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Nitrofurantoin, phenazopyridine, and the superoxide-response regulon soxRS of Escherichia coli
J Infect Chemother DOI 10.1007/s10156-013-0635-4
ORIGINAL ARTICLE
Nitrofurantoin, phenazopyridine, and the superoxide-response regulon soxRS of Escherichia coli Carlos F. Ama´bile-Cuevas • Jose´ Luis Arredondo-Garcı´a
Received: 6 May 2013 / Accepted: 10 June 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract Nitrofurantoin and phenazopyridine are two drugs commonly used against urinary tract infections. Both compounds exert oxidative damage in patients deficient in glucose-6-phosphate dehydrogenase. This study was done to assess the interactions of these drugs with the soxRS regulon of Escherichia coli, a superoxide-defense system (that includes a nitroreductase that yields the active metabolite of nitrofurantoin) involved in antibiotic multiresistance. The effects of either nitrofurantoin or phenazopyridine, upon strains with different soxRS genotypes, were measured as minimum inhibitory concentrations (MICs) and growth curves. Also, the ability of these drugs to induce the expression of a soxS’::lacZ gene fusion was assessed. The effect of antibiotics in the presence of phenazopyridine, paraquat (a known soxRS inducer), or an efflux inhibitor, was measured using the disk diffusion method. A strain constitutively expressing the soxRS regulon was slightly more susceptible to nitrofurantoin, and more resistant to phenazopyridine, compared to wild-type and soxRS-deleted strains, during early treatment, but 24-h MICs were the same (8 mg/l nitrofurantoin, 1,000 mg/l phenazopyridine) for all strains. Both compounds were capable of inducing the expression of a soxS’::lacZ fusion, but less than paraquat. Subinhibitory concentrations of phenazopyridine increased the antimicrobial effect of ampicillin, chloramphenicol, tetracycline, and nitrofurantoin. The induction or
C. F. Ama´bile-Cuevas (&) Fundacio´n Lusara, Apartado Postal 8-895, 08231 Mexico City, Mexico e-mail: [email protected] J. L. Arredondo-Garcı´a Instituto Nacional de Pediatrı´a, Mexico City, Mexico
constitutive expression of the soxRS regulon seems to be a disadvantage for E. coli during nitrofurantoin exposure; but might be an advantage during phenazopyridine exposure, indicating that the latter compound could act as a selective pressure for mutations related to virulence and antibiotic multi-resistance. Keywords Antibiotic resistance Escherichia coli Nitrofurantoin Phenazopyridine soxRS Urinary tract infection
Introduction Several drugs can be used in the therapy of lower urinary tract infections (UTI). Of these, two are old and their precise mechanism of action is mostly unknown: the antimicrobial drug nitrofurantoin (NF, patented in 1952), and the urospecific analgesic phenazopyridine (PP, patented in 1927–1928). NF is used against lower UTI, and PP is often used along with an antibiotic, occasionally NF itself, also in the management of UTI, particularly in developing countries. Although it is commonly accepted that PP is devoid of antibacterial properties [1], NF is known to be a sort of prodrug, requiring an enzyme-mediated reaction to generate derivatives [2] that are involved in various kinds of damage to the bacterial cell. These notions are, however, controversial: one of the earliest reports on PP states a ‘‘bacteriostatic action against Staphylococcus aureus and Escherichia coli’’ at 600-mg doses [3]; and a paper on the mechanisms of action of NF reports one that ‘‘does not require the production of reactive NF metabolites by bacterial reductases’’ [4]. In addition to being used in urinary tract ailments, NF and PP have in common being compounds capable of inducing oxidative injury to erythrocytes,
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especially in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency [5]. Our first objective was to assess the interactions of each of these compounds, with the response of Escherichia coli to superoxide stress governed by the soxRS genes. The products of these genes act sequentially: first, SoxR becomes a transcriptional activator of soxS, in the presence of superoxide stress (likely because of diminishing NADPH concentrations [6]); SoxS, in turn, activates the expression of defense and repair mechanisms that include a Mn-containing superoxide-dismutase, NADPH-replenishing G6PD, DNA-repairing endonuclease IV, and the xenobiotic efflux system AcrAB, among many others [7]. A second objective was to explore the potential paradox between NF action and the soxRS response: one of the two reductases, NfsA, that turn NF into an antimicrobially active compound [8], is under the control of soxRS [9]. However, at least two other soxRS-controlled genes (acrAB, mentioned above; and micF, encoding an antisense RNA that post-transcriptionally inhibits the expression of OmpF, hence diminishing the permeability of the outer membrane) mediate unspecific antibiotic resistance. Therefore, although the induction of the soxRS system (or its constitutive expression caused by mutations) could potentially increase the conversion of NF into an active compound, it could also provide the means for resisting its antimicrobial effects.
