Fierce poison to others: the phenomenon of bacterial dependence on antibiotics.

Beyond the development of resistance, the effects of antibiotics on bacteria and microbial communities are complex and far from exhaustively studied. In the context of the current global antimicrobial resistance crisis, understanding the adaptive and physiological responses of bacteria to antimicrobials is of paramount importance along with the development of new therapies. Bacterial dependence on antibiotics is a phenomenon in which antimicrobials instead of eliminating the pathogens actually provide a boost for their growth. This trait comprises an extreme example of the complexities of responses elicited by microorganisms to these drugs. This compelling evolutionary trait was readily described along with the first wave of antibiotics use and dependence to various antimicrobials has been reported. Nevertheless, current molecular characterizations have been focused on dependence on vancomycin, linezolid and colistin, three critically important antibiotics frequently used as last resource therapy for multi resistant pathogens. Outstanding advances have been made in understanding the molecular basis for the dependence to vancomycin, including specific mutations involved. Regarding linezolid and colistin, the general physiological components affected by the dependence, namely ribosomes and membrane function respectively, have been established. Nonetheless the implications of antibiotic dependence in clinically relevant features, such as virulence, epidemics, relationship with development of resistance, diagnostics and therapy effectiveness require clarification. This review presents a brief introduction of the phenomenon of bacterial dependence to antibiotics and a summary on early and current research concerning the basis for this trait. Furthermore, the available information on the effect of dependence in key clinical aspects is discussed. The studies performed so far underline the need to fully disclose the biological and clinical significance of this trait in pathogens to successfully assess its role in resistance and to design adjusted therapies.

The Assembly of Bacteria Living in Natural Environments Shapes Neuronal Integrity and Behavioral Outputs in Caenorhabditis elegans.

Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. Thus, the effects of environmental microbes on the nematodes' life-history traits are likely to contribute to the general health of the biosphere. Caenorhabditis elegans is an excellent model to study the behavioral and physiological outputs of microbial diets. However, the effects of complex natural bacterial assemblies have only recently been reported, as most studies have been carried out with monoxenic cultures of laboratory-reared bacteria. Here, we quantified the physiological, phenotypic, and behavioral traits of C. elegans feeding on two bacteria that were coisolated with wild nematodes from a soil sample. These bacteria were identified as a putative novel species of Stenotrophomonas named Stenotrophomonas sp. strain Iso1 and a strain of Bacillus pumilus designated Iso2. The distinctive behaviors and developmental patterns observed in animals fed with individual isolates changed when bacteria were mixed. We studied in more depth the degeneration rate of the touch circuit of C. elegans and show that B. pumilus alone is protective, while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite contents of each isolate and their combination identified NAD+ as being potentially neuroprotective. In vivo supplementation shows that NAD+ restores neuroprotection to the mixes and also to individual nonprotective bacteria. Our results highlight the distinctive physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates on nematodes. IMPORTANCE Do behavioral choices depend on animals' microbiota? To answer this question, we studied how different bacterial assemblies impact the life-history traits of the bacterivore nematode C. elegans using isolated bacteria found in association with wild nematodes in Chilean soil. We identified the first isolate, Iso1, as a novel species of Stenotrophomonas and isolate Iso2 as Bacillus pumilus. We find that worm traits such as food choice, pharyngeal pumping, and neuroprotection, among others, are dependent on the biota composition. For example, the neurodegeneration of the touch circuit needed to sense and escape from predators in the wild decreases when nematodes are fed on B. pumilus, while its coculture with Stenotrophomonas sp. eliminates neuroprotection. Using metabolomics analysis, we identify metabolites such as NAD+, present in B. pumilus yet lost in the mix, as being neuroprotective and validated their protective effects using in vivo experiments.

IurV, Encoded by ORF VCA0231, Is Involved in the Regulation of Iron Uptake Genes in Vibrio cholerae.

The pathogen Vibrio cholerae has multiple iron acquisition systems which allow bacteria to exploit a variety of iron sources across the different environments on which it thrives. The expression of such iron uptake systems is highly regulated, mainly by the master iron homeostasis regulator Fur but also by other mechanisms. Recently, we documented that the expression of many of the iron-responsive genes is also modulated by riboflavin. Among them, the open reading frame VCA0231, repressed both by riboflavin and iron, encodes a putative transcriptional regulator of the AraC/XylS family. Nonetheless, the genes or functions affected by this factor are unknown. In the present study, a series of in silico analyses was performed in order to identify the putative functions associated with the product of VCA0231. The STRING database predicted many iron uptake genes as functional partners for the product of VCA0231. In addition, a genomic neighborhood analysis with the Enzyme Function Initiative tools detected many Pfam families involved in iron homeostasis genetically associated with VCA0231. Moreover, a phylogenetic tree showed that other AraC/XylS members known to regulate siderophore utilization in bacteria clustered together and the product of VCA0231 localized in this cluster. This suggested that the product of VCA0231, here named IurV, is involved in the regulation of iron uptake processes. RNAseq was performed to determine the transcriptional effects of a deletion in VCA0231. A total of 52 genes were overexpressed and 21 genes were downregulated in response to the iurV deletion. Among these, several iron uptake genes and other iron homeostasis-related genes were found. Six gene ontology (GO) functional terms were enriched in the upregulated genes, of which five were related to iron metabolism. The regulatory pattern observed in the transcriptomics of a subset of genes was independently confirmed by quantitative real time PCR analysis. The results indicate that IurV is a novel regulator of the AraC/XylS family involved in the repression of iron uptake genes. Whether this effect is direct or indirect remains to be determined.

