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ABSTRACTIntroductionRecurrent pericarditis (RP) is a debilitating disease that has an underlying autoinflammatory pathophysiology mediated by cytokine interleukin (IL)-1. Rilonacept, a recombinant dimeric fusion protein that blocks IL-1α and IL-1β signaling has emerged as a valuable therapeutic option of RP. Rilonacept has been evaluated in Phase 2 and 3 clinical trials and was recently approved for RP treatment.Areas coveredThis article reviews available clinical trials assessing the efficacy and safety of rilonacept for the treatment of RP.Expert opinionFindings from the Rhapsody study) trial suggest that rilonacept represents a promising new therapy for those patients with colchicine resistant or glucocorticoid-dependent disease. Treatment leads to rapid clinical response, with a median resolution of symptoms in 5 days, normalization of C-reactive protein (CRP) in a median of 7 days, and successful weaning from glucocorticoids. This novel therapy also reduces recurrence rates compared with placebo. Rilonacept has also demonstrated a good safety profile, with the most common adverse events including injection-site reactions and upper respiratory tract infections. This anti-IL 1 agent has emerged as an efficacious treatment for RP, with potential use for glucocorticoid-free regimens and as monotherapy. Future trials are needed to explore these treatment options and to clarify the appropriate therapy duration.

Pedobacter cryoconitisstrain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A–H (1–8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1–8were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitrocytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 μg/mL, respectively. Furthermore, compound 2had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 μg/mL, respectively. Compound 6was tested in an E. coliliposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

We have developed compounds with a promising activity against Acinetobacter baumanniiand Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumanniiand P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1in complex with Escherichia coliGyrB24 and (S)-27in complex with A. baumanniiGyrB23 and P. aeruginosaGyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27were improved by fine-tuning of lipophilicity. In particular, analogs of 27with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faeciumand multidrug resistant (MDR) Staphylococcus aureusstrains [best compound minimal inhibitory concentrations (MICs): range, <0.03125–0.25 μg/mL] and against the Gram-negatives Acinetobacter baumanniiand Klebsiella pneumoniae(best compound MICs: range, 1–4 μg/mL). Lead compound 7awas identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7ain complex with Pseudomonas aeruginosaGyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7aand 7hshowed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumanniiand several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7ain a mouse model of vancomycin-intermediate S. aureusthigh infection was also demonstrated.

Introduction:Obesity is a major risk factor for HFpEF. Metabolic surgery has proven to be effective in reducing heart failure incidence.Hypothesis:This study examined the impact of metabolic surgery on LV structure and function in patients with high risk for HFpEF.Methods:Patients undergoing metabolic surgery between March 2005 and February 2019 with pre- and post-operative TTE imaging, a preserved EF (>=50%) and a H2FPEF score >=5 were recruited for this study. A H2FPEF score of ≥5 is considered a high possibility of HFpEF diagnosis.Results:Table 1 shows baseline characteristics of the 126 patients that met the entry requirements. The mean age was 65.5 years (SD 8.9) and 64.3% were female. Table 2 outlines the clinical and echocardiographic variables before and after metabolic surgery. There was a significant decrease in mean body mass index (BMI) post operatively, from 47.7 to 36.6 (P<0.001), along with a number of statistically significant improvements in cardiovascular risk factors including a decrease in mean systolic blood pressure (BP) from 138 to 131 mmHg (P=0.005) and mean diastolic BP from 75 to 71 mmHg (P=0.014). There were improvements seen in the LV structure and function with the LV mass decreasing from 247.8 g to 220.7 g (P<0.001), and both the mean septal wall (1.4 cm to 1.3cm, P<0.001) and posterior wall (1.2 cm to 1.1 cm, P=0.007) thicknesses decreasing. There was also an improvement in the average LV global longitudinal strain (GLS) from -15.6 to -17.7% (P<0.001).Conclusions:This study demonstrates that for patients at high risk of HFPEF, metabolic surgery was associated with improvements in LV structure and function, including LVGLS.

ABSTRACTThe small-colony-variant (SCV) phenotype of Staphylococcus aureushas been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureusstrain and its counterpart with an SCV phenotype both in vitroand in vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureuspathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log10in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.

ABSTRACTResistance to fusidic acid in Staphylococcus aureusis caused by mutation of the elongation factor G (EF-G) drug target (FusA class) or by expression of a protein that protects the drug target (FusB and FusC classes). Recently, two novel genetic classes of small-colony variants (SCVs) were identified among fusidic acid-resistant mutants selected in vitro (FusA-SCV and FusE classes). We analyzed a phylogenetically diverse collection of fusidic acid-resistant bacteremia isolates to determine which resistance classes were prevalent and whether these were associated with particular phylogenetic lineages. Each isolate was shown by DNA sequencing and plasmid curing to carry only one determinant of fusidic acid resistance, with approximately equal frequencies of the FusA, FusB, and FusC genetic classes. The FusA class (mutations in fusA) were distributed among different phylogenetic types. Two distinct variants of the FusC class (chromosomal fusCgene) were identified, and FusC was also distributed among different phylogenetic types. In contrast, the FusB class (carrying fusBon a plasmid) was found in closely related types. No FusE-class mutants (carrying mutations in rplF) were found. However, one FusA-class isolate had multiple mutations in the fusAgene, including one altering a codon associated with the FusA-SCV class. SCVs are frequently unstable and may undergo compensatory evolution to a normal growth phenotype after their initial occurrence. Accordingly, this normal-growth isolate might have evolved from a fusidic acid-resistant SCV. We conclude that at least three different resistance classes are prevalent among fusidic acid-resistant bacteremia isolates of S. aureus.

ABSTRACTSmall-colony variants (SCVs) of Staphylococcus aureusare associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureusisolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB, the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menBmutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 × 10−8per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menBsequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menBcause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureusisolates.

Small-colony variants (SCVs) of Staphylococcus aureus are associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureus isolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB, the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menB mutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 x 10–8per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menB sequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menB cause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureus isolates.

ABSTRACTSmall-colony variants (SCVs) of Staphylococcus aureusare a slow-growing subpopulation whose phenotypes can include resistance to aminoglycosides, defects in electron transport, and enhanced persistence in mammalian cells. Here we show that a subset of mutants selected as SCVs by reduced susceptibility to aminoglycosides are resistant to the antibiotic fusidic acid (FA) and conversely that a subset of mutants selected for resistance to FA are SCVs. Mutation analysis reveals different genetic classes of FA-resistant SCVs. One class, FusA-SCVs, have amino acid substitution mutations in the ribosomal translocase EF-G different from those found in classic FusA mutants. Most of these mutations are located in structural domain V of EF-G, but some are in domain I or III. FusA-SCVs are auxotrophic for hemin. A second class of FA-resistant SCVs carry mutations in rplF, coding for ribosomal protein L6, and are designated as FusE mutants. FusE mutants fall into two phenotypic groups: one auxotrophic for hemin and the other auxotrophic for menadione. Accordingly, we have identified new genetic and phenotypic classes of FA-resistant mutants and clarified the genetic basis of a subset of S. aureusSCV mutants. A clinical implication of these data is that FA resistance could be selected by antimicrobial agents other than FA.

