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Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences., Genome editing methods are limited by the inability to selectively edit repetitive sequences. Here the authors demonstrate precise editing of a repetitive genetic element, a ribosome, while avoiding edits to native sites sharing identical sequence.

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.

A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new in vitro diagnostic nucleic acid amplification test (NAAT) for the detection of Mycoplasma genitalium., A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new in vitro diagnostic nucleic acid amplification test (NAAT) for the detection of Mycoplasma genitalium. Seven urogenital specimen types (n = 11,556) obtained from 1,778 females, aged 15 to 74 years, and 1,583 males, aged 16 to 82 years, were tested with the Aptima Mycoplasma genitalium assay, an investigational transcription-mediated amplification (TMA) NAAT for the detection of M. genitalium 16S rRNA. Infected status for enrolled subjects was established using results obtained from testing either self-collected vaginal swab or clinician-collected male urethral swab specimens with a composite reference method consisting of three transcription-mediated amplification NAATs targeting unique regions of M. genitalium 16S or 23S rRNA. M. genitalium prevalence was 10.2% in females and 10.6% in males; prevalence was high in both symptomatic and asymptomatic subjects for both sexes. Compared to the subject infected status standard, the investigational test had sensitivity and specificity estimates, respectively, of 98.9% and 98.5% for subject-collected vaginal swabs, 92.0% and 98.0% for clinician-collected vaginal swabs, 81.5% and 98.3% for endocervical swabs, 77.8% and 99.0% for female urine, and 98.2% and 99.6% for male urethral swabs, 88.4% and 97.8% for self-collected penile meatal swabs, and 90.9% and 99.4% for male urine specimens. For all seven specimen types, within-specimen positive and negative agreements between the investigational test and the composite reference standard ranged from 94.2% to 98.3% and from 98.5 to 99.9%, respectively. These results provide clinical efficacy evidence for the first FDA-cleared NAAT for M. genitalium detection in the United States.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila . Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

International audience; Objectives: A previous study on 12 in vitro -selected azithromycin-resistant Legionella~pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia~coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L.~pneumophila . Methods: The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L.~pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. Results: We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L.~pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. Conclusions: LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L.~pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.

International audience; Objectives: We investigated the epidemiological, clinical, microbiological and genetic characteristics of linezolid-resistant (LZR) Staphylococcus capitis isolates from French ICUs, and compared them with LZR S. capitis isolates from other European countries. Methods: All LZR isolates were subjected to antimicrobial susceptibility testing (AST) and the presence of cfr and optrA genes as well as mutations in the 23S rRNA and ribosomal proteins were investigated using specific PCR with sequencing. The genetic relationship between isolates was investigated using PFGE and WGS. Epidemiological data concerning LZR S. capitis were collected retrospectively in French microbiology laboratories. Results: Twenty-one LZR isolates were studied: 9 from France, 11 from Greece and 1 from Finland. All were resistant to methicillin and aminoglycosides. In addition, this unusual AST profile was identified in S. capitis isolates from seven French hospitals, and represented up to 12% of the S. capitis isolates in one centre. A G2576T mutation in 23S rRNA was identified in all isolates; cfr and optrA genes were absent. All isolates belonged to the same clone on the basis of their PFGE profiles, whatever their geographical origin. WGS found at most 212 SNPs between core genomes of the LZR isolates. Conclusions: We identified and characterized an LZR S. capitis clone disseminated in three European countries, harbouring the same multiple resistance and a G2576T mutation in the 23S rRNA. The possible unrecognized wider distribution of this clone, belonging to a species classically regarded as a low-virulence skin colonizer, is of major concern not least because of the increasing use of oxazolidinones.

It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 extracts and incubated with chemically mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here we report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β(3)-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β(3)-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.

International audience; Multiresistance in staphylococci constitutes a major challenge for the antimicrobial chemotherapy of invasive infections such as bacteraemia or bone and joint infections (BJIs). A nationwide prospective study was performed to detect antimicrobial resistance trends among staphylococci causing invasive infections. Between October 2011 and February 2012, 367 meticillin-resistant Staphylococcus aureus (MRSA) and 695 coagulase-negative staphylococci (CoNS) were collected from 37 French hospitals, mainly from bacteraemia (59.9%) and osteoarticular infections (29.0%). Minimum inhibitory concentrations (MICs) were determined by broth microdilution, and specific screening and confirmation tests were performed to detect heterogeneous vancomycin-intermediate S. aureus (hVISA). Staphylococcal isolates exhibiting a linezolid MIC\textgreater4 mg/L were further characterised to determinate their clonal relationships and the mechanism of resistance. MRSA exhibited additional resistances, including levofloxacin (82% associated resistance), gentamicin (13.6%), fusidic acid (13.6%) and rifampicin (6.5%), compromising oral step-down therapy in BJIs. Only two hVISA strains (0.5%) were identified. Among the CoNS, mainly Staphylococcus epidermidis (506/695; 72.8%), resistance to first- and second-line agents was more common. Linezolid resistance was identified in 10 CoNS (1.4%). The most frequent linezolid resistance mechanism was the G2576T mutation in 23S rDNA (9/10). For the first time in France, the cfr gene was found in five related sequence type 2 (ST2) S. epidermidis from two different hospitals, in association with ribosomal RNA and L3 ribosomal protein mutations. These national data must be considered when selecting empirical treatment for invasive staphylococcal infections. Moreover, the emergence and spread of linezolid-resistant CoNS carrying the cfr gene is of concern.

