Microbiol Immunol 2014; 58: 96–102 doi: 10.1111/1348-0421.12124
ORIGINAL ARTICLE
Effect of moxifloxacin on survival, lipid peroxidation and inflammation in immunosuppressed rats with soft tissue infection caused by Stenotrophomonas maltophilia Orestis Ioannidis1, Basilios Papaziogas1, Panagiotis Tsiaousis1, George Paraskevas2, Evangelos J. Giamarellos-Bourboulis3 and Ioannis Koutelidakis1 Second Surgical Department, School of Medicine, “G. Gennimatas” General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Department of Anatomy, Medical School, Aristotle University of Thessaloniki, Thessaloniki and 3Fourth Department of Internal Medicine, University of Athens Medical School, Athens, Greece
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ABSTRACT In order to investigate the effect of moxifloxacin on survival, lipid peroxidation and inflammation in immunosuppressed rats with soft tissue infection caused by Stenotrophomonas maltophilia, 144 white male Wistar rats were randomized into six groups: Groups A and B received saline or moxifloxacin once per day, respectively; Groups C and D received saline or moxifloxacin twice per day, respectively, and Groups E and F received saline or moxifloxacin three times per day, respectively. Blood samples were taken at 6 and 30 hr after administration of S. maltophilia. Malonodialdehyde (MDA), WBC counts, bacterial tissue overgrowth, serum concentrations of moxifloxacin and survival were assessed. Survival analysis proved that treatment with moxifloxacin every 8 hr was accompanied by longer survival than occurred in any other group. Tissue cultures 30 hr after bacterial challenge showed considerably less bacterial overgrowth in the spleens and lungs of moxifloxacin-treated than in salinetreated animals, but not in their livers. At 6 hr there were no statistically significant differences between groups. However, at 30 hr, MDA concentrations were significantly greater (P ¼ 0.044) and WBC counts significantly lower (P ¼ 0.026) in group D than in group C. No statistically significant variations were observed between the other groups. Moxifloxacin possibly stimulates lipid peroxidation and enhances phagocytosis, as indicated by MDA production and survival prolongation, without being toxic, as indicated by WBC count. Therefore, under the appropriate conditions, moxifloxacin has a place in treatment of infections in immunosuppressed patients and of infections caused by S. maltophilia. Key words
immunosuppression, malonodialdehyde, moxifloxacin.
The number of immunosuppressed patients is constantly increasing as a result of increased frequency of organ transplantation, increased use of chemotherapy to treat malignancies and of immunomodulative drugs to treat autoimmune diseases and increased incidence of
diseases that cause immunodeficiency. Infections in these patients pose significant clinical problems regarding both diagnosis and treatment. Soft tissue infections, which may be nosocomial or community infections, present with higher frequency
Correspondence Orestis Ioannidis, Second Surgical Department, School of Medicine, “G. Gennimatas” General Hospital, Aristotle University of Thessaloniki, Alexandrou Mihailidi 13, 54640 Thessaloniki, Greece. Tel: þ30 2310845470; fax: þ30 2310551301; email: [email protected] Received 12 June 2013; revised 1 December 2013; accepted 17 December 2013. List of Abbreviations: ER, endoplasmic reticulum; JNK, c-Jun N-terminal kinases; MAP, mitogen-activated protein; MDA, malonodialdehyde; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; THP-1, human acute monocytic leukemia cell line.
