The macrophage-bacterium mismatch in persister formation.

Many pathogens are hard to eradicate, even in the absence of genetically detectable antimicrobial resistance mechanisms and despite proven antibiotic susceptibility. The fraction of clonal bacteria that temporarily elude effective antibiotic treatments is commonly known as 'antibiotic persisters.' Over the past decade, there has been a growing body of research highlighting the pivotal role played by the cellular host in the development of persisters. In parallel, this research has also sought to elucidate the molecular mechanisms underlying the formation of intracellular antibiotic persisters and has demonstrated a prominent role for the bacterial stress response. However, questions remain regarding the conditions leading to the formation of stress-induced persisters among a clonal population of intracellular bacteria and despite an ostensibly uniform environment. In this opinion, following a brief review of the current state of knowledge regarding intracellular antibiotic persisters, we explore the ways in which macrophage functional heterogeneity and bacterial phenotypic heterogeneity may contribute to the emergence of these persisters. We propose that the degree of mismatch between the macrophage permissiveness and the bacterial preparedness to invade and thrive intracellularly may explain the formation of stress-induced nonreplicating intracellular persisters.

Spatio-temporal variations in chemical pollutants found among urban deposits match changes in thiopurine S-methyltransferase-harboring bacteria tracked by the tpm metabarcoding approach.

The bTPMT (bacterial thiopurine S-methyltransferase), encoded by the tpm gene, can detoxify metalloid-containing oxyanions and xenobiotics. The hypothesis of significant relationships between tpm distribution patterns and chemical pollutants found in urban deposits was investigated. The tpm gene was found conserved among eight bacterial phyla with no sign of horizontal gene transfers but a predominance among gammaproteobacteria. A DNA metabarcoding approach was designed for tracking tpm-harboring bacteria among polluted urban deposits and sediments recovered for more than six years in a detention basin (DB). This DB recovers runoff waters and sediments from a zone of high commercial activities. The PCR products from DB samples led to more than 540,000 tpm reads after DADA2 or MOTHUR bio-informatic manipulations that were allocated to more than 88 and less than 634 sequence variants per sample. The tpm community patterns were significantly different between the recent urban deposits and those that had accumulated for more than 2 years in the DB, and between those of the DB surface and the DB settling pit. These groups of samples had distinct mixture of priority pollutants. Significant relationships between tpm ordination patterns, sediment accumulation time periods and location, and concentrations in PAH, chlorpyrifos, and 4-nonylphenols (NP) were observed. These correlations matched the higher occurrences of, among others, Aeromonas, Pseudomonas, and Xanthomonas tpm-harboring bacteria in recent urban DB deposits more contaminated with chrysene and alkylphenol ethoxylates. Highly significant drops in tpm reads allocated to Aeromonas species were recorded in the oldest DB sediments accumulating naphthalene and metallic pollutants. Degraders of urban pollutants such as P. aeruginosa and P. putida showed conserved distribution patterns over time but P. syringae phytopathogens were more abundant in the oldest sediments. TPMT-harboring bacteria can be used to assess the incidence of high risk priority pollutants on environmental systems.

Metal oxide nanoparticles for the decontamination of toxic chemical and biological compounds.

For several years, the international context is deeply affected by the use of chemical and biological weapons. The use of CBRN (Chemical Biological Radiological Nuclear) threat agents from military stockpiles or biological civilian industry demonstrate the critical need to improve capabilities of decontamination for civilians and military. Physical decontamination systems that operate only by adsorption and displacement such as Fuller's Earth, have the drawback of not neutralizing hazardous agents, giving place to cross contaminations. Consequently, the development of a formulation based on metal oxide nanoparticles attracts considerable interest, since they offer physicochemical properties that allow them to both adsorb and degrade toxic compounds. Thus, the aim of this study is to found metal oxide nanoparticles with a versatile activity on both chemical and biological toxic agents. Therefore, several metal oxides such as MgO, TiO2, CeO2, ZnO and ZrO2 were characterized and their decontamination kinetics of less-toxic surrogate of VX, paraoxon, were studied in vitro. To determine the antimicrobial activity of these nanoparticles, simulants of biological terrorist threat were used by performing a 3-hours decontamination kinetics. This proof-of-concept study showed that MgO is the only one that exhibits both chemical and antibacterial actions but without sporicidal activity.

