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Keywords:

  • Proteus mirabilis;
  • ciprofloxacin;
  • antioxidant systems;
  • resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study investigates new aspects of the possible role of antioxidant defenses in the mechanisms of resistance to ciprofloxacin in Proteus mirabilis. Four ciprofloxacin-resistant variants (CRVs), selected in vitro by repeated cultures in a sub-minimum inhibitory concentration (MIC) concentration of ciprofloxacin, attained different levels of antibiotic resistance and high Ferric reducing antioxidant power, with 10−6 frequencies. However, no mutations occurred in positions 83 or 87 of gyrA, 464 or 466 of gyrB, or 78, 80 or 84 of parC, suggesting that resistance took place without these typical mutations in DNA gyrase or topoisomerase IV. Assays with ciprofloxacin and the pump inhibitor carbonyl cyanide m-chlorophenylhydrazone showed that in addition to the antioxidant mechanisms, the influx/efflux mechanism also contributed to the increase in the resistance to ciprofloxacin in one CRV. Moreover, lipid oxidation to malondialdehyde and protein oxidation to carbonyls and advanced oxidation protein products were higher in sensitive than in the resistant strains, as a new factor involved in the mechanisms of resistance in P. mirabilis. The oxidative stress cross-resistance to telluride in CRVs enhanced the role of the antioxidants in the ciprofloxacin resistance of P. mirabilis, which was reinforced during the assays of reduction of susceptibility to ciprofloxacin by glutathione and ascorbic acid.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The primary mechanisms of resistance to fluoroquinolones are mutations that result in the alteration of the target proteins, DNA gyrase and topoisomerase IV, and decreased intracellular drug accumulation due to drug efflux or changes in outer membrane proteins (Piddock, 1999; Ruiz, 2003).

Results previously obtained in our laboratory have indicated that several antibiotics, including ciprofloxacin (CIP), stimulate the production of reactive oxygen species (ROS) in bacterial cells (Becerra & Albesa, 2002; Albesa et al., 2004). In addition, Goswami et al. (2006) concluded that the antibacterial action of fluoroquinolones involves ROS, such as superoxide anions and hydrogen peroxide. Furthermore, Kohanski et al. (2007) showed that the three major classes of bactericidal drugs utilize a common mechanism of killing, as they stimulate the production of lethal doses of hydroxyl radicals. The role of ROS in antibiotic action was related to resistance (Dwyer et al., 2009; Kohanski et al., 2010).

Nevertheless, although protection against oxidative stress by antioxidant has been reported (Koziol et al., 2005; Goswami et al., 2006; Páez et al., 2010), the participation of antioxidant defenses in the resistance to antibiotics needs to be clarified.

The investigation of the physiological relation between oxidative stress and antibiotic resistance was first stimulated by genetic studies. Various authors observed that bacterial antioxidants are present in both sensitive and resistant strains, but in the latter, regulons of defenses against the oxidative stress, such as soxS, are enhanced. It was also observed that the superoxide SoxRS regulon confers increased resistance to chemically unrelated antibiotics (Miller & Sulavik, 1996). A proportion of the high-level fluoroquinolone-resistant Escherichia coli clinical isolates that display the Mar phenotype have been shown to constitutively increase the expression of soxS genes (Maneewannakul & Levy, 1996; Oethinger et al., 1998). In subsequent investigations it was shown that exposure to oxidative stress induced both the soxS operon and the mar operon of multi-antibiotic resistance (Wick & Egli, 2004).

In this work, we obtained resistant strains of Proteus mirabilis by induction with repeated cultures in a sub-MIC concentration of CIP, with the purpose of producing CIP-resistant variants (CRVs) without mutations in gyr A or gyr B of DNA gyrase and without mutation in par C of topoisomerase IV. We then explored the mechanisms of resistance to CIP by efflux/influx mechanisms, as well as by antioxidant defenses by ferric reducing antioxidant power (FRAP) assay, together with oxidation of lipids and proteins, to detect whether CRVs could have changes in the oxidative stress pathways. The present work added new data about CIP accumulation in P. mirabilis, and also about lipid peroxidation, oxidation of proteins to carbonyls and degradation to advanced oxidation protein products (AOPP). The reduction of susceptibility to CIP by means of glutathione (GSH) or ascorbic acid (AA), and the relation of resistance with the level of oxidation of macromolecules, are aspects that have not been investigated previously in this bacterium.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

Proteus mirabilis isolates S1, S2 and R3 were collected from three different patients with urinary infections who had been treated at Hospital Tránsito Cáceres de Allende, Córdoba, Argentina. Isolates S1 and S2 were sensitive to CIP with a minimum inhibitory concentration (MIC) of 0.125 and 2 μg mL−1, respectively, whereas isolate R3 was resistant to this antibiotic (MIC > 128 μg mL−1).