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in immunocompromised patients. In these patients there is also an increased rate of isolation of uncommon microorganisms, the treatment of which is difficult because of their resistance to multiple antibiotics. Stenotrophomonas maltophilia is among these infrequently isolated pathogens; it is a non-fermentative, oxidase-negative, motile, aerobic, gram-negative bacillus (1, 2). It has an increasingly important role as a hospital-acquired pathogen associated with significant case/fatality ratios in certain patient groups, particularly severely debilitated or immunocompromised patients (1, 2). S. maltophilia has been mainly associated with the following clinical syndromes: respiratory tract infection, bacteremia, endocarditis, soft tissue infections and nosocomial infections. Predisposing risk factors associated with S. maltophilia infection are prior antibiotic therapy, central venous catheterization, neutropenia, cytotoxic chemotherapy, malignancy, prolonged hospitalization and admission to an intensive care unit. Treatment of S. maltophilia infections is complicated because the bacterium is resistant to many broadspectrum anti-microbial agents (1). S. maltophilia shows inherent resistance to virtually all classes of beta-lactams, including extended-spectrum penicillins, third-generation cephalosporins and carbapenems. Furthermore, it is resistant to aminoglycosides and shows variable susceptibility to fluoroquinolones. The antibiotic agent of choice has been considered to be trimethoprim– sulfamethoxazole; however, lately strains of the bacterium resistant to this combination have emerged (1–3). Thus, there is an imperative need to assess the efficacy of other antibiotics. Many studies to test the susceptibility of S. maltophilia to various anti-microbial agents have been performed (4–15). One class of antibiotics that has received increased attention for its potential usage in the treatment of S. maltophilia infections is fluoroquinolones (4–15). Of the quinolones studied, moxifloxacin seems to be one of the most potent (4–12). Moxifloxacin is a third-generation quinolone, active both against gram-positive and gram-negative microorganisms and also against anaerobic bacteria (16, 17). The susceptibility rates of S. maltophilia to moxifloxacin vary between 80% and 96.4% (4–12). Another study showed activity of moxifloxacin against isolates resistant to trimethoprim– sulfamethoxazole (10). Although these studies have tested the in vitro activity of moxifloxacin, in vivo evidence is lacking. The aim of this study was to investigate the effect of moxifloxacin on the survival of immunocompromised rats with soft tissue infection caused by S. maltophilia. © 2013 The Societies and Wiley Publishing Asia Pty Ltd
MATERIALS AND METHODS All experiments were performed in the Laboratory of Surgical Research of the Second Surgical Clinic of the Aristotle University of Thessaloniki after approval by the Prefecture of Thessaloniki according to law 86/609 EEC of the European Union. In all, 144 male white Wistar rats aged 18–22 weeks and weighing 300–400 g were studied. The animals were kept in stainless steel cages of five animals per cage, in a temperature range of 18–22 °C, with relative humidity 55–65% and a light:dark cycle of 12:12 hr. The rats had free access to food and water (ad libitum). The rats were rendered immunocompromised with cyclophosphamide. Under general anesthesia using ether, neutropenia was induced by intraperitoneal administration of cyclophosphamide 100 mg/kg on the first day and 150 mg/kg on the third day. On the fifth day, 1 108 CFU/kg of S. maltophilia was administered i.m. into the right thigh under general anesthesia with ether. The organisms had been isolated from the bloodstream of a patient with severe sepsis. The minimum inhibitory concentration of moxifloxacin against this isolate as assessed by a microdilution technique was 0.03 mg/mL. The rats were randomized into six groups of 24 mice each. In all groups, therapy was given i.v. into the tail vein under aseptic conditions starting 1 hr after bacterial challenge. The groups were treated as follows: Group A received 1 mL of saline once a day for 7 days; Group B received 12 mg/kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG, Athens, Greece) once a day for 7 days; Group C received 1 mL of saline every 12 hr each day for 7 days; Group D received 12 mg/kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG) every 12 hr for 7 days; Group E received 1 mL of saline every 8 hr for 7 days; and Group F received 12 mg/ kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG) every 8 hr for 7 days. Twelve rats from each group were used to investigate survival. After administration of S. maltophilia, the rats were monitored every 12 hr for 7 days. The other 12 rats in each group were used to investigate changes related to lipid peroxidation and inflammation by taking blood samples of 5 mL each from the inferior vena cava 6 hr (six rats per group) and 30 hr (six rats per group) after administration of S. maltophilia. MDA was used as a marker of lipid peroxidation and WBC counts as a marker of inflammation. Of the 5 mL of blood that was collected, 2 mL was placed in EDTA tubes (Greiner Bio-One, Monroe, NC, USA) and used for immediate WBC counts using a Genesis auto counter (Coulter Electronics, Miami, FL, USA). The remaining 3 mL was placed in toxin-free tubes 97
