Antimicrobial Resistance: Fitness gains hamper efforts to tackle drug resistance

  1. Shanta Dutta  Is a corresponding author
  1. National Institute of Cholera and Enteric Diseases, India

The effectiveness of the antibiotics used to treat diseases caused by bacteria is increasingly being limited by the spread of resistance to these drugs. For a long time it was thought that most of the mutations that lead to antimicrobial resistance in bacteria led to a reduction in fitness, which meant that the resistant bacteria did not grow as fast as those that were susceptible to antimicrobials. These fitness costs are important because they determine how quickly resistance is likely to develop, or to disappear if antibiotic use is discontinued (Andersson and Hughes, 2010). However, resistant bacteria often develop secondary mutations that can compensate for these fitness costs, and these mutations allow the bacteria to thrive, even in antimicrobial free environments (Lenski, 1998). When this occurs, resistance is likely to continue even if antibiotic use is stopped. This is why public health bodies around the world are so worried by the spread of antibiotic resistance.

Typhoid fever is caused by eating food or drinking water contaminated with Salmonella Typhi bacteria. According to the World Heath Organisation (WHO), the drug of choice for treating typhoid fever is ciprofloxacin, which belongs to the fluoroquinolone family of antibiotics (WHO, 2003). However, resistance to the fluoroquinolones is increasing all over the world (Parry and Beeching, 2009). Now, in eLife, Stephen Baker of the Oxford University Clinical Research Unit in Vietnam and co-workers in Vietnam and the UK have compared the fitness of various mutants of Salmonella Typhi that are resistant to fluoroquinolones with the fitness of a strain that is still susceptible to these drugs (Baker et al., 2013).

The fluoroquinolones target enzymes that untangle knots in bacterial genomes. Resistance to these antibiotics in Salmonella Typhi involves changes in these target enzymes that reduce the ability of the drugs to bind to them. These changes occur by substitution mutations in two genes, gyrA and/or parC, that encode these enzymes. Advances in site-directed mutagenesis allowed Baker and co-workers to construct eleven fluoroquinolone-resistant mutants and one susceptible control mutant with known nucleotide substitutions from a susceptible, weakened laboratory strain of Salmonella Typhi. The resistant mutants were generated by substitutions at two positions in the gyrA gene and one in the parC gene, which were introduced in combinations to create single, double or triple mutants. Seven of the eleven lab-engineered mutants have been found in clinical isolates, whilst the remaining four had not been observed in nature before.

To start each in vitro competition assay, a series of cultures containing equal numbers of bacteria from the susceptible parent strain and one of the 12 lab-engineered mutant strains were prepared. These cultures were then grown in the absence of any antibiotics for several generations. Every 24 hr, some of each culture mixture was removed and analysed (Figure 1). More culture was also diluted into fresh antibiotic-free growth media, and the entire process was repeated for a period of 15 days. Colony counting was used to calculate the number of bacterial generations during each period.

Fluoroquinolone-resistant mutants of Salmonella Typhi vs susceptible strains.

Fluoroquinolone-resistant mutants (red) with either one, two or three mutations in the gyrA and parC genes were cultured with a fluoroquinolone-susceptible parent strain (white) in liquid media that did not contain any antibiotics. Every 24 hr, over a period of 15 days, these growth cultures were subcultured into fresh antibiotic-free media, and samples were taken for colony counting and DNA extraction. Pyrosequencing of extracted DNA was used to determine the frequency of each mutation and the parent strain at each time-point, and to calculate the fitness of each mutant compared to the parent strain. Increases in fitness allowed some single and double mutants (red arrow) outcompeted the parent strain, while a reduction in fitness meant that the triple mutant lost out to the parent strain (blue arrow).

Whether each mutant had a selective advantage or disadvantage was determined from the frequency of the engineered mutated genes at each time point, which was measured by specific pyrosequencing with the extracted DNA. Statistical models were used to calculate the selection coefficient and fitness difference of each mutant when compared to the parent strain. Since the fate of a particular mutation depends on its interaction with its environment, including all the other genes found in the genome (Cordell, 2002), the effects of interactions between genes, often known as ‘epistasis’, were also calculated for the double and triple mutants.

Interestingly, six resistant mutants (two single mutants and the four double mutants tested) could outcompete the susceptible Salmonella Typhi parent strain. This indicates that these mutations conferred increased fitness, even in the absence of the antimicrobial. This goes against the dogma that resistance mutations always have a fitness cost, and can explain the frequent occurrence of certain resistant mutants in the antibiotic free environment. The single mutant with highest selective advantage (called S83F) contributed 1.3% more offspring per generation than the parent strain, and the most competitive double mutant (S83F-D87N, which has two changes within the gyrA gene) contributed 7.4% more offspring per generation. In contrast, the triple mutant (S83F-D87G-S80I) contributed 1% less offspring per generation than the parent strain: this indicates a negative interaction between these mutations, with the third mutation wiping out the fitness advantage of the double mutants.

