Salicylate induction of phenotypic resistance to quinolones in Serratia marcescens

Mercedes Berlanga and Miguel Viñas*

Microbiology Unit, Campus of Bellvitge, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, E-08907 Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The influence of salicylic acid on the permeability and susceptibility of Serratia marcescens to both nalidixic acid and ciprofloxacin was studied, as well as the effect of salicylate on outer membrane proteins and lipopolysaccharide. As salicylic acid concentration increased, ciprofloxacin accumulation decreased with a concomitant, previously observed, reduction in the porin content of the outer membrane. Resistance to ciprofloxacin and nalidixic acid was enhanced when bacteria grew in the presence of salicylic acid.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The introduction of fluoroquinolones more than 10 years ago offered clinicians orally or parenterally administrable compounds with a broad spectrum of activity and unprecedented therapeutic results in a wide range of infections. Extensive use and misuse of these compounds in both human and veterinary medicine has led to the emergence and spread of resistant bacteria. Widely varying percentages of resistance to fluoroquinolones have been described in several bacterial species, such as Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens. Several reports have been published on the effect of salicylic acid on antimicrobial susceptibility.1,2 Salicylate and antibiotics are often administered simultaneously, and subsequent high levels of both drugs can compromise their effectiveness. It has been shown that salicylate and other membranepermeating weak acids, such as acetate and benzoate, increase the resistance of Escherichia coli to several antibiotics, including ampicillin, tetracycline, chloramphenicol, nalidixic acid and cephalosporins.3,4 This has been attributed to two phenomena: a weak acid effect, possibly due to an increase in the membrane potential and some additional, uncharacterized effect related to salicylate structure.5 However, suppression of porin synthesis in bacteria grown in the presence of salicylate has been reported.6 Recently, we examined the mechanisms of quinolone uptake by S. marcescens: accumulation of quinolones through the outer membrane in both lipopolysaccharide-deficient and porin-deficient mutants. It was shown that quinolones with a low relative hydrophobicity coefficient seemed to pass preferentially through the water-filled Omp3 porin channels (M. Berlanga and M. Viñas, unpublished data). Our previous studies also identify an efflux pump in Serratia spp. that evacuates ciprofloxacin but not nalidixic acid (M. Berlanga and M. Viñas, manuscript in preparation). Previously, the effect of therapeutic concentrations of salicylate on susceptibility to ß-lactams and aminoglycosides was studied in this species.2 Here, we examine the effect of salicylate at therapeutic concentration on permeability and susceptibility of S. marcescens to ciprofloxacin and nalidixic acid.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The bacterial strain used in this study was Serratia marcescens NIMA, wild type. It was maintained on trypticase soy agar (TSA) slants and cultured in tryptone soy broth (TSB) before experiments. Antimicrobial susceptibility of the NIMA strain was determined previously.2 Media were purchased from Difco (Detroit, MI, USA). Antibiotics used were ciprofloxacin and nalidixic acid (gift from Cenavisa, S.A. laboratories, Reus, Spain). Salicylic acid was purchased from Sigma (St Louis, MO, USA). MICs were determined by the broth dilution method. Overnight cultures in TSB were diluted 1000-fold in fresh broth and 5 µL of the bacterial suspension was inoculated in TSB containing serial dilutions of the antimicrobial agents. MICs were determined after 18 h incubation at 37°C and defined as the minimum concentration of antibiotic that inhibits growth. The bactericidal index (BI), was calculated following Morrissey7 [antibiotic concentrations (in mg/L) from 0.06 to 2.60 and 5 to 50 for ciprofloxacin and nalidixic acid respectively]. The efficiency of plating (EOP) in different conditions was estimated according to Aumercier et al.3 Briefly, a fresh overnight culture of NIMA was grown in TSB at 37°C, diluted 1:3 in Ringer solution and plated on to TSA with increasing concentrations of salicylate and two concentrations of antibiotics (0.06 mg/L and 0.125 mg/L ciprofloxacin and 5 mg/L and 10 mg/L nalidixic acid). The EOP was the titre of cfu obtained from the test plates divided by the titre from control plates lacking both antibiotics and salicylate. The accumulation of quinolone was measured using the method of Mortimer & Piddock.8 Briefly, bacteria were cultured until the OD600 reached 0.5–0.7. Cells were harvested by centrifugation at 4000g for 15 min at room temperature, suspended in PBS pH 7.5 and washed twice. Finally, pellets were suspended in 1/10 volume of PBS. Aliquots of 1 mL were prepared and maintained at 37°C. Quinolone was added to each tube to reach 10 mg/L and then incubation for 0.5, 1, 3, 6 and 12 min was accomplished. After incubation, a rapid centrifugation (13000g at 4°C for 1 min) was carried out. Bacteria were washed in cold PBS and suspended in 1 mL of HCl–glycine 0.1 M pH 3 and incubated for 2 h. This suspension was centrifuged at 11000g for 5 min to remove cellular debris and antibiotic concentration measured in a Hitachi F-2000 spectrofluorimeter.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The antibacterial activity of quinolones is the result of both their ability to pass through the outer membrane and their capacity to inhibit target enzymes, such as DNA gyrase and topoisomerase IV. Susceptibility to ciprofloxacin and nalidixic acid was not affected by salicylate at therapeutic concentrations (TableGo). However, BI values in vitro depended on the presence of salicylate (BI is an index of bactericidal activity at a clinically achievable concentration). EOPs showed a decrease in the susceptibility to quinolones concomitant with an increase in salicylate concentration. Saturation occurred at 5 mM salicylate (Figure 1Go). After subculturing in the absence of salicylate bacteria recover their original susceptibility patterns (data not shown). These results are in agreement with those demonstrating that salicylate induced reversible ‘phenotypic antibiotic resistance’.