Transcriptomics reveals a cross-modulatory effect between riboflavin and iron and outlines responses to riboflavin biosynthesis and uptake in Vibrio cholerae.

Vibrio cholerae, a pandemic diarrheagenic bacterium, is able to synthesize the essential vitamin riboflavin through the riboflavin biosynthetic pathway (RBP) and also to internalize it through the RibN importer. In bacteria, the way riboflavin biosynthesis and uptake functions correlate is unclear. To gain insights into the role of the riboflavin provision pathways in the physiology of V. cholerae, we analyzed the transcriptomics response to extracellular riboflavin and to deletions of ribD (RBP-deficient strain) or ribN. Many riboflavin-responsive genes were previously reported to belong to the iron regulon, including various iron uptake genes. Real time PCR analysis confirmed this effect and further documented that reciprocally, iron regulates RBP and ribN genes in a riboflavin-dependent way. A subset of genes were responding to both ribD and ribN deletions. However, in the subset of genes specifically affected in the ∆ribD strain, the functional terms protein folding and oxidation reduction process were enriched, as determined by a Gene Ontology analysis. In the gene subset specifically affected in the ∆ribN strain, the cytochrome complex assembly functional term was enriched. Results suggest that iron and riboflavin interrelate to regulate its respective provision genes and that both common and specific effects of biosynthesized and internalized riboflavin exist.

Contribution of riboflavin supply pathways to Vibrio cholerae in different environments.

BACKGROUND: The waterborne diarrheagenic bacterium Vibrio cholerae, cause of the pandemic cholera disease, thrives in a variety of environments ranging from estuarine waters to the human intestinal tract. This species has two ways to obtain the essential micronutrient riboflavin, de novo biosynthesis and environmental uptake through the RibN importer. The way these functions interrelate to fulfill riboflavin needs in different conditions in this species is unknown. RESULTS: This study analyzed the contributions of riboflavin biosynthesis and transport to the culturability of Vibrio cholerae in river and seawater in vitro and in the Caenorhabditis elegans nematode host model. Elimination of the ribD riboflavin biosynthetic gene renders the bacteria riboflavin-auxotrophic, while a ribN mutant strain has no growth defect in minimal media. When growing in river water, deletion of ribD causes an impairment in culturability. In this condition, the ∆ribN strain has a defect to compete against a wild type strain but outcompetes the ∆ribD strain. The latter effect is inverted by the addition of riboflavin to the water. In contrast, growth in seawater causes a loss in culturability independent of riboflavin biosynthesis or transport. In the C. elegans model, only the ∆ribD strain is attenuated. CONCLUSION: Results indicate that while riboflavin biosynthesis seems to outweigh riboflavin uptake, the latter may still provide a selective advantage to V. cholerae in some environments.

Overlapping riboflavin supply pathways in bacteria.

Riboflavin derivatives are essential cofactors for a myriad of flavoproteins. In bacteria, flavins importance extends beyond their role as intracellular protein cofactors, as secreted flavins are a key metabolite in a variety of physiological processes. Bacteria obtain riboflavin through the endogenous riboflavin biosynthetic pathway (RBP) or by the use of importer proteins. Bacteria frequently encode multiple paralogs of the RBP enzymes and as for other micronutrient supply pathways, biosynthesis and uptake functions largely coexist. It is proposed that bacteria shut down biosynthesis and would rather uptake riboflavin when the vitamin is environmentally available. Recently, the overlap of riboflavin provisioning elements has gained attention and the functions of duplicated paralogs of RBP enzymes started to be addressed. Results point towards the existence of a modular structure in the bacterial riboflavin supply pathways. Such structure uses subsets of RBP genes to supply riboflavin for specific functions. Given the importance of riboflavin in intra and extracellular bacterial physiology, this complex array of riboflavin provision pathways may have developed to contend with the various riboflavin requirements. In riboflavin-prototrophic bacteria, riboflavin transporters could represent a module for riboflavin provision for particular, yet unidentified processes, rather than substituting for the RBP as usually assumed.