Ciprofloxacin-resistant variants

CRVs 1X, 1Y, 2X and 2Y derived from the sensitive parental isolates S1 and S2, were obtained in vitro by repeated cultures in a sub-MIC concentration of CIP, and the last passage was plated in Mueller–Hinton agar plates containing 4 μg mL−1 of CIP according to Aiassa et al. (2010). The MIC for these CRVs was determined after propagation in CIP-free medium for 20 days. Strains which maintained their values of MIC were considered to be CRVs.

Oxidative stress cross-resistance to CIP and telluride in CRVs

Oxidative stress was investigated by Nitro Blue Tetrazolium (NBT) assay; 0.4 mL of bacteria suspension (OD600 nm 1.0) in sodium phosphate buffer (PBS, pH 7.0) was incubated with 64 μg mL−1 telluride or 4 μg mL−1 CIP, and 0.5 mL of 1 mg mL−1 NBT for 30 min at 37 °C. After the addition of 0.1 mL of 0.1 M HCl, the tubes were centrifuged and the sediments of bacteria were treated with 0.4 mL of dimethylsulfoxide (DMSO) to extract the reduced NBT; finally 0.8 mL of phosphate-buffered saline (PBS) was added and the optical density was determined at 575 nm.

Oxidative stress resistance in terms of survivability was studied by determining the number of colony-forming units (CFU) mL−1, with living bacteria being determined by colony counts in cultures of cystine lactose electrolyte-deficient containing 200 μg mL−1 telluride at 37 °C compared to plates without telluride.

PCR amplification and sequence analysis of gyrA,gyrB and parC genes

Genomic DNA was purified with Wizard® Genomic DNA Purification Kit (Promega), according to the technical manual. Sequences of gyrA, gyr B and parC of P. mirabilis ATCC 29906 strain were used as referential CIP-sensitive bacteria, and the P. mirabilis clinical CIP-resistant isolate R3 was used as a positive control. The quinolone resistance-determining region (QRDR) domains of the gyrA, gyrB and parC genes were amplified according to a method described previously by Weigel et al. (2002) using the following primer sets:

gyrA for 5′CCAGATGT(A/C/T)CG(A/C/T)GATGG

gyrA rev 5′ACGAAATCAAC(G/C)GT(C/T)TCTTTTTC

gyrB for 5′TGA(C/T)GATGC(G/C/A)CG(T/C)GAAGG

gyrB rev 5′CGTACG(A/G)ATGTG(C/A)GA(G/A)CC

gyrB sec 5′CCACATCCGTCATGATAA

parC for 5′TTGCC(A/T)TTTAT(C/T)GG(G/T)GATGG

parC rev 5′ CGCGC(A/T)GGCAGCATTTT(A/T)GG

PCR amplifications were performed under the following conditions: 5 min at 95 °C, 35 cycles of 45 s at 95 °C, 20 s at 47.7 °C (for gyrA), 54 °C (for gyrB) or 52 °C (for parC), 30 s at 72 °C, and a final extension of 7 min at 72 °C. The PCR products were cleaned with a Gel purification kit (Qiagen) and directly sequenced (Macrogene Corp.). With the exception of the gyrB reverse sequence, degenerate PCR primers were also used as sequencing primers.

Nucleotide sequence accession numbers

The partial DNA sequences of gyrA, gyrB and parC from P. mirabilis ATCC 29906 were submitted to GenBank and assigned the accession numbers AF397169 (gyrA), AF503506 (gyrB) and AF363611 (parC).