Materials and methods Strains and media Escherichia coli K12 strains with three different soxRS genotypes were used: GC4468, carrying wild-type (wt) soxRS genes; DJ901, a derivative of GC4468 but with the soxRS genes deleted (DsoxRS); and JTG1052, carrying allele soxR101 (soxRc), that constitutively expresses soxS, hence the entire regulon [10]; and also, strain TN521, a derivative of DJ901 carrying a prophage with a wild-type soxR gene, and a soxS’::lacZ fusion; and TN531, the same as TN521 but without the soxR gene [11]. All these strains were a kind gift from B. Demple. Cells were grown on liquid or solid LB media [except for antibiotic susceptibility assays, performed on liquid or solid Mueller–Hinton media (MH; Fluka)].
MH broth, incubated for 24 h at 35 °C without agitation: the NF series was 128, 64, 32, 16, 8, 4, 2, and 1 lg/ml; the PP series was 1,000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 lg/ml. Additionally, cells were treated with subinhibitory concentrations of NF (5 lg/ml) or PP (100 lg/ml), in MH broth incubated at 35 °C, agitated at 200 rpm, sampling each hour, diluting and plating on LB agar; plates were incubated at 35 °C for 18 h, and colonies were counted. Measurement of soxS’::lacZ induction The activity of b-galactosidase was measured as described by Miller [12], after treating strains TN521 and TN531 with subinhibitory concentrations of NF or PP (paraquat, PQ, a redox-cycling, superoxide generating agent, and a known inducer of the soxRS regulon [7, 11], was used as positive control), for 30 min at 35 °C, agitated at 200 rpm. In experiments with PP, cells were first washed twice in ice-cold Z buffer, as PP interferes with the measuring of hydrolyzed ONPG at 420 nm. Effect of PP or efflux-pump inhibitor upon the activity of NF and other antibiotics The activity of ampicillin, chloramphenicol, tetracycline, and NF was measured using the disk diffusion method (Bauer-Kirby) on MH agar plates with or without PP (100 lg/ml), PQ (50 lM), or Phe-Arg-b-naphthylamide (PAbN, at 50 lM [13], a known inhibitor of xenobiotic efflux mediated by AcrAB, a member of the soxRS regulon [7]). Overnight cultures were diluted in fresh MH broth, incubated at 35 °C until OA600 of 0.5, pretreated with PP 100 lg/ml or PQ 50 lM for 30 min, and streaked on MH agar plates; antibiotic disks were applied, and plates were incubated at 35 °C for 18 h.
Results MICs of NF and PP The growth of all three strains with different soxRS genotypes was completely inhibited by NF at 8 lg/ml after a 24-h incubation. MIC of PP was 1,000 lg/ml for all three soxRS strains as well, although turbidity of JTG1052 was slightly higher at 500 lg/ml (not shown).
Antibacterial activity of NF and PP Growth curves Minimum inhibitory concentrations (MIC) of NF (Sigma) and PP (3-phenylazo-2,6-diaminopyridine hydrochloride; Alfa Aesar) were assessed by twofold serial dilution on
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Hourly monitoring bacterial growth in the presence of subinhibitory concentrations of NF (5 lg/ml) or PP
J Infect Chemother
Fig. 1 Effect of nitrofurantoin (NF) (left) or phenazopyridine (PP) (right) upon growth of Escherichia coli strains with different soxRS genotype. NF nitrofurantoin 5 lg/ml, PP phenazopyridine 100 lg/ml, GC strain GC4468 (soxRS-wt), DJ strain DJ901 (DsoxRS), JTG strain
JTG1052 (soxRc). Symbols indicate geometric means of three independent experiments, along with standard deviations, sometimes falling within the symbol (NF and PP values separated for clarity)
(100 lg/ml) showed the differential effects of these compounds, depending on the soxRS genotype. NF does not seem to affect the growth of strain DJ901 (DsoxRS), slightly diminishes the growth rate of GC4468 (soxRS-wt), and completely inhibits the growth of JTG1052 (soxRc) for about 2 h (Fig. 1, left). On the other hand, PP slightly impairs the growth of JTG1052; kills about 70 % of the initial population of GC4468 within the first hour, resuming growth afterward; and kills about 80 % of the initial population of DJ901 within the first 2 h, resuming growth afterward (Fig. 1, right).
Table 1 Induction of soxS’::lacZ fusion by nitrofurantoin (NF) and phenazopyridine (PP) (b-galactosidase activity, in Miller unitsa) TN521 (soxR?, soxS’::lacZ)
Treatment
TN531 (DsoxR, soxS’::lacZ)
None
161.5 (9.6)
172.6 (8.8)
PQb 50 lM
155.0 (10.3)
1078.8 (34.6)
NF 2 lg/ml PP 100 lg/ml
156.7 (7.7) 159.9 (8.1)
241.6 (12.0) 510.9 (43.9)
a
Mean (standard deviation) of three independent experiments
b
Paraquat, a known inducer of the soxRS regulon, was used as a positive control
Induction of soxS’::lacZ fusion by NF and PP After a 30-min treatment with subinhibitory concentrations of NF or PP, b-galactosidase activity resulting from the induction of a soxS’::lacZ fusion was measured (Table 1). Strain TN531, lacking a functional soxR gene, failed to respond to either drug. Strain TN521, having a wild-type soxR gene, showed a very slight induction when treated with NF 2 lg/ml, whereas PP 100 lg/ml reached about half the induction obtained with 50 lM PQ [lower concentrations of either NF (1 lg/ml) or PP (50 lg/ml), did not induce the expression of the gene fusion; higher concentrations inhibited the growth of these DsoxRS strains, and the expression of the soxS’::lacZ fusion; data not shown]. As expected, PQ treatment had no effect upon the expression of the soxS’::lacZ fusion in strain TN531 but produced a sixfold induction in strain TN521.
