Preferences regarding antibiotic treatment and the role of antibiotic resistance: A discrete choice experiment.

OBJECTIVES: To identify preferences of the Swedish public regarding antibiotic treatment characteristics and the relative weight of antibiotic resistance in their treatment choices. METHODS: A questionnaire including a discrete choice experiment questionnaire was answered by 378 Swedish participants. Preferences of the general public regarding five treatment characteristics (attributes) were measured: contribution to antibiotic resistance, cost, side effects, failure rate and treatment duration. Latent class analysis models were used to determine attribute-level estimates and heterogeneity in preferences. Relative importance of the attributes and willingness to pay for antibiotics with a lower contribution to antibiotic resistance were calculated from the estimates. RESULTS: All attributes influenced participants' preferences for antibiotic treatment. For the majority of participants, contribution to antibiotic resistance was the most important attribute. Younger respondents found contribution to antibiotic resistance more important in their choice of antibiotic treatments. Choices of respondents with lower numeracy, higher health literacy and higher financial vulnerability were influenced more by the cost of the antibiotic treatment. Older respondents with lower financial vulnerability and health literacy, and higher numeracy found side effects to be most important. CONCLUSIONS: All attributes can be considered as potential drivers of antibiotic use by lay people. Findings also suggest that the behaviour of lay people may be influenced by concerns over the rise of antibiotic resistance. Therefore, stressing individual responsibility for antibiotic resistance in clinical and societal communication has the potential to affect personal decision making.

Predictable Phenotypes of Antibiotic Resistance Mutations.

Antibiotic-resistant bacteria represent a major threat to our ability to treat bacterial infections. Two factors that determine the evolutionary success of antibiotic resistance mutations are their impact on resistance level and the fitness cost. Recent studies suggest that resistance mutations commonly show epistatic interactions, which would complicate predictions of their stability in bacterial populations. We analyzed 13 different chromosomal resistance mutations and 10 host strains of Salmonella enterica and Escherichia coli to address two main questions. (i) Are there epistatic interactions between different chromosomal resistance mutations? (ii) How does the strain background and genetic distance influence the effect of chromosomal resistance mutations on resistance and fitness? Our results show that the effects of combined resistance mutations on resistance and fitness are largely predictable and that epistasis remains rare even when up to four mutations were combined. Furthermore, a majority of the mutations, especially target alteration mutations, demonstrate strain-independent phenotypes across different species. This study extends our understanding of epistasis among resistance mutations and shows that interactions between different resistance mutations are often predictable from the characteristics of the individual mutations.IMPORTANCE The spread of antibiotic-resistant bacteria imposes an urgent threat to public health. The ability to forecast the evolutionary success of resistant mutants would help to combat dissemination of antibiotic resistance. Previous studies have shown that the phenotypic effects (fitness and resistance level) of resistance mutations can vary substantially depending on the genetic context in which they occur. We conducted a broad screen using many different resistance mutations and host strains to identify potential epistatic interactions between various types of resistance mutations and to determine the effect of strain background on resistance phenotypes. Combinations of several different mutations showed a large amount of phenotypic predictability, and the majority of the mutations displayed strain-independent phenotypes. However, we also identified a few outliers from these patterns, illustrating that the choice of host organism can be critically important when studying antibiotic resistance mutations.

Mechanisms and consequences of bacterial resistance to antimicrobial peptides.

Cationic antimicrobial peptides (AMPs) are an intrinsic part of the human innate immune system. Over 100 different human AMPs are known to exhibit broad-spectrum antibacterial activity. Because of the increased frequency of resistance to conventional antibiotics there is an interest in developing AMPs as an alternative antibacterial therapy. Several cationic peptides that are derivatives of AMPs from the human innate immune system are currently in clinical development. There are also ongoing clinical studies aimed at modulating the expression of AMPs to boost the human innate immune response. In this review we discuss the potential problems associated with these therapeutic approaches. There is considerable experimental data describing mechanisms by which bacteria can develop resistance to AMPs. As for any type of drug resistance, the rate by which AMP resistance would emerge and spread in a population of bacteria in a natural setting will be determined by a complex interplay of several different factors, including the mutation supply rate, the fitness of the resistant mutant at different AMP concentrations, and the strength of the selective pressure. Several studies have already shown that AMP-resistant bacterial mutants display broad cross-resistance to a variety of AMPs with different structures and modes of action. Therefore, routine clinical administration of AMPs to treat bacterial infections may select for resistant bacterial pathogens capable of better evading the innate immune system. The ramifications of therapeutic levels of exposure on the development of AMP resistance and bacterial pathogenesis are not yet understood. This is something that needs to be carefully studied and monitored if AMPs are used in clinical settings.