Accumulation of CIP

CIP uptake was assayed by the method of Giraud et al. (1999) with some modifications. Bacteria suspended in PBS to OD600 nm ~1.2 were equilibrated for 10 min at 37 °C. After the addition of CIP to a final concentration of 10 μg mL−1, 0.5 mL samples were removed at different time intervals. Five minutes after this addition, the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) 100 μM was added to the reaction mixture. The samples were diluted in 1 mL of ice-cold PBS and centrifuged for 5 min at 5600 g. The pellet was washed once with 1 mL of ice-cold PBS and resuspended in 1 mL of 0.1 M glycine hydrochloride (pH 3.0) for 1 h at room temperature. The samples were then centrifuged at 5600 g for 10 min and the fluorescence of the supernatant was measured with a YASCO FP-777 spectrofluorimeter at excitation and emission wavelengths of 278.5 and 448.5 nm, respectively. The concentration of CIP in the supernatant was calculated by comparison with a standard curve for CIP in 0.1 M glycine hydrochloride. The results were expressed as nanograms of CIP incorporated mg−1 of protein.

The ferric reducing antioxidant power (FRAP) method

The FRAP assay (Benzie & Strain, 1999) was adapted to measure the antioxidant capacity of P. mirabilis. A volume of 100 μL of bacterial suspensions (OD600 nm ~1) was incubated with 125 μL of 3.1 mg mL−1 of 2,4,6-tripyridyl-1,3,5-triazine (TPTZ) in 40 mM HCl, 125 μL of FeCl3·6H2O 5.4 mg mL−1 and 1.25 mL of 300 mM acetate buffer (pH 3.6). Absorbances were determined at 593 nm and expressed as μM of FeSO4 mg−1 protein.

Protein quantification

The concentration of proteins in bacterial suspension was determined by Folin–Ciocalteau assay (Stauffer, 1975).

Lipid peroxidation

Bacterial suspensions of 1 mL were incubated with CIP or using PBS (control). The incubations were stopped at 2 h with 1 mL of TCA 35% (p/v) in the absence of light. After 20 min, 1 mL of 0.5% (p/v) thiobarbituric acid was added and the samples were heated to 80 °C for 30 min. An ice bath was used to cool the samples, which were centrifuged at 1500 g and the absorbance of the supernatant was determined at 535 nm. A calibration curve of malondialdehyde (MDA) solutions was applied to estimate lipid oxidation. MDA levels were expressed as nmol MDA mg−1 protein.

Carbonyl residues

Bacterial suspensions of 3 mL were incubated with 0.5 mL of CIP or PBS (control) for 2 h. After that, 1 mL of the samples was treated with 1 mL of 0.1% 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl for 1 h. The proteins were precipitated in 5% trichloroacetic acid (TCA), centrifuged 20 min at 10 000 g, and the supernatant discarded. Samples were extracted three times with 1 mL ethanol/ethylacetate (1 : 1, v/v) to remove any remaining residual of DNPH. The precipitate was dissolved in 6 M guanidine hydrochloride solution in PBS and incubated for 30 min at 37 °C. The insoluble debris was removed by centrifugation, and the absorbance was measured at 364.5 nm. Results were expressed as mm of residues of carbonyl mg−1 protein and calculated using a molar extinction coefficient of 22 mol−1 cm−1 for aliphatic hydrazones (Witko-Sarsat et al., 1998).

Advanced oxidation protein products

Proteus mirabilis suspensions were prepared from 18-h cultures at 35 °C in Trypticase Soya Broth (TSB). Aliquots of 5 mL of the sample were incubated with 0.5 mL of CIP or with PBS (control) for 2 h. Then, 1 mL of the samples or 1 mL of 50 μM chloramine T (standard) was treated with 50 μL of 1.16 M KI and 0.1 mL of acetic acid. The absorbance at 340 nm was applied to estimate the AOPP concentrations, which were expressed as μM L−1 of chloramine-T equivalents (Witko-Sarsat et al., 1998).

Evaluation of the effect of addition of exogenous antioxidants on the sensitivity of P. mirabilis to CIP by MIC determination

CIP MIC was determined by the broth dilution method as outlined by the Clinical and Laboratory Standards Institute (CLSI), in the presence or absence of the antioxidants 10 mM GSH or 10 mM ascorbic acid in the culture medium.

Statistical determinations

Statistical analysis was performed using anova, with < 0.05 taken as statistically significant. The experiments were repeated at least three times, and the means and standard deviations were calculated.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Susceptibility of CRVs to CIP

Four CRVs (1X, 1Y, 2X and 2Y) with attained resistance (MICs of 16, 4, 8 and 4 μg mL−1 respectively) were obtained from two sensible clinical P. mirabilis S1 and S2, by repeated cultures with a sub-inhibitory concentration of CIP. The resistance frequency provoked by a sub-MIC concentration of CIP was 10−6 and this resistant population was evaluated and compared with the respective parental sensible strains.