Effect of different soxRS genotypes, PQ, PP, or PAbN upon activity of NF and other antibiotics The effects of different antibiotics, measured as inhibitory halos by the disk diffusion method, are shown in Table 2. The constitutive expression of the soxRS regulon in strain JTG1052 resulted in a reduction of 14 % in the halo formed by ampicillin and chloramphenicol, and was without detectable effect in NF or tetracycline activity. PQ increased the size of the inhibitory halo of NF 11–18 %, decreased this effect for ampicillin, chloramphenicol, and tetracycline upon strain GC4468 (soxRS wt), and increased it upon strain DJ901 (DsoxRS). PP increased the size of the inhibitory halo of all antibiotics tested 9–18 % upon all three strains. PAbN, an efflux-pump inhibitor, decreased the size of the halo of NF upon DJ901, but increased it upon JTG1052; it increased the size of the halos of
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J Infect Chemother Table 2 Effect of different soxRS genotypes, and of PP, paraquat (PQ), or Phe-Arg-b-naphthylamide (PAbN), upon the effect of various antibioticsa GC4468 (soxRS-wt) –
PP
PQ
JTG1052 (soxRc)
DJ901 (DsoxRS) PAbN
–b
PP
PQ
PAbN
–b
PP
PQ
PAbN
NF
1
1.18
1.23
1.00
1 (1.06)
1.16
1.26
0.87
1 (1.00)
1.11
1.14
1.08
AM
1
1.10
0.90
0.75
1 (0.99)
1.09
1.23
0.86
1 (0.86)
1.13
0.97
0.89
CM TE
1 1
1.18 1.17
0.77 0.93
1.24 0.97
1 (1.04) 1 (1.05)
1.15 1.09
1.12 1.15
1.20 0.90
1 (0.86) 1 (0.97)
1.08 1.09
0.82 0.94
1.31 1.01
a
Measured as inhibitory halos using the disk diffusion method, relative to halos obtained on each strain for each antibiotic without additional treatment. For strain GC4468: NF nitrofurantoin, 20.7 mm; AM ampicillin, 20.7 mm; CM chloramphenicol, 27.0 mm; TE tetracycline, 24.1 mm. Results of a typical experiment of three independent ones; differences between experiments were ±0.5 mm
b
Values in parentheses are relative to results of strain GC4468
chloramphenicol in all strains. Interestingly, PAbN decreased the size of the halos of ampicillin 11–25 % in all strains.
Discussion The superoxide response governed by soxRS in E. coli has been implicated in antibiotic resistance phenotypes [14]. Although some of these phenotypes do not reach a resistance considered to be ‘‘clinically relevant’’ (i.e., that do not surpass resistance breakpoint values established for clinical microbiology laboratories), this low-level resistance does play a significant role both directly in some clinical conditions and indirectly when adding up to a fullresistance phenotype [15]. The soxRS response system contributes also to bacterial virulence by conferring resistance to nitric oxide produced by activated macrophages [16]. Moreover, SoxS controls the expression of znuCB, part of the zinc transport system ZnuACB, which is crucial for the persistence of E. coli in a murine pyelonephritis model [17]. Agents or conditions that induce the expression of this regulon, and/or select for constitutive mutants, could therefore yield a more resistant, more virulent microorganism. The soxRS genes have been detected in a variety of gram-negative bacteria, although their regulatory role and the genes under their control vary greatly among species. Resistance to NF is known to have a fitness cost in E. coli [18], as most resistant strains lack either nitroreductase activity necessary for the generation of NF reactive intermediates. These enzymes seem to catalyze the divalent reduction of organic nitro compounds, quinones, and dyes, preventing their univalent reduction, hence diminishing the production of superoxide radicals caused by the redox cycling of these compounds [9]. This fitness cost, along with the ubiquitous damage caused by NF, has been pointed out as the reason for a low prevalence of resistance [19]. Results shown here extend this notion: the
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constitutive expression of the soxRS regulon, that yields a multi-resistance phenotype, increases the antibacterial activity of NF in the very short term (Fig. 1), perhaps because of increased activation of NF by the overexpression of nfsA. However, this does not reflect as changes in 24-h MIC. Perhaps the increased activation of NF is compensated by increased efflux caused by the overexpression of AcrAB; PAbN increases by 8 % the activity of NF upon strain JTG1052, measured as inhibitory halos (Table 2). Simultaneous exposure of soxRS-wt and soxRc strains (but not of DsoxRS strain) to PQ reduces the activity of other antibiotics, but increases the activity of NF; this could be explained again by the overexpression of nfsA, yielding more NF active metabolite. However, the activity of NF alone upon soxRS-wt and soxRc strains was identical, measured as inhibitory halos at 18 h. PQ is also a marginal inducer of the marRAB regulon [20] that includes the other nitroreductase involved in NF activity, NfsB (or NfnB [21]), which could account for the increased activity of NF in the presence of PQ, even upon a soxRc strain, but not acting alone while only the soxRS regulon was overexpressed. NF itself poorly induces the expression of a soxS’::lacZ fusion at low, subinhibitory concentrations. Overall, contrasting to other antibiotics, the antimicrobial activity of NF is not reduced (and is marginally augmented) by the overexpression or induction of the soxRS regulon; hence, NF does not seems capable of selecting for soxRc mutants that could have a multiresistant, more virulent phenotype. Interestingly, marRAB, a regulon that overlaps extensively with soxRS, and is also involved in antibiotic multi-resistance phenotypes [14], governs the expression of NfsB, as mentioned earlier. It seems likely that the induction or constitutive expression of the marRAB regulon would have the same effects upon the activity of NF as that reported here for the soxRS regulon. The effect of PP is of particular clinical relevance. It seems to induce the expression of a soxS’::lacZ fusion, but only at a concentration nearly inhibitory, in the short term,
