Mycobacterium ulcerans Experimental Dormancy.

Whether Mycobacterium ulcerans, the etiological agent of Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, has not been established. Six M. ulcerans strains were exposed to a progressive depletion in oxygen for 2 months, using the Wayne model of dormancy previously described for M. tuberculosis, and further examined by microscopy after staining of dynamic, dormant, and dead mycobacteria (DDD staining), microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls. Mycobacterium ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile red-stained intracellular lipid droplets and a 14- to 20-day regrowth when exposed to ambient air, consistent with dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD staining and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, warrants further investigations in the natural ecological niches where M. ulcerans thrive as well as in Buruli ulcer lesions.

Fecal Excretion of Mycobacterium leprae, Burkina Faso.

Mycobacterium leprae was detected by optical microscopy, fluorescent in situ hybridization, and molecular detection in feces collected for the diagnosis of Entamoeba coli enteritis in a leprosy patient in Burkina Faso. This observation raises questions about the role of fecal excretion of M. leprae in the natural history and diagnosis of leprosy.

Methanobrevibacter smithii Archaemia in Febrile Patients With Bacteremia, Including Those With Endocarditis.

BACKGROUND: The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. METHODS: Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. RESULTS: PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. CONCLUSIONS: Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria.

Fluorescent Hybridization of Mycobacterium leprae in Skin Samples Collected in Burkina Faso.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d'Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Translocation of Mycobacterium tuberculosis after experimental ingestion.

Human tuberculosis is a life-threatening infection following the inhalation of Mycobacterium tuberculosis, while the closely related bacteria Mycobacterium bovis and Mycobacterium canettii are thought to be transmitted by ingestion. To explore whether M. tuberculosis could also infect individuals by ingestion, male BALBc mice were fed 2 x 106 CFUs of M. tuberculosis Beijing or phosphate-buffered saline as a negative control, over a 28-day experiment. While eight negative control mice remained disease-free, M. tuberculosis was identified in the lymph nodes and lungs of 8/14 mice and in the spleens of 4/14 mice by microscopy, PCR-based detection and culture. Whole-genome sequencing confirmed the identity of the inoculum and the tissue isolates. In these genetically identical mice, the dissemination of M. tuberculosis correlated with the results of the culture detection of four intestinal bacteria. These observations indicate that ingested M. tuberculosis mycobacteria can translocate, notably provoking lymphatic tuberculosis.

Blood Culture-Negative Cardiovascular Infection in a Patient With Multiple Sclerosis.

A patient with multiple sclerosis presented with seronegative C. burnetii endocarditis diagnosed using C. burnetii-specific polymerase chain reaction and fluorescence in situ hybridization on cardiovascular biopsy. This case supports the necessity of a systematic polymerase chain reaction testing of removed cardiac valves because blood culture-negative endocarditis can be pauci-symptomatic, and serological tests can be negative in cases of immunosuppression.

Decrypting the environmental sources of Mycobacterium canettii by high-throughput biochemical profiling.

Mycobacterium canettii is a smooth bacillus related to the Mycobacterium tuberculosis complex. It causes lymph nodes and pulmonary tuberculosis in patients living in countries of the Horn of Africa, including Djibouti. The environmental reservoirs of M. canettii are still unknown. We aimed to further decrypt these potential reservoirs by using an original approach of High-Throughput Carbon and Azote Substrate Profiling. The Biolog Phenotype profiling was performed on six clinical strains of M. canettii and one M. tuberculosis strain was used as a positive control. The experiments were duplicated and authenticated by negative controls. While M. tuberculosis metabolized 22/190 (11%) carbon substrates and 3/95 (3%) nitrogen substrates, 17/190 (8.9%) carbon substrates and three nitrogen substrates were metabolized by the six M. canettii strains forming the so-called corebiologome. A total at 16 carbon substrates and three nitrogen substrates were metabolized in common by M. tuberculosis and the six M. canettii strains. Moreover, at least one M. canettii strain metabolized 36/190 (19%) carbon substrates and 3/95 (3%) nitrogen substrates for a total of 39/285 (13%) substrates. Classifying these carbon and nitrogen substrates into ten potential environmental sources (plants, fruits and vegetables, bacteria, algae, fungi, nematodes, mollusks, mammals, insects and inanimate environment) significantly associated carbon and nitrogen substrates metabolized by at least one M. canettii strain with plants (p = 0.006). These results suggest that some plants endemic in the Horn of Africa may serve as ecological niches for M. canettii. Further ethnobotanical studies will indicate plant usages by local populations, then guiding field microbiological investigations in order to prove the definite environmental reservoirs of this opportunistic tuberculous pathogen.