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Table. MICs (mg/L) and bactericidal index (BI) for Serratia marcescens of ciprofloxacin and nalidixic acid with/without therapeutic concentration of salicylic acid
 


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Figure 1. EOP of Serratia marcescens NIMA at different salicylate concentrations in the presence of (a) ciprofloxacin: {square}, ciprofloxacin 0.06 mg/L; {triangleup}, ciprofloxacin 0.125 mg/L; {diamondsuit}, control; and (b) nalidixic acid: {square}, nalidixic acid 5 mg/L; {triangleup}, nalidixic acid 10 mg/L; {diamondsuit}, control.

 
Three major putative porins (Omp1, Omp2 and Omp3) have been described in S. marcescens.9 It has been shown that synthesis of Omp3 by S. marcescens is abolished, or at least severely repressed, by concentrations of salicylate of 3 mM or higher.2 The lipopolysaccharide (LPS) of NIMA includes the three typical regions, core, lipid A and lateral oligosaccharides (O-antigen). No differences in electrophoretic profiles of Omp or LPS were observed in cells grown in salicylate at therapeutic (1 mM) concentrations (data not shown). The kinetics of ciprofloxacin and nalidixic acid accumulation are shown in Figure 2Go. Whereas salicylic acid had no effect on nalidixic acid accumulation, a significant decrease in the uptake of ciprofloxacin was detected especially at high salicylate concentrations. It should be noted that salicylic acid alone (labelled as control) did not affect the EOPs of the strain studied.



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Figure 2. (a) Accumulation of nalidixic acid (10 mg/L external concentration) in the presence of therapeutic (1 mM, {triangleup}) and high concentration (10 mM, x) of salicylic acid. (b) Accumulation of ciprofloxacin (10 mg/L external concentration) in the presence of therapeutic (1 mM, {triangleup}) and high concentration (10 mM, x) of salicylic acid.

 
In principle, hydrophobic quinolones such as nalidixic acid might be able to pass through the lipid bilayer while hydrophilic quinolones such as ciprofloxacin cross the outer membrane mainly via porins.8 Thus, differences in the accumulation of hydrophobic and hydrophilic quinolones can be explained by reference to these two pathways. It has been shown that bacteria growing in the presence of salicylate produce fewer porins, which would affect their permeability to ciprofloxacin and lead to decreased intracellular accumulation, and subsequently lower susceptibility. Our results are consistent with this explanation, since salicylate has a clear effect on ciprofloxacin accumulation but no effect on nalidixic acid accumulation. Moreover, the fact that porin synthesis is inhibited by salicylate6 correlates with the decreased OM permeability to hydrophilic quinolones and the subsequent decreased susceptibility.10 This is also consistent with the diminished susceptibility to ciprofloxacin in the presence of salicylate. However, the electrophoretic profile of these OMPs was not affected by these low salicylate concentrations (data not shown), in fact the unchanged electrophoretic profiles in outer membrane proteins of S. marcescens at lower salicylate concentrations (150 mg/L, i.e. 1 mM) has already been described.2 Further analysis of porin synthesis in the presence of salicylate will be required to investigate whether changes in susceptibility are due to membrane phenomena or whether further mechanisms such as transcriptional changes in the mar operon could be involved.