J Infect Chemother
for the DsoxRS strain carrying the gene construct. This result was expected, as PP is known to cause oxidative damage in G6PD-deficient cells. At subinhibitory concentrations, PP increases the effect of all antibiotics tested, disregarding the soxRS genotype, indicating a mechanism independent of superoxide stress. Although the MIC of PP is high, unlikely to be reached at routine clinical doses, lower concentrations could potentially select for soxRc mutants, as they seem less susceptible to the short-term bactericidal effects of PP, compared to soxRS-wt and DsoxRS strains. Taken together, these results strongly point to the need to reevaluate the pertinence of using PP, especially in the context of an UTI, as PP seems likely to act as a selective pressure for virulent, resistant bacteria. However, it is necessary to emphasize that these results were attained at high PP concentrations. The actual urinary concentration of PP is controversial: a 1970 paper, not aimed at assessing it, reports that 0–4 h after a 200-mg dose, only 1.0–1.5 lg/ml of unchanged PP was found in urine [22]; the authors themselves contrast these data with previous reports of 45 % elimination of unchanged PP in urine. Later, it was reported that 42.5 % of PP administered to humans is excreted in the urine within 0–4 h, and 60.3 % in 8 h, mostly as the 5-hydroxy metabolite [23], but without providing concentration values. The typical orange-to-red coloring of urine after taking PP [1] was only noticed in vitro at PP concentrations of several hundred micrograms per milliliter (data not shown); the urine color is likely caused by metabolites in vivo. PP maximum plasma concentration after taking a 200-mg dose is 63–65 ng/ml, reached at 9.4–9.6 h [24], indicating an extensive metabolism and/or excretion. Whether actual PP urinary concentrations, or its metabolites, have the effects reported here, remains to be ascertained. In any case, PP is clearly one of those drugs that would not have been approved for clinical use under current standards. Aside from our results, and of the questionable relevance of diminishing the symptoms of UTI [25], PP is ‘‘reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals’’ [26]. Conflict of interest
None.
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3. Goener A, Haley FL. Chemical and bacteriological studies of pyridium. Am J Clin Pathol. 1932;2:379–89. 4. McOsker CC, Fitzpatrick PM. Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens. J Antimicrob Chemother. 1994;33(suppl A):23–30. 5. Bloom JC, Brandt JT. Toxic responses of the blood. In: Klaassen CD, editor. Toxicology, the basic science of poisons. New York: McGraw-Hill; 2008. p. 455–84. 6. Krapp AR, Humbert MV, Carrillo N. The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content. Microbiology. 2011;157:957–65. 7. Ama´bile-Cuevas CF, Demple B. Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon. Nucleic Acids Res. 1991;19:4479–84. 8. Race PR, Lovering AL, Green GM, Ossor A, White SA, Searle PF, et al. Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. J Biol Chem. 2005;280:13256–64. 9. Liochev SI, Hausladen A, Fridovich I. Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli. Proc Natl Acad Sci USA. 1999;96:3537–9. 10. Greenberg JT, Monach P, Chou JH, Josephy PD, Demple B. Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci USA. 1990;87:6181–5. 11. Nunoshiba T, Demple B. Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide. Cancer Res. 1993;53:3250–2. 12. Miller JH. A short course in bacterial genetics. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1992. 13. Hasdemir UO, Chevalier J, Nordmann P, Page`s J-M. Detection and prevalence of active drug efflux mechanism in various multidrug-resistant Klebsiella pneumoniae strains from Turkey. J Clin Microbiol. 2004;42:2701–6. 14. Demple B, Ama´bile-Cuevas CF. Multiple resistance mediated by individual genetic loci. In: Ama´bile-Cuevas CF, editor. Multiple drug resistant bacteria. Wymondham: Horizon Scientific Press; 2003. p. 61–80. 15. Ama´bile-Cuevas CF. Antibiotic resistance: from Darwin to Lederberg to Keynes. Microb Drug Resist. 2013;19:73–87. 16. Nunoshiba T, de Rojas-Walker T, Wishnok JS, Tannenbaum SR, Demple B. Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. Proc Natl Acad Sci USA. 1993;90:9993–7. 17. Warner DM, Levy SB. SoxS increases the expression of the zinc uptake system ZnuACB in an Escherichia coli murine pyelonephritis model. J Bacteriol. 2012;194:1177–85. 18. Sandegren L, Lindqvist A, Kahlmeter G, Andersson DI. Nitrofurantoin resistance mechanisms and fitness cost. J Antimicrob Chemother. 2008;62:495–503. 19. Arredondo-Garcı´a JL, Ama´bile-Cuevas CF. High resistance prevalence towards ampicillin, co-trimoxazole and ciprofloxacin, among uropathogenic Escherichia coli isolates in Mexico City. J Infect Dev Ctries. 2008;2:350–3. 20. Martin RG, Jair KW, Wolf RE, Rosner JL. Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli. J Bacteriol. 1996;178:2216–23. 21. Barbosa TM, Levy SB. Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in class II promoter. Mol Microbiol. 2002;45:191–202. 22. Hollifield RD, Conklin JD. A method for determining nitrofurantoin in urine in the presence of phenazopyridine hydrochloride and its metabolites. Clin Chem. 1970;16:335–8.
