Bovine mastitis caused by rapid-growth environmental mycobacteria

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

Reply to Pavlik et al. Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on “Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797”

We appreciate the valuable comment of Pavlik et al. [...]

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.

Non-tuberculous Mycobacterial Infection of the Musculoskeletal System Detected at Two Tertiary Medical Centres in Henan, China, 2016–2020

Non-tuberculous mycobacterial (NTM) infection of the musculoskeletal system is rare but poses a grave threat to public health. These infections yield non-specific symptoms that remain undetected until the development of the later stages of the disease. In this study, we performed a retrospective review of 25 cases of musculoskeletal NTM infection at two tertiary medical centres over a 5-year period to determine the clinical features and improve the current clinical diagnosis and treatment. The most common mycobacterial species detected were Mycobacterium fortuitum in eleven patients, Mycobacterium abscessus in eight patients, Mycobacterium houstonense in three patients, Mycobacterium avium in two patients, and Mycobacterium smegmatis in one patient. The sites of infection included the limbs and joints, most commonly the knee (ten patients) and foot (six patients). The median duration from the onset of symptoms to diagnosis was 2.5 months (0.8–13.5 months). Deep sinus tracts extending to the surgical site were observed in 60% of the patients (15/25), and granulomatous inflammation and granulomatous inflammation with necrosis occurred in 60% of the patients (15/25). All patients underwent surgical treatment for infection control, and all patients, except one, received antimycobacterial therapy based on drug sensitivity assays. The median duration of the antimicrobial chemotherapy was 5 months (range: 3–20 months). At the final follow-up, 24 patients presented with absence of recurrence and one patient succumbed owing to heart failure after debridement. Our findings highlight the importance of vigilance and improvements in the diagnostic methods for musculoskeletal NTM infection. Aggressive surgical treatment and antimycobacterial drug treatment can help achieve satisfactory results.

Development of a nucleic acid chromatography assay for the detection of commonly isolated rapidly growing mycobacteria

Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM). Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal. Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM. Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus , Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense , Mycobacterium fortuitum , Mycobacterium chelonae and Mycobacterium peregrinum . The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay. Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1. Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.

Clinical Relevance and Environmental Prevalence of Mycobacterium fortuitum Group Members. Comment on Mugetti et al. Gene Sequencing and Phylogenetic Analysis: Powerful Tools for an Improved Diagnosis of Fish Mycobacteriosis Caused by Mycobacterium fortuitum Group Members. Microorganisms 2021, 9, 797

Mycobacterium fortuitum group (MFG) members are able to cause clinical mycobacteriosis in fish and other animals including humans. M. alvei, M. arceuilense, M. brisbanense, M. conceptionense, M. fortuitum, M. peregrinum, M. porcinum, M. senegalense, M. septicum, and M. setense were isolated from fish with mycobacteriosis. In other animals only three MFG species have been isolated: M. arceuilense from camels’ milk, M. farcinogenes from cutaneous infections often described as “farcy”, and M. fortuitum from different domestic and wild mammals’ species. Out of 17, only 3 MFG species (M. arceuilense, M. lutetiense and M. montmartrense) have never been reported in humans. A total of eight MFG members (M. alvei, M. brisbanense, M. conceptionense, M. fortuitum subsp. acetamidolyticum, M. houstonense, M. peregrinum, M. porcinum, and M. septicum) have been isolated from both pulmonary and extrathoracic locations. In extrathoracic tissues five MFG species (M. boenickei, M. farcinogenes, M. neworleansense, M. senegalense, and M. setense) have been diagnosed and only one MFG member (M. fortuitum subsp. acetamidolyticum) has been isolated from pulmonary infection.