Interaction of Antibiotic Mixing and Temperature: History
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Subjects: Biology
Contributor:
Sharon Azams

Use of antibiotics for the treatment and prevention of bacterial infections in humans, agri- and aquaculture as well as livestock rearing leads to antibiotic pollution of fresh water and these antibiotics have an impact on free-living bacteria. While we know which antibiotics are most common in natural environments such as rivers and streams, there is considerable uncertainty regarding antibiotics’ interactions with one another and the effect of abiotic factors such as temperature. 

  • Pseudomonas fluorescens
  • dose–response
  • ED50
  • additive models
  • independent action
  • concentration addition
  • antibiotics
  • temperature

1. Introduction

Freshwater micro-organisms are exposed to ever-increasing levels of antibiotic pollution [1], and some antibiotics have been shown to occur in particularly high concentrations in the environment [2]. Antibiotics such as ciprofloxacin, ofloxacin and sulfamethoxazole are prevalent in European surface waters and frequently measured in concentrations of around 0.01 µg/mL (and in much higher concentrations near wastewater effluents) [2]. Standard approaches to estimating the toxicity/efficacy of antibiotics include constructing dose–response curves for single antibiotics [3], estimating parameters such as minimum inhibitory concentration (MIC) [4] and assessing the half maximal effective concentration (EC50) [3]. These methods are important when it comes to exploring the full range of antibiotic toxicity but typically ignore that antibiotics occur as mixtures in polluted environments and that their effects are also density and temperature dependent [2][5].
A complex angle to antibiotic pollution in the environment is that organisms are faced with ‘antibiotic cocktails’; different antibiotics, often of different functional classes, are typically detected simultaneously in fresh water (e.g., [2][6]). The individual concentrations of antibiotics that are measured in the environment might be low, but the combined concentrations could result in significant toxicity and antibiotic resistance as the latter can be the consequence of weak, non-lethal selective pressures such as low levels of antibiotics [7][8]. Chemicals in mixtures potentially interact with each other, which can lead to synergy (the same inhibition is achieved at lower combined concentrations of the mixed antibiotic than for each single antibiotic), additivity (mixed or single effects are identical at the same concentration) or antagonism/suppression (less inhibition is achieved in a mixture than for single antibiotics) [9][10]. It is therefore essential to investigate the potential interactions between antibiotics in the environment [11][12]. It is not straightforward to predict the effects of antibiotic mixtures because the drug effects are dose dependent, non-linear and can affect physiology as well as behavioral traits such as virulence [13]. Therefore, an understanding of the dose–response pattern is required, together with effects of interacting factors such as population densities and temperature [14].
Antibiotics affect different parts of bacterial processes (e.g., protein synthesis or mRNA transcription), which are also influenced by abiotic factors such as pH, nutrient availability or temperature [14]. Despite this, there is limited understanding of how physiological adaptation and stress responses to abiotic stressors affect drug susceptibility in bacteria. While, as a whole, life on Earth can be found across a temperature range of about 150 °C [15], organisms have evolved to grow at their niche-specific ‘optimal’ temperature and changes in growth conditions can be expected to interact with the effects of exogenous stressors such as antibiotics because of organisms’ physiologies [16]. Temperature therefore is a key factor to include in antibiotic studies because both chemical reactions and the metabolic activity of organisms are governed by strict physical laws [16]. In aquatic ecology, theoretical frameworks that include temperature and traits of organisms are established and can explain how communities respond to temperature changes (e.g., [17]). These temperature changes might trigger physiological responses that can be beneficial or detrimental to an organism’s response to antibiotic-induced stress.
As a case in point, Cruz-Loya and colleagues (2019) showed that bacterial response to antibiotic–temperature interaction is complex and mechanism dependent [14]. The response to DNA gyrase-inhibiting ciprofloxacin exposure is linked to the cellular cold shock response (as both cold temperature and gyrase inhibition inhibit unwinding of DNA), while exposure to drugs leading to protein misfolding (usually a feature of higher temperature [15]) is ameliorated by heat shock responses [14]. This context dependency of antibiotic tolerance and its link to temperature is a current area of research, but most studies that address the interaction of antibiotics and temperature (reviewed in [5]) test extreme temperature ranges that are not realistic for free-living bacteria.
Although it is obvious that water temperature, antibiotic type and concentration as well as antibiotic mixture interact in their effects on bacteria, a more mechanistic understanding of these interactions is lacking. Here, we used an experimental approach to explore these drivers on bacterial growth response in Pseudomonas fluorescens. First, we determined MICs and concentration-dependent growth inhibition of four antibiotics at higher temperature (25 °C and 30 °C). As a second step, we combined two antibiotics (ciprofloxacin and ofloxacin) in tandem (with a focus on concentrations below the MIC and EC50 values, i.e., potentially sub-lethal conditions) at a range of environmental temperatures (from 15 to 25 °C).

