. conceptionense in an immunocompetent patient that was identified by a sequencing analysis of 16S rRNA, hps65, and rpoB genes.