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Although the bovine tuberculosis (TB) agent, Mycobacterium bovis , may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis , resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS 6110 element upstream of the phoPR locus may completely revert the phoPR-bovis –associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR -mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

Mycobacteriophages—bacteriophages infecting Mycobacterium hosts—contribute substantially to our understanding of viral diversity and evolution, provide resources for advancing Mycobacterium genetics, are the basis of high-impact science education programs, and show considerable therapeutic potential. Over 10,000 individual mycobacteriophages have been isolated by high school and undergraduate students using the model organism Mycobacterium smegmatis mc 2 155 and 2,100 have been completely sequenced, giving a high-resolution view of the phages that infect a single common host strain. The phage genomes are revealed to be highly diverse and architecturally mosaic and are replete with genes of unknown function. Mycobacteriophages have provided many widely used tools for Mycobacterium genetics including integration-proficient vectors and recombineering systems, as well as systems for efficient delivery of reporter genes, transposons, and allelic exchange substrates. The genomic insights and engineering tools have facilitated exploration of phages for treatment of Mycobacterium infections, although their full therapeutic potential has yet to be realized.

The Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in mycobacteria plays a crucial role in pathogenesis and immune evasion. These proteins characterized by unique structures with conserved sequences. This study elucidated the specific immunological functions of MMAR_1296 from marine mycobacterium. Expressing MMAR_1296 in Mycobacterium smegmatis (M. smegmatis) led to significant alterations in bacterial morphology, as well as reduced survival of M. smegmatis under adverse in vitro conditions and within macrophages. Furthermore, transcriptome analysis of mouse macrophages indicated that natural immunity-related pathways were upregulated in the group infected with M. smegmatis recombinantly expressing MMAR_1296. Moreover, the mycobacterium Growth Inhibition Assays(MGIA)in mice demonstrated that M. smegmatis expressing MMAR_1296 exerted a significant inhibitory effect against Mycobacterium abscessus (M. abscessus) and Mycobacterium marinum (M. marinum) infections. Immunization challenge experiments in mice further confirmed its protective effects, showing a reduction in organ bacterial loads by 1 log10 value compared to the positive control group. These findings indicate that MMAR_1296 is a promising vaccine candidate for M. abscessus and M. marinum. Given that PE/PPE protein family is also a crucial component of Mycobacterium tuberculosis (M. tuberculosis) antigens, further exploration of sequence functions based on MMAR_1296 could reveal broader applications of PE/PPE proteins family for M. tuberculosis treatment. This study supported vaccine development targeting PE/PPE proteins in mycobacteria and paves the way for broader applications.

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Free-living amoebae (FLA) are described as environmental reservoirs for some bacteria able to resist their phagocytosis. In the environment, the fate of Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) responsible for bovine tuberculosis and paratuberculosis, respectively, remains poorly understood and is considered potentially problematic in the eradication and control of these diseases. We hypothesize that FLA may play a role in the persistence of Mbo and Map in the environment. In this study, 90 samples were collected from herds affected by one or both diseases to investigate the diversity of amoeba and their associated bacteria. Metabarcoding analyses revealed that Acanthamoeba, Copromyxa, Naegleria, and Vermamoeba were the most represented genera of FLA, with Pseudomonadota being the bacteria most commonly found associated with FLA. Although no Mbo and Map DNA were identified by sequencing, traces were detected by ddPCR (digital droplet PCR), specifically targeting these bacteria. In conclusion, we described a wide diversity of FLA and associated bacteria in this environment. It also suggests that Map and Mbo could be associated, even weakly, with FLA in the environment. However, this needs to be confirmed by detecting a highest amount of DNA and, if possible, cultivable Map and/or Mbo associated with these environmental FLA.

The evolution of tubercle bacilli parallels a route from environmental Mycobacterium kansasii , through intermediate “ Mycobacterium canettii ”, to the modern Mycobacterium tuberculosis complex. Cell envelope outer membrane lipids change systematically from hydrophilic lipooligosaccharides and phenolic glycolipids to hydrophobic phthiocerol dimycocerosates, di- and pentaacyl trehaloses and sulfoglycolipids. Such lipid changes point to a hydrophobic phenotype for M. tuberculosis sensu stricto . Using Congo Red staining and hexadecane-aqueous buffer partitioning, the hydrophobicity of rough morphology M. tuberculosis and Mycobacterium bovis strains was greater than smooth “ M. canettii ” and M. kansasii . Killed mycobacteria maintained differential hydrophobicity but defatted cells were similar, indicating that outer membrane lipids govern overall hydrophobicity. A rough M. tuberculosis H37Rv Δ papA1 sulfoglycolipid-deficient mutant had significantly diminished Congo Red uptake though hexadecane-aqueous buffer partitioning was similar to H37Rv. An M. kansasii , Δ MKAN27435 partially lipooligosaccharide-deficient mutant absorbed marginally more Congo Red dye than the parent strain but was comparable in partition experiments. In evolving from ancestral mycobacteria, related to “ M. canettii ” and M. kansasii , modern M. tuberculosis probably became more hydrophobic by increasing the proportion of less polar lipids in the outer membrane. Importantly, such a change would enhance the capability for aerosol transmission, affecting virulence and pathogenicity.

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 μM against Mycobacterium tuberculosis, SI = 9.8). Here, we found that the compound is bactericidal towards Mycobacterium smegmatis, resulting in greater than 5 Log10 reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 μM (4X MIC) concentration. Growth inhibition (MIC = 50 μM) and reduction in cfu could also be observed against a clinical isolate of Mycobacterium abscessus. Stepwise isolation of resistant mutants of M. smegmatis was conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibits Mtb topoisomerase I and Mtb gyrase with IC50 of 0.24 and 27 μM, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC50 for inhibition of Mtb topoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of Mtb topoisomerase I.

The World Health Organization gave great attention to Mycobacterium tuberculosis , especially its zoonotic impact. Dromedary camels in Arabian countries are of great importance, as well as awareness of production and health. Little was known about the occurrence of M. tuberculosis among Arabian camels. Out of 88 samples were collected from necropsied male camels aged 5–6.5 years after the slaughter process resident in Cairo abattoir. Isolation of Mycobacteria was achieved on Middle Brook 7H10 agar with special supplements, and then the suspected colonies were assessed by their specific aspects. Lungs and lymph nodes were processed for histopathology. Molecular characterization was carried out by both conventional amplification ( Mycobacterium bovis mpb70, M. tuberculosis - Pan Mycobacterium 16S rRNA) tracked by sanger sequencing; and bacterial 16S rRNA V3–V4 hypervariable region was amplified then it was followed by Mi-seq Ilumina. Moringa oliefera’s oil was analyzed by GC–MS. The antimycobacterial potential of M. oliefera was conducted by In vitro tetrazolium microplate assay (TEMA). In silico docking mode of action and prediction were studied. Mycobacterium was isolated from 9.4% (3/32) of the lung samples and 2.4% (1/41) of the recovered lymph node samples. The isolated strains had ideal culture characteristics of Mycobacterium . Sanger sequencing identified the M. tuberculosis variant bovis DRC-EG-CAMEL PQ036932. Mi-seq Illumina revealed abundant sequence readings belonging to ancestral Actinobacteria and Micromonosporaceae. In vitro testing showed that the Moringa oleifera methanol leaf extract had antimicrobial activity with MIC ranging from 7.8 to 32 µg/ml, and the seed oil showed inhibitory effects at 50% (v/v) ( P value < 0.05). In silico docking of ferulic acid against M. tuberculosis variant bovis ribosomal protein S1 showed an affinity score of − 5.95 kcal/mol with one hydrogen bond. While squalene lipoprotein LprF exhibited a professional affinity score of − 6.11 kcal/mol with seventeen hydrophobic π-interactions. Mycobacterium tuberculosis variant bovis is measured to prevail in the Arabian camels. However, this study provided a detailed examination of Mycobacterium in camels, offering practical solutions to combat this pathogen and mitigate the effects of infection or zoonotic impacts on other animals and humans. Sanger sequencing is more recommended for Mycobacterium identification. Moringa oliefera’s potential anti-mycobacterial effect through either leaves or oil might be achieved for humans and animals as a different strategy for medicinal plants’ role. It might be a new insight into the struggle and the adverse effects of tuberculosis. In the upcoming research, therapeutic compounds could be separated from M. oliefera .

Mycobacterium abscessus is an important infectious agent highly associated with drug resistance and treatment failure. We investigated the drug resistance situation of M. abscessus in Northeast Thailand and the possible genetic basis for this. Sixty-eight M. abscessus clinical isolates were obtained from 26 patients at Srinagarind Hospital during 2012-2016. Drug susceptibility tests and sequencing of erm(41), rrl and rrs genes were performed. Mycobacterium abscessus was resistant to 11/15 antibiotics (nearly 100% resistance in each case). Partial susceptibility to four antibiotics was found (amikacin, tigecycline, clarithromycin and linezolid). Non-massiliense subspecies were significantly associated with clarithromycin resistance (p<0.0001) whereas massiliense subspecies were associated with tigecycline resistance (p = 0.028). Inducible clarithromycin resistance was seen in 22/68 (32.35%) isolates: 21 of these isolates (95.45%) belonged to non-massiliense subspecies and resistance was explicable by the T28C mutation in erm(41). Inducible clarithromycin resistance was found in one isolate of the massiliense subspecies. Acquired clarithromycin resistance explicable by the A2271G/C mutation of rrl was seen in only 7/16 (43.75%) of strains. Inducible and acquired resistance mechanisms can be interchangeable during the course of infection. Rrs mutations were not associated with amikacin resistance in our study. Antibiotic resistance in subspecies of M. abscessus was reported from Northeast Thailand. Known resistance-associated mutations cannot explain all of the resistance patterns observed.

Mycobacterium decipiens is a newly identified species with high genomic similarity to M. tuberculosis. We report a cutaneous M. decipiens infection in a patient in France who had inflammatory bowel disease being treated with anti-tumor necrosis factor-α therapy. The infection was successfully treated with an oral antimicrobial regimen.