Incidence of hydrological, chemical, and physical constraints on bacterial pathogens, Nocardia cells, and fecal indicator bacteria trapped in an urban stormwater detention basin in Chassieu, France.

The nature and fate of urban contaminants washed by stormwater events and accumulating in a detention basin (DB) were investigated. Relations between bacterial and chemical contaminants of trapped urban sediments, and field parameters were analyzed. Fecal indicators and some pathogens known to be environmentally transmitted (Nocardia, Pseudomonas aeruginosa, and Aeromonas caviae) were tracked, and their persistence investigated. Six sampling campaigns were carried out over 3 years, using five sites including a settling chamber (SC). Aerosolized bacteria at these sites were also monitored. Deposits in the basin were made of fine particles and their content in chemical pollutants was found highly variable. High polycyclic aromatic hydrocarbon (PAH) contents were measured but only three pesticides, over 22, were detected. Deposits were significantly contaminated by fecal indicator bacteria (FIB), P. aeruginosa, A. caviae, and by Nocardia. Only A. caviae showed significant numbers in aerosolized particles recovered over the detention basin. Nocardia spp. cells heavily contaminated the SC. The efficacy of the detention basin at reducing bacterial counts per rain event and over time were estimated. A slight drop in the counts was monitored for fecal indicators but not for the other bacterial groups. Hydrodynamic parameters had a strong impact on the distribution and features of the deposits. Multiple factors impacted the fate of FIB, P. aeruginosa, A. caviae, and Nocardia cells, but in a group dependent manner. Nocardia counts were found positively correlated with volatile organic matter. FIB appeared highly efficient colonizers of the DB.

Bacteriome genetic structures of urban deposits are indicative of their origin and impacted by chemical pollutants.

Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16 S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits.

Virulence test using nematodes to prescreen Nocardia species capable of inducing neurodegeneration and behavioral disorders.

BACKGROUND: Parkinson's disease (PD) is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain-some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. Nocardia are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system (CNS) invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are N. farcinica, N. nova, N. brasiliensis and N. cyriacigeorgica. Some studies have highlighted the ability of N. cyriacigeorgica to induce Parkinson's disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. METHODS: Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. RESULTS: The nematode Caenorhabditis elegans allowed us to demonstrate that Nocardia strains belonging to N. cyriacigeorgica and N. farcinica species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of Nocardia strains to induce neurodegeneration. These behavioral disorders were induced by N. cyriacigeorgica species (N. cyriacigeorgica GUH-2 and N. cyriacigeorgica 44484) and N. farcinica 10152. DISCUSSION: We conclude that C. elegans is a good model for detecting Nocardia strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to characterize the induced behavioral disorders and bacterial dissemination.

The Nocardia cyriacigeorgica GUH-2 genome shows ongoing adaptation of an environmental Actinobacteria to a pathogen's lifestyle.

BACKGROUND: Nocardia cyriacigeorgica is recognized as one of the most prevalent etiological agents of human nocardiosis. Human exposure to these Actinobacteria stems from direct contact with contaminated environmental matrices. The full genome sequence of N. cyriacigeorgica strain GUH-2 was studied to infer major trends in its evolution, including the acquisition of novel genetic elements that could explain its ability to thrive in multiple habitats. RESULTS: N. cyriacigeorgica strain GUH-2 genome size is 6.19 Mb-long, 82.7% of its CDS have homologs in at least another actinobacterial genome, and 74.5% of these are found in N. farcinica. Among N. cyriacigeorgica specific CDS, some are likely implicated in niche specialization such as those involved in denitrification and RuBisCO production, and are found in regions of genomic plasticity (RGP). Overall, 22 RGP were identified in this genome, representing 11.4% of its content. Some of these RGP encode a recombinase and IS elements which are indicative of genomic instability. CDS playing part in virulence were identified in this genome such as those involved in mammalian cell entry or encoding a superoxide dismutase. CDS encoding non ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) were identified, with some being likely involved in the synthesis of siderophores and toxins. COG analyses showed this genome to have an organization similar to environmental Actinobacteria. CONCLUSION: N. cyriacigeorgica GUH-2 genome shows features suggesting a diversification from an ancestral saprophytic state. GUH-2 ability at acquiring foreign DNA was found significant and to have led to functional changes likely beneficial for its environmental cycle and opportunistic colonization of a human host.