The influence of resource availability on planktonic and biofilm microbial community membership is poorly understood. Heterotrophic bacteria derive some to all of their organic carbon (C) from photoautotrophs while simultaneously competing for inorganic nutrients such as phosphorus (P) or nitrogen (N). Therefore, inputs have the potential to shift the competitive balance of aquatic microbial communities by increasing the resource space available to heterotrophic bacteria (more C) while decreasing the resource space available to algae (less mineral nutrients due to increased competition from osmotrophic heterotrophs). To test how resource dynamics affect membership of planktonic communities and assembly of biofilm communities we amended a series of flow-through mesocosms with C and P to alter the availability of C among treatments. Each mesoscosm was fed with unfiltered seawater and incubated with sterile glass substrate for biofilm formation. We used 454 pyrosequencing of bacterial 16S and 23S plastid genes to ask how resource driven shifts in the pool size of each community affected community membership and structure. The highest C treatment had the lowest planktonic algal abundance yet highest planktonic bacterial abundance and highest biofilm biomass. Resource amendments did not have a significant effect on alpha diversity in either the planktonic or biofilm communities. Rather the biofilm communities consistently had higher alpha diversity than the planktonic communities in all mesocosms. Bacterioplankton and biofilm membership was distinct in all but the highest C treatment where biofilm and planktonic communities increasingly resembled each other over time.Unlike the bacteria, algal biofilm and plankton communities displayed distinct microbial membership and structure in all treatments including the highest C treatment. Our results suggest that even though resource amendments affect community membership, microbial lifestyle (biofilm or planktonic) places a significanlty stronger constraint on community assembly and membership.

Several molecular platforms can identify bacteria associated with bloodstream infections but require positive culture bottles as starting material. Here, we describe results of screening 1140 blood cultures at 8h postinoculation, from 918 eligible adults being evaluated for bloodstream infection. DNA was extracted and analyzed by 16S and/or 23S rRNA real-time PCR/pyrosequencing. Compared to culture, PCR/pyrosequencing displayed 90.9% sensitivity, 99.6% specificity, 95.7% positive predictive value, and 99.1% negative predictive value. Overall concordance rate was 98.9% (1127/1140). In 4 cases with molecular-positive/culture-negative results, medical chart reviews provided evidence of identical bacteria from subsequent blood or concomitant urine/sputum cultures. Nine culture-positive/molecular-negative cases were associated with either polymicrobial growth, grew only in the anaerobic bottle of the clinical pair, and/or were detected by PCR/pyrosequencing after 8h. In summary, this approach accurately detected and identified bacteria in ~91% of culture-confirmed cases significantly sooner than the phenotypic identification was available, having the potential to improve antibiotic stewardship.

Hyphomonas neptunium is a marine prosthecate α-proteobacterium currently classified as a member of the order ‘Rhodobacterales’. Although this classification is supported by 16S rRNA gene sequence phylogeny, 23S rRNA gene sequence analysis, concatenated ribosomal proteins, HSP70 and EF-Tu phylogenies all support classifying Hyphomonas neptunium as a member of the Caulobacterales instead. The possible reasons why the 16S rRNA gene sequence gives conflicting results in this case are also discussed.

Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

The aim of this multicentre study was to analyse the characteristics of patients with bloodstream infections due to staphylococcal strains resistant to linezolid. This was a retrospective case-case-control study of patients hospitalised in three large teaching hospitals in Italy. A linezolid-resistant (LIN-R) Staphylococcus spp. group and a linezolid-susceptible (LIN-S) Staphylococcus spp. group were compared with control patients to determine the clinical features and factors associated with isolation of LIN-R strains. All LIN-R Staphylococcus spp. strains underwent molecular typing. Compared with the LIN-S group, central venous catheters were the main source of infection in the LIN-R group. The LIN-R and LIN-S groups showed a similar incidence of severe sepsis or septic shock, and both showed a higher incidence of these compared with the control group. Overall, patients in the LIN-R group had a higher 30-day mortality rate. Multivariate analysis found previous linezolid therapy, linezolid therapy14 days, antibiotic therapy in the previous 30 days, antibiotic therapy14 days, previous use of at least two antibiotics and hospitalisation in the previous 90 days as independent risk factors associated with isolation of a LIN-R strain. The G2576T mutation in domain V of 23S rRNA was the principal mechanism of resistance; only one strain of Staphylococcus epidermidis carried the cfr methylase gene (A2503), together with L4 insertion (71GGR72) and L3 substitution (H146Q). LIN-R strains are associated with severe impairment of clinical conditions and unfavourable patient outcomes. Reinforcement of infection control measures may have an important role in preventing these infections.

