Phenotypic and genomic hallmarks of a novel, potentially pathogenic rapidly growing Mycobacterium species related to the Mycobacterium fortuitum complex.

Previously, we have identified a putative novel rapidly growing Mycobacterium species, referred to as TNTM28, recovered from the sputum of an apparently immunocompetent young man with an underlying pulmonary disease. Here we provide a thorough characterization of TNTM28 genome sequence, which consists of one chromosome of 5,526,191 bp with a 67.3% G + C content, and a total of 5193 predicted coding sequences. Phylogenomic analyses revealed a deep-rooting relationship to the Mycobacterium fortuitum complex, thus suggesting a new taxonomic entity. TNTM28 was predicted to be a human pathogen with a probability of 0.804, reflecting the identification of several virulence factors, including export systems (Sec, Tat, and ESX), a nearly complete set of Mce proteins, toxin-antitoxins systems, and an extended range of other genes involved in intramacrophage replication and persistence (hspX, ahpC, sodA, sodC, katG, mgtC, ClpR, virS, etc.), some of which had likely been acquired through horizontal gene transfer. Such an arsenal of potential virulence factors, along with an almost intact ESX-1 locus, might have significantly contributed to TNTM28 pathogenicity, as witnessed by its ability to replicate efficiently in macrophages. Overall, the identification of this new species as a potential human pathogen will help to broaden our understanding of mycobacterial pathogenesis.

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.

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.

Non-pigmented rapidly growing mycobacteria smooth and rough colony phenotypes pathogenicity evaluated using in vitro and experimental models.

Non-pigmented rapidly growing mycobacteria (NPRGM) are widely distributed in water, soil and animals. It has been observed an increasing importance of NPRGM related-infections, particularly due to the high antimicrobial resistance. NPRGM have rough and smooth colony phenotypes, and several studies have showed that rough colony variants are more virulent than smooth ones. However, other studies have failed to validate this observation. In this study, we have performed two models, invitro and in vivo, in order to assess the different pathogenicity of these two phenotypes. We used collection and clinical strains of Mycobacteriumabscessus, Mycobacterium fortuitum and Mycobacteriumchelonae. On the invitro model (macrophages), phagocytosis was higher for M. abscessus and M. fortuitum rough colony variant strains when compared to smooth colony variants. However, we did not find differences with colonial variants of M. chelonae. Survival of Galleriamellonella larvae in the experimental model was lower for M. abscessus and M. fortuitum rough colony variants when compared with larvae infected with smooth colony variants. We did not find differences in larvae infected with M. chelonae.Results of our in vivo study correlated well with the experimental model. This fact could have implications on the interpretation of the clinical significance of the NPRGM isolate colonial variants.

A case of pleural effusion caused by Mycobacterium fortuitum and Mycobacterium mageritense coinfection.

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. CASE PRESENTATION: 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.

Mycobacterium fortuitum infection of a hemodialysis catheter in a pediatric patient.

This case report discusses a pediatric patient who developed a hemodialysis catheter line infection from an uncommon etiology, Mycobacterium fortuitum. The initial presentation revealed a well appearing patient with a slow growing skin lesion near the site of the hemodialysis catheter. The treatment course was complicated by resistance to initial antibiotics leading to continued spread of the lesion. The diagnosis was confirmed via skin biopsy of the lesion that required 2 weeks to grow the atypical Mycobacterium. Treatment was successful after hemodialysis catheter removal, transition to peritoneal dialysis, and a prolonged antibiotic course. Mycobacterium fortuitum is a rare cause of dialysis catheter infections that is resistant to standard antibiotic treatment. Treatment success is improved after removal of the dialysis catheter and prolonged antibiotics based on susceptibilities. This case highlights the importance of keeping atypical Mycobacterium in the differential for patients with slow growing skin lesions near dialysis catheter sites with resistance to initial treatment.

Natural history of Mycobacterium fortuitum pulmonary infection presenting with migratory infiltrates: a case report with microbiological analysis.