Nine Cases of Methanogenic Archaea in Refractory Sinusitis, an Emerging Clinical Entity.

The authors report the cases of 9 patients eventually diagnosed with methanogenic archaea refractory or recalcitrant chronic rhinosinusitis, a condition known to involve various anaerobic bacteria but in which the role of methanogenic archaea is unknown. The authors retrospectively searched these microorganisms by PCR in surgically-collected sinusal pus specimens from patients diagnosed with refractory sinusitis, defined by the persistance of sinus inflammation and related-symptoms for more than 12 weeks despite appropriate treatment. Of the 116 tested sinus surgical specimens, 12 (10.3%) from 9 patients (six females, three males; aged 20-71 years) were PCR-positive. These specimens were further investigated by fluorescence in-situ hybridization, PCR amplicon-sequencing and culture. Methanobrevibacter smithii was documented in four patients and Methanobrevibacter oralis in another four, one of whom was also culture-positive. They were associated with a mixed flora including Gram-positive and Gram-negative bacteria. In the latter patient, "Methanobrevibacter massiliense" was the sole microorganism detected. These results highlight methanogenic archaea as being part of a mixed anaerobic flora involved in refractory sinusitis, and suggest that the treatment of this condition should include an antibiotic active against methanogens, notably a nitroimidazole derivative.

Co-culture models illustrate the digestion of Gemmata spp. by phagocytes.

Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes' cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.

FISHing Mycobacterium tuberculosis Complex by Use of a rpoB DNA Probe Bait.

Routine staining of sputum specimens does not identify acid-fast bacilli as Mycobacterium tuberculosis with utmost precision, limiting its usability as a confirmatory test for pulmonary tuberculosis. We have combined Ziehl-Neelsen staining and fluorescence in situ hybridization (FISH) to detect M. tuberculosis in sputum specimens. We have developed a new fluorescent oligonucleotide rpoBMTC probe (5'-Alexa-555-AGCGGGGTGATGTCAACCCAG-3') targeting the M. tuberculosis complex rpoB gene. In silico alignment yielded 100% match for M. tuberculosis complex mycobacteria, 66.6% to 47.6% for other bacteria, and no significant hits for viruses and eukaryotes. Negative binding of rpoBMTC probe to the top six respiratory tract bacterial pathogens and to Mycobacterium abscessus and Mycobacterium avium experimentally confirmed its specificity. As for sensitivity, rpoBMTC-FISH detected 103 CFU/ml M. tuberculosis as confirmed by successful detection of M. tuberculosis in artificially seeded sputum samples. The application of rpoBMTC-FISH to 116 routine sputum specimens yielded a detection of M. tuberculosis in all of the 31 Ziehl-Neelsen-positive and culture-positive specimens, and no detection of M. tuberculosis in the 85 M. tuberculosis-negative specimens. These data established the proof of concept that rpoBMTC-FISH alone or combined with Ziehl-Neelsen staining can specifically "FISH out" M. tuberculosis complex mycobacteria in sputum samples collected from patients suspected of pulmonary mycobacteriosis. We are implementing this probe for the routine and specific detection of M. tuberculosis complex bacteria in sputum exhibiting acid-fast mycobacteria.

Fluorescence in situ hybridization microscopic detection of Bacilli Calmette Guérin mycobacteria in aortic lesions: A case report.

RATIONALE: To improve the diagnosis of life-threatening Bacilli Calmette Guérin (BCG) arterial aneurysm in patients treated by intravesical instillation of BCG vaccine as adjunctive therapy for non-muscular bladder carcinoma, is a life-threatening condition. Its diagnosis remains cumbersome. PATIENT CONCERNS: One patient with a history of intravesical BCG installation presented with aortic aneurysm with routine microscopic examination after Ziehl-Neelsen staining remaining negative. DIAGNOSES: We used fluorescence in situ hybridization (FISH) to target the Mycobacterium tuberculosis complex rpob gene in a fresh aortic specimen. FISH yielded fluorescent mycobacteria in aortic lesions; mycobacteria were further confirmed as Mycobacterium bovis BCG mycobacteria by polymerase chain reaction (PCR) sequencing. INTERVENTIONS: The patient benefited from an antituberculous treatment combining rifampicin, isoniazid, and ethambunol. OUTCOME: A 9-month follow-up indicated a favorable outcome. LESSONS: This case report teaches that FISH targeting the M tuberculosis complex rpoB gene should be incorporated in the laboratory investigation of aortic aneurysm in patients with a history of bladder carcinoma.