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25. Pergialiotis V, Arnos P, Mavros MN, Pitsouni E, Athanasiou S, Falagas ME. Urinary tract analgesics for the treatment of patients with acute cystitis: where is the clinical evidence? Expert Rev Anti Infect Ther. 2012;10:875–9. 26. National Toxicology Program. Phenazopyridine hydrochloride. Rep Carcinog. 2011;12:341–2.
ORIGINAL ARTICLE
Nitrofurantoin, phenazopyridine, and the superoxide-response regulon soxRS of Escherichia coli Carlos F. Ama´bile-Cuevas • Jose´ Luis Arredondo-Garcı´a
Received: 6 May 2013 / Accepted: 10 June 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract Nitrofurantoin and phenazopyridine are two drugs commonly used against urinary tract infections. Both compounds exert oxidative damage in patients deficient in glucose-6-phosphate dehydrogenase. This study was done to assess the interactions of these drugs with the soxRS regulon of Escherichia coli, a superoxide-defense system (that includes a nitroreductase that yields the active metabolite of nitrofurantoin) involved in antibiotic multiresistance. The effects of either nitrofurantoin or phenazopyridine, upon strains with different soxRS genotypes, were measured as minimum inhibitory concentrations (MICs) and growth curves. Also, the ability of these drugs to induce the expression of a soxS’::lacZ gene fusion was assessed. The effect of antibiotics in the presence of phenazopyridine, paraquat (a known soxRS inducer), or an efflux inhibitor, was measured using the disk diffusion method. A strain constitutively expressing the soxRS regulon was slightly more susceptible to nitrofurantoin, and more resistant to phenazopyridine, compared to wild-type and soxRS-deleted strains, during early treatment, but 24-h MICs were the same (8 mg/l nitrofurantoin, 1,000 mg/l phenazopyridine) for all strains. Both compounds were capable of inducing the expression of a soxS’::lacZ fusion, but less than paraquat. Subinhibitory concentrations of phenazopyridine increased the antimicrobial effect of ampicillin, chloramphenicol, tetracycline, and nitrofurantoin. The induction or
C. F. Ama´bile-Cuevas (&) Fundacio´n Lusara, Apartado Postal 8-895, 08231 Mexico City, Mexico e-mail: [email protected] J. L. Arredondo-Garcı´a Instituto Nacional de Pediatrı´a, Mexico City, Mexico
constitutive expression of the soxRS regulon seems to be a disadvantage for E. coli during nitrofurantoin exposure; but might be an advantage during phenazopyridine exposure, indicating that the latter compound could act as a selective pressure for mutations related to virulence and antibiotic multi-resistance. Keywords Antibiotic resistance Escherichia coli Nitrofurantoin Phenazopyridine soxRS Urinary tract infection
Introduction Several drugs can be used in the therapy of lower urinary tract infections (UTI). Of these, two are old and their precise mechanism of action is mostly unknown: the antimicrobial drug nitrofurantoin (NF, patented in 1952), and the urospecific analgesic phenazopyridine (PP, patented in 1927–1928). NF is used against lower UTI, and PP is often used along with an antibiotic, occasionally NF itself, also in the management of UTI, particularly in developing countries. Although it is commonly accepted that PP is devoid of antibacterial properties [1], NF is known to be a sort of prodrug, requiring an enzyme-mediated reaction to generate derivatives [2] that are involved in various kinds of damage to the bacterial cell. These notions are, however, controversial: one of the earliest reports on PP states a ‘‘bacteriostatic action against Staphylococcus aureus and Escherichia coli’’ at 600-mg doses [3]; and a paper on the mechanisms of action of NF reports one that ‘‘does not require the production of reactive NF metabolites by bacterial reductases’’ [4]. In addition to being used in urinary tract ailments, NF and PP have in common being compounds capable of inducing oxidative injury to erythrocytes,
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J Infect Chemother
especially in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency [5]. Our first objective was to assess the interactions of each of these compounds, with the response of Escherichia coli to superoxide stress governed by the soxRS genes. The products of these genes act sequentially: first, SoxR becomes a transcriptional activator of soxS, in the presence of superoxide stress (likely because of diminishing NADPH concentrations [6]); SoxS, in turn, activates the expression of defense and repair mechanisms that include a Mn-containing superoxide-dismutase, NADPH-replenishing G6PD, DNA-repairing endonuclease IV, and the xenobiotic efflux system AcrAB, among many others [7]. A second objective was to explore the potential paradox between NF action and the soxRS response: one of the two reductases, NfsA, that turn NF into an antimicrobially active compound [8], is under the control of soxRS [9]. However, at least two other soxRS-controlled genes (acrAB, mentioned above; and micF, encoding an antisense RNA that post-transcriptionally inhibits the expression of OmpF, hence diminishing the permeability of the outer membrane) mediate unspecific antibiotic resistance. Therefore, although the induction of the soxRS system (or its constitutive expression caused by mutations) could potentially increase the conversion of NF into an active compound, it could also provide the means for resisting its antimicrobial effects.
