The clinical utility of rifamycins against non-tuberculous mycobacterial (NTM) disease is limited by intrinsic drug resistance achieved by ADP-ribosyltransferase Arr. By blocking the site of ribosylation, we recently optimized a series of analogs with substantially improved potency against Mycobacterium abscessus. Here, we show that a representative member of this series is significantly more potent than rifabutin against major NTM pathogens expressing Arr, providing a powerful medicinal chemistry approach to expand the antimycobacterial spectrum of rifamycins. IMPORTANCE Lung disease caused by a range of different species of non-tuberculous mycobacteria (NTM) is difficult to cure. The rifamycins are very active against Mycobacterium tuberculosis, which causes tuberculosis (TB), but inactive against many NTM species. Previously, we showed that the natural resistance of the NTM Mycobacterium abscessus to rifamycins is due to enzymatic inactivation of the drug by the bacterium. We generated chemically modified versions of rifamycins that prevent inactivation by the bacterium and thus become highly active against M. abscessus. Here, we show that such a chemically modified rifamycin is also highly active against several additional NTM species that harbor the rifamycin inactivating enzyme found in M. abscessus, including M. chelonae, M. fortuitum, and M. simiae. This finding expands the potential therapeutic utility of our novel rifamycins to include several currently difficult-to-cure NTM lung disease pathogens beyond M. abscessus.

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.

BACKGROUND: Nontuberculous mycobacteria (NTM) are environmental bacterium that may cause and/or compound respiratory diseases in humans. There are over a hundred NTM species with varying pathogenicity\'s Therefore, it is necessary to characterize the populations at risk for each species.
METHODS: Demographic (age, sex, and state of residence) and microbiological data from 2014 were extracted from Mississippi, Missouri, and Ohio disease surveillance systems. NTM species with > 50 reports were included in the analysis. Patient sex, age, and incidence rates were generated for each of the following NTM species: M. abscessus, M. avium complex (MAC), M. chelonae, M. fortuitum, M. gordonae, M. kansasii, M. mucogenicum, and M. peregrinum.
RESULTS: Analysis by sex showed that M. chelonae,M. fortuitum, M. gordonae,and M. kansasii had significantly higher rates in males than females. Age was not associated with patient rates for several specific NTM species e.g., M. chelonae. Mississippi had the highest patient\' rates for M. avium, M. gordonae, M. kansasii, and M. chelonae. Ohio had the highest patient\' rates for M. abscessus, M. mucogenicum, and M. peregrinum. The highest patient\'s rate for M. fortuitum was observed in Missouri.
CONCLUSIONS: This study showed that NTM infection occurred more frequently in males. The highest rates were observed in Mississippi for most of the NTMs studied. Age was not a strong risk factor for some of the NTM species.

BACKGROUND: Non-tuberculous mycobacteria cause chronic pulmonary infection, but pleuritis and pleural effusion are rarely associated with infection with non-tuberculous mycobacteria, especially rapid-growing mycobacteria.
METHODS: A 68-year-old woman with rheumatoid arthritis who was using prednisone, azathioprine, and certolizumab pegol presented complaining of fever, dry cough, and night sweats for the past 2 weeks. Chest examination revealed bilateral opacity that was more pronounced on her right side. Bronchoalveolar lavage fluid and pleural effusion fluid were obtained, and revealed coinfection with Mycobacterium fortuitum and Mycobacterium mageritense. Imipenem/cilastatin, levofloxacin, and minocycline were prescribed for 6 months, and the patient was well and asymptomatic for the subsequent 6 months.
CONCLUSIONS: This is the first case report describing pleural effusion associated with coinfection with two different mycobacterial species. If the species cannot be identified, the possibility of mycobacterial coinfection should be considered.

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.

There has been an increasing awareness of the rapidly growing mycobacteria (RGM), of which numerous species and phylogenetic groups are clearly established human pathogens. It is important to appropriately distinguish RGM from other mycobacteria, as first-line antituberculous drugs are ineffective for their treatment. Variability in susceptibility of RGM is seen in relation to species, different geographical areas, and time. Therefore, we conducted a study to speciate the isolates of RGM and perform antimicrobial susceptibility testing. The study was carried out in the department of microbiology of a tertiary care hospital. This study included 40 isolates of RGM obtained from clinical specimens from suspected cases of extrapulmonary tuberculosis. Forty isolates of RGM were speciated by phenotypic methods and drug susceptibility testing was done by broth microdilution method. Of the 40 isolates of RGM, 55% belonged to Mycobacterium fortuitum group, 35% were M. smegmatis group, and 10% were M. chelonae-abscessus group. In M. fortuitum group, sensitivity was seen to amikacin (13.63%), cefoxitin (18.18%), imipenem (31.81%), ceftriaxone (22.72%), and cotrimoxazole (31.81%). Only 14.28% and 7.14% of M. smegmatis were sensitive to cotrimoxazole and amikacin, respectively. M. chelonae-abscessus group was resistant to all the antibiotics tested and showed only intermediate sensitivity to amoxicillin-clavulanic acid (50%) and gatifloxacin (25%). A variability in sensitivity to different antimicrobials exists in all groups. Hence, it is advisable to perform antimicrobial susceptibility test before commencement of therapy.

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens.

The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 °C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 °C as compared to 30 °C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 °C as compared to 37 and 42 °C. Mycobacterium tuberculosis developed strong biofilm at 37 °C and no biofilm at 30 and 42 °C in Sauton\'s media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton\'s medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multi-layered, three-dimensional structure.

A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei to diminish unspecific reactions As M. fortuitum is one of the most frequent environmental mycobacteria, the purpose of this pilot study was to evaluate its use as an alternative for the preadsorption in ELISAs for paratuberculosis. Results suggest that M. fortuitum can be an alternative instead of or associated to M. phlei with comparable results (κ > 0.8) to conventional ELISAs using M. phlei as a preadsorption antigen.