Abstract

Results and Discussion

The concentrations of the antibiotics used for the transcriptomic studies were selected just below a decrease in the growth rate of P. aeruginosa was observed (arrows in Fig. 1a).

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Fig. 1.

Effect of antibiotics on biofilm formation and motility of P. aeruginosa. (a) The effects of different concentrations of antibiotics (tobramycin, Left; tetracycline, Center; ciprofloxacin; Right) on the formation of static biofilms was measured as described in ref. 33. Red, planktonic growth; black, growth-forming biofilm. The mean value and standard deviations of eight different replicates are shown. Standard deviation bar is not shown when its size was lower than the size of the symbol in the graph. The arrows indicate the concentrations of antibiotics used for the experiments with microarrays. (b) Effect of antibiotics in P. aeruginosa motility. Swarming (Upper) and swimming (Lower) motility was measured as described in ref. 34. Growth culture media were different as the one used in the biofilm assay and the experiments with microarrays, and thus antibiotic concentrations also were different. The concentrations used were 0.015 mg/liter ciprofloxacin, 0.5 mg/liter tetracycline, and 0.25 mg/liter tobramycin.

As shown in Tables 1 and ​and2,2, treatment with each antibiotic produced changes in the expression of a specific subset of genes, although there was a common type of response in some cases. Overall, tobramycin treatment induced the expression (fold change >2) of 9 of the 555 genes present in the microarray (1.6%) and repressed the expression of 31 (5.6%). Ciprofloxacin induced the expression of 8 (1.4%) genes and repressed the expression of 21 (3.8%). Finally, tetracycline induced the expression of 8 (1.4%) genes and repressed the expression of 18 (3.2%). It is known that quinolones trigger the SOS response and the expression of pyocin genes in P. aeruginosa (9). Thus, the observed induction of those genes by ciprofloxacin (Table 1) confirms the reliability of our analysis.

Some of the genes with changes in their level of expression have a role in relevant traits for bacterial chronic colonization and virulence such as iron uptake, response to oxidative stress, motility, biofilm formation, and cytotoxicity. Thus, functional assays were preformed to address whether bacterial phenotypes changed accordingly. Noteworthy, the formation of static biofilm (Fig. 1a) increased when bacteria were incubated in the presence of all three antibiotics tested. Increase in biofilm formation attributable to tobramycin treatment has been reported recently and considered specific for aminoglycoside antibiotics, because no effect was detected with members of other antibiotic classes, such as carbenicillin, chloramphenicol, or polymyxin (2). We confirmed that tobramycin induced the formation of biofilm and found that tetracycline and ciprofloxacin had the same effect at concentrations below their minimal inhibitory concentrations (MICs). Searching the literature suggests that a similar phenomenon may occur for other antibiotics. For instance, it has been shown that β-lactams induce the synthesis of colanic acid in Escherichia coli (10). Because colanic acid is involved in adhesion to surfaces in this bacterial species (11), it can be predicted that β-lactams might induce biofilm formation in E. coli.

Most previously published works on the effect of antibiotics on bacterial physiology assay only one or just a few antibiotic concentrations, frequently above minimal inhibitory concentrations. In these cases, the observed changes deal mainly with the mechanisms involved in antibiotic-mediated growth inhibition or killing. However, we showed that lower (nonlethal) subminimal inhibitory concentrations of antibiotics might result in more subtle changes in bacterial physiology, eventually involving different “signaling” cell-to-cell effects with specific consequences in the collective behavior of the bacterial population. In fact, although the effect on biofilm formation seemed to be common for all three antibiotics tested, some other changes in bacterial physiology were antibiotic-specific. Swimming and swarming are two important systems of bacterial motility and probably related with the pathogenic process in CF patients. When the effect of antibiotics in these motility types was explored, clear differences were observed among the different antibacterials (Fig. 1b): We did not detect any effect on motility in the case of bacteria growing in the presence of tetracycline, whereas a reduction in both types of motility was observed in the case of ciprofloxacin. Noteworthy, the aminoglycoside tobramycin induced both swimming and swarming of P. aeruginosa. Again, this finding indicates that subinhibitory antibiotic concentrations do not necessarily produce a burden on bacterial physiology but in some occasions may enhance some potentially adaptive characteristics useful for colonization of specific environments. Indeed, in agreement with the idea that antibiotics might trigger some phenotypes that can be advantageous for the ecological behavior of bacterial populations, it has been described that subinhibitory antibiotic concentrations might increase mutation frequency in P. aeruginosa (12), favoring adaptive radiation (13) and allowing more efficient colonization of heterogeneous environments by this opportunistic pathogen.