Materials and methods Strains and media Escherichia coli K12 strains with three different soxRS genotypes were used: GC4468, carrying wild-type (wt) soxRS genes; DJ901, a derivative of GC4468 but with the soxRS genes deleted (DsoxRS); and JTG1052, carrying allele soxR101 (soxRc), that constitutively expresses soxS, hence the entire regulon [10]; and also, strain TN521, a derivative of DJ901 carrying a prophage with a wild-type soxR gene, and a soxS’::lacZ fusion; and TN531, the same as TN521 but without the soxR gene [11]. All these strains were a kind gift from B. Demple. Cells were grown on liquid or solid LB media [except for antibiotic susceptibility assays, performed on liquid or solid Mueller–Hinton media (MH; Fluka)].
MH broth, incubated for 24 h at 35 °C without agitation: the NF series was 128, 64, 32, 16, 8, 4, 2, and 1 lg/ml; the PP series was 1,000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 lg/ml. Additionally, cells were treated with subinhibitory concentrations of NF (5 lg/ml) or PP (100 lg/ml), in MH broth incubated at 35 °C, agitated at 200 rpm, sampling each hour, diluting and plating on LB agar; plates were incubated at 35 °C for 18 h, and colonies were counted. Measurement of soxS’::lacZ induction The activity of b-galactosidase was measured as described by Miller [12], after treating strains TN521 and TN531 with subinhibitory concentrations of NF or PP (paraquat, PQ, a redox-cycling, superoxide generating agent, and a known inducer of the soxRS regulon [7, 11], was used as positive control), for 30 min at 35 °C, agitated at 200 rpm. In experiments with PP, cells were first washed twice in ice-cold Z buffer, as PP interferes with the measuring of hydrolyzed ONPG at 420 nm. Effect of PP or efflux-pump inhibitor upon the activity of NF and other antibiotics The activity of ampicillin, chloramphenicol, tetracycline, and NF was measured using the disk diffusion method (Bauer-Kirby) on MH agar plates with or without PP (100 lg/ml), PQ (50 lM), or Phe-Arg-b-naphthylamide (PAbN, at 50 lM [13], a known inhibitor of xenobiotic efflux mediated by AcrAB, a member of the soxRS regulon [7]). Overnight cultures were diluted in fresh MH broth, incubated at 35 °C until OA600 of 0.5, pretreated with PP 100 lg/ml or PQ 50 lM for 30 min, and streaked on MH agar plates; antibiotic disks were applied, and plates were incubated at 35 °C for 18 h.
Results MICs of NF and PP The growth of all three strains with different soxRS genotypes was completely inhibited by NF at 8 lg/ml after a 24-h incubation. MIC of PP was 1,000 lg/ml for all three soxRS strains as well, although turbidity of JTG1052 was slightly higher at 500 lg/ml (not shown).
Antibacterial activity of NF and PP Growth curves Minimum inhibitory concentrations (MIC) of NF (Sigma) and PP (3-phenylazo-2,6-diaminopyridine hydrochloride; Alfa Aesar) were assessed by twofold serial dilution on
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Hourly monitoring bacterial growth in the presence of subinhibitory concentrations of NF (5 lg/ml) or PP
J Infect Chemother
Fig. 1 Effect of nitrofurantoin (NF) (left) or phenazopyridine (PP) (right) upon growth of Escherichia coli strains with different soxRS genotype. NF nitrofurantoin 5 lg/ml, PP phenazopyridine 100 lg/ml, GC strain GC4468 (soxRS-wt), DJ strain DJ901 (DsoxRS), JTG strain
JTG1052 (soxRc). Symbols indicate geometric means of three independent experiments, along with standard deviations, sometimes falling within the symbol (NF and PP values separated for clarity)
(100 lg/ml) showed the differential effects of these compounds, depending on the soxRS genotype. NF does not seem to affect the growth of strain DJ901 (DsoxRS), slightly diminishes the growth rate of GC4468 (soxRS-wt), and completely inhibits the growth of JTG1052 (soxRc) for about 2 h (Fig. 1, left). On the other hand, PP slightly impairs the growth of JTG1052; kills about 70 % of the initial population of GC4468 within the first hour, resuming growth afterward; and kills about 80 % of the initial population of DJ901 within the first 2 h, resuming growth afterward (Fig. 1, right).