Objectives: To evaluate the mutant prevention concentrations (MPCs) of ciprofloxacin for two susceptible and one first-step gyrA resistant mutant Escherichia coli strains in an in vitro kinetic model and to identify the pharmacodynamic index that best predicts prevention of resistance emergence.Methods: An in vitro kinetic model was used to measure MPC with static antibiotic concentrations and to test different dosing profiles to study pharmacokinetics/pharmacodynamics indices important to prevent the growth of resistant mutants. In one set of kinetic experiments the starting concentration was equal to the MPC and the T > MPC was varied before antibiotic dilution was begun. In a second set of kinetic experiments Cmax was varied and dilution of the antibiotic was started at time zero.Results: From the static experiments we calculated MPC values of 0.128 mg/L for both the susceptible strains (16× MIC) and 0.188 mg/L (4× MIC) for the first-step resistant (gyrA) strain. The kinetic experiments showed that the T > MPC needed to prevent the growth of resistant bacteria was shorter with an increased Cmax. When resistance was selected, several subpopulations with different levels of susceptibility to ciprofloxacin emerged.Conclusions: Neither T > MPC nor Cmax proved to be single correlates for preventing resistance development. For the two investigated wild-type strains, an AUC/MPC ratio of ≥22 was the single pharmacodynamic index that predicted prevention of resistant mutant development.

ABSTRACTA new animal model for the purpose of studying superficial infections is presented. In this model an infection is established by disruption of the skin barrier by partial removal of the epidermal layer by tape stripping and subsequent application of the pathogens Staphylococcus aureusand Streptococcus pyogenes. The infection and the infection route are purely topical, in contrast to those used in previously described animal models in mice, such as the skin suture-wound model, where the infection is introduced into the deeper layers of the skin. Thus, the present model is considered more biologically relevant for the study of superficial skin infections in mice and humans. Established topical antibiotic treatments are shown to be effective. The procedures involved in the model are simple, a feature that increases throughput and reproducibility. This new model should be applicable to the evaluation of novel antimicrobial treatments of superficial infections caused by S. aureusand S. pyogenes.

ABSTRACTResistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coliis associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coliUTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marORand acrRwere sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marORbut not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. colistrains that develop resistance to fluoroquinolones.

ABSTRACTMutations in the translation elongation factor G (EF-G) make Salmonella entericaserovar Typhimurium resistant to the antibiotic fusidic acid. Fusrmutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide. We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase). These catalases require the iron-binding cofactor heme for their activity. Fusrmutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fusrmutants to hydrogen peroxide is abolished by the presence of δ-aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis. Fluorescence measurements show that the level of heme in a Fusrmutant is significantly lower than it is in the wild type. Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration. We found that the rate of respiration is reduced significantly in Fusrmutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fusrmutants to oxidative stress. Taken together, these results suggest that Fusrmutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fusrmutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide. We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents.

Mutants of Salmonella enterica serovar Typhimurium resistant to fusidic acid (Fusr) have mutations in fusA, the gene encoding translation elongation factor G (EF-G). Most Fusr mutants have reduced fitness in vitro and in vivo, in part explained by mutant EF-G slowing the rate of protein synthesis and growth. However, some Fusr mutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. As shown here, Fusr mutants could be similarly ranked in their relative fitness in mouse infection models, in a macrophage infection model, in their relative hypersensitivity to hydrogen peroxide in vivo and in vitro, and in the amount of RpoS production induced upon entry into the stationary phase. We identify a reduced ability to induce production of RpoS (sigmas) as a defect associated with Fusr strains. Because RpoS is a regulator of the general stress response, and an important virulence factor in Salmonella, an inability to produce RpoS in appropriate amounts can explain the low fitness of Fusr strains in vivo. The unfit Fusr mutants also produce reduced levels of the regulatory molecule ppGpp in response to starvation. Because ppGpp is a positive regulator of RpoS production, we suggest that a possible cause of the reduced levels of RpoS is the reduction in ppGpp production associated with mutant EF-G. The low fitness of Fusr mutants in vivo suggests that drugs that can alter the levels of global regulators of gene expression deserve attention as potential antimicrobial agents.

