Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

The human parasite Leishmania uses adaptive gene rearrangements and amplification involving repeated sequences on a genome-wide scale as one strategy to adapt to a changing environment.

Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate. Whole-genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.

No abstract

Recently, two orthologues of the Drosophila homeobox Cut gene, Cux-1 and Cux-2, have been identified as restricted molecular markers of upper layer (II-IV) neurons in the murine cerebral cortex. We show that during early postnatal life, from P0 to P10, Cux-1 and Cux-2 mRNA are coexpressed in all primary sensory cortices. Antisera to Cux-1 and Cux-2 immunoreactivities preferentially label neurons in the barrel walls of the primary somatosensory cortex (S1). Subsequently, Cux-1 remains enriched in sensory cortices, whereas Cux-2 expression enlarges to comprise the frontal and insular areas. The laminar distribution of Cux-1 and Cux-2 differs: Cux-1 follows a layer IV to layer II decreasing gradient of expression, whereas Cux-2 expression is homogeneous across layers IV-II. No colocalization was found with GABA and birth dating experiments showed that Cux-1-positive neurons in layer IV are born during a restricted period, E13.5-E14.5, suggesting that Cux-1 is a useful molecular marker of the glutamatergic neurons of layer IV. We examined Cux-1 and Cux-2 in barrel-defective mouse strains, the VMAT2 KO, the MAOA KO, and the Adcyl 1 brl strain. A normal expression level of Cux-1 and Cux-2 was found in layer IV, despite the lack of segregation of the neurons as barrels. Conversely, in Reeler mice, Cux-1 and Cux-2 had a distinct laminar distribution: the Cux-1-positive neurons had an inverted deep localization, whereas the Cux-2-positive neurons were distributed throughout the cortical thickness, suggesting that Cux-2 expression is more widely expressed in the inverted cortex of reeler mutants. Our results indicate that Cux-1 is a useful marker of the layer IV neurons in S1, and that Cux-1 and Cux-2 are differently regulated in the upper layers of the cerebral cortex.

Durancin and reuterin were effective inhibitors of Clostridium difficile, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus. The combination of durancin and reuterin was highly synergistic against C. difficile (fractional inhibitory concentration index = 0.2). Durancin/vancomycin combination was synergistic against S. aureus ATCC 700699 (fractional inhibitory concentration index = 0.3). Conclusion & future perspective: Durancin 61A alone or combined with other bacteriocins or antibiotics may therefore provide a possible therapeutic option for the treatment of infections by these pathogens.

Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

This first report about tigecycline resistance mechanisms in S. pneumoniae revealed that, in contrast to Gram-negative species, for which efflux appears central for tigecycline resistance, resistance in the pneumococcus occurs through mutations related to ribosomes.