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.

Investigation of a Mycobacterium fortuitum catheter-related bloodstream infection in an oncology unit.

We describe a case of healthcare-associated bloodstream infection due to Mycobacterium fortuitum. Whole-genome sequencing showed that the same strain was isolated from the shared shower water of the unit. Nontuberculous mycobacteria frequently contaminate hospital water networks. Preventative actions are needed to reduce the exposure risk for immunocompromised patients.

Mycobacterium fortuitum abortion in a sow.

Two aborted Chester White pig fetuses were presented to a veterinary diagnostic laboratory in Illinois. Postmortem examination identified no gross abnormalities. Histologic evaluation revealed multifocal necrosis of chorionic epithelial cells, coalescing areas of mineralization in the placenta, and focal accumulations of viable and degenerate neutrophils in the lung. Intra- and extracellular acid-fast bacilli were identified in the lesions in both the placenta and lungs. Bacterial culture of stomach contents yielded heavy growth of Mycobacterium fortuitum, a rapidly growing nontuberculous mycobacterium (NTM), which was further confirmed through whole-genome sequencing. NTM are opportunistic pathogens commonly found in the soil and in contaminated water supplies. In animals, M. fortuitum is typically introduced through cutaneous wounds leading to infections limited to the skin, with systemic infection being uncommon. To our knowledge, abortion caused by M. fortuitum has not been reported previously.

A case of Mycobacterium fortuitum prosthetic joint infection successfully treated medically without prosthesis explantation or joint debridement.

A 58-year-old man with a history of end-stage degenerative joint disease developed a postsurgical infection at the right hip 4 weeks after hip replacement surgery. He underwent surgical washout of the right hip without opening the joint capsule. Arthrocentesis returned positive for Mycobacterium fortuitum He was started on antibiotics with the recommendation to remove the prosthesis. The prosthesis was retained. Based on antimicrobial susceptibilities, he was treated with 4 weeks of intravenous therapy using cefoxitin and amikacin and later switched to oral ciprofloxacin and doxycycline for 5 additional months. Eighteen months from his initial hip replacement surgery, he continues to do well. Joint aspiration culture is important to make a diagnosis of prosthetic joint infection (PJI) when periprosthetic culture is not available. In the absence of serious systemic or comorbid joint conditions, PJI due to M. fortuitum can be managed medically without having to remove the prosthesis or debride the joint.

M. fortuitum-induced CNS-pathology: Deciphering the role of canonical Wnt signaling, blood brain barrier components and cytokines.

Molecular underpinning of mycobacteria-induced CNS-pathology is not well understood. In the present study, zebrafish were infected with Mycobacterium fortuitum and the prognosis of CNS-pathogenesis studied. We observed M. fortuitum triggers extensive brain-pathology. Evans blue extravasation demonstrated compromised blood-brain barrier (BBB) integrity. Further, decreased expression in tight-junction (TJ) and adherens junction complex (AJC) genes were noted in infected brain. Wnt-signaling has emerged as a major player in host-mycobacterial immunity but its involvement/role in brain-infection is not well studied. Sustained expression of wnt2, wnt3a, fzd5, lrp5/6 and ß-catenin, with concordant decline in degradation complex components axin, gsk3ß and ß-catenin regulator capn2a were observed. The surge in ifng1 and tnfa expression preceding il10 and il4 suggested cytokine-interplay critical in M. fortuitum-induced brain-pathology. Therefore, we suggest adult zebrafish as a viable model for studying CNS-pathology and using the same, conclude that M. fortuitum infection is associated with repressed TJ-AJC gene expression and compromised BBB permeability. Our results implicate Wnt/ß-catenin pathway in M. fortuitum-induced CNS-pathology wherein Th1-type signals facilitate bacterial clearance and Th2-type signals prevent the disease sequel.

Use of Negative-Pressure Wound Therapy With Instillation and Dwell in Breast Reconstruction.

SUMMARY: The use of negative-pressure wound therapy (NPWT) has expanded over the last 3 decades, paralleled and documented by an increase in research. This article discusses the evolution and current applications of NPWT in modern breast reconstruction. Negative-pressure wound therapy with instillation and dwell (NPWTi-d) technology can be used to remove infectious material, facilitate salvaging compromised tissue, and stabilize the soft-tissue environment. Published consensus NPWTi-d guidelines can aid in treatment selection and implementation of this new technology. The therapeutic approach of simultaneously removing infectious material and actively improving mastectomy flap perfusion and thickness is a burgeoning concept, and illustrative cases are presented. NPWTi-d preliminary use has led to reconstruction salvage with reproducible early experience and outcomes, and it is hoped that it will raise interest and awareness of this promising application of the technology to improve breast reconstruction outcomes.

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus.

The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2-endoplasmic reticulum (ER) stress-store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum.

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca2+)c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O2•-) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca2+)c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Mycobacterial Airsacculitis Caused by Mycobacterium fortuitum in a Southern Rockhopper Penguin (Eudyptes chrysocome).