2.Current Insights

In this study,  interactions between temperature and antibiotics at sub-MIC concentrations for P. fluorescens were investigated and showed that this interaction was twofold: at low and very high temperatures (possibly outside the organisms’ temperature optimum), the antibiotic mixtures showed increased synergy, yet overall temperature increased antibiotic efficacy for single antibiotics. A striking result of our study was that antibiotic mixtures had lethal effects even when the concentrations added together were below their respective individual toxicity in a realistic antibiotic pollution scenario.
The latter result was unexpected because ciprofloxacin and ofloxacin both belong to the fluoroquinolone class and are not expected to act in synergy. Antibiotics with different target action are more likely to show interactions when combined—either as synergy [18] or as antagonism/suppression [10]—and there is now growing evidence that both synergy and antagonism are a common feature of antibiotics, and generally pharmaceuticals and other stressors, in mixtures [10][19][20]. Focusing on antibiotic mixing is important as risk assessments and ecotoxicological tests are based on single compounds but antibiotics, in common with all pharmaceuticals, do not occur as isolated and pure substances in the environment and they should be regarded as a multi-component chemical mixture [2]. A growing body of literature shows that mixtures of pollutants can have different effects compared to single compounds and that the joint effect of such chemical cocktails is often higher than the toxicity of each individual compound [12][21][22]. For example, González-Pleiter et al. (2013) demonstrated that the combined effect can be ‘more than the sum of the parts’ by testing the effects of antibiotics in mixtures including a mixture of erythromycin and tetracycline that had particular strong synergistic effects on cyanobacteria [11]. However, knowledge about the toxicity of antibiotic mixtures is still limited and ignoring possible mixture effects might underestimate the actual impact of antibiotics in the environment [18].
Low- and high-temperature-dependent synergistic effects were observed in inhibiting bacterial growth, suggesting antibiotic-specific off-target effects that only affect the bacteria once they enter a certain temperature range. This is in line with emerging literature on antibiotic mixing and temperature [5][14][22] that highlights the context dependency of antibiotic efficacy and it alludes to the fact that stressors such as chemicals, temperature or pH interact [5]—especially when levels are reached that are outside or ‘on the edges’ of the organism’s tolerance breadth [14][23]. Complicating matters, these interactions ‘play out’ on different levels of biological organization—from subcellular to the individual and population level. For example, while it is intuitive that low concentrations of antibiotics change populations because they can provoke resistance in bacteria [8], even more complex mechanisms are at play here and antibiotic stress below lethal levels can result in bacterial strains with narrowed temperature breadth and shifted temperature optima [23]—resulting in individuals/populations that are more susceptible to stressors.
In this study, EC50 values of single antibiotics decreased with temperature and explanations for this pattern include that both uptake [24] and metabolism [16] of antibiotics increase with temperature. Additionally, high temperatures enhance the toxicity of contaminants (yet, at the same time, enhance the rates of chemical degradation [25]). Further, synergistic effects are possible such as both temperature and (some) antibiotics influencing protein folding and synthesis [26][27]. Cruz-Loya and colleagues (2019) found that cellular responses to temperature stress have likely been evolutionarily co-opted to also respond to many classes of antibiotic stress [14]. A further factor is that a population-level effect could come into play in a nutrient-limited environment, as a rise in temperature results in increased population density and potentially competition for resources. In our experiments, the assays with higher population density resulted in lower EC50 values of ciprofloxacin and ofloxacin compared to the experiment with lower bacterial densities. Density-dependent effects in bacteria could therefore be explored more when it comes to antibiotic assays and studies, as is indeed the case for other driving forces of evolution in bacteria such as time, space or disturbance (but see, e.g., [28]).
Interactions among different stressors [20] are at the core of unexpected ecological impact because interactions can lessen or amplify the direct signal effect of each stressor [29]. In this vein, adaptation to both temperature and antibiotics is another future research avenue and a strong focus is needed on sub-lethal antibiotic concentrations as highlighted above. Changes in environmental temperature ‘hit’ multicellular organisms in ‘acute’ ways (such as species extinctions or range shifts [30]) but also shape microbial communities despite their seemingly immediate capability to adapt. For instance, bacterial strains adapted to high temperatures can be more sensitive to certain antibiotics [14] and generally temperature can alter the average body size of microbes (e.g., [31]) and this in turn affects metabolic rates [16][32][33].

This entry is adapted from the peer-reviewed paper 10.3390/life11121435

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