New Resistance Mutations Linked to Decreased Susceptibility to Solithromycin in Streptococcus pneumoniae Revealed by Chemogenomic Screens.

Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm(B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm(B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef(E)/mel and in the vicinity of the ribosome binding site of erm(B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations.

Azithromycin resistance mutations in Streptococcus pneumoniae as revealed by a chemogenomic screen.

We report on the combination of chemical mutagenesis, azithromycin selection and next-generation sequencing (Mut-Seq) for the identification of small nucleotide variants that decrease the susceptibility of Streptococcus pneumoniae to the macrolide antibiotic azithromycin. Mutations in the 23S ribosomal RNA or in ribosomal proteins can confer resistance to macrolides and these were detected by Mut-Seq. By concentrating on recurrent variants, we could associate mutations in genes implicated in the metabolism of glutamine with decreased azithromycin susceptibility among S. pneumoniae mutants. Glutamine synthetase catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition is shown to sensitize S. pneumoniae to antibiotics. A mutation affecting the ribosomal-binding site of a putative ribonuclease J2 is also shown to confer low-level resistance. Mut-Seq has the potential to reveal chromosomal changes enabling high resistance as well as novel events conferring more subtle phenotypes.

Gain- and Loss-of-Function Screens Coupled to Next-Generation Sequencing for Antibiotic Mode of Action and Resistance Studies in Streptococcus pneumoniae.

Two whole-genome screening approaches are described for studying the mode of action and the mechanisms of resistance to trimethoprim (TMP) in the Gram-positive Streptococcus pneumoniae The gain-of-function approach (Int-Seq) relies on a genomic library of DNA fragments integrated into a fucose-inducible cassette. The second approach, leading to both gain- and loss-of-function mutation, is based on chemical mutagenesis coupled to next-generation sequencing (Mut-Seq). Both approaches pointed at the drug target dihydrofolate reductase (DHFR) as a major resistance mechanism to TMP. Resistance was achieved by dhfr overexpression either through the addition of fucose (Int-Seq) or by mutations upstream of the gene (Mut-Seq). Three types of mutations increased expression by disrupting a predicted Rho-independent terminator upstream of dhfr Known and novel DHFR mutations were also detected by Mut-Seq, and these were functionally validated for TMP resistance. The two approaches also suggested that an increase in the metabolic flux from purine synthesis to GTP and then to folate can modulate the susceptibility to TMP. Finally, we provide evidence for a novel role of the ABC transporter PatAB in TMP susceptibility. Our genomic screens highlighted novel aspects on the mode of action and mechanisms of resistance to antibiotics.

Inductors and regulatory properties of the genomic island-associated fru2 metabolic operon of Streptococcus agalactiae.

The fru2 metabolic operon of Streptococcus agalactiae encodes the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) enzyme II complex Fru2 (EIIBFru2 , EIIAFru2 , and EIICFru2 ); Fru2 R, a transcriptional activator with PTS regulatory domains (PRDs); a d-allulose-6-phosphate 3-epimerase; a transaldolase; and a transketolase. We showed that the transcription of fru2 is induced during the stationary phase of growth in complex media and during incubation in human cerebrospinal or amniotic fluids. d-allose and d-ribose are environmental signals governing this induction. PTSFru2 is involved in the activation of the fru2 promoter, and the histidine-67 of EIIAFru2 and the cysteine-9 of EIIBFru2 are important for this function. The activation of fru2 is also controlled by Fru2 R. The histidine-243 in the PRD1 domain, the histidine-323 in the PRD2 domain, the cysteine-400 in the EIIB-like domain, and the histidine-549 in the EIIA-like domain are important for the function of Fru2 R. Fru2 R binds to a DNA region containing palindromic sequences upstream of the identified transcriptional start site. EIIBFru2 interacts physically with the C-terminal part of Fru2 R (expressing the EIIB-like and EIIA-like motifs) and with EIIAFru2 . We propose a model of regulation of fru2 depending on the presence of an activatory carbohydrate in the growth medium.

The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival.

The Lmb protein of Streptococcus agalactiae is described as an adhesin that binds laminin, a component of the human extracellular matrix. In this study, we revealed a new role for this protein in zinc uptake. We also identified two Lmb homologs, AdcA and AdcAII, redundant binding proteins that combine with the AdcCB translocon to form a zinc-ABC transporter. Expression of this transporter is controlled by the zinc concentration in the medium through the zinc-dependent regulator AdcR. Triple deletion of lmb, adcA, and adcAII, or that of the adcCB genes, impaired growth and cell separation in a zinc-restricted environment. Moreover, we found that this Adc zinc-ABC transporter promotes S. agalactiae growth and survival in some human biological fluids, suggesting that it contributes to the infection process. These results indicated that zinc has biologically vital functions in S. agalactiae and that, under the conditions tested, the Adc/Lmb transporter constitutes the main zinc acquisition system of the bacterium. IMPORTANCE: A zinc transporter, composed of three redundant binding proteins (Lmb, AdcA, and AdcAII), was characterized in Streptococcus agalactiae This system was shown to be essential for bacterial growth and morphology in zinc-restricted environments, including human biological fluids.

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic Escherichia coli is encoded on a genomic island in specific lineages of Streptococcus agalactiae.

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults.

Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice.

Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure™ Gram Positive DNA Purification Kit, QIAamp® DNA Stool Mini Kit; NucliSENS® easyMAG®, ZR Fecal DNA MiniPrep™, FastDNA® SPIN Kit for Feces and FastDNA® SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA® SPIN Kit for Soil.

New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface.

Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.