The bacterial thiopurine methyltransferase tellurite resistance process is highly dependent upon aggregation properties and oxidative stress response.

Bacterial thiopurine methyltransferases (bTPMTs) can favour resistance towards toxic tellurite oxyanions through a pathway leading to the emission of a garlic-like smell. Gene expression profiling completed by genetic, physiological and electron microscopy analyses was performed to identify key bacterial activities contributing to this resistance process. Escherichia coli strain MG1655 expressing the bTPMT was used as a cell model in these experiments. This strain produced a garlic-like smell which was found to be due to dimethyl telluride, and cell aggregates in culture media supplemented with tellurite. Properties involved in aggregation were correlated with cell attachment to polystyrene, which increased with tellurite concentrations. Gene expression profiling supported a role of adhesins in the resistance process with 14% of the tellurite-regulated genes involved in cell envelope, flagella and fimbriae biogenesis. Other tellurite-regulated genes were, at 27%, involved in energy, carbohydrate and amino acid metabolism including the synthesis of antioxidant proteins, and at 12% in the synthesis of transcriptional regulators and signal transduction systems. Escherichia coli mutants impaired in tellurite-regulated genes showed ubiquinone and adhesins synthesis, oxidative stress response, and efflux to be essential in the bTPMT resistance process. High tellurite resistance required a synergistic expression of these functions and an efficient tellurium volatilization by the bTPMT.

Genome sequence of the human- and animal-pathogenic strain Nocardia cyriacigeorgica GUH-2.

The pathogenic strain Nocardia cyriacigeorgica GUH-2 was isolated from a fatal human nocardiosis case, and its genome was sequenced. The complete genomic sequence of this strain contains 6,194,645 bp, an average G+C content of 68.37%, and no plasmids. We also identified several protein-coding genes to which N. cyriacigeorgica's virulence can potentially be attributed.

The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt.

RcnA is an efflux pump responsible for Ni and Co detoxification in Escherichia coli. The expression of rcnA is induced by Ni and Co via the metallo-regulator RcnR. In the present work, the functioning of the promoter-operator region of rcnR and rcnA was investigated using primer extension and DNAse I footprinting experiments. We show that the promoters of rcnR and rcnA are convergent and that apo-RcnR binds on symmetrically located sequences in this intergenic region. Moreover, RcnR DNA binding is specifically modulated by one Ni or Co equivalent and not by other metals. In addition to rcnA, RcnR controls expression of its own gene in response to Ni and Co, but the two genes are differentially expressed.

Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria.

The phytostimulatory alphaproteobacterium Azospirillum lipoferum 4B exhibits the plant-beneficial gene acdS, which enables deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Here, we show that acdS is in the vicinity of acdR, a homolog to leucine-responsive regulator lrp, in A. lipoferum 4B and most other acdS+ Proteobacteria. Unlike in Beta- and Gammaproteobacteria, acdS (and acdR) is preferentially located on symbiotic islands and plasmids in Alphaproteobacteria. In A. lipoferum 4B, acdS was mapped on a 750-kb plasmid that is lost during phenotypic variation, whereas other phytobeneficial genes such as nifH (associative nitrogen fixation) are maintained. In Proteobacteria, the phylogenies of acdR and acdS were largely but not totally congruent, despite physical proximity of the genes, regardless of whether DNA or deduced protein sequences were used. Potential Lrp, cAMP receptor protein (CRP) and fumarate-nitrate reduction regulator (FNR) binding sites were evidenced in the acdS promoter regions of strain 4B and most of 46 other acdS+ Proteobacteria. Indeed, transcriptional and enzymatic analyses done in vitro pointed to the involvement of Lrp- and FNR-like transcriptional up-regulation of ACC deaminase activity in A. lipoferum 4B. This is the first synteny, phylogenetic, and functional analysis of factors modulating acdS expression in Azospirillum plant growth-promoting rhizobacterium.

