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Environmental decontamination and water disinfection practices are hallmarks of disease prevention and control in agricultural and public health settings. Informed fit-to-purpose biocontainment is thus dependent on methodologies accurately assessing microbial burden and viability. Also, rigorous evaluation of the efficacy of biocontrol measures implies monitoring microbial inactivation after decontamination/disinfection procedures. In this study, we used flow cytometry coupled with a resuscitation protocol to monitor the metabolic inactivation of bacteria capable of entering non-cultivable states, after the application of a chlorine-based water disinfectant. For this purpose, we used Mycobacterium bovis BCG as a model of slow-growing bacteria able to enter dormancy and representing a multi-host pathogen in a zoonotic disease system—animal tuberculosis—thriving both across temperate and semi-arid regions and involving environmental contamination. The biocide activity of a commercial sodium dichloroisocyanurate (NaDCC) disinfectant against M. bovis BCG was evaluated through mock environmental matrix tests. Using the manufacturer-recommended dosage of NaDCC, BCG cells were apparently inactivated after 24 h upon exposure. However, we show via flow cytometry that, upon exposure to optimal growth conditions, mycobacterial cells were able to regain metabolic activity shortly after, highlighting a sublethal effect of NaDCC at the recommended commercial dosage due to reversible BCG cell damage. In contrast, increasing twice the disinfectant dosage completely inactivated BCG cells after 24 h of exposure, with full irreversible loss of metabolic activity. Methodological workflows based on conventional culture or PCR would have missed the detection of these dormant subpopulations that were in fact able to resume growth when following the recommendations of a commercial disinfectant. This study highlights the superior, high-resolution value of single-cell approaches, such as flow cytometry, to accurately assess the activity of biocides against metabolically heterogeneous and dormant pathogenic bacteria with environmental cycles, supporting data-driven prioritization of environmental management and disinfection options in contaminated vulnerable settings. [ABSTRACT FROM AUTHOR]

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention. [ABSTRACT FROM AUTHOR]

Non-tuberculous mycobacteria (NTM) are paradigmatic colonizers of the total environment, circulating at the interfaces of the atmosphere, lithosphere, hydrosphere, biosphere, and anthroposphere. Their striking adaptive ecology on the interconnection of multiple spheres results from the combination of several biological features related to their exclusive hydrophobic and lipid-rich impermeable cell wall, transcriptional regulation signatures, biofilm phenotype, and symbiosis with protozoa. This unique blend of traits is reviewed in this work, with highlights to the prodigious plasticity and persistence hallmarks of NTM in a wide diversity of environments, from extreme natural milieus to microniches in the human body. Knowledge on the taxonomy, evolution, and functional diversity of NTM is updated, as well as the molecular and physiological bases for environmental adaptation, tolerance to xenobiotics, and infection biology in the human and non-human host. The complex interplay between individual, species-specific and ecological niche traits contributing to NTM resilience across ecosystems are also explored. This work hinges current understandings of NTM, approaching their biology and heterogeneity from several angles and reinforcing the complexity of these microorganisms often associated with a multiplicity of diseases, including pulmonary, soft-tissue, or milliary. In addition to emphasizing the cornerstones of knowledge involving these bacteria, we identify research gaps that need to be addressed, stressing out the need for decision-makers to recognize NTM infection as a public health issue that has to be tackled, especially when considering an increasingly susceptible elderly and immunocompromised population in developed countries, as well as in low- or middle-income countries, where NTM infections are still highly misdiagnosed and neglected. [ABSTRACT FROM AUTHOR]

Recently, we unveiled taxonomical and functional differences in Egyptian mongoose (Herpestes ichneumon) gut microbiota across sex and age classes by microbial profiling. In this study, we generate, through culturomics, extended baseline information on the culturable bacterial and fungal microbiome of the species using the same specimens as models. Firstly, this strategy enabled us to explore cultivable microbial community differences across sexes and to ascertain the influence exerted by biological and environmental contexts of each host in its microbiota signature. Secondly, it permitted us to compare the culturomics and microbial profiling approaches and their ability to provide information on mongoose gut microbiota. In agreement with microbial profiling, culturomics showed that the core gut cultivable microbiota of the mongoose is dominated by Firmicutes and, as previously found, is able to distinguish sex- and age class-specific genera. Additional information could be obtained by culturomics, with six new genera unveiled. Richness indices and the Shannon index were concordant between culture-dependent and culture-independent approaches, highlighting significantly higher values when using microbial profiling. However, the Simpson index underlined higher values for the culturomics-generated data. These contrasting results were due to a differential influence of dominant and rare taxa on those indices. Beta diversity analyses of culturable microbiota showed similarities between adults and juveniles, but not in the data series originated from microbial profiling. Additionally, whereas the microbial profiling indicated that there were several bioenvironmental features related to the bacterial gut microbiota of the Egyptian mongoose, a clear association between microbiota and bioenvironmental features could not be established through culturomics. The discrepancies found between the data generated by the two methodologies and the underlying inferences, both in terms of β-diversity and role of bioenvironmental features, confirm that culture-independent, sequence-based methods have a higher ability to assess, at a fine scale, the influence of abiotic and biotic factors on the microbial community composition of mongoose' gut. However, when used in a complementary perspective, this knowledge can be expanded by culturomics. [ABSTRACT FROM AUTHOR]

The Egyptian mongoose (Herpestes ichneumon) is a medium-size carnivore that, in Europe, is restricted to Iberia. The bio-ecology of this species remains to be elucidated in several dimensions, including gut microbiota that is nowadays recognized as a fundamental component of mammals. In this work, we investigated the gut microbiota of this herpestid by single-molecule real-time sequencing of twenty paired male (n = 10) and female (n = 10) intestinal samples. This culture-independent approach enabled microbial profiling based on 16S rDNA and investigation of taxonomical and functional features. The core gut microbiome of the adult subpopulation was dominated by Firmicutes, Fusobacteria, Actinobacteria, and Proteobacteria. Eight genera were uniquely found in adults and five in non-adults. When comparing gut bacterial communities across sex, four genera were exclusive of females and six uniquely found in males. Despite these compositional distinctions, alpha- and beta-diversity analyses showed no statistically significant differences across sex or between adult and non-adult specimens. However, when function was inferred, males presented a significantly higher abundance of amino acid and citrate cycle metabolic pathways, compared to the significant overrepresentation in females of galactose metabolic pathways. Additionally, adults exhibited a significantly higher abundance of cationic antimicrobial peptide resistance pathways, while non-adults bared a significant overrepresentation of two-component systems associated with antibiotic synthesis, flagellin and biofilm production, and chemotaxis control. This study adds new insights into the mongoose bio-ecology palette, highlighting taxonomical and functional microbiome dissimilarities across sex and age classes, possibly related to primary production resources and life-history traits that impact on behavior and diet. [ABSTRACT FROM AUTHOR]