P. aeruginosa strains can be either cytoinvasive or cytotoxic (14). Type III secretion (T3S) has a predominant role in the cytotoxic phenotype of this bacterial species. T3S is a system by which bacterial pathogens can deliver effectors directly into the cytoplasm of their eukaryotic host cell (15). The T3S system (T3SS) regulon is composed by different operons coding the proteins involved in the secretion and the effectors. Expression of the genes forming the regulon is triggered by ExsA (16), a transcriptional activator that autoregulates its own expression by a feedback mechanism. It is known that the expression of the T3SS is induced by the contact of the bacteria with its host cell. Calcium deprivation also induces T3S in P. aeruginosa. However, the physiological inductor of the expression of T3S remains unknown. Besides these specific inductors, it has been found recently that metabolic disbalance or overexpression of multidrug efflux pumps also might produce changes in T3S (17). Because antibiotics exposure was expected to produce a general burden to bacterial physiology that might affect T3S, we have measured the expression the gene exsA, coding ExsA, the transcriptional activator of the T3SS in P. aeruginosa, and the gene exoS, coding the T3SS-secreted exotoxin ExoS, in conditions that induce T3S. Surprisingly, incubation with tetracycline (1 mg/liter) increased the expression of T3S genes (Fig. 2a), whereas no changes were observed for the other antibiotics (data not shown). To confirm these results with an in vivo model, we measured the cytotoxicity produced by P. aeruginosa cells grown in the presence of antibiotics on a macrophage cell line. As shown in Fig. 2b, the presence of tetracycline increased cytotoxicity of P. aeruginosa by nearly 4-fold. We did not detect any effect when the added antibiotics were ciprofloxacin or tobramycin (data not shown). When tetracycline was added alone to the macrophage cell line, we did not observe any cytotoxic effect either, indicating that the effect is attributable to the bacteria growing in the presence of the antibiotic. Paradoxical increases in wound infection have been recorded during tetracycline therapy, which have been attributed to tetracycline inhibition of host wound-healing mechanisms (18, 19). Our observations might suggest that subinhibitory concentrations of tetracyclines also might increase bacterial pathogenesis. Quinolone antibiotics previously have been shown to increase toxicity in Shigella, but as a result of bacteriophage induction (20); our work is an example showing that subinhibitory concentrations of antibiotics may induce the expression of chromosomal genes encoding specific bacterial virulence factors.

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Fig. 2.

Effect of tetracycline on the expression of T3SS and the cytotoxicity of P. aeruginosa. (a) The effect of tetracycline on the expression of the T3SS genes exsA and exoS was estimated by semiquantitative RT-PCR as described in ref. 17, by using rpsL as a control of RNA load. (b) The effect of tetracycline on the cytotoxicity of P. aeruginosa on a macrophage cell line was measured as described in ref. 17. Three experiments, each one consisting of eight replicates are shown. For each assay, median values and standard deviations are presented. White, cytotoxicity of P. aeruginosa without antibiotics; gray, cytotoxicity of P. aeruginosa in the presence of tetracycline.

An unexpected residual excess of teleological thinking has influenced the evolution of biological sciences during the last few decades. Although this approach can contribute to the establishment of potential scenarios of research, it frequently leads to wrong interpretations when applied to the experimental results. Antibiotics have been searched on purpose as inhibitors (either bactericidal or bacteriostatic) of bacterial growth, and we have tended to consider that microbes are using them to play the same function in nature. The automatic application of a classic Darwinian view supported the idea that such bacterial killer compounds could play an important role in the fight between microbial competitors for colonizing the same niche. It is obvious that many primary or secondary metabolites, not only antibiotics, might exert different effects at different concentrations. For instance, antibiotics that inhibit bacterial topoisomerases might, at low concentrations, produce changes in DNA supercoiling (21, 22). Because DNA supercoiling regulates expression of virulence determinants in some bacterial species (23), a general change in the virulent properties in the presence of subinhibitory concentrations of these antibiotics might be expected.

Our results support the notion that antibiotics provide a good example of hormesis (7, 8), as they have a very different effect on susceptible bacteria depending on their actual concentration. The results shown in the present work and others (2, 24) indicate that subinhibitory concentrations of antibiotics can produce specific changes in the behavior of susceptible bacteria. In fact, it recently has been demonstrated that rifampicin induces specific modulation of transcription in Salmonella typhimurium in a promoter-dependent manner, without the need of global regulators or the bacterial global stress response (35). It has been stated that this could be just a defensive response to subinhibitory antibiotic concentrations (2, 25). Opposite to that view, we believe that these results, more than reflecting bacterial competition, reflect bacterial cooperation and offer more information on the actual ecological and evolutionary role of antibiotics in nature. Some arguments against the role of antibiotics as weapons were earlier raised, including the low concentrations of free antibiotics present in soils compared with laboratory conditions and the fact that the highest antibiotic production is usually at the stationary growth phase not at the exponential one, when competition for resources would expected to concentrate. Let us imagine that antibiotic-producing microorganisms in nature could serve as an organizing force in integrated microbial communities, helping, for instance, nonantibiotic members of the community to colonize surfaces (by biofilm formation) or to fight against predator Protistae (cytotoxicity). We thus could start to envisage a Copernican turn-about for the role of antibiotics in nature: from weapons involved in microbial struggle for life to collective regulators of the homeostasis of microbial communities. Weapons and signals always have been linked in nature [armament-ornament duality (26)]. Our results show that antibiotics constitute a paradigmatic example of this general evolution trend. This view is supported by other work in addition to ours (27) and also might result in a reconsideration of the unexpected, and therefore unexplored, effects on microbial biosphere resulting from the release of industrial antibiotics or biocides in nature (27, 28), including those used to feed animal for their ability to promote growth.

Materials and Methods

Supplementary Material

Acknowledgments

Abbreviations

Footnotes

References