Phase variation and genomic architecture changes in Azospirillum.

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.

Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography.

Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in plant growth-promoting rhizobacteria (PGPR) and phytosymbiotic bacteria, but the diversity of the corresponding gene (acdS) is poorly documented. Here, acdS sequences were obtained by screening putative ACC deaminase sequences listed in databases, based on phylogenetic properties and key residues. In addition, acdS was sought in 71 proteobacterial strains by PCR amplification and/or hybridization using colony dot blots. The presence of acdS was confirmed in established AcdS+ bacteria and evidenced noticeably in Azospirillum (previously reported as AcdS-), in 10 species of Burkholderia and six Burkholderia cepacia genomovars (which included PGPR, phytopathogens and opportunistic human pathogens), and in five Agrobacterium genomovars. The occurrence of acdS in true and opportunistic pathogens raises new questions concerning their ecology in plant-associated habitats. Many (but not all) acdS+ bacteria displayed ACC deaminase activity in vitro, including two Burkholderia clinical isolates. Phylogenetic analysis of partial acdS and deduced AcdS sequences evidenced three main phylogenetic clusters, each gathering pathogens and plant-beneficial strains of contrasting geographic and habitat origins. The acdS phylogenetic tree was only partly congruent with the rrs tree. Two clusters gathered both Betaprotobacteria and Gammaproteobacteria, suggesting extensive horizontal transfers of acdS, noticeably between plant-associated Proteobacteria.

Physical organization of phytobeneficial genes nifH and ipdC in the plant growth-promoting rhizobacterium Azospirillum lipoferum 4VI.

The physical organization of phytobeneficial genes was investigated in the plant growth-promoting rhizobacterium Azospirillum lipoferum 4VI by hybridization screening of a bacterial artificial chromosome (BAC) library. Pulsed-field gel electrophoresis gave an estimated 5.7-Mb genome size for strain 4VI and a coverage level of 9 for the BAC library. The phytobeneficial genes nifH (associative nitrogen fixation) and ipdC (synthesis of the phytohormone indoleacetic acid) are chromosomal, but no BAC clone containing both genes was found, pointing to the absence of any genetic island containing nifH and ipdC. A 11.8-kb fragment containing nifH was analyzed. Neighboring genes implicated in nitrogen fixation (nifH, draT, draG) or not (arsC, yafJ and acpD) were organized as in A. brasilense. In contrast, the region located downstream of acpD contained four housekeeping genes (i.e. genes encoding DapF-, MiaB- and FtsY-like proteins, as well as gene amn) and differed totally from the one found in A. brasilense.

Selection of unusual actinomycetal primary sigma70 factors by plant-colonizing Frankia strains.

Functional adaptations of sigma70 transcriptional factors led to the emergence of several paralogous lineages, each one being specialized for gene transcription under particular growth conditions. Screening of a Frankia strain EaI-12 gene library by sigma70 DNA probing allowed the detection and characterization of a novel actinomycetal primary (housekeeping) sigma70 factor. Phylogenetic analysis positioned this factor in the RpoD cluster of proteobacterial and low-G+C-content gram-positive factors, a cluster previously free of any actinobacterial sequences. sigma70 DNA probing of Frankia total DNA blots and PCR screening detected one or two rpoD-like DNA regions per species. rpoD matched the conserved region in all of the species tested. The other region was found to contain sigA, an alternative primary factor. sigA appeared to be strictly distributed among Frankia species infecting plants by the root hair infection process. Both genes were transcribed by Frankia strain ACN14a grown in liquid cultures. The molecular phylogeny of the sigma70 family determined with Frankia sequences showed that the alternative actinomycetal factors and the essential ones belonged to the same radiation. At least seven distinct paralogous lineages were observed among this radiation, and gene transfers were detected in the HrdB actinomycetal lineage.