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.

BACKGROUND: Injection-related abscesses are a common complication in clinical practice, but the identification of infected bacteria might be difficult.
METHODS: A 51-year-old female patient was admitted to the hospital due to a lump on her right buttock that emerged after receiving intramuscular injections to treat left shoulder joint pain. The lump gradually enlarged into a 3.0 to 4.5 cm mass at the time of admission with symptoms such as skin redness, itching, and pain.
METHODS: The patient received ultrasonic and other laboratory examinations. Laboratory results from the drainage indicated that the infection was caused by a rapidly growing mycobacteria and was confirmed as Mycobacterium fortuitum by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
METHODS: The patient was treated with antibiotics for 12 days after incision and drainage of the abscess in the right buttock. Local dressings were changed regularly. A migration lesion that appeared 3 days after treatment was drained and cleaned when it matured.
RESULTS: The lesion substantially decreased in size and the patient was discharged after 2 months of treatment.
CONCLUSIONS: Rapidly growing mycobacteria are rare but important pathogens that should be considered in patients with injection-related abscesses. Early identification and appropriate treatment can result in a favorable prognosis.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17β-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

We aimed to evaluate the activity of PBTZ169 and pretomanid against non-tuberculous mycobacteriosis (NTM) in vitro and in vivo.
The minimum inhibitory concentrations (MICs) of 11 antibiotics, against slow-growing mycobacteria (SGMs) and rapid-growing mycobacteria (RGMs) were tested using the microplate alamarBlue assay. The in vivo activities of bedaquiline, clofazimine, moxifloxacin, rifabutin, PBTZ169 and pretomanid against four common NTMs were assessed in murine models.
PBTZ169 and pretomanid had MICs of >32 μg/mL against most NTM reference and clinical strains. However, PBTZ169 was bactericidal against Mycobacterium abscessus (3.33 and 1.49 log10 CFU reductions in the lungs and spleen, respectively) and Mycobacterium chelonae (2.29 and 2.24 CFU reductions in the lungs and spleen, respectively) in mice, and bacteriostatic against Mycobacterium avium and Mycobacterium fortuitum. Pretomanid dramatically decreased the CFU counts of M. abscessus (3.12 and 2.30 log10 CFU reductions in the lungs and spleen, respectively), whereas it showed moderate inhibition of M. chelonae and M. fortuitum. Bedaquiline, clofazimine, and moxifloxacin showed good activities against four NTMs in vitro and in vivo. Rifabutin did not inhibit M. avium and M. abscessus in mice.
PBTZ169 appears to be a candidate for treating four common NTM infections. Pretomanid was more active against M. abscessus, M. chelonae and M. fortuitum than against M. avium.

BACKGROUND: 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1‑dehydrogenation and regulating metabolic flux.
RESULTS: Five 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ‑dihydroxy‑20‑methyl‑pregna‑4‑en‑3‑one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9‑OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol.
CONCLUSIONS: KstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare.
The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism.
Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2\')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus.
Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.

BACKGROUND: Nontuberculous mycobacterium (NTM) refers to all mycobacteria except Mycobacterium tuberculosis and Mycobacterium leprae, also known as environmental Mycobacterium. The patients with lung cancer and NTM are somewhat special; the two diseases are inevitably influenced by each other. It brings difficulties and challenges to the choice of treatment. Recently, cancer immunotherapy has been considered one of the pillars for the treatment of lung cancer. However, the clinical experience in the application of immune checkpoint inhibitors is scarce for lung cancer patients with pulmonary tuberculosis, and lung cancer with NTM is even more rare. Although it ameliorates lung cancer, immunotherapy with immune checkpoint inhibitors presents complications of infectious diseases, including tuberculosis and NTM.
METHODS: A 61-year-old male patient visited a doctor in May 2019. His admitting diagnoses were: (1) Cancer of the left lung with a pathological diagnosis of poorly differentiated non-small cell carcinoma, likely poorly differentiated adenocarcinoma, clinical stage IIIb (T3N3M0); and (2) Mycobacterium fortuitum (M. fortuitum) infection. We chose to proceed with pembrolizumab treatment. After two treatment cycles, a chest computed tomography scan showed a new irregular subpleural mass in the anterior segment of the left upper lobe of the lung, a reduction in the mediastinal enlarged lymph node, and no other obvious changes. Next, an ultrasound-guided biopsy of the new tumor was performed. Pathological examination showed that a large number of carbon particles were deposited in the alveolar tissue with histiocyte reaction and multinucleated giant cell formation. The tuberculosis (TB) specialist suggested that anti-TB therapy be combined with continued antitumor treatment. The patient continued to be treated with pembrolizumab. After 14 cycles, the lesion shrunk by 79%, there was no recurrence of M. fortuitum infection, and there were no intolerable adverse reactions.
CONCLUSIONS: We have observed that in cases of lung cancer complicated with M. fortuitum infection, opportunistic pathogen infection recurrence can be overcome, and immunotherapy is most beneficial when TB doctors and oncologists cooperate to closely observe dynamic changes in M. fortuitum and lung cancer. Treatment should be maintained with low dosage anti-TB drugs after general anti-TB chemotherapy for 1 year; this may prevent opportunistic pathogen infection recurrence during immunotherapy.