The effect of salicylate has been attributed to a weak acid effect that possibly leads to an increase in the membrane potential and to some additional effect.5 Moreover, some authors suggest that the effect of salicylate is due to derepression of the mar operon, which is negatively regulated by MarB protein.1 Salicylate induces the mar operon by binding marR, causing an increase in marA, a transcriptional activator, and increasing the expression of several genes, some of which produce antibiotic and oxidative stress resistance or control microbial metabolism and pathogenesis.

We do not know whether the mar operon is overexpressed in NIMA when salicylate is present, in these conditions ciprofloxacin accumulation decreases. Puig et al.2 demonstrated that salicylate increases the MICs of ampicillin and several cephalosporins, it decreases the MICs of kanamycin and cephalothin, but has no effect on the MIC of chloramphenicol. Salicylate induces resistance to chloramphenicol, tetracycline, ampicillin, cephalosporins and nalidixic acid in E. coli; Cohen et al.1 pointed out that salicylate increases drug resistance even in mar-inactivated strains. So salicylate must also be able to induce drug resistance via a mar-independent pathway. In our case it seems unlikely that mar overexpression could lead to phenotypic resistance. The low-level resistance to quinolones and ß-lactams induced by salicylate allows bacterial survival. Surviving cells would then have the potential to mutate spontaneously to higher, more clinically relevant levels of resistance to fluoroquinolones. Recently, evidence has been reported demonstrating that salicylate increases the frequency with which a susceptible strain of S. aureus mutates to become more resistant to ciprofloxacin.11

The use of salicylic acid can reduce the uptake and bactericidal activity of ciprofloxacin. This may not be serious enough to compromise treatment, but it may allow the survival of a few bacteria, thus favouring the emergence of clinically significant levels of resistance.


    Acknowledgments
 
The help of Robin Rycroft in the preparation of the manuscript is gratefully acknowledged. This work was supported by grants PB94-0910 and PM98-0189 from the DGICYT (Spanish Ministry of Education and Culture).


    Notes
 
* Corresponding author. Tel: +34-93-402-4250; Fax: +34-93-402-4258; E-mail: mvinyas{at}bell.ub.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cohen, S. P., Foulds, J. J., Levy, S. B. & Rosner, L. (1993). Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and mar-independent pathway. Journal of Bacteriology 175, 7856–62.[Abstract]

2 . Puig, M., Palomar, J., Loren, J. G. & Viñas, M. (1995). Modification by analgesics of the susceptibility to antibiotics in Serratia marcescens. Microbiologica 18, 385–90.[ISI][Medline]

3 . Foulds, J., Murray, D. M., Chai, T. & Rosner, J. L. (1989). Decreased permeation of cephalosporins through the outer membrane of Escherichia coli grown in salicylates. Antimicrobial Agents and Chemotherapy 33, 412–7.[ISI][Medline]

4 . Rosner, J. L. (1985). Non heritable resistance to chloramphenicol and other antibiotics induced by salicylates and other chemotactic repellents in Escherichia coli K12. Proceedings of the National Academy of Sciences, USA 82, 8771–4.[Abstract]

5 . Aumercier, M., Murray, D. M. & Rosner, J. L. (1990). Potentiation of susceptibility to aminoglycosides by salicylate in Escherichia coli. Antimicrobial Agents and Chemotherapy 34, 786–91.[ISI][Medline]

6 . Sawai, T., Hirano, S. & Yamaguchi, A. (1987). Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumoniae and Serratia marcescens. FEMS Microbiology Letters 40, 233–7.[ISI]

7 . Morrissey, I. (1997). Bactericidal index: a new way to assess quinolone bactericidal activity in vitro. Journal of Antimicrobial Chemotherapy 39, 713–7.[Abstract]

8 . Mortimer, P. G. S. & Piddock, L. V. J. (1991). A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 28, 639–53.[Abstract]

9 . Puig, M., Fusté, M. C. & Viñas, M. (1993). Outer membrane proteins from Serratia marcescens. Canadian Journal of Microbiology 39, 108–11.[ISI][Medline]

10 . Piddock, L. J. V., Hall, M. C. & Walters, R. N. (1991). Phenotypic characterization of quinolone-resistant mutants of Enterobacteriaceae selected from wild type, gyrA type and multiply-resistant (marA) type strains. Journal of Antimicrobial Chemotherapy 28, 185–98.[Abstract]

11 . Gustafson, J. E., Candelaria, P. V., Fisher, S. A., Goodridge, J. P., Lichocik, T. M., McWilliams, T. M. et al. (1999). Growth in the presence of salicylate increases fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 990–2.[Abstract/Free Full Text]

Received 6 December 1999; returned 22 February 2000; revised 15 March 2000; accepted 19 April 2000