 In fact, a recently published work has shown that specific subinhibitory concentrations of the aminoglycoside tobramycin enhances the formation of bacterial biofilms (2), evidence suggesting that antibiotic-induced reduction of bacterial fitness (and virulence) is not always the result upon antibiotic exposure. To address in more detail the effect of subinhibitory concentrations of antibiotics in bacterial physiology and virulence, we have used as a model Pseudomonas aeruginosa, one of the major opportunistic pathogens producing infections in hospitalized and cystic fibrosis (CF) patients (3). CF is one of the most prevalent genetic inheritable diseases (4). People suffering CF have long-term P. aeruginosa chronic colonization of the bronchial tree (years), with recurrent infections that result in lung deterioration and finally in the death of the patient (5). Because these people are usually under antibiotic treatment, P. aeruginosa grows in their highly compartmentalized bronchial environment in the presence of gradients formed by widely variable concentrations of antibiotics. Learning the response of P. aeruginosa to subinhibitory concentrations of antibiotics is thus a relevant task to understanding the biological responses of this bacterium in patients under treatment (6). To that goal, we have developed a P. aeruginosa subgenomic DNA microarray containing 555 genes selected as relevant for the development of chronic colonization and infection, antibiotic resistance, transcriptional regulation, and stress response. The effect of three antimicrobial agents belonging to different structural families, namely tetracycline, tobramycin, and ciprofloxacin, on the transcription of virulence-related determinants of P. aeruginosa was analyzed by using this subgenomic array. To further understand the effect of antibiotics on P. aeruginosa pathophysiology, functional analyses on the production of a number of determinants relevant for the virulence of this pathogen also were performed.
Here we show that specific antibiotics at given concentrations may increase expression of bacterial virulence determinants. This result has clear implications for the treatment of infectious diseases. In addition, our results offer more information for understanding the ecological role of antibiotics in nature. In this regard, antibiotics are good examples of hormesis (7, 8). At high concentrations, they are bacterial killers, whereas at low concentrations, they produce specific changes that might eventually favor the behavior of susceptible bacteria in nature.
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).
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 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.
Effect of tetracycline on the expression of T3SS and the cytotoxicity of P