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Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to reduce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhIJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics (chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to resistance development by up-regulation of an array of efflux pumps. [ABSTRACT FROM AUTHOR]

Antibiotic resistance can be acquired by mutation or horizontal transfer of a resistance gene, and generally an acquired mechanism results in a predictable increase in phenotypic resistance. However, recent findings suggest that the environment and/or the genetic context can modify the phenotypic expression of specific resistance genes/mutations. An important implication from these findings is that a given genotype does not always result in the expected phenotype. This dissociation of genotype and phenotype has important consequences for clinical bacteriology and for our ability to predict resistance phenotypes from genetics and DNA sequences. A related problem concerns the degree to which the genes/mutations currently identified in vitro can fully explain the in vivo resistance phenotype, or whether there is a significant additional amount of presently unknown mutations/genes (genetic 'dark matter') that could contribute to resistance in clinical isolates. Finally, a very important question is whether/how we can identify the genetic features that contribute to making a successful pathogen, and predict why some resistant clones are very successful and spread globally? In this review, we describe different environmental and genetic factors that influence phenotypic expression of antibiotic resistance genes/mutations and how this information is needed to understand why particular resistant clones spread worldwide and to what extent we can use DNA sequences to predict evolutionary success. [ABSTRACT FROM AUTHOR]

Objectives: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the β-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism.Methods: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action.Results: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele.Conclusions: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux. [ABSTRACT FROM AUTHOR]

Objectives: In silico pharmacokinetic/pharmacodynamic (PK/PD) models can be developed based on data from in vitro time-kill experiments and can provide valuable information to guide dosing of antibiotics. The aim was to develop a mechanism-based in silico model that can describe in vitro time-kill experiments of Escherichia coli MG1655 WT and six isogenic mutants exposed to ciprofloxacin and to identify relationships that may be used to simplify future characterizations in a similar setting.Methods: In this study, we developed a mechanism-based PK/PD model describing killing kinetics for E. coli following exposure to ciprofloxacin. WT and six well-characterized mutants, with one to four clinically relevant resistance mutations each, were exposed to a wide range of static ciprofloxacin concentrations.Results: The developed model includes susceptible growing bacteria, less susceptible (pre-existing resistant) growing bacteria, non-susceptible non-growing bacteria and non-colony-forming non-growing bacteria. The non-colony-forming state was likely due to formation of filaments and was needed to describe data close to the MIC. A common model structure with different potency for bacterial killing (EC50) for each strain successfully characterized the time-kill curves for both WT and the six E. coli mutants.Conclusions: The model-derived mutant-specific EC50 estimates were highly correlated (r(2) = 0.99) with the experimentally determined MICs, implying that the in vitro time-kill profile of a mutant strain is reasonably well predictable by the MIC alone based on the model. [ABSTRACT FROM AUTHOR]

Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Objectives The evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism. Methods We created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains. Results Compensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation. Conclusions The compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations. [ABSTRACT FROM PUBLISHER]

Much of what we currently know about the genetics and evolution of antibiotic-resistance is based on selections with lethal drug concentrations that allow the detection of rare mutants with strong phenotypes. These data may be misleading with regard to the evolution of antibiotic resistance in natural environments, because bacteria are frequently exposed to concentration gradients of antibiotics. A significant part of antibiotic-resistance evolution may occur when bacteria are exposed to non-lethal concentrations of drug. High-resolution competition assays show that resistance mutations are rapidly enriched, and selected de novo, at very low antibiotic concentrations. Genomic analysis is providing a better understanding of how frequent and small-effect mutations selected at very low antibiotic concentrations contribute to the step-wise development of antibiotic resistance. [ABSTRACT FROM AUTHOR]

FusE mutants are fusidic acid-resistant small colony variants (SCVs) of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants. [ABSTRACT FROM AUTHOR]

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations. [ABSTRACT FROM AUTHOR]

Objectives To determine whether phylogenetic type is associated with the development of multidrug resistance to antibiotics. Methods Urinary tract infection (UTI) isolates from three hospitals in Pakistan were collected over a period of 10 months, and analysed in terms of causative bacterial species and drug susceptibility. Results Multidrug resistance was widespread and resistance frequencies were >50% for several of the most commonly used antibiotics, including ciprofloxacin and third-generation cephalosporins for Escherichia coli isolates. The great majority of E. coli isolates remained susceptible to meropenem and fosfomycin. Sixty E. coli isolates were analysed in detail to determine correlations between resistance phenotypes and genotypes, mutation rates and phylogenetic group. Most isolates had elevated mutation rates, suggesting this was being selected. The majority of ciprofloxacin-resistant isolates carried a specific set of mutations in the quinolone resistance-determining region of gyrA and parC (S83L, D87N, S80I and E84V). In addition, 67% of the ciprofloxacin-resistant E. coli isolates carried one or more horizontally transmissible determinants of resistance to ciprofloxacin, including aac(6′)-Ib-cr, qepA, qnrA and qnrB. There was a significant correlation between resistance to third-generation cephalosporins, being an extended-spectrum β-lactamase producer, being resistant to ciprofloxacin and belonging to phylogenetic group B2. Conclusions The data suggest that features of the bacterial genotype might facilitate the development of multidrug resistance in particular lineages. Better understanding of the mechanistic basis for correlations between drug resistance and genotype could potentially be exploited to develop molecular tools for the prediction of resistance development. [ABSTRACT FROM PUBLISHER]

Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions. [ABSTRACT FROM AUTHOR]

It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin, nalidixic acid, and chlorate resistance and Lac+ reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V, and SamAB) and RpoS. Constitutive derepression of the LexA regulon by a lexA(def) mutation had no effect on Lac+ reversion rates but increased the other three mutation rates up to 11-fold, and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. The increase in mutation rates in the lexA(def) mutant required the presence of the LexA-controlled UvrB protein and endonucleases UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial overexpression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with overexpression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion DNA polymerases by inactivation of LexA, or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by derepression of the LexA regulon alone. [ABSTRACT FROM AUTHOR]

Examines how starvation affects the rates of formation of several types of mutations resulting in auxotrophy, phototrophy, and antibiotic resistance in Salmonella typhimurium. Methods used in the experiments conducted; Results obtained; Discussion of results.

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype. [ABSTRACT FROM AUTHOR]

We have used quantitative immunoblotting to estimate the amount of EF-Tu in a variety of S. typhimurium strains with wild-type, mutant, insertionally inactivated or piasmid-borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild-type strain, at moderate to fast growth rates, our data show that EF-Tu makes up 8-9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild-type EF-Tu level, irrespective of which tuf gene remains active, or whether that gene Is wild-type or a kirromycin-resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction In elongation rate relative to the level of EF-Tu we calculate that in glucose minimal medium the in vivo saturation level of wild-type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non-viable on minimal medium when the level of EF-Tu is reduced. [ABSTRACT FROM AUTHOR]

We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate. [ABSTRACT FROM AUTHOR]

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66»Cys, Ile207»Ser, Ile207»Asn, and Ala327»Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth. [ABSTRACT FROM AUTHOR]

Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012–0.016 mg/L and 0.2–0.3 mg/L, respectively, giving a mean ± standard deviation mutant prevention index (MPI = MPC/MIC) of 17.7 ± 1.1. The mean MPI PRA of the 14 mutant strains was 19.2 ± 12, and the mean MPI across all 17 strains was 18.9 ± 10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7 h to mimic in vivo conditions, an initial concentration of PRA of 1.6–2.4 mg/L (8–10× MPC), giving a PRA AUC/MPC ratio of 73–92, and a T >MPC of 21–23 h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo. [ABSTRACT FROM AUTHOR]