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International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
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Short Communication
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The aerobic activity of metronidazole against anaerobic bacteria Q1
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Niokhor Dione, Saber Khelaifia, Jean-Christophe Lagier, Didier Raoult ∗ Aix-Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, 13005 Marseille, France
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6 18
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10
Article history: Received 24 December 2014 Accepted 29 December 2014
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Keywords: Metronidazole Antioxidants Aerobic susceptibility testing In vitro activity MALDI-TOF/MS
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1. Introduction
12 13 14 15 16
Recently, the aerobic growth of strictly anaerobic bacteria was demonstrated using antioxidants. Metronidazole is frequently used to treat infections caused by anaerobic bacteria; however, to date its antibacterial activity was only tested in anaerobic conditions. Here we aerobically tested using antioxidants the in vitro activities of metronidazole, gentamicin, doxycycline and imipenem against 10 common anaerobic and aerobic bacteria. In vitro susceptibility testing was performed by the disk diffusion method, and minimum inhibitory concentrations (MICs) were determined by Etest. Aerobic culture of the bacteria was performed at 37 ◦ C using Schaedler agar medium supplemented with 1 mg/mL ascorbic acid and 0.1 mg/mL glutathione; the pH was adjusted to 7.2 by 10 M KOH. Growth of anaerobic bacteria cultured aerobically using antioxidants was inhibited by metronidazole after 72 h of incubation at 37 ◦ C, with a mean inhibition diameter of 37.76 mm and an MIC of 1 g/mL; however, strains remained non-sensitive to gentamicin. No growth inhibition of aerobic bacteria was observed after 24 h of incubation at 37 ◦ C with metronidazole; however, inhibition was observed with doxycycline and imipenem used as controls. These results indicate that bacterial sensitivity to metronidazole is not related to the oxygen tension but is a result of the sensitivity of the micro-organism. In future, both culture and antibiotic susceptibility testing of strictly anaerobic bacteria will be performed in an aerobic atmosphere using antioxidants in clinical microbiology laboratories. © 2015 Published by Elsevier B.V.
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Metronidazole and its derivatives are known for their antiparasitic, bactericidal and archaeacidal activity against anaerobic bacteria and methanogenic archaea [1]. These antibiotics are mainly active in anaerobic or microaerophilic conditions [2]; however, their mechanism of action has not yet been determined accurately. A study suggests that after crossing the cell wall by passive transport, metronidazole and its derivatives are converted by the bacterial or archaeal intracellular enzymes to a nitro free radical [3]. To the best of our knowledge, the in vitro activity of metronidazole and its derivatives has only been tested in anaerobic or microaerophilic conditions [4]. However, La Scola et al. recently demonstrated that it was possible to cultivate anaerobic bacteria in aerobic conditions using antioxidants [5]; therefore, we questioned the aerobic activity of metronidazole against anaerobic bacteria in this culture condition. In this work, we tested aerobically, as a proof
∗ Corresponding author. Present address: Aix-Marseille Université, URMITE, UM63 CNRS 7278, IRD 198, INSERM 1095, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France. Tel.: +33 4 91 32 43 75; fax: +33 4 91 38 77 72. E-mail address: [email protected] (D. Raoult).
of concept, the in vitro activity of metronidazole against anaerobic and aerobic bacteria cultured from human microbiota.
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2. Materials and methods
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2.1. Bacterial strains
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In this study, the five most frequent anaerobic and aerobic bacteria isolated in the clinical microbiology laboratory of Timone Hospital (Marseille, France) were tested for their sensitivity to metronidazole [6]. The anaerobic bacteria tested were Bacteroides thetaiotaomicron CSUR P766, Bacteroides fragilis CSUR P863, Finegoldia magna CSUR P588, Fusobacterium nucleatum CSUR P1151 and Parvimonas micra CSUR P875. The aerobic bacteria tested were Escherichia coli CSUR P729, Staphylococcus aureus CSUR P749, Staphylococcus epidermidis CSUR P748, Klebsiella pneumoniae CSUR P931 and Pseudomonas aeruginosa CSUR P1003. All of the strains tested were isolated in the laboratory as part of a culturomics study as previously described [7,8]. Aerobic and anaerobic bacterial strains were routinely subcultured using 5% sheep blood agar medium (bioMérieux, Marcy-l’Étoile, France). Anaerobic bacteria were incubated under an anaerobic atmosphere using a gas pack
http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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Table 1 Diameter of zones of growth inhibition of the antibiotics used, and minimum inhibitory concentrations (MICs) of metronidazole and gentamicin. Strain
Inhibition diameter (mm) Doxycycline
Imipenem
MIC (g/mL) Metronidazole
Metronidazole a
Aerobic bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis
14.6 15.2 15.4 21.7 37
Anaerobic bacteria Bacteroides thetaiotaomicron Bacteroides fragilis Finegoldia magna Fusobacterium nucleatum Parvimonas micra
37.6 ± 0.5 42.6 ± 0.3 37.6 ± 0.8 37.6 ± 0.1 32.6 ± 0.15
b
Gentamicin
Positive control
R-medium
Positive control
R-medium
26.9 26.3 20.2 37 36.9
R R R R R
R R R R R
R R R R R
R R R R R
2 3 3 3 2
37.8 ± 0.8 42.4 ± 2.5 42 ± 0.9 37.6 ± 0.3 41.7 ± 1.7
35.7 ± 0.5 36.5 ± 0.3 37 ± 0.2 37.2 ± 0.7 36.4 ± 0.1
37.6 ± 0.9 36 ± 0.5 37.6 ± 0.4 37.4 ± 0.15 37.6 ± 0.1
1 0.25 0.19 0.25 0.125
1 0.25 0.19 0.25 0.125
R R R R R
R, Resistant (no inhibition). a The positive control was incubated anaerobically for anaerobic bacteria and aerobically for aerobic bacteria. b R-medium was incubated aerobically for aerobic and anaerobic bacteria.
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system (bioMérieux) at 37 ◦ C. Aerