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The MORDOR I trial 1 , conducted in Niger, Malawi and Tanzania, demonstrated that mass azithromycin distribution to preschool children reduced childhood mortality 1 . However, the large but simple trial design precluded determination of the mechanisms involved. Here we examined the gut microbiome of preschool children from 30 Nigerien communities randomized to either biannual azithromycin or placebo. Gut microbiome γ-diversity was not significantly altered ( P  = 0.08), but the relative abundances of two Campylobacter species, along with another 33 gut bacteria, were significantly reduced in children treated with azithromycin at the 24-month follow-up. Metagenomic analysis revealed functional differences in gut bacteria between treatment groups. Resistome analysis showed an increase in macrolide resistance gene expression in gut microbiota in communities treated with azithromycin ( P  = 0.004). These results suggest that prolonged mass azithromycin distribution to reduce childhood mortality reduces certain gut bacteria, including known pathogens, while selecting for antibiotic resistance. Mass antibiotic administration to preschool children in Nigerien communities alters the relative abundances of 35 bacterial species in the gut microbiome, including enteric pathogens, but also increases expression of macrolide resistance genes.

Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

In response to environmental stress the model cyanobacterium, Synechocystis sp. PCC6803 can switch from a planktonic state to autoaggregation and biofilm formation. The precise mechanism of this transition remains unknown. Here we investigated the role of a candidate two-component regulatory system (TCS) in controlling morphological changes, as a way to understand the intermediate molecular steps that are part of the signaling pathway. A bacterial two-hybrid assay showed that the response regulator Rre6 formed a TCS together with a split histidine kinase consisting of Hik36 and Hik43. Individual disruption mutants displayed autoaggregation in a static culture. In contrast, unlike in the wild type, high salinity did not induce biofilm formation in Δhik36 , Δhik43 and Δrre6 . The expression levels of exopolysaccharide (EPS) production genes were higher in Δhik36 and Δhik43 , compared with the wild type, but lower in Δrre6 , suggesting that the TCS regulated EPS production in Synechocystis . Rre6 interacted physically with the motor protein PilT2, that is a component of the type IV pilus system. This interaction was enhanced in a phosphomimic version of Rre6. Taken together, Hik36–Hik43–Rre6 function as an upstream component of the pili-related signal transduction cascade and control the prevention of cell adhesion and biofilm formation.

Antibiotic treatment generally results in the selection of resistant bacterial strains, and the dynamics of resistance evolution is dependent on complex interactions between cellular components. To better characterize the mechanisms of antibiotic resistance and evaluate its dependence on gene regulatory networks, we performed systematic laboratory evolution of Escherichia coli strains with single-gene deletions of 173 transcription factors under three different antibiotics. This resulted in the identification of several genes whose deletion significantly suppressed resistance evolution, including arcA and gutM . Analysis of double-gene deletion strains suggested that the suppression of resistance evolution caused by arcA and gutM deletion was not caused by epistatic interactions with mutations known to confer drug resistance. These results provide a methodological basis for combinatorial drug treatments that may help to suppress the emergence of resistant pathogens by inhibiting resistance evolution.

Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium’s primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.

The qnr genes are plasmid‐borne fluoroquinolone‐resistance determinants widespread in Enterobacteriaceae. Three families of qnr determinants ( qnrA , B and S ) have been described, but little is known about their expression and regulation. Two new determinants, qnrC and qnrD , have been found recently. Here, we describe the characterization of the qnrB2 promoter and the identification of a LexA‐binding site in the promoter region of all qnrB alleles. LexA is the central regulator of the SOS response to DNA damage. We show that qnrB2 expression is regulated through the SOS response in a LexA/RecA‐dependent manner, and that it can be induced by the quinolone ciprofloxacin, a known inducer of the SOS system. This is the first description of direct SOS‐dependent regulation of an antibiotic‐resistance mechanism in response to the antibiotic itself.

Riboswitches—RNA motifs that regulate gene expression in response to binding of specific ligands—have been identified for many small-molecule metabolites. An ATP-binding element in the ydaO mRNA of Bacillus subtilis provides the first example of an ATP-responsive riboswitch. We report what is to our knowledge the first natural RNA that regulates gene expression in response to intracellular ATP. Using a biochemical screen, we found that several putative riboswitches bind ATP in vitro . The ydaO motif specifically bound ATP and regulated expression of endogenous and reporter genes in response to ATP concentrations in Bacillus subtilis. This discovery demonstrates a role for RNAs in regulating gene expression in response to energy balance in bacteria.

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human beta-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

The present study was undertaken to investigate growth kinetics and time-dependent change in global expression of Escherichia coli O157∶H7 Sakai upon an abrupt downshift in water activity (aw). Based on viable count data, shifting E. coli from aw 0.993 to aw 0.985 or less caused an apparent loss, then recovery, of culturability. Exponential growth then resumed at a rate characteristic for the aw imposed. To understand the responses of this pathogen to abrupt osmotic stress, we employed an integrated genomic and proteomic approach to characterize its cellular response during exposure to a rapid downshift but still within the growth range from aw 0.993 to aw 0.967. Of particular interest, genes and proteins with cell envelope-related functions were induced during the initial loss and subsequent recovery of culturability. This implies that cells undergo remodeling of their envelope composition, enabling them to adapt to osmotic stress. Growth at low aw, however, involved up-regulating additional genes and proteins, which are involved in the biosynthesis of specific amino acids, and carbohydrate catabolism and energy generation. This suggests their important role in facilitating growth under such stress. Finally, we highlighted the ability of E. coli to activate multiple stress responses by transiently inducing the RpoE and RpoH regulons to control protein misfolding, while simultaneously activating the master stress regulator RpoS to mediate long-term adaptation to hyperosmolality. This investigation extends our understanding of the potential mechanisms used by pathogenic E. coli to adapt, survive and grow under osmotic stress, which could potentially be exploited to aid the selection and/or development of novel strategies to inactivate this pathogen.

Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor. We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB(-) mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 +/- 1 d versus 7 +/- 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not. The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.