A 21-year-old male southern rockhopper penguin (Eudyptes chrysocome) was presented with a chronic history of intermittently decreased appetite, lethargy, and regurgitation. On the external physical examination, the bird was determined to be in fair-to-thin body condition with the complete blood count and plasma chemistry panel being largely unremarkable. Full-body radiographic images were considered normal, and gastroscopy showed only mild gastritis and duodenitis. The penguin was euthanatized shortly thereafter due to acute onset of respiratory distress. During the gross necropsy examination, the bird had severe airsacculitis with thick, yellow-to-tan, moist granular plaques adhering to the surface of many air sacs, as well as regional contiguous pneumonia. Intralesional acid-fast bacilli were observed in histologic sections of air sac tissue, and polymerase chain reaction of the affected air sacs was positive for Mycobacterium fortuitum. This clinical case study describes mycobacteriosis in a sub-Antarctic penguin and to the best of the authors' knowledge, the first reported isolation of M fortuitum from a penguin.

Mycobaterium fortuitum disseminated infection in an immunocompetent patient without predisposing factors.

Most Mycobacterium fortuitum infections described involve direct inoculation through skin lesions. We describe the case of a patient without risk factors who presented with an intracranial mass and a pulmonary infection with M. fortuitum As M. fortuitum are rarely pathogens, there is little knowledge about the optimal treatment and outcome of such infections: what is the best mode of administration, what is the best therapy duration and is surgery always required are some of the unanswered questions. In our patient, surgical removal of the mass associated with a 1-year antimycobacterial therapy led to a full recovery. Even though M. fortuitum was rapidly identified in sputum, it was initially considered non-pathogenic and the definitive diagnosis required almost 6 weeks of investigations. New molecular techniques will probably lead to more identifications of M. fortuitum in the next few years and a better knowledge of their possible pathogenicity and optimal treatment.

Misleading of the diagnosis of Mycobacterium attributed lung diseases to malignancy due to smear, culture and PCR negative results: A lesson from a case report.

The incidence of non-tuberculous mycobacteria (NTM) attributed diseases are rising and they are responsible for an increasing proportion of mycobacterial diseases, worldwide. However, their diagnosis is still a big challenge. In this study, a 77-year-old diabetic woman with familial history of lung cancer and 40 pack/year smoking history was presented. She described significant weight loss, shortness of breath, yellow productive sputum, fever, and chills from 4 months ago. The empirical antibiotic therapy didn't cause a significant improvement in the patient's health condition. Also, the sputum smear, culture, and polymerase chain reaction-based (PCR) tests were negative for Mycobacterium tuberculosis (MTB). Computed tomography scanning identified a consolidation at the right upper lobe which was susceptible to malignancy. Non-caseous granulomatous inflammation with the presence of acid-fast bacillus was detected in the biopsies. Therefore, the patient's sputum was reexamined. Although PCR was negative, both smear and culture became positive. PCR-based amplification of a 596 bp fragment of 16S rRNA gene of the isolated bacteria, followed by almost full 16S rRNA sequencing, identified the Mycobacterium fortuitum strain. No malignant cell was detected at pathology evaluations. Due to the increase of NTM attributed diseases which can exhibit negative PCR for MTB and low reliability of negative results of sputum smear and culture, multiple repetitions of the sputum evaluations and, utilizing from 16S rRNA sequencing is recommended to diagnose NTM related lung disease.

CFTR Depletion Confers Hypersusceptibility to Mycobacterium fortuitum in a Zebrafish Model.

The Mycobacterium fortuitum complex comprises several closely related species, causing pulmonary and extra-pulmonary infections. However, there is very limited knowledge about the disease pathogenesis involved in M. fortuitum infections, particularly due to the lack of suitable animal models. Using the zebrafish model, we show that embryos are susceptible to M. fortuitum infection in a dose-dependent manner. Furthermore, zebrafish embryos form granulomas from as early as 2 days post-infection, recapitulating critical aspects of mycobacterial pathogenesis observed in other pathogenic species. The formation of extracellular cords in infected embryos highlights a previously unknown pathogenic feature of M. fortuitum. The formation of large corded structures occurs also during in vitro growth, suggesting that this is not a host-adapted stress mechanism deployed during infection. Moreover, transient macrophage depletion led to rapid embryo death with increased extracellular cords, indicating that macrophages are essential determinants of M. fortuitum infection control. Importantly, morpholino depletion of the cystic fibrosis transmembrane conductance regulator (cftr) significantly increased embryo death, bacterial burden, bacterial cords and abscesses. There was a noticeable decrease in the number of cftr-deficient infected embryos with granulomas as compared to infected controls, suggesting that loss of CFTR leads to impaired host immune responses and confers hypersusceptiblity to M. fortuitum infection. Overall, these findings highlight the application of the zebrafish embryo to study M. fortuitum and emphasizes previously unexplored aspects of disease pathogenesis of this significant mycobacterial species.