Table 1 Induction of soxS’::lacZ fusion by nitrofurantoin (NF) and phenazopyridine (PP) (b-galactosidase activity, in Miller unitsa) TN521 (soxR?, soxS’::lacZ)
Treatment
TN531 (DsoxR, soxS’::lacZ)
None
161.5 (9.6)
172.6 (8.8)
PQb 50 lM
155.0 (10.3)
1078.8 (34.6)
NF 2 lg/ml PP 100 lg/ml
156.7 (7.7) 159.9 (8.1)
241.6 (12.0) 510.9 (43.9)
a
Mean (standard deviation) of three independent experiments
b
Paraquat, a known inducer of the soxRS regulon, was used as a positive control
Induction of soxS’::lacZ fusion by NF and PP After a 30-min treatment with subinhibitory concentrations of NF or PP, b-galactosidase activity resulting from the induction of a soxS’::lacZ fusion was measured (Table 1). Strain TN531, lacking a functional soxR gene, failed to respond to either drug. Strain TN521, having a wild-type soxR gene, showed a very slight induction when treated with NF 2 lg/ml, whereas PP 100 lg/ml reached about half the induction obtained with 50 lM PQ [lower concentrations of either NF (1 lg/ml) or PP (50 lg/ml), did not induce the expression of the gene fusion; higher concentrations inhibited the growth of these DsoxRS strains, and the expression of the soxS’::lacZ fusion; data not shown]. As expected, PQ treatment had no effect upon the expression of the soxS’::lacZ fusion in strain TN531 but produced a sixfold induction in strain TN521.
Effect of different soxRS genotypes, PQ, PP, or PAbN upon activity of NF and other antibiotics The effects of different antibiotics, measured as inhibitory halos by the disk diffusion method, are shown in Table 2. The constitutive expression of the soxRS regulon in strain JTG1052 resulted in a reduction of 14 % in the halo formed by ampicillin and chloramphenicol, and was without detectable effect in NF or tetracycline activity. PQ increased the size of the inhibitory halo of NF 11–18 %, decreased this effect for ampicillin, chloramphenicol, and tetracycline upon strain GC4468 (soxRS wt), and increased it upon strain DJ901 (DsoxRS). PP increased the size of the inhibitory halo of all antibiotics tested 9–18 % upon all three strains. PAbN, an efflux-pump inhibitor, decreased the size of the halo of NF upon DJ901, but increased it upon JTG1052; it increased the size of the halos of
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J Infect Chemother Table 2 Effect of different soxRS genotypes, and of PP, paraquat (PQ), or Phe-Arg-b-naphthylamide (PAbN), upon the effect of various antibioticsa GC4468 (soxRS-wt) –
PP
PQ
JTG1052 (soxRc)
DJ901 (DsoxRS) PAbN
–b
PP
PQ
PAbN
–b
PP
PQ
PAbN
NF
1
1.18
1.23
1.00
1 (1.06)
1.16
1.26
0.87
1 (1.00)
1.11
1.14
1.08
AM
1
1.10
0.90
0.75
1 (0.99)
1.09
1.23
0.86
1 (0.86)
1.13
0.97
0.89
CM TE
1 1
1.18 1.17
0.77 0.93
1.24 0.97
1 (1.04) 1 (1.05)
1.15 1.09
1.12 1.15
1.20 0.90
1 (0.86) 1 (0.97)
1.08 1.09
0.82 0.94
1.31 1.01
a
Measured as inhibitory halos using the disk diffusion method, relative to halos obtained on each strain for each antibiotic without additional treatment. For strain GC4468: NF nitrofurantoin, 20.7 mm; AM ampicillin, 20.7 mm; CM chloramphenicol, 27.0 mm; TE tetracycline, 24.1 mm. Results of a typical experiment of three independent ones; differences between experiments were ±0.5 mm
b
Values in parentheses are relative to results of strain GC4468
chloramphenicol in all strains. Interestingly, PAbN decreased the size of the halos of ampicillin 11–25 % in all strains.