Since Salmonella Typhi lives within host cells during an infection, and is restricted to humans, the results obtained in this in vitro experimental system using a weakened, lab strain would not be the same with wild strain and ex vivo or in vivo models. However, as acknowledged by Baker et al., all models have their own inherent limitations and currently there are no available systems (except humans and primates) that exactly mimic Salmonella Typhi infection cycle.

The process of evolution in any organism is determined by the order in which mutations occur (Kimura, 1985). Baker et al. have concluded that single mutations in gyrA gene that reduce susceptibility to fluoroquinolones (especially the S83F mutation) provide the starting point for the evolution of more resistant double mutants, which maintain the resistance, even in the absence of antibiotics. This would contribute to the global spread of fluoroquinolone-resistant Salmonella Typhi, and would also undermine efforts to control the spread of drug-resistant typhoid through the prudent use of antimicrobial drugs.

References

    1. Lenski RE
    (1998)
    Bacterial evolution and the cost of antibiotic resistance
    International Microbiology 1:265–270.
    1. WHO
    (2003) Background document: the diagnosis, prevention and treatment of typhoid fever
    19–23, Background document: the diagnosis, prevention and treatment of typhoid fever, Geneva, World Health Organization, Available at: , http://apps.who.int/iris/bitstream/10665/68122/1/WHO_V-B_03.07_eng.pdf.

Article and author information

Author details

  1. Shanta Dutta

    Bacteriology Division, National Institute of Cholera and Enteric Diseases, Kolkata, India
    For correspondence
    shanta1232001@yahoo.co.in
    Competing interests
    The author declares that no competing interests exist.

Publication history

  1. Version of Record published: December 10, 2013 (version 1)

Copyright

© 2013, Dutta

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Shanta Dutta
(2013)
Antimicrobial Resistance: Fitness gains hamper efforts to tackle drug resistance
eLife 2:e01809.
https://doi.org/10.7554/eLife.01809

Further reading

  1. Resistance to antimicrobial drugs is not driven solely by the excessive use of these drugs.

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    Background:

    Few national-level studies have evaluated the impact of ‘hybrid’ immunity (vaccination coupled with recovery from infection) from the Omicron variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

    Methods:

    From May 2020 to December 2022, we conducted serial assessments (each of ~4000–9000 adults) examining SARS-CoV-2 antibodies within a mostly representative Canadian cohort drawn from a national online polling platform. Adults, most of whom were vaccinated, reported viral test-confirmed infections and mailed self-collected dried blood spots (DBSs) to a central lab. Samples underwent highly sensitive and specific antibody assays to spike and nucleocapsid protein antigens, the latter triggered only by infection. We estimated cumulative SARS-CoV-2 incidence prior to the Omicron period and during the BA.1/1.1 and BA.2/5 waves. We assessed changes in antibody levels and in age-specific active immunity levels.

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    Spike levels were higher in infected than in uninfected adults, regardless of vaccination doses. Among adults vaccinated at least thrice and infected more than 6 months earlier, spike levels fell notably and continuously for the 9-month post-vaccination. In contrast, among adults infected within 6 months, spike levels declined gradually. Declines were similar by sex, age group, and ethnicity. Recent vaccination attenuated declines in spike levels from older infections. In a convenience sample, spike antibody and cellular responses were correlated. Near the end of 2022, about 35% of adults above age 60 had their last vaccine dose more than 6 months ago, and about 25% remained uninfected. The cumulative incidence of SARS-CoV-2 infection rose from 13% (95% confidence interval 11–14%) before omicron to 78% (76–80%) by December 2022, equating to 25 million infected adults cumulatively. However, the coronavirus disease 2019 (COVID-19) weekly death rate during the BA.2/5 waves was less than half of that during the BA.1/1.1 wave, implying a protective role for hybrid immunity.

    Conclusions:

    Strategies to maintain population-level hybrid immunity require up-to-date vaccination coverage, including among those recovering from infection. Population-based, self-collected DBSs are a practicable biological surveillance platform.

    Funding:

    Funding was provided by the COVID-19 Immunity Task Force, Canadian Institutes of Health Research, Pfizer Global Medical Grants, and St. Michael’s Hospital Foundation. PJ and ACG are funded by the Canada Research Chairs Program.