[DELAYED PERSISTENT BREAST IMPLANT INFECTION WITH MYCOBACTERIUM FORTUITUM].

INTRODUCTION: Non-Tuberculous Mycobacteria (NTM) are opportunistic environmental pathogens that can produce a wide range of diseases, including infection of the skin and soft tissues. Mycobacterium fortuitum is a predominant causative agent of postsurgical wound infection, especially in breast surgery. Peri-prosthetic infection due to Mycobacterium fortuitum are uncommon, but increasingly reported. This report summarizes the case of a young healthy female who underwent bilateral breast mastopexy with insertion of silicone implants. Shortly after the procedure she presented with clinical signs of wound infection that failed to heal completely in spite of repeated drainage and empiric antibiotic therapy. Additional microbiological investigation allowed for a diagnosis of Mycobacterium fortuitum. A prolonged course of anti-mycobacterial therapy, combined with removal of the implants initiated eradication of the infection and enabled re-implantation of the prosthesis. This case report underscores the importance of awareness to this type of pathogen especially in cases of exudative infection with sterile cultures. Timely identification can lead to prompt therapy of patients preventing further complications, costs and remaining aesthetic damage.

Mycobacterium fortuitum Prosthetic Joint Infection After Total Hip Arthroplasty: A Case Report.

CASE: A 57-year-old man presented with a Mycobacterium fortuitum prosthetic joint infection (PJI) after right total hip arthroplasty refractory to the initial revision surgery and cement spacer placement. The patient was subsequently treated with 2-stage total joint arthroplasty revision surgery using an antibiotic-laden spacer customized to include meropenem and delayed reimplantation to allow for prolonged, systemic antimicrobial treatment with multiple antimicrobials, including levofloxacin and linezolid. CONCLUSIONS: There is little evidence to guide practitioners in the diagnosis and treatment of PJI caused by rare, rapidly growing mycobacteria (RGM) such as M. fortuitum. This case demonstrates a successful strategy for the treatment of RGM PJI.

Successful bedaquiline-containing antimycobacterial treatment in post-traumatic skin and soft-tissue infection by Mycobacterium fortuitum complex: a case report.

BACKGROUND: Mycobacterium fortuitum complex is a group of rapidly growing nontuberculous mycobacteria (NTM) associated with skin and soft-tissue infections after surgery or trauma. Treatment of NTM is challenging, due to resistance to multiple antimycobacterial agents. Bedaquiline is a diarylquinoline that inhibits mycobacterial ATP-synthase. The drug has recently been approved for the treatment of multidrug-resistant tuberculosis and evidence of its in vitro efficacy against NTM, including Mycobacterium fortuitum complex, has been published. CASE PRESENTATION: A 20-year-old Caucasian woman with chronic skin and soft tissue infection in the lower leg following a traffic accident in Vietnam underwent a tedious journey of healthcare visits, hospital admissions, empiric antimicrobial treatments, surgical debridement and plastic reconstruction before definite diagnosis of Mycobacterium fortuitum complex-infection was established by culture from a tissue biopsy and targeted antimycobacterial therapy was administered. Histopathological examination revealed granulomatous purulent inflammation, which strongly supported the diagnosis. Genotypic identification was performed and broth microdilution for susceptibility testing showed macrolide resistance. Five weeks of induction treatment with intravenous amikacin, imipenem / cilastin, and oral levofloxacin was administered, followed by all-oral treatment with bedaquiline combined with levofloxacin for four months, which was well-tolerated and led to persistent healing with scars but without signs of residual infection. CONCLUSIONS: Bedaquiline is a promising novel agent for NTM treatment, although clinical data are limited and trials evaluating efficacy, safety, and resistance of bedaquiline are required. To our knowledge, this is the first reported case of successful in vivo use of bedaquiline for a skin and soft tissue infection caused by Mycobacterium fortuitum complex.

Odontogenic cutaneous sinus tracts due to infection with nontuberculous mycobacteria: a report of three cases.

BACKGROUND: Soft tissue or skin infections due to nontuberculous mycobacteria (NTM) have been reported frequently and are mostly associated with trauma or cosmetic interventions like plastic surgery. However, infection with NTM as a result of a dental procedure have rarely been described and the lack of clinical suspicion and a clear clinical manifestation makes diagnosis challenging. CASE PRESENTATION: We report on three patients with a facial cutaneous sinus tract of dental origin, due to an infection with respectively Mycobacterium fortuitum, M. abscessus and M. peregrinum. The infection source was the dental unit waterlines (DUWLs), which were colonized with NTM. CONCLUSIONS: Water of the DUWL can pose a health risk. This report emphasizes the need for quality control and certification of water flowing through DUWLs, including the absence of NTM. Our report also shows the need for a rapid recognition of NTM infections and accurate laboratory diagnosis in order to avoid long-term ineffective antibiotic treatment.