ABSTRACTMutants of Salmonella entericaserovar Typhimurium resistant to fusidic acid (Fusr) have mutations in fusA, the gene encoding translation elongation factor G (EF-G). Most Fusrmutants have reduced fitness in vitro and in vivo, in part explained by mutant EF-G slowing the rate of protein synthesis and growth. However, some Fusrmutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. As shown here, Fusrmutants could be similarly ranked in their relative fitness in mouse infection models, in a macrophage infection model, in their relative hypersensitivity to hydrogen peroxide in vivo and in vitro, and in the amount of RpoS production induced upon entry into the stationary phase. We identify a reduced ability to induce production of RpoS (σs) as a defect associated with Fusrstrains. Because RpoS is a regulator of the general stress response, and an important virulence factor in Salmonella, an inability to produce RpoS in appropriate amounts can explain the low fitness of Fusrstrains in vivo. The unfit Fusrmutants also produce reduced levels of the regulatory molecule ppGpp in response to starvation. Because ppGpp is a positive regulator of RpoS production, we suggest that a possible cause of the reduced levels of RpoS is the reduction in ppGpp production associated with mutant EF-G. The low fitness of Fusrmutants in vivo suggests that drugs that can alter the levels of global regulators of gene expression deserve attention as potential antimicrobial agents.

ABSTRACTEscherichia colistrains from patients with uncomplicated urinary tract infections were examined by DNA sequencing for fluoroquinolone resistance-associated mutations in six genes: gyrA, gyrB, parC, parE, marOR, and acrR. The 54 strains analyzed had a susceptibility range distributed across 15 dilutions of the fluoroquinolone MICs. There was a correlation between the fluoroquinolone MIC and the number of resistance mutations that a strain carried, with resistant strains having mutations in two to five of these genes. Most resistant strains carried two mutations in gyrAand one mutation in parC. In addition, many resistant strains had mutations in parE, marOR, and/or acrR. No (resistance) mutation was found in gyrB. Thus, the evolution of fluoroquinolone resistance involves the accumulation of multiple mutations in several genes. The spontaneous mutation rate in these clinical strains varied by 2 orders of magnitude. A high mutation rate correlated strongly with a clinical resistance phenotype. This correlation suggests that an increased general mutation rate may play a significant role in the development of high-level resistance to fluoroquinolones by increasing the rate of accumulation of rare new mutations.

Inversions and translocations distinguish the genomes of closely related bacterial species, but most of these rearrangements preserve the relationship between the rearranged fragments and the axis of chromosome replication. Within species, such rearrangements are found less frequently, except in the case of clinical isolates of human pathogens, where rearrangements are very frequent

The crystal structure of Thermus thermophiluselongation factor G (EF-G) carrying the point mutation His573Ala was determined at a resolution of 2.8 Å. The mutant has a more closed structure than that previously reported for wild-type EF-G. This is obtained by a 10° rigid rotation of domains III, IV and V with regard to domains I and II. This rotation results in a displacement of the tip of domain IV by approximately 9 Å. The structure of domain III is now fully visible and reveals the double split β-α-β motif also observed for EF-G domain V and for several ribosomal proteins. A large number of fusidic acid resistant mutations found in domain III have now been possible to locate. Possible locations for the effector loop and a possible binding site for fusidic acid are discussed in relation to some of the fusidic acid resistant mutations.

The tufA and tufB genes in Salmonella typhimuriumco-evolve by recombination and exchange of genetic material. A model is presented which predicts that co-evolution is achieved by gene conversions and chromosomal inversions. Analysis of recombinants reveals that conversion and inversion each occur with similar rates and each depends on RecBCD activity. The model predicts sequence structures for different classes of post-recombination tuf genes. Sequence analysis reveals the presence of each of these structures and classes, with a predicted bias in the absence of mismatch repair. An implication of these data is that co-evolution of gene families can be linked with the generation of chromosomal rearrangements.