Oxidative stress cross-resistance to CIP and telluride in CRVs

The NBT assay showed a smaller increase of ROS in CRVs with CIP than in parental strains (Fig. 1a). Moreover, oxidative stress cross-resistance to telluride was induced by successive subcultures in CIP (Fig. 1b), as 1X, 1Y, 2X and 2Y exhibited a three- to eight-fold decrease in ROS stimuli with enhanced survivability in the presence of telluride. Also, CRVs exhibited a smaller reduction of CFU mL−1 in the presence of this oxidant agent (8-, 11.8-, 1.5- and 1.1-fold decrease in 1X, 1Y, 2X and 2Y, respectively) compared with sensitive parental strains (57.7-fold decrease in S1 and 25.7-fold decrease in S2). In addition, the MIC to telluride was still increased eight-fold in CRVs (data not shown).

image

Figure 1. Increase of ROS by: (a) 4 μg mL−1CIP and (b) 64 μg mL−1 telluride, during NBT reaction in parental strains S1 and S2 sensitive to CIP, compared with the percentage of ROS increase in the CRVs 1X, 1Y, 2X and 2Y resistant to CIP.

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PCR amplification and sequencing of QRDRs of gyrA, gyrB and parC genes

PCR amplification and direct sequencing of gyrA, gyrB and parC of P. mirabilis showed no mutations in any CRVs, thus demonstrating sequences unaltered from those occurring in the parental isolates and the P. mirabilis ATCC 29906 strain in the QRDR regions (Table 1). In contrast, mutations in GyrA, GyrB and ParC appeared in the codons for S83, E466 and S80-E84, respectively, in the CIP-resistant clinical isolate R3.

Table 1. QRDR amino acid mutations of GyrA,GyrB and ParC, and CIP susceptibility in Proteus mirabilis parental isolates and CRVs
Amino acid mutations at indicated position in QRDRa
PositionGyrAGyrBParCMIC μg mL−1
Strains8387464466788084 
  1. a

    Amino acid mutations were identified by comparing them with the sequences of Proteus mirabilis ATCC 29906.

ATCC 29906SESEGSE0.125
S10.125
1X16
1Y4
S22
2X8
2Y4
R3REIK> 128

Accumulation of CIP

The possible involvement of an active efflux mechanism in CIP resistance of P. mirabilis CRVs was evaluated (Fig. 2a,b). Previous antibiotic accumulation at the addition of CCCP appeared to be less in the CRVs than in sensitive parent strains. However, the addition of CCCP induced a significant increase in accumulated CIP in 1X; this suggested that an active efflux process limits the accumulation of CIP by the cells in a much more efficient way in 1X than in the parent strain. In addition, sensitive strain S2 and the CRVs 2X and 2Y did not differ significantly in terms of accumulation of CIP with CCCP.

image

Figure 2. (a) Accumulation of ciprofloxacin (CIP) by isolate S1 (■), CRV 1X (●) and 1Y (▲). (b) Accumulation of CIP by strain S2 (□), CRV 2X (○) and 2Y (∆) determined by a classical fluorimetric method. CCCP (100 μM) was added at the time indicated by the arrow. Results are expressed as ng CIP mg−1 of protein.

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Ferrous reduction antioxidant potency

The antioxidant capacity of P. mirabilis determined by FRAP, was significantly higher in CRVs showing greater MICs (1X and 2X), revealing a close correlation between CIP resistance and FRAP (Fig. 3).

image

Figure 3. Antioxidant power determined by FRAP assay in Proteus mirabilis isolates S1, S2 and CRVs. Results are expressed as μM of FeSO4 mg−1 of protein. *< 0.05 vs. parental control strains S1 and S2.

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Lipid and protein oxidation

Lipid oxidation to MDA increased with CIP in both sensitive parental strains and decreased in CRVs (Fig. 4a). Additionally, in absence of antibiotic, MDA was higher in S1, the strain with a lower MIC. Moreover, the oxidization of proteins to carbonyls and AOPP in the presence of CIP increased more in S1 and S2 than in the CRVs 1X, 1Y, 2X and 2Y (Fig. 4b,c).

image

Figure 4. (a) Lipid oxidation to MDA, (b) oxidation of proteins to carbonyls and (c) degradation of oxidized proteins to AOPP in Proteus mirabilis parental and CRV strains without CIP (□) and with 4 μg mL−1CIP for 2 h (image). *< 0.05.