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(Vacutainer; Becton Dickinson, Cockeysville, MD, USA), centrifuged and the supernatant serum placed in the same tubes and frozen at 70 °C until measurement of MDA. MDA measurement was performed by the thiobarbituric acid method and analyzed by HPLC (Agilent Technologies, New York, NY, USA). More precisely, 100 mL of each sample was added to 900 mL of trichloroacetic acid 20% (Sigma, St. Louis, MO, USA) and stirred for 30 sec with Vortex. Next, the solution was centrifuged at 13,000 g for 10 min at 4 °C, 0.5 mL of supernatant added to 2 mL of thiobarbituric acid 0.2% (Sigma) and the resultant mixture incubated at 90 °C for 60 min. Following centrifugation at 3500 g for 10 min, 10 mL of the supernatant was injected into an HPLC system. The chromatographic column used was 150 mm long, of internal diameter 4.6 mm and with pores of 5 mm (Zorbax Eclipse XDB-C18; Agilent Technologies). The motile phase was a mixture of 60% phosphate potassium buffer 50 mM and 40% methanol. The flow rate was 1 mL/min, temperature 37 °C and pressure 100– 105 mm Hg. Using a fluorescence detector, the emission of every sample at 553 nm was measured after stimulation of the sample at 515 nm. Determination of MDA concentrations was made by comparing the emission values with a titration curve of known concentrations of 1,1,3,3-tetramethoxypropane (Merck, Darmstadt, Germany). When the animals were killed 30 hr after bacterial challenge, segments of the liver, spleen and lower lobe of the right lung were excised under aseptic conditions with sterile blades and collected into separate sterile containers. The segments were weighed and homogenized with a tissue grinder in a volume of 1 mL of sodium chloride 0.9%. They were then serially diluted 1:10 four times with sodium chloride 0.9%. A volume of 0.1 mL of each dilution was plated onto MacConkey agar and incubated for 48 hr at 35 °C. At the end of incubation, the number of colonies of each dilution was counted and multiplied by the appropriate dilution factor. Colony counts are expressed as the log10 of CFU/g of tissue. The lower limit of detection was 10 CFU/g. In order to assess the serum concentrations of moxifloxacin achieved with the administered doses, a 12 mg/kg dose was administered to 12 healthy rats. Six of these rats were killed 15 and six more 30 min postinfusion and blood collected from their inferior vena cavae. The blood was centrifuged and concentrations of moxifloxacin measured in serum after analysis by HPLC as described previously (18). All measurements are expressed as mean values 1 SD. To compare results, after checking the normality, the independent samples t-test we used for variables with normal distribution and the non-parametric Mann– 98
Whitney test for variables that did not have normal distribution. Survival was assessed by the Kaplan–Meier test and comparison of survival by the log-rank test. P values below 0.05 were considered statistically significant. Statistical analysis was made using the SPSS (Statistical Package for Social Sciences, version 14.0; Chicago, IL, USA).
RESULTS As shown in Figure 1, treatment with moxifloxacin every 8 hr (Group F) was accompanied by longer survival than in any other group. Tissue cultures 30 hr after bacterial challenge showed considerably less bacterial growth in the spleens and lungs of moxifloxacin-treated animals than in the other groups, but not in their livers (Fig. 2). Mean SD concentrations of moxifloxacin 15 min postinfusion were 2.76 0.14 mg/ and 1.95 1.19 mg/mL 30 min post-infusion. The reduced bacterial growth after treatment with moxifloxacin did not involve all organs, indicating the possibility that part of moxifloxacin’s activity is mediated by modulation of inflammation. This possibility was assessed by measuring concentrations of circulating MDA and WBC counts. At 6 hr after administration of S. maltophilia, there were no statistically significant differences in absolute WBC counts between the groups receiving moxifloxacin
Fig. 1. Survival of groups of rats subjected to the following treatments: Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Statistical comparisons: Log-rankA versus B ¼ 1.118, P ¼ 0.277; log-rankC versus D ¼ 5.295, P ¼ 0.021; log-rankE versus F ¼ 24.832, P < 0.0001; log-rankB versus F ¼ 7.501, P < 0.0001; log-rankD versus F ¼ 4.023, P ¼ 0.045.
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Moxifloxacin & S. maltophilia infection
Fig. 2. Tissue bacterial overgrowth. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding saline-treated group.
and those receiving saline (Fig. 3). However, at 30 hr the animals that had received two and three doses of moxifloxacin had lower WBCs than the animals that had received saline; this difference was significant only between Groups C and D, which had received two doses. Similar differences were found for circulating MDA (Fig. 4).