The antibiotic kirromycin inhibits protein synthesis by binding to EF‐Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tufmutations and identified thirteen single amino acid substitutions at seven different sites in EF‐Tu. These have been mapped onto the 3D structures of EF‐Tu·GTP and EF‐Tu·GDP. In the active GTP form of EF‐Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

The first isolation of EF-Tu mutations in Salmonella typhimurium is reported. The mutations were isolated by selecting for resistance to the antibiotic mocimycin (= kirromycin). The mocimycin resistant phenotype is the result of mutations in each of two genes, tufA and tufB. Strains mutant in only one of the two tuf genes are sensitive to mocimycin. The spontaneous mutation rate of each of the two tuf genes to a mocimycin resistant phenotype differs by an order of magnitude. tufA maps at minute 71–72, closely linked to rpsL. tufB maps at minute 88–89, closely linked to rpoB. These map positions correspond to the locations of tufA and tufB in E. coli.

We have previously identified 20 different fusidic acid-resistant alleles offusA, encoding mutant forms of the ribosomal translocase EF-G. One of these, P413L, is used here as the starting point in selections for internal revertants, identifying 20 different pseudo-wild-type forms of EF-G. We have also identified two alleles offusApreviously isolated as suppressors of 4.5 S RNA deficiency. All of these mutants are analysed in terms of their effects on the structural dynamics of EF-G. Most mutation conferring fusidic acid-resistance interfere with conformational changes of EF-G, but some may be located at a possible fusidic acid binding site. Revertants of the P413L mutations restore the function of EF-G with or without affecting the level of resistance to fusidic acid. The revertant mutations probably restore the balance between the GDP and GTP conformations of EF-G off the ribosome, and most of them are located close to the interface between the G domain and domain II. The procedure for the isolation of pseudo-wild-type forms of EF-G can be used to direct evolution progressively away from the wild-type while still maintaining the essential functions of EF-G.

Five single amino acid substitution variants of EF-Tu from Salmonella typhimuriumwere tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu 120Gln, Gln 124Arg and Tyr 160(Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu·GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, kcat/ KMfor the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu 120Gln, Gln 124Arg and Tyr 160Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu·GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln 124Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant ( kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr 160Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I–III interface of EF-Tu.

The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tufmutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu·GTP and EF-Tu·GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

Five single amino acid substitution variants of EF‐Tu from Salmonella typhimuriumwere tested for their ability to promote poly(U)‐translation in vitro. The substitutions are Leu120Gln, Gln124Arg and Tyr160(Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF‐Tu·GTP configuration. The different EF‐Tu variants exhibit a spectrum of phenotypes. First, kcat/KMfor the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120Gln, Gln124Arg and Tyr160Cys. Second, this reduction is caused by a defect in the interaction between these EF‐Tu variants and aminoacyl‐tRNA during translation. Third, in four cases out of five the affinity of the complex between EF‐Tu·GTP and aminoacyl‐tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30‐fold lower than in the wild‐type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (kd) for the release of GDP from EF‐Tu is increased 6‐fold by the Tyr160Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I–III interface of EF‐Tu.

The genes coding for the translation factor EF-Tu,tufAandtufBare separated by over 700 kb on the circular chromosome ofSalmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons betweenS. typhimuriumandEscherichia colisuggest that within each species the twotufgenes are evolving in concert. Here we show that each of theS. typhimurium tufgenes can transfer genetic information to the other. In our genetic system the transfers are seen as non-reciprocal, i.e. as gene conversion events. However, the mechanism of recombination could be reciprocal, with sister chromosome segregation and selection leading to the isolation of a particular class of recombinant. The amount of sequence information transferred in individual recombination events varies, but can be close to the entire length of the gene. The recombination is RecABCD-dependent, and is opposed by MutSHLU mismatch repair. In the wild-type, this type of recombination occurs at a rate that is two or three orders of magnitude greater than the nucleotide substitution rate. The rate of recombination differs by six orders of magnitude between arecAand amutSstrain. Mismatch repair reduces the rate of this recombination 1000-fold. The rate of recombination also differs by one order of magnitude depending on whichtufgene is donating the sequence selected for. We discuss three classes of model that could, in principle, account for the sequence transfers: (1)tufmRNA mediated recombination; (2) non-allelic reciprocal recombination involving sister chromosomes; (3) non-allelic gene conversion involving sister chromosomes, initiated by a double-strand break close to onetufgene. Although the mechanism remains to be determined, the effect on the bacterial cells istufgene sequence homogenisation. This recombination phenomenon can account for the concerted evolution of thetufgenes.