BACKGROUND: Presence of Mycobacterium fortuitum in respiratory tracts usually indicates mere colonization or transient infection, whereas true pulmonary infection occurs in patients with gastroesophageal disease. However, little is known about the diagnostic indications for true M. fortuitum pulmonary infection and the natural history of the disease. CASE PRESENTATION: A 59-year-old man was referred to our hospital for treatment against M. fortuitum pulmonary infection. Fifteen years before the referral, he underwent total gastrectomy, after which he experienced esophageal reflux symptoms. After the referral, the patient was closely monitored without antimicrobial therapy because of mild symptoms and no pathological evidence of M. fortuitum pulmonary infection. During the observation, chest imaging showed migratory infiltrates. Two years after the referral, his lung biopsy specimen revealed foamy macrophages and multinucleated giant cells, indicating lipoid pneumonia. However, he was continually monitored without any treatment because there was no evidence of nontuberculous mycobacterial infection. Four years after the referral, he developed refractory pneumonia despite receiving adequate antibiotic therapy. After confirmation of granulomatous lesions, multiple antimicrobial therapy for M. fortuitum resulted in a remarkable improvement with no exacerbation for over 5 years. Random amplified polymorphic DNA polymerase chain reaction analysis revealed identical M. fortuitum strains in seven isolates from six sputum and one intestinal fluid specimens obtained during the course of the disease. CONCLUSIONS: We have described a patient with M. fortuitum pulmonary infection who presented with migratory infiltrates. The pathological evidence and microbiological analysis suggested that M. fortuitum pulmonary infection was associated with lipoid pneumonia and chronic exposure to gastrointestinal fluid. Therefore, physicians should carefully monitor patients with M. fortuitum detected from lower respiratory tract specimens and consider antimicrobial therapy for M. fortuitum infection when the patient does not respond to adequate antibiotic therapy against common pneumonia pathogens.

Nontuberculous mycobacteria pathogenesis and biofilm assembly.

Nontuberculous mycobacteria (NTM) are emergent pathogens whose importance in human health has been gaining relevance after being recognized as etiological agents of opportunist infections in HIV patients. Currently, NTM are recognized as etiological agents of several respiratory and extra-respiratory infections of immune-competent individuals. The environmental nature of NTM together with the ability to assemble biofilms on different surfaces plays a key role on their pathogenesis. In the present work the ability of three fast-growing NTM (Mycobacterium smegmatis, Mycobacterium fortuitum and Mycobacterium chelonae) to persist within a model of human alveolar macrophages was evaluated. Most often human infections with NTM occur by contact with the environment. Biofilms can work as environmental reservoirs. For this reason, it was decided to evaluate the ability of NTM to assemble biofilms on different surfaces. Scanning electron microscopy was used to elucidate the biofilm structure. The ability to assemble biofilms was connected with the ability to spread on solid media known as sliding. Biofilm assembly and intracellular persistence seems to be ruled by different mechanisms.

Mycobacterium fortuitum prosthetic valve endocarditis: a case for the pathogenetic role of biofilms.

BACKGROUND: Prosthetic valve endocarditis presents unique challenges for both diagnosis and treatment. A potential role of biofilm has been hypothesized in the pathogenesis of these infections. METHODS: A patient with infective endocarditis involving a stentless (Freestyle) porcine prosthetic aortic valve with annular abscess and paravalvular leak 8 months after implantation is reported. RESULTS: The infected valve did not show vegetations or perforations, but histiocytic inflammation was seen along the endocardial surfaces of the valve. Auramine-rhodamine staining revealed many acid-fast organisms associated with the inflammation. There was also an acellular matrix material with ultrastructural features of biofilm. Blood cultures grew Mycobacterium fortuitum, a biofilm-associated microbe. CONCLUSIONS: The role of biofilm in prosthetic valve endocarditis is discussed. The importance of microscopy for prosthetic valves, even when no vegetations are present, is highlighted along with correlation of pathologic findings with culture results.

An outbreak of Mycobacterium fortuitum cutaneous infection associated with mesotherapy.

