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Abstract Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization‐related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas‐1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65‐related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina‐MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species‐level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.

Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.

Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods. [ABSTRACT FROM AUTHOR]

We prospectively studied 298 patients with cystic fibrosis (mean age 11.3 years; range 2 months to 32 years; sex ratio, 0.47) for nontuberculous mycobacteria in respiratory samples from January 1, 1996, to December 31, 1999. Mycobacterium abscessus was by far the most prevalent nontuberculous mycobacterium: 15 patients (6 male, 9 female; mean age 11.9 years; range 2.5–22 years) had at least one positive sample for this microorganism (versus 6 patients positive for M. avium complex), including 10 with >3 positive samples (versus 3 patients for M. avium complex). The M. abscessus isolates from 14 patients were typed by pulsed-field gel electrophoresis: each of the 14 patients harbored a unique strain, ruling out a common environmental reservoir or person-to-person transmission. Water samples collected in the cystic fibrosis center were negative for M. abscessus. This major mycobacterial pathogen in children and teenagers with cystic fibrosis does not appear to be acquired nosocomially.

Objective: Differentiation between non-tuberculous mycobacteria ( NTM) and Mycobacterium tuberculosis complex (MTBC) is crucial for case management with the appropriate antimycobacterials. This study was undertaken in three West and Central African countries to understand NTM associated with pulmonary tuberculosis in the sub-region., Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from solid and liquid cultures were analysed. The isolates were tested for drug susceptibility, and MTBC were confirmed using IS 6110 . All IS 6110 -negative isolates were identified by 65-kilodalton heat shock protein ( hsp65 ) gene amplification, DNA sequencing and BLAST analysis., Results: Overall, the prevalence of NTM was 16/503 (3.2%), distributed as 2/202 (1%) in Nigeria, 2/158 (1.3%) in Cameroon and 12/143 (8.4%) in Ghana. The main NTM isolates included 5/16 (31.3%) M. fortuitum , 2/16 (12.5%) M. intracellulare and 2/16 (12.5%) M. engbaekii. Eight (57.1%) of the 14 previously treated patients harboured NTM (odds ratio 0.21, 95% confidence interval 0.06-0.77; P =0.021). Three multi-drug-resistant strains were identified: M. engbaekii, M. fortuitum and M. intracellulare., Conclusion: NTM were mainly found among individuals with unsuccessful treatment. This highlights the need for mycobacterial species differentiation using rapid molecular tools for appropriate case management, as most are resistant to routine first-line antimycobacterials., Competing Interests: None declared., (© 2022 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.)

Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database  was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65 -, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required. [ABSTRACT FROM AUTHOR]