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Effects of antioxidants on CIP sensitivity of P. mirabilis

Table 2 shows that the incorporation of GSH or AA to culture media reduced the susceptibility of all P. mirabilis CRVs to CIP, as there was an evident increase of MIC in isolates S1, S2 and in all the CRVs after incubation with both antioxidants.

Table 2. Effects of antioxidants GSH and AA on the MIC of the CIP determined by the broth dilution method
StrainMIC (μg mL−1)
CIPCIP + GSH 10 mMCIP + AA 10 mM
S10.12510.5
1X166432
1Y41616
S2244
2X83216
2Y41616

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The mechanisms involved in the resistance to CIP can be best interpreted by considering the different aspects that may be implicated in the antibacterial mechanism of action. The molecular mechanisms underlying resistance to fluoroquinolones in P. mirabilis include mutations in the target enzymes DNA gyrase and topoisomerase IV (Ser-83 in GyrA, Ser-464 in GyrB and Ser-80 in ParC) and over-expression of endogenous multidrug efflux pumps (Weigel et al., 2002; Saito et al., 2006). Therefore, the results obtained, indicated that MICs of up to 16 μg mL−1 were displayed in the P. mirabilis CRVs, without typical mutations in DNA gyrase or topoisomerase IV genes. In addition, accumulation studies with CCCP indicated that the influx/efflux mechanisms could contribute to the increase in the resistance of the CRVs to CIP only in 1X.

In this work, an increase in FRAP was proposed as another factor involved in resistance. Previous results of elevated superoxide dismutase and GSH in CRVs (Aiassa et al., 2010) led to the investigation of the antioxidant capacity, as FRAP involves the combined or total reducing power of electron-donating antioxidants (Benzie & Strain, 1996; Litescu et al., 2011). FRAP is also an assay employed in different cellular extracts to measure the antioxidant capacity of different compounds, including antioxidant peptides (Nilsson et al., 2005; Di Bernardini et al., 2011), alpha-lipoic acid and vitamins that can be found in bacteria (Schlesier et al., 2002; Piechota & Goraca, 2009), as validated by several studies (Huang et al., 2005; Thaipong et al., 2006; Magalhães et al., 2008). These antecede even more the investigation of CIP action on biofilm (Aiassa et al., 2007), which indicated that enzymatic and non-enzymatic antioxidant systems may have a role in the defensive reaction against the oxidative stress caused by CIP in P. mirabilis.

Although the pathways that are derived in the end products of oxidation were characterized for other substances (Krisko & Radman, 2010; Chen et al., 2011), the biomarkers of oxidative pathways of lipid and proteins, such as MDA, carbonyls and AOPP, were not investigated in investigations of the action of CIP in P. mirabilis. We therefore studied these products of oxidation and observed that sensitive strains suffer more oxidation of these macromolecules compared with resistant bacteria.

In agreement with the present work, mutants with constitutive expression of antibiotic resistance genes (marA), over-expressed genes of resistance to oxidative stress (soxS) (Kern et al., 2000). In the same way, a sub-inhibitory concentration of CIP resulted in strains of Staphylococcus aureus in which no mutations were found in the QRDR of gyrA or gyrB (Tattevin et al., 2009). Consequently, the results obtained in this work reinforce physiologically these genetics investigations, suggesting that antioxidant defense might be another factor in the resistance to CIP.

Finally, and in order to try to investigate further the idea that antioxidant defenses may constitute an additional antibiotic resistance mechanism, complementary assays with exogenous antioxidants GSH and AA were performed. The results indicate that when acting as antioxidants, GSH and AA might interfere at any step of the oxidative action of CIP, which could be associated to resistance to this antibiotic.

Summing up, the present study suggests that the antioxidant defenses can contribute to the other factors that regulate the susceptibility to CIP, such as influx/efflux mechanisms observed only in strain 1X. To our knowledge, this is the first study that has analyzed FRAP, MDA, carbonyls and AOPP in relation to CIP resistance of P. mirabilis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This investigation was supported by PICTO 36163 (FONCYT), SECYT-UNC, Agencia de Promoción Científica y Tecnológica, Agencia Córdoba de Promoción Científica y Técnica, and Secretaría de Ciencia y Técnica from Universidad Nacional de Córdoba. The authors thank CONICET for support of Virginia Aiassa as a postgraduate fellow. We also thank Dr Paul Hobson, a native English speaker, for revision of the manuscript.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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