Discussion The superoxide response governed by soxRS in E. coli has been implicated in antibiotic resistance phenotypes [14]. Although some of these phenotypes do not reach a resistance considered to be ‘‘clinically relevant’’ (i.e., that do not surpass resistance breakpoint values established for clinical microbiology laboratories), this low-level resistance does play a significant role both directly in some clinical conditions and indirectly when adding up to a fullresistance phenotype [15]. The soxRS response system contributes also to bacterial virulence by conferring resistance to nitric oxide produced by activated macrophages [16]. Moreover, SoxS controls the expression of znuCB, part of the zinc transport system ZnuACB, which is crucial for the persistence of E. coli in a murine pyelonephritis model [17]. Agents or conditions that induce the expression of this regulon, and/or select for constitutive mutants, could therefore yield a more resistant, more virulent microorganism. The soxRS genes have been detected in a variety of gram-negative bacteria, although their regulatory role and the genes under their control vary greatly among species. Resistance to NF is known to have a fitness cost in E. coli [18], as most resistant strains lack either nitroreductase activity necessary for the generation of NF reactive intermediates. These enzymes seem to catalyze the divalent reduction of organic nitro compounds, quinones, and dyes, preventing their univalent reduction, hence diminishing the production of superoxide radicals caused by the redox cycling of these compounds [9]. This fitness cost, along with the ubiquitous damage caused by NF, has been pointed out as the reason for a low prevalence of resistance [19]. Results shown here extend this notion: the
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constitutive expression of the soxRS regulon, that yields a multi-resistance phenotype, increases the antibacterial activity of NF in the very short term (Fig. 1), perhaps because of increased activation of NF by the overexpression of nfsA. However, this does not reflect as changes in 24-h MIC. Perhaps the increased activation of NF is compensated by increased efflux caused by the overexpression of AcrAB; PAbN increases by 8 % the activity of NF upon strain JTG1052, measured as inhibitory halos (Table 2). Simultaneous exposure of soxRS-wt and soxRc strains (but not of DsoxRS strain) to PQ reduces the activity of other antibiotics, but increases the activity of NF; this could be explained again by the overexpression of nfsA, yielding more NF active metabolite. However, the activity of NF alone upon soxRS-wt and soxRc strains was identical, measured as inhibitory halos at 18 h. PQ is also a marginal inducer of the marRAB regulon [20] that includes the other nitroreductase involved in NF activity, NfsB (or NfnB [21]), which could account for the increased activity of NF in the presence of PQ, even upon a soxRc strain, but not acting alone while only the soxRS regulon was overexpressed. NF itself poorly induces the expression of a soxS’::lacZ fusion at low, subinhibitory concentrations. Overall, contrasting to other antibiotics, the antimicrobial activity of NF is not reduced (and is marginally augmented) by the overexpression or induction of the soxRS regulon; hence, NF does not seems capable of selecting for soxRc mutants that could have a multiresistant, more virulent phenotype. Interestingly, marRAB, a regulon that overlaps extensively with soxRS, and is also involved in antibiotic multi-resistance phenotypes [14], governs the expression of NfsB, as mentioned earlier. It seems likely that the induction or constitutive expression of the marRAB regulon would have the same effects upon the activity of NF as that reported here for the soxRS regulon. The effect of PP is of particular clinical relevance. It seems to induce the expression of a soxS’::lacZ fusion, but only at a concentration nearly inhibitory, in the short term,
J Infect Chemother
for the DsoxRS strain carrying the gene construct. This result was expected, as PP is known to cause oxidative damage in G6PD-deficient cells. At subinhibitory concentrations, PP increases the effect of all antibiotics tested, disregarding the soxRS genotype, indicating a mechanism independent of superoxide stress. Although the MIC of PP is high, unlikely to be reached at routine clinical doses, lower concentrations could potentially select for soxRc mutants, as they seem less susceptible to the short-term bactericidal effects of PP, compared to soxRS-wt and DsoxRS strains. Taken together, these results strongly point to the need to reevaluate the pertinence of using PP, especially in the context of an UTI, as PP seems likely to act as a selective pressure for virulent, resistant bacteria. However, it is necessary to emphasize that these results were attained at high PP concentrations. The actual urinary concentration of PP is controversial: a 1970 paper, not aimed at assessing it, reports that 0–4 h after a 200-mg dose, only 1.0–1.5 lg/ml of unchanged PP was found in urine [22]; the authors themselves contrast these data with previous reports of 45 % elimination of unchanged PP in urine. Later, it was reported that 42.5 % of PP administered to humans is excreted in the urine within 0–4 h, and 60.3 % in 8 h, mostly as the 5-hydroxy metabolite [23], but without providing concentration values. The typical orange-to-red coloring of urine after taking PP [1] was only noticed in vitro at PP concentrations of several hundred micrograms per milliliter (data not shown); the urine color is likely caused by metabolites in vivo. PP maximum plasma concentration after taking a 200-mg dose is 63–65 ng/ml, reached at 9.4–9.6 h [24], indicating an extensive metabolism and/or excretion. Whether actual PP urinary concentrations, or its metabolites, have the effects reported here, remains to be ascertained. In any case, PP is clearly one of those drugs that would not have been approved for clinical use under current standards. Aside from our results, and of the questionable relevance of diminishing the symptoms of UTI [25], PP is ‘‘reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals’’ [26]. Conflict of interest
None.
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