The effects of side chain modification and chirality in linezolid-like 1,2,4-oxadiazoles have been studied to design new potent antibacterials against Gram-positive multidrug-resistant pathogens. The adopted strategy involved a molecular modelling approach, the synthesis and biological evaluation of new designed compounds, enantiomers separation and absolute configuration assignment. Experimental determination of the antibacterial activity of the designed (S)-1-((3-(4-(3-methyl-1,2,4-oxadiazol-5- yl)phenyl)-oxazolidin-2-one-5-yl)methyl)-3-methylthiourea and (S)-1-((3-(3-fluoro-4-(3-methyl-1,2,4- oxadiazol-5-yl)phenyl)-oxazolidin-2-one-5-yl)methyl)-3-methylthiourea against multidrug resistant linezolid bacterial strains was higher than that of linezolid.

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.

Theoretical data for electron impact excitation of neutral helium in the He (2(1,3)S) states are reviewed and a preferred data set is established for excitation to the He (n(1,3)L) states with n=2-4. Such a set of data was presented in a FOM report. The present work is an improvement made possible by new theoretical data, in particular the convergent close coupling data of Bray and Fursa, valid for the entire impact energy range of interest.

The constitutive or inducible macrolide–lincosamide–streptogramin (MLS) phenotype of 30 erm (B)-positive Streptococcus pyogenes isolates was determined by different methods and under various growth conditions and correlated to the sequence of the 5′-untranslated regions of erm (B). The MLS phenotype of one-third of the isolates could not be classified. In liquid medium, some of these isolates responded to induction only during the logarithmic phase of growth, while others expressed clindamycin resistance even under noninducing conditions. By increasing the growth rate, we observed a shift from a constitutive towards an inducible pattern of resistance. All data were confirmed by analysis of the 23S rRNA methylation level. The erm (B)-5′-untranslated region was 99% similar in sequence. In erm (B)-positive S. pyogenes , the MLS phenotype is strongly influenced by culture conditions and control of its expression does not depend exclusively on the sequence of the erm (B)-5′-untranslated region.

Experimental data for electron impact excitation of neutral helium in its ground state are reviewed critically. A preferred dataset is established and combined with theoretical close coupling approximation data below the ionization threshold and Born approximation data at high energy. Results are presented in figures and tables. Maxwell averaged collision strengths are also tolerated.

Experimental data for electron impact excitation of neutral helium in its ground state are reviewed critically. A preferred dataset is established and combined with theoretical close coupling approximation data below the ionization threshold and Born approximation data at high energy. Results are presented in figures and tables. Maxwell averaged collision strengths are also tolerated.

Experimental data for electron impact excitation of neutral helium in its ground state are reviewed critically. A preferred dataset is established and combined with theoretical close coupling approximation data below the ionization threshold and Born approximation data at high energy. Results are presented in figures and tables. Maxwell averaged collision strengths are also tolerated.

The emerging enteric pathogen Campylobacter concisus is associated with prolonged diarrhea and inflammatory bowel disease. Previous studies have shown that C. concisus strains are very genetically diverse. Nevertheless, C. concisus strains have been divided into two genomospecies, where GS1 strains have been isolated predominantly from healthy individuals, while the GS2 cluster consists of isolates primarily from diarrheic individuals. The aim of the present study was to determine the genetic diversity of C. concisus isolates from Danish diarrheic patients. Multilocus sequence typing using the loci aspA, atpA, glnA, gltA, glyA, ilvD and pgm, as well as genomospecies based on specific differences in the 23S rRNA, was used to characterize 67 isolates (63 fecal and 4 oral), from 49 patients with different clinical presentations (29 with diarrhea, eight with bloody diarrhea, seven with collagenous colitis and five with Crohn’s disease). MLST revealed a high diversity of C. concisus with 53 sequence types (STs), of which 52 were identified as ‘new’ STs. Allele sequences showed more than 90 % similarity between isolates, with only four outliers. Dendrogram profiles of each allele showed a division into two groups, which more or less correlated with genomospecies A and genomospecies B. However, in contrary to previous results, this subgrouping had no association to the clinical severity of disease. Electronic supplementary material The online version of this article (doi:10.1186/s13099-016-0126-0) contains supplementary material, which is available to authorized users.

Theoretical data for electron impact excitation of neutral helium in the He (2(1,3)S) states are reviewed and a preferred data set is established for excitation to the He (n(1,3)L) states with n=2-4. Such a set of data was presented in a FOM report. The present work is an improvement made possible by new theoretical data, in particular the convergent close coupling data of Bray and Fursa, valid for the entire impact energy range of interest.