DISCUSSION Our finding suggest that survival benefit after experimental soft tissue infection by S. maltophilia in immunocompromised rats is correlated with the frequency of administration. This could be caused by a direct bactericidal action of moxifloxacin against S. maltophilia but could also be attributable, at least in part, to an immunomodulatory effect of moxifloxacin. Despite the fact that moxifloxacin lacks direct antifungal activity, Shalit et al. have reported it has an immunomodulatory effect in a model of immunocompromised mice bronchopneumonia caused by Candida albicans (19). © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Shalit et al. were the first to study the immunomodulatory effects of moxifloxacin (20). They concluded that administration of moxifloxacin to mice with cyclophosphamide-induced leukopenia reverses some of cyclophosphamide’s immunosuppressive effects, as indicated by higher WBC counts, greater colony-stimulating activity and increased granulocyte macrophage colonystimulating factor in treated animals compared with controls. Araujo et al. reported that moxifloxacin significantly decreased secretion of TNF-a and IL-1a by human monocytes stimulated with LPS (21). In another study, Choi et al. found that moxifloxacin decreases production of TNF-a and of IL-6 by human peripheral blood mononuclear cells after stimulation with LPS, lipoteichoic acid and heat-killed bacteria (22). In addition, Weiss et al. have shown that moxifloxacin has an anti-inflammatory effect on activated human monocytic cells, specifically decreasing production of TNF-a, IL-8 and IL-1b and inhibiting activation of NFkΒ, ER kinase and JNK (23). Werber et al. have also reported not only inhibition of activation of NF-kΒ, ER kinase and JNKs, but also of mitogen-activated protein 99
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leukemia cell line) monocytes (27). They observed an increase in release of nitric oxide and hydrogen peroxide within the first hour followed by a decrease in lipid peroxidation after 4 hr. Moxifloxacin seems to initially activate monocytes in order to kill bacteria by releasing ROS and lysosomal hydrolytic enzymes. At a later stage, reversal of the effects of moxifloxacin on free radical generation and hydrolytic enzymes reportedly occurs, resulting in suppression of lipid peroxidation (27). In addition, Attia observed significantly greater serum MDA production in mice that received lomefloxacin than in controls (28). Also, Rampal et al. have shown that ofloxacin increases lipid peroxidation in rabbits (29). In addition, various studies have shown that phototoxicity of quinolones is caused by increased lipid peroxidation, possibly due to increased production of free radicals (30– 33). In the present study, the increased MDA concentrations are a result of increased lipid peroxidation, possibly caused by moxifloxacin-induced direct increase
Fig. 3. White blood cell count (per mm3) at 6 and 30âhr in all groups. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding saline-treated group.
kinase after treating human respiratory epithelial cells with moxifloxacin in vitro (24). Furthermore, Williams et al. found that moxifloxacin decreases interferon, INFg and IL-4 expression on Th1 and Th2 cells in a dosedependent manner without altering Th1/Th2 ratios (25). Finally, Blau et al. have shown that moxifloxacin inhibits activation of IL-8, IL-6, NF-kΒ, ER kinase and JNK in cystic fibrosis epithelial cells (26). In the present study, moxifloxacin administration increased MDA production in the group that received two doses per day. MDA is a side product of enzymatic lipid peroxidation process and a final product of nonenzymatic lipid peroxidation. Infection with these bacteria initiates non-enzymatic lipid peroxidation; for such initiation, the presence of free radicals is necessary. Hall et al. studied the effects of moxifloxacin on free radical generation, cytokine immunomodulatory mediators and hydrolytic enzyme activities in zymogen Astimulated human THP-1 (human acute monocytic 100
Fig. 4. MDA values (in µM) at 6 and 30âhr in all groups. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding salinetreated group.
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Moxifloxacin & S. maltophilia infection
in production of free radicals. Also, moxifloxacin has been found to initiate phagocytosis through stimulation of the membrane to release cyclic adenosine monophosphate and increase activity of protein kinase C (27). Moxifloxacin activates monocytes to kill microorganisms by bactericidal or ROS mechanisms of phagocytosis of bacteria (27). The presence of free radicals increases the cidal effect provided the free radicals are not cleared by the cells’ protective mechanisms (27). We therefore suggest that the increased MDA concentrations are a result of increased lipid peroxidation by free radicals, probably linked with increased phagocytosis. To the best of our knowledge, the present study is the first published report that has assessed certain markers, such as MDA for lipid peroxidation and WBC counts for inflammation, to evaluates the effect of moxifloxacin in the lipid peroxidation process and in inflammation of immunocompromised rats with soft tissue infection by S. maltophilia. Also, we attempted to correlate these markers with the general effect of moxifloxacin on the progress of the infection by measuring survival. In conclusion, administration of two doses of moxifloxacin increases lipid peroxidation, as indicated by increased MDA concentrations. It may also enhance phagocytosis in immunosuppressed rats, though without associated toxic effects, as shown by decreased numbers of WBCs, and increased survival. Thus, moxifloxacin could become a valuable drug for treating both infection in immunosuppressed patients and infections caused by S. maltophilia. However, more studies are needed to evaluate the immunomodulatory effects of moxifloxacin in clinical settings and its potential value for treating immunosuppressed patients and f S. maltophilia infections.
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DISCLOSURE The authors declare that they have no conflict of interest.