Improving predictions of the risk of resistance development against new and old antibiotics.

The methods used today by academic researchers and the pharmaceutical industry to assess the risk of emergence of resistance, for example during development of new antibiotics or when assessing an old antibiotic, are sub-optimal. Even though easy to perform, the presently used serial passage procedures, minimal prevention concentration measurements and determination of mutation rates in vitro are generally providing inadequate knowledge for risk assessment and making decisions to continue/discontinue drug development. These methods need to be complemented and replaced with more relevant methods such as determination of whether resistance genes already pre-exist in various metagenomes, and the likelihood that these genes can transfer into the relevant pathogens and be stably maintained. Furthermore, to determine the risk of emergence of mutationally conferred resistance the fitness effect of the resistance mechanism is key, as this parameter will determine the ability of the resistant mutants to be maintained and enriched in the host after they have emerged. This information combined with knowledge of bacterial population sizes and growth and killing dynamics at relevant infection sites should allow for better forecasting of the risk of resistance emerging in clinical settings.

Poxvirus use a "gene accordion" to tune out host defenses.

A multistep process of gene amplification, mutation, and reduction allows poxvirus to overcome host antiviral defenses. The mechanism speeds genetic adaptation and promises to be broadly applicable in many biological settings.

Little evidence for reversibility of trimethoprim resistance after a drastic reduction in trimethoprim use.

OBJECTIVES: The worldwide rapid increase in antibiotic-resistant bacteria has made efforts to prolong the lifespan of existing antibiotics very important. Antibiotic resistance often confers a fitness cost in the bacterium. Resistance may thus be reversible if antibiotic use is discontinued or reduced. To examine this concept, we performed a 24 month voluntary restriction on the use of trimethoprim-containing drugs in Kronoberg County, Sweden. METHODS: The intervention was performed on a 14 year baseline of monthly data on trimethoprim resistance and consumption. A three-parameter mathematical model was used to analyse the intervention effect. The prerequisites for reversion of resistance (i.e. fitness cost, associated resistance and clonal composition) were studied on subsets of consecutively collected Escherichia coli from urinary tract infections. RESULTS: The use of trimethoprim-containing drugs decreased by 85% during the intervention. A marginal but statistically significant effect on the increase in trimethoprim resistance was registered. There was no change in the clonal composition of E. coli and there was no measurable fitness cost associated with trimethoprim resistance in clinical isolates. The frequency of associated antibiotic resistances in trimethoprim-resistant isolates was high. CONCLUSIONS: A lack of detectable fitness cost of trimethoprim resistance in vitro together with a strong co-selection of other antibiotics could explain the rather disappointing effect of the intervention. The result emphasizes the low possibility of reverting antibiotic resistance once established and the urgent need for the development of new antibacterial agents.

Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor.

R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types.

Bacterial genome size reduction by experimental evolution.

Bacterial evolution toward endosymbiosis with eukaryotic cells is associated with extensive bacterial genome reduction and loss of metabolic and regulatory capabilities. Here we examined the rate and process of genome reduction in the bacterium Salmonella enterica by a serial passage experimental evolution procedure. The initial rate of DNA loss was estimated to be 0.05 bp per chromosome per generation for a WT bacterium and approximately 50-fold higher for a mutS mutant defective in methyl-directed DNA mismatch repair. The endpoints were identified for seven chromosomal deletions isolated during serial passage and in two separate genetic selections. Deletions ranged in size from 1 to 202 kb, and most of them were not associated with DNA repeats, indicating that they were formed via RecA-independent recombination events. These results suggest that extensive genome reduction can occur on a short evolutionary time scale and that RecA-dependent homologous recombination only plays a limited role in this process of jettisoning superfluous DNA.

Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori.

Among the several factors that affect the appearance and spread of acquired antibiotic resistance, the mutation frequency and the biological cost of resistance are of special importance. Measurements of the mutation frequency to rifampicin resistance in Helicobacter pylori strains isolated from dyspeptic patients showed that approximately 1/4 of the isolates had higher mutation frequencies than Enterobacteriaceae mismatch-repair defective mutants. This high mutation frequency could explain why resistance is so frequently acquired during antibiotic treatment of H. pylori infections. Inactivation of the mutS gene had no substantial effect on the mutation frequency, suggesting that MutS-dependent mismatch repair is absent in this bacterium. Furthermore, clarithromycin resistance conferred a biological cost, as measured by a decreased competitive ability of the resistant mutants in mice. In clinical isolates this cost could be reduced, indicating that compensation is a clinically relevant phenomenon that could act to stabilize resistant bacteria in a population.

Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus.

Fusidic acid resistance resulting from mutations in elongation factor G (EF-G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid-resistant strains have been identified that carry multiple mutations in EF-G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness-compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.

An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium.

Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end-product. This regulation is conferred mainly at the translational level and involves a cobalamin-induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation. A combined structural and mutational analysis shows that a cis-acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine-Dalgarno sequence in the 5'-untranslated region (5'-UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin. The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin. This interaction destabilizes the RNA hairpin and allows ribosome binding. In the presence of cobalamin, the enhancer function is inhibited. As a result, the RBS hairpin forms and prevents translation initiation. Several additional RNA hairpins in the 5'-UTR were also identified and are suggested to be important for repression. The above data suggest that normal cobalamin repression of the cob operon requires that the 5'-UTR has a defined secondary and tertiary structure.

Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance.

Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production.

To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth. J774-A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.

The biological cost of antibiotic resistance.

The frequency and rates of ascent and dissemination of antibiotic resistance in bacterial populations are anticipated to be directly related to the volume of antibiotic use and inversely related to the cost that resistance imposes on the fitness of bacteria. The data available from recent laboratory studies suggest that most, but not all, resistance-determining mutations and accessory elements engender some fitness cost, but those costs are likely to be ameliorated by subsequent evolution.

Novel Salmonella typhimurium properties in host--parasite interactions.

Inflammatory bowel disease (IBD) comprises different diseases in the gastrointestinal tract in human, of which Crohn's disease (CD) and ulcerative colitis (UC) are the most prominent. A key factor in the etiology of IBD is the chronic inflammatory process, and a large body of evidence suggests that the transcription factor nuclear factor-kappa B (NF-kappaB) is the key regulator of responses determining the clinical inflammatory condition. Recent findings using antisense oligonucleotides provide direct evidence that the p65 subunit of NF-kappaB plays a central role in chronic intestinal inflammation. It has previously been shown that the Gram negative bacteria Yersinia pseudotubercolosis targets the eukaryotic signal transduction pathway(s) that lead to NF-kappaB activation (and thus avoid an anti-bacterial inflammatory response). In this paper, growth-based selected Salmonella typhimurium clones have been used to generate a clearer picture of the molecular mechanisms involved in host-parasite interactions. From the results presented here, S. typhimurium and Y. pseudotubercolosis may use the same mechanism to block NF-kappaB activation, following host cell infection. A new adaptational feature could also be shown, where a growth-based selected bacteria avoided the normally induced translocation of NF-kappaB in host cells.

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium.

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.

Evidence that gene amplification underlies adaptive mutability of the bacterial lac operon.

Adaptive mutability is the apparent alteration in specificity or rate of mutability seen in bacteria during stress. A model is proposed by which gene amplification during selective growth can give the appearance of adaptive mutability without requiring any change in mutability. The model is based on two assumptions, that a mutant lac locus with residual function allows growth if its copy number is increased, and that true reversion events are made more likely by replication of chromosomes with many copies of the locus. Apparent directed mutability, its recombination requirement, and its apparent independence of cell growth are all accounted for by the model. Evidence is provided for the required residual function and gene amplification.

[Antibiotic resistance here to stay? Compensatory mutations restore virulence of resistant bacteria]. / Antibiotikaresistens här för att stanna? Kompensatoriska mutationer återställer resistenta bakteriers virulens.

Bacterial antibiotic resistance has increased alarmingly because of overuse of antibiotics both in humans and animals. One way of reversing this development is to reduce the use of antibiotics, thus promoting the disappearance of the resistant bacteria already present in humans and the environment. This approach is based on the assumption that resistance is conferred at the cost of impaired survival fitness in the absence of antibiotics, as compared with sensitive strains. It seems to be generally true that resistant bacteria are less fit than the respective sensitive strains, which suggests that resistance may be reversible. However, a complicating factor is the frequent finding in resistant strains of various types of compensatory mutations that restore fitness without concomitant loss of resistance. Thus, second-site compensatory mutations may allow resistant strains to persist and compete successfully with sensitive strains even in an environment depleted of antibiotics. It is concluded in the article that, if compensatory mutations are as common in clinical settings as they are in the laboratory, many types of resistance will be irreversible.

Virulence of antibiotic-resistant Salmonella typhimurium.

We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice. Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice. The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance. Such second-site compensatory mutations were more common then reversion to the sensitive wild type. We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs. Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.