OBJECTIVE: We describe an outbreak of Mycobacterium fortuitum cutaneous infections associated with mesotherapy in La Rioja, Spain. DESIGN: Descriptive epidemiology. SETTING: Private practice. PATIENTS OR OTHER PARTICIPANTS: Case subjects were customers of a single beauty salon who were treated with mesotherapy injections. INTERVENTION(S): Two skin biopsies were taken from each patient. RESULTS: Over the designated period, 138 women received mesotherapy. Of these women, 39, or 28.3%, developed lesions ultimately thought to be caused by Mycobacterium fortuitum infection. The number of lesions per patient varied from 3 to 20 in the most severe case. Most of the lesions were indurated, erythematous or violaceous papules, some progressing to become fluctuant boils with suppuration, fistulization and scarring. The individual lesions varied in diameter from 0.5 to 6 cm. Two patients (5.1%) developed inguinal or axillary adenopathy. Two others presented with fever. One reported muscular pain. In 12 of the 39 cases, M. fortuitum was isolated from the wound cultures. The patients were all successfully treated with clarithromycin and levofloxacin. CONCLUSIONS: We identified a large outbreak of rapidly growing mycobacterial lesions among women who received mesotherapy injections in a single beauty salon.

[Microbiological properties of M. porcinum isolated from a patient with impairments in IL-12/IFN-gamma pathway].

PURPOSE: Mycobacterium porcinum has been successfully isolated from the patient with abnormal signal transduction pathway of IL12/IFN-gamma. The properties of each bacterium were determined by conventional identification methods, DNA sequencing analysis and MIC assay. MATERIALS AND METHODS: M. porcinum was isolated 7 times from 1996 to 2007 from cervical lymph node, axillary lymph nodes, inguinal lymph node, brachial lymph node and site of a tumor of the patient. In another occasion, mycobacteria were isolated from lavage fluid of the endoscope in routine inspection. Using these mycobacteria, M. porcinum (ATCC33776) and M. fortuitum (ATCC6841), the conventional identification method and MIC assay were carried out. For analyses of the DNA sequencing (rpoB, dnaJ and hsp65), the ATCC type strain of mycobacteria (11 strains) which are closely related to M. porcinum were also used. RESULTS AND DISCUSSION: DNA sequencing analyses of the 7 samples isolated from the patient, were concurrently identical in 3 different genes. Drug susceptibility test showed that 7 isolates had no marked change. In conventional identification analyses, M. porcinum (ATCC33776), M. fortuitum (ATCC6841), and M. porcinum that were isolated in 1996, were able to grow at 42 degrees C. However, 6 isolates that were isolated after 1999, did not grow at 42 degrees C. The colony detectable days of these 7 strains changed from 3 to 7. Over the time, the morphology of each colony changed from smooth to rough. Though the initial isolate had the ability to utilize mannitol, the later 4 isolates had no such ability.

A transposon insertion mutant of Mycobacterium fortuitum attenuated in virulence and persistence in a murine infection model that is complemented by Rv3291c of Mycobacterium tuberculosis.

Mycobacterium fortuitum is a non-tubercular fast growing pathogenic mycobacteria whose virulence factors have not been studied. Infection of M. fortuitum ATCC 6841 in a murine infection model leads to spinning of the head in 8-12 days after infection, 20-25% mortality and a constant bacillary load in the kidney of mice, suggesting persistence. From a TnphoA insertion library, a mutant MT13 was isolated which was attenuated in virulence with lesser bacterial burden, milder and delayed spinning of the head and no mortality of mice. The significant feature of the mutant was its failure to persist in kidney and thus the persistent bacillary load characteristic exhibited by the wild type strain was not observed. The insertion of transposon in MT13 was mapped in a region of the genome, which showed homology to Rv3291c of M. tuberculosis, annotated as a transcriptional regulatory factor and reported to be up regulated in nutrient starvation and anaerobic persistent states. Complementation of MT13 with rv3291c resulted in restoration of wild type characteristics including persistence in kidney suggesting the role of a Rv3291c homolog in the virulence and persistence of M. fortuitum.

Wound infections due to Mycobacterium fortuitum after polypropylene mesh inguinal hernia repair.

Mycobacterium fortuitum complex is a group of rapidly growing mycobacteria (RGM). These opportunistic pathogens are frequently associated with infections related to surgical procedures involving biomaterials. Two cases of Mycobacterium fortuitum infection occurred in a prospective study of inguinal hernia prosthesis repairs. These infections differed from those caused by other bacteria in terms of pathogenic mechanisms, clinical manifestation and resistance to both prophylactic and therapeutic antibiotics.