Bacterial chromosomes frequently carry multiple copies of genes at separate chromosomal locations. In Salmonella, these include the 7 rrnoperons and the duplicate tufgenes.

ABSTRACTWeak mutators are common among clinical isolates of Escherichia coli. We show that the relative mutation rate and the “evolvability of fluoroquinolone resistance” are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.

A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufBwere easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.

ABSTRACTGenes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufAgene in the Escherichia coligenome with its extant and ancestral homologs. The extant homologs represented tufvariants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tufsequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tufgenes are transferable to the E. coligenome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coligenome. However, when the tufBgene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tufwith its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tufhomologs inserted into the E. coligenome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tufpartially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCEHorizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tufwith ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tufsequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tufwith its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

ABSTRACTStaphylococcus aureusis known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD+, thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections.IMPORTANCESmall colony variants (SCVs) of Staphylococcus aureusare a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

&lt;it>Objectives&lt;/it>: To measure the mutant prevention concentration (MPC) of ciprofloxacin for a set of urinary tract infection (UTI) &lt;it>Escherichia coli&lt;/it> isolates with different levels of susceptibility and determine whether MPC can be predicted from MIC. &lt;it>Methods&lt;/it>: MPC was defined as the lowest ciprofloxacin concentration that prevented the growth of resistant colonies when 10&lt;cross-ref type="bib" refid="r10">10&lt;/cross-ref> bacteria were spread on solid medium and incubated for 96 h at 37°C. MIC was measured by Etest. Bacteria surviving (persisting) at MPC were isolated and quantified from agar plugs taken after 96 h. The genes &lt;it>hipA&lt;/it> and &lt;it>hipB&lt;/it> were amplified by PCR from persisters and sequenced. &lt;it>Results&lt;/it>: Isolates with MICs above the NCCLS breakpoint for ciprofloxacin resistance (4 mg/L) typically have MPCs greater than 32 mg/L. Isolates with MICs below the breakpoint for ciprofloxacin susceptibility (1 mg/L) have MPCs up to 5 mg/L. MPC/MIC is ∼16 for most susceptible isolates but there are several notable exceptions (MPC/MIC > 100). Resistant colonies arising one dilution step below MPC often had MIC > MPC. In every case tested, a proportion of cells survived (persisted), but did not grow into colonies, at MPC, without any increase in MIC. &lt;it>Conclusions&lt;/it>: MPCs were determined for all ciprofloxacin-susceptible isolates. MPC is not accurately predicted from MIC. Colonies selected below MPC frequently have MIC > MPC, suggesting multiple mutations. A small fraction of cells from all strains tested survived for 96 h at MPC, without any associated increase in MIC. These survivors/persisters are not &lt;it>hipAB&lt;/it> mutants.

We report a novel mutation, C1066U in 16S rRNA which was selected for resistance to spectinomycin, an antibiotic which inhibits ribosomal translocation. The minimal inhibitory concentration (MIC) of spectinomycin determined for this mutant (15 μg/ml) is greater than with the wild-type plasmid (5 μg/ml) but lower than with the well known C1192U mutation (>80 μg/ml). The C1066U mutation also increases the cells sensitivity to fusidic acid, another antibiotic which Inhibits translation at the translocation stage, whereas C1192U is unchanged relative to the wild type. We discuss why the acquisition of resistance to one of these drugs Is often associated with hypersensitivity to the other.