UNASSIGNED: Mycobacterium fortuitum is a rapidly growing non-tuberculous mycobacterium (NTM) with weak pathogenicity. Here, we present a rare case of disseminated M. fortuitum and Talaromyces marneffei coinfection in a human immunodeficiency virus (HIV) negative patient.
UNASSIGNED: A 28-year-old female was admitted to our hospital due to 2 months of swelling of lymph nodes on the right side of her cervix, accompanied by repeated low fever for more than 1 month. Biopsy of the right cervical lymph node and endobronchial ultrasound-guided transbronchial fine needle aspiration (EBUS-TBNA) both suggested granulomatous inflammation. The bacterial culture and mycobacteria examination of the lesion as well as HIV antibody test were all negative. Disseminated T. marneffei infection was diagnosed by the quantitative polymerase chain reaction (qPCR) results from the blood showing 1798 copies/ul. In the meantime, treatment with amphotericin B combined with cefoxitin was administered for suspected NTM infection. However, the once-dropped fever recurred and the lymph nodes continued to swell. Metagenomics next-generation sequencing (mNGS) detection of the lymph nodes indicated M. fortuitum. After combination treatment with amphotericin B, voriconazole, linazolamide, and imipenem, the patient\'s body temperature returned to normal, the lymph node swelling was gradually reduced, and the lung lesion was absorbed.
UNASSIGNED: We report the first case of an HIV-negative patient diagnosed with disseminated M. fortuitum and T. marneffei coinfection with nonspecific clinical manifestation, in order to heighten awareness of these infections.

Background: Endophthalmitis caused by Mycobacterium fortuitum as a complication of cataract surgery is rare in China and globally. This study aimed to report the clinical features, surgical treatment strategies, antibiotic susceptibilities and treatment outcomes in patients with endophthalmitis caused by Mycobacterium fortuitum. Patients and methods: A case series of 15 patients (15 eyes) with post-cataract endophthalmitis caused by Mycobacterium fortuitum treated at the Ophthalmology Clinic of Shengjing Hospital between June 2008 and June 2017 was reviewed retrospectively. Information about Mycobacterium fortuitum culture results, clinical features, surgical procedures, and final visual acuity were obtained. Results: The vitreous samples were all positive. These eyes underwent total capsulectomy and intraocular lens explantation and vitrectomy combined with intravitreal and intravenous injections of antibiotics. Finally, 14 eyes had controlled inflammation and retained eyeballs. One eye had panophthalmitis and enucleation of the eyeball. Presenting visual acuity ranged from counting fingers to no light perception. Final visual acuity was better than 20/400 in 4 of 15 eyes (26.67%), 20/400 to hand motions in 7 of 15 eyes (46.67%), and light perception to no light perception in 4 of 15 eyes (26.67%) at 6-month follow-up visit. Conclusion: Mycobacterium fortuitum might be an etiologic agent in postoperative endophthalmitis. The Mycobacterium fortuitum endophthalmitis might be treated with early vitrectomy and targeted antibiotic therapy approach.

With the rising utilization of cardiovascular implantable electronic devices (CIEDs), infections secondary to device implantation are increasingly encountered. Staphylococcus aureus and coagulase-negative staphylococci are usually the predominant causative organisms. A CIED infection due to non-tuberculous mycobacteria (NTM) is extremely rare.
A 68-year-old man was admitted to our hospital with a history of pain and swelling at his cardiac resynchronization therapy-defibrillator (CRT-D) pocket site, for 4 days. The CRT-D had been implanted 2 weeks prior. The exudate smear was positive for acid-fast bacilli and culture results revealed rapidly growing nontuberculous mycobacteria (RGM). After an urgent removal of the device followed by 1 year of antibiotic treatment, the patient was completely cured. A new device was finally implanted, 3 years later.
Infections caused by nontuberculous mycobacteria following the implantation of cardiac devices are very rare. The typical manifestations of post-implantation CIED infections caused by RGMs include an early onset, with local redness, swelling, and spontaneous drainage. Systemic symptoms such as fever, chills, and fatigue are absent. Mycobacterium fortuitum is the most common species of RGM implicated in CIED infections, the manifestations of which usually appear within several weeks of the implantation procedure. An urgent removal of the device and appropriate antibiotic therapy are essential therapeutic measures. This is the first such reported case, in which the patient has been re-implanted with another device at the same site, after achieving a complete cure. We followed-up the patient for an additional 3 years and observed that the patient remained free of infection. Our case report shows that though an RGM infection is rare and difficult to treat, it can be completely cured. In addition, we demonstrated that it is subsequently possible to safely re-implant a CIED for the patient, at the same site.