Pacemaker infection due to Mycobacterium fortuitum: the role of universal 16S rRNA gene PCR and sequencing.

Rapidly growing mycobacteria are a rare cause of pacemaker infection. A low index of suspicion and conventional diagnostic methods may delay diagnosis. We present a review of the literature and report a case of pacemaker infection due to Mycobacterium fortuitum rapidly detected by universal 16S rRNA gene polymerase chain reaction and sequencing.

Genome-wide RNAi screen for host factors required for intracellular bacterial infection.

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.

Pacemaker infection due to Mycobacterium fortuitum.

Pacemaker infection with Mycobacterium fortuitum has not been reported previously. We describe a case of pacemaker generator pocket infection and intravascular lead endocarditis due to Mycobacterium fortuitum. The entire pacing system was removed and the patient was treated successfully with a multidrug regimen for a total of 6 months.

Delayed post-operative wound infections due to non-tuberculous Mycobacterium.

BACKGROUND & OBJECTIVE: The non-tuberculous mycobacteria (NTM) have emerged as important opportunistic pathogens of human beings in the recent years. The NTM are rapid growing mycobacteria (RGM), which include Mycobacterium fortuitum and M. chelonae and grouped as M. fortuitum-chelonae complex. Though there are reports on isolation of NTM from various parts of India, information on its occurrence in northeastern India is lacking. We therefore undertook this preliminary investigation to report on the occurrence of NTM-associated with non-healing postoperative wound infections that did not respond to antibiotics used for pyogenic infections and having sterile routine aerobic cultures in patients from northeastern part of India. METHODS: Pus/discharge from 25 patients with delayed onset of post-operative wound infections not responding to antibiotics used for pyogenic infections were collected and examined for isolation and identification of the causative agents. RESULTS: Of the 25 pus/discharge specimens examined, 20 revealed growth of non-tuberculous Mycobacterium spp. All the isolates were identified as M. fortuitum-chelonae complex. Of these only 10 samples revealed acid-fast bacilli (AFB) on direct examination of Ziehl-Neelsen stained smears from the specimens. All cases where direct smear was positive for AFB were also positive for Mycobacterium culture. INTERPRETATION & CONCLUSION: The results of the present study indicated that non-tuberculous mycobacterial post-operative wound infection was fairly common in northeastern India. Thus, mycobacterial infections should be considered in wounds that show delayed healing and do not respond to antibiotics used for acute pyogenic infections. Further, 80 per cent of the specimens yielded the growth of AFB in cultures as against only 40 per cent positive in the ZN stained direct smears. This indicates the possibility of missing a mycobacterial wound infection if only direct smears are taken for diagnosis.

Mastitis por Mycobacterium fortuitum en una paciente HIV negativa / Mastitis due to Mycobacterium fortuitum in an HIV negative patient

A case of a 39 year old HIV negative female patient with a Mycobacterium fortuitum mastitis without previous pathogenic history is reported. She was treated on the bases of drug-susceptibility testing and bibliographic empirical evidence with kanamycin, doxicicline, ciprofloxacin and trimetoprim-sulfametoxazol. A complete remission of her lesions was obtained after 15 months of treatment. Lesions due to this rapidly growing mycobacterium, diagnosis and treatment are commented.

Se presenta el caso de una paciente HIV negativa de 39 años con una mastitis por Mycobacterium fortuitum, sin antecedentes patogénicos previos. Fue tratada en base a las pruebas de susceptibilidad a antibióticos y quimioterápicos y a la evidencia empírica citada por la bibliografía, con kanamicina, doxiciclina, ciprofloxacina y trimetoprima-sulfametoxazol. Se obtuvo la remisión completa de sus lesiones luego de 15 meses de tratamiento. Se comenta la capacidad de producir lesiones de esta micobacteria de crecimiento rápido, su diagnóstico y tratamiento

Clinical and laboratory features of Mycobacterium porcinum.

Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776(T)) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776(T). Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, D-mannitol, i-myo-inositol, and catalase at 68 degrees C. They were negative for L-rhamnose and D-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776(T) gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant D-sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.