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ORIGINAL ARTICLE
Effect of moxifloxacin on survival, lipid peroxidation and inflammation in immunosuppressed rats with soft tissue infection caused by Stenotrophomonas maltophilia Orestis Ioannidis1, Basilios Papaziogas1, Panagiotis Tsiaousis1, George Paraskevas2, Evangelos J. Giamarellos-Bourboulis3 and Ioannis Koutelidakis1 Second Surgical Department, School of Medicine, “G. Gennimatas” General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Department of Anatomy, Medical School, Aristotle University of Thessaloniki, Thessaloniki and 3Fourth Department of Internal Medicine, University of Athens Medical School, Athens, Greece
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ABSTRACT In order to investigate the effect of moxifloxacin on survival, lipid peroxidation and inflammation in immunosuppressed rats with soft tissue infection caused by Stenotrophomonas maltophilia, 144 white male Wistar rats were randomized into six groups: Groups A and B received saline or moxifloxacin once per day, respectively; Groups C and D received saline or moxifloxacin twice per day, respectively, and Groups E and F received saline or moxifloxacin three times per day, respectively. Blood samples were taken at 6 and 30 hr after administration of S. maltophilia. Malonodialdehyde (MDA), WBC counts, bacterial tissue overgrowth, serum concentrations of moxifloxacin and survival were assessed. Survival analysis proved that treatment with moxifloxacin every 8 hr was accompanied by longer survival than occurred in any other group. Tissue cultures 30 hr after bacterial challenge showed considerably less bacterial overgrowth in the spleens and lungs of moxifloxacin-treated than in salinetreated animals, but not in their livers. At 6 hr there were no statistically significant differences between groups. However, at 30 hr, MDA concentrations were significantly greater (P ¼ 0.044) and WBC counts significantly lower (P ¼ 0.026) in group D than in group C. No statistically significant variations were observed between the other groups. Moxifloxacin possibly stimulates lipid peroxidation and enhances phagocytosis, as indicated by MDA production and survival prolongation, without being toxic, as indicated by WBC count. Therefore, under the appropriate conditions, moxifloxacin has a place in treatment of infections in immunosuppressed patients and of infections caused by S. maltophilia. Key words
immunosuppression, malonodialdehyde, moxifloxacin.
The number of immunosuppressed patients is constantly increasing as a result of increased frequency of organ transplantation, increased use of chemotherapy to treat malignancies and of immunomodulative drugs to treat autoimmune diseases and increased incidence of
diseases that cause immunodeficiency. Infections in these patients pose significant clinical problems regarding both diagnosis and treatment. Soft tissue infections, which may be nosocomial or community infections, present with higher frequency
Correspondence Orestis Ioannidis, Second Surgical Department, School of Medicine, “G. Gennimatas” General Hospital, Aristotle University of Thessaloniki, Alexandrou Mihailidi 13, 54640 Thessaloniki, Greece. Tel: þ30 2310845470; fax: þ30 2310551301; email: [email protected] Received 12 June 2013; revised 1 December 2013; accepted 17 December 2013. List of Abbreviations: ER, endoplasmic reticulum; JNK, c-Jun N-terminal kinases; MAP, mitogen-activated protein; MDA, malonodialdehyde; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, reactive oxygen species; THP-1, human acute monocytic leukemia cell line.
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Moxifloxacin & S. maltophilia infection
in immunocompromised patients. In these patients there is also an increased rate of isolation of uncommon microorganisms, the treatment of which is difficult because of their resistance to multiple antibiotics. Stenotrophomonas maltophilia is among these infrequently isolated pathogens; it is a non-fermentative, oxidase-negative, motile, aerobic, gram-negative bacillus (1, 2). It has an increasingly important role as a hospital-acquired pathogen associated with significant case/fatality ratios in certain patient groups, particularly severely debilitated or immunocompromised patients (1, 2). S. maltophilia has been mainly associated with the following clinical syndromes: respiratory tract infection, bacteremia, endocarditis, soft tissue infections and nosocomial infections. Predisposing risk factors associated with S. maltophilia infection are prior antibiotic therapy, central venous catheterization, neutropenia, cytotoxic chemotherapy, malignancy, prolonged hospitalization and admission to an intensive care unit. Treatment of S. maltophilia infections is complicated because the bacterium is resistant to many broadspectrum anti-microbial agents (1). S. maltophilia shows inherent resistance to virtually all classes of beta-lactams, including extended-spectrum penicillins, third-generation cephalosporins and carbapenems. Furthermore, it is resistant to aminoglycosides and shows variable susceptibility to fluoroquinolones. The antibiotic agent of choice has been considered to be trimethoprim– sulfamethoxazole; however, lately strains of the bacterium resistant to this combination have emerged (1–3). Thus, there is an imperative need to assess the efficacy of other antibiotics. Many studies to test the susceptibility of S. maltophilia to various anti-microbial agents have been performed (4–15). One class of antibiotics that has received increased attention for its potential usage in the treatment of S. maltophilia infections is fluoroquinolones (4–15). Of the quinolones studied, moxifloxacin seems to be one of the most potent (4–12). Moxifloxacin is a third-generation quinolone, active both against gram-positive and gram-negative microorganisms and also against anaerobic bacteria (16, 17). The susceptibility rates of S. maltophilia to moxifloxacin vary between 80% and 96.4% (4–12). Another study showed activity of moxifloxacin against isolates resistant to trimethoprim– sulfamethoxazole (10). Although these studies have tested the in vitro activity of moxifloxacin, in vivo evidence is lacking. The aim of this study was to investigate the effect of moxifloxacin on the survival of immunocompromised rats with soft tissue infection caused by S. maltophilia. © 2013 The Societies and Wiley Publishing Asia Pty Ltd
MATERIALS AND METHODS All experiments were performed in the Laboratory of Surgical Research of the Second Surgical Clinic of the Aristotle University of Thessaloniki after approval by the Prefecture of Thessaloniki according to law 86/609 EEC of the European Union. In all, 144 male white Wistar rats aged 18–22 weeks and weighing 300–400 g were studied. The animals were kept in stainless steel cages of five animals per cage, in a temperature range of 18–22 °C, with relative humidity 55–65% and a light:dark cycle of 12:12 hr. The rats had free access to food and water (ad libitum). The rats were rendered immunocompromised with cyclophosphamide. Under general anesthesia using ether, neutropenia was induced by intraperitoneal administration of cyclophosphamide 100 mg/kg on the first day and 150 mg/kg on the third day. On the fifth day, 1 108 CFU/kg of S. maltophilia was administered i.m. into the right thigh under general anesthesia with ether. The organisms had been isolated from the bloodstream of a patient with severe sepsis. The minimum inhibitory concentration of moxifloxacin against this isolate as assessed by a microdilution technique was 0.03 mg/mL. The rats were randomized into six groups of 24 mice each. In all groups, therapy was given i.v. into the tail vein under aseptic conditions starting 1 hr after bacterial challenge. The groups were treated as follows: Group A received 1 mL of saline once a day for 7 days; Group B received 12 mg/kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG, Athens, Greece) once a day for 7 days; Group C received 1 mL of saline every 12 hr each day for 7 days; Group D received 12 mg/kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG) every 12 hr for 7 days; Group E received 1 mL of saline every 8 hr for 7 days; and Group F received 12 mg/ kg of moxifloxacin in a volume of 1 mL (Avelox, Bayer Hellas AG) every 8 hr for 7 days. Twelve rats from each group were used to investigate survival. After administration of S. maltophilia, the rats were monitored every 12 hr for 7 days. The other 12 rats in each group were used to investigate changes related to lipid peroxidation and inflammation by taking blood samples of 5 mL each from the inferior vena cava 6 hr (six rats per group) and 30 hr (six rats per group) after administration of S. maltophilia. MDA was used as a marker of lipid peroxidation and WBC counts as a marker of inflammation. Of the 5 mL of blood that was collected, 2 mL was placed in EDTA tubes (Greiner Bio-One, Monroe, NC, USA) and used for immediate WBC counts using a Genesis auto counter (Coulter Electronics, Miami, FL, USA). The remaining 3 mL was placed in toxin-free tubes 97
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(Vacutainer; Becton Dickinson, Cockeysville, MD, USA), centrifuged and the supernatant serum placed in the same tubes and frozen at 70 °C until measurement of MDA. MDA measurement was performed by the thiobarbituric acid method and analyzed by HPLC (Agilent Technologies, New York, NY, USA). More precisely, 100 mL of each sample was added to 900 mL of trichloroacetic acid 20% (Sigma, St. Louis, MO, USA) and stirred for 30 sec with Vortex. Next, the solution was centrifuged at 13,000 g for 10 min at 4 °C, 0.5 mL of supernatant added to 2 mL of thiobarbituric acid 0.2% (Sigma) and the resultant mixture incubated at 90 °C for 60 min. Following centrifugation at 3500 g for 10 min, 10 mL of the supernatant was injected into an HPLC system. The chromatographic column used was 150 mm long, of internal diameter 4.6 mm and with pores of 5 mm (Zorbax Eclipse XDB-C18; Agilent Technologies). The motile phase was a mixture of 60% phosphate potassium buffer 50 mM and 40% methanol. The flow rate was 1 mL/min, temperature 37 °C and pressure 100– 105 mm Hg. Using a fluorescence detector, the emission of every sample at 553 nm was measured after stimulation of the sample at 515 nm. Determination of MDA concentrations was made by comparing the emission values with a titration curve of known concentrations of 1,1,3,3-tetramethoxypropane (Merck, Darmstadt, Germany). When the animals were killed 30 hr after bacterial challenge, segments of the liver, spleen and lower lobe of the right lung were excised under aseptic conditions with sterile blades and collected into separate sterile containers. The segments were weighed and homogenized with a tissue grinder in a volume of 1 mL of sodium chloride 0.9%. They were then serially diluted 1:10 four times with sodium chloride 0.9%. A volume of 0.1 mL of each dilution was plated onto MacConkey agar and incubated for 48 hr at 35 °C. At the end of incubation, the number of colonies of each dilution was counted and multiplied by the appropriate dilution factor. Colony counts are expressed as the log10 of CFU/g of tissue. The lower limit of detection was 10 CFU/g. In order to assess the serum concentrations of moxifloxacin achieved with the administered doses, a 12 mg/kg dose was administered to 12 healthy rats. Six of these rats were killed 15 and six more 30 min postinfusion and blood collected from their inferior vena cavae. The blood was centrifuged and concentrations of moxifloxacin measured in serum after analysis by HPLC as described previously (18). All measurements are expressed as mean values 1 SD. To compare results, after checking the normality, the independent samples t-test we used for variables with normal distribution and the non-parametric Mann– 98
Whitney test for variables that did not have normal distribution. Survival was assessed by the Kaplan–Meier test and comparison of survival by the log-rank test. P values below 0.05 were considered statistically significant. Statistical analysis was made using the SPSS (Statistical Package for Social Sciences, version 14.0; Chicago, IL, USA).
RESULTS As shown in Figure 1, treatment with moxifloxacin every 8 hr (Group F) was accompanied by longer survival than in any other group. Tissue cultures 30 hr after bacterial challenge showed considerably less bacterial growth in the spleens and lungs of moxifloxacin-treated animals than in the other groups, but not in their livers (Fig. 2). Mean SD concentrations of moxifloxacin 15 min postinfusion were 2.76 0.14 mg/ and 1.95 1.19 mg/mL 30 min post-infusion. The reduced bacterial growth after treatment with moxifloxacin did not involve all organs, indicating the possibility that part of moxifloxacin’s activity is mediated by modulation of inflammation. This possibility was assessed by measuring concentrations of circulating MDA and WBC counts. At 6 hr after administration of S. maltophilia, there were no statistically significant differences in absolute WBC counts between the groups receiving moxifloxacin
Fig. 1. Survival of groups of rats subjected to the following treatments: Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Statistical comparisons: Log-rankA versus B ¼ 1.118, P ¼ 0.277; log-rankC versus D ¼ 5.295, P ¼ 0.021; log-rankE versus F ¼ 24.832, P < 0.0001; log-rankB versus F ¼ 7.501, P < 0.0001; log-rankD versus F ¼ 4.023, P ¼ 0.045.
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Moxifloxacin & S. maltophilia infection
Fig. 2. Tissue bacterial overgrowth. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding saline-treated group.
and those receiving saline (Fig. 3). However, at 30 hr the animals that had received two and three doses of moxifloxacin had lower WBCs than the animals that had received saline; this difference was significant only between Groups C and D, which had received two doses. Similar differences were found for circulating MDA (Fig. 4).
DISCUSSION Our finding suggest that survival benefit after experimental soft tissue infection by S. maltophilia in immunocompromised rats is correlated with the frequency of administration. This could be caused by a direct bactericidal action of moxifloxacin against S. maltophilia but could also be attributable, at least in part, to an immunomodulatory effect of moxifloxacin. Despite the fact that moxifloxacin lacks direct antifungal activity, Shalit et al. have reported it has an immunomodulatory effect in a model of immunocompromised mice bronchopneumonia caused by Candida albicans (19). © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Shalit et al. were the first to study the immunomodulatory effects of moxifloxacin (20). They concluded that administration of moxifloxacin to mice with cyclophosphamide-induced leukopenia reverses some of cyclophosphamide’s immunosuppressive effects, as indicated by higher WBC counts, greater colony-stimulating activity and increased granulocyte macrophage colonystimulating factor in treated animals compared with controls. Araujo et al. reported that moxifloxacin significantly decreased secretion of TNF-a and IL-1a by human monocytes stimulated with LPS (21). In another study, Choi et al. found that moxifloxacin decreases production of TNF-a and of IL-6 by human peripheral blood mononuclear cells after stimulation with LPS, lipoteichoic acid and heat-killed bacteria (22). In addition, Weiss et al. have shown that moxifloxacin has an anti-inflammatory effect on activated human monocytic cells, specifically decreasing production of TNF-a, IL-8 and IL-1b and inhibiting activation of NFkΒ, ER kinase and JNK (23). Werber et al. have also reported not only inhibition of activation of NF-kΒ, ER kinase and JNKs, but also of mitogen-activated protein 99
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leukemia cell line) monocytes (27). They observed an increase in release of nitric oxide and hydrogen peroxide within the first hour followed by a decrease in lipid peroxidation after 4 hr. Moxifloxacin seems to initially activate monocytes in order to kill bacteria by releasing ROS and lysosomal hydrolytic enzymes. At a later stage, reversal of the effects of moxifloxacin on free radical generation and hydrolytic enzymes reportedly occurs, resulting in suppression of lipid peroxidation (27). In addition, Attia observed significantly greater serum MDA production in mice that received lomefloxacin than in controls (28). Also, Rampal et al. have shown that ofloxacin increases lipid peroxidation in rabbits (29). In addition, various studies have shown that phototoxicity of quinolones is caused by increased lipid peroxidation, possibly due to increased production of free radicals (30– 33). In the present study, the increased MDA concentrations are a result of increased lipid peroxidation, possibly caused by moxifloxacin-induced direct increase
Fig. 3. White blood cell count (per mm3) at 6 and 30âhr in all groups. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding saline-treated group.
kinase after treating human respiratory epithelial cells with moxifloxacin in vitro (24). Furthermore, Williams et al. found that moxifloxacin decreases interferon, INFg and IL-4 expression on Th1 and Th2 cells in a dosedependent manner without altering Th1/Th2 ratios (25). Finally, Blau et al. have shown that moxifloxacin inhibits activation of IL-8, IL-6, NF-kΒ, ER kinase and JNK in cystic fibrosis epithelial cells (26). In the present study, moxifloxacin administration increased MDA production in the group that received two doses per day. MDA is a side product of enzymatic lipid peroxidation process and a final product of nonenzymatic lipid peroxidation. Infection with these bacteria initiates non-enzymatic lipid peroxidation; for such initiation, the presence of free radicals is necessary. Hall et al. studied the effects of moxifloxacin on free radical generation, cytokine immunomodulatory mediators and hydrolytic enzyme activities in zymogen Astimulated human THP-1 (human acute monocytic 100
Fig. 4. MDA values (in µM) at 6 and 30âhr in all groups. Group A, 0.9% normal saline daily; Group B, moxifloxacin daily; Group C, normal 0.9% saline twice daily; Group D, moxifloxacin twice daily; Group E, 0.9% saline 8 hourly; Group F, moxifloxacin 8 hourly. Asterisks denote significant differences with the corresponding salinetreated group.
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Moxifloxacin & S. maltophilia infection
in production of free radicals. Also, moxifloxacin has been found to initiate phagocytosis through stimulation of the membrane to release cyclic adenosine monophosphate and increase activity of protein kinase C (27). Moxifloxacin activates monocytes to kill microorganisms by bactericidal or ROS mechanisms of phagocytosis of bacteria (27). The presence of free radicals increases the cidal effect provided the free radicals are not cleared by the cells’ protective mechanisms (27). We therefore suggest that the increased MDA concentrations are a result of increased lipid peroxidation by free radicals, probably linked with increased phagocytosis. To the best of our knowledge, the present study is the first published report that has assessed certain markers, such as MDA for lipid peroxidation and WBC counts for inflammation, to evaluates the effect of moxifloxacin in the lipid peroxidation process and in inflammation of immunocompromised rats with soft tissue infection by S. maltophilia. Also, we attempted to correlate these markers with the general effect of moxifloxacin on the progress of the infection by measuring survival. In conclusion, administration of two doses of moxifloxacin increases lipid peroxidation, as indicated by increased MDA concentrations. It may also enhance phagocytosis in immunosuppressed rats, though without associated toxic effects, as shown by decreased numbers of WBCs, and increased survival. Thus, moxifloxacin could become a valuable drug for treating both infection in immunosuppressed patients and infections caused by S. maltophilia. However, more studies are needed to evaluate the immunomodulatory effects of moxifloxacin in clinical settings and its potential value for treating immunosuppressed patients and f S. maltophilia infections.
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DISCLOSURE The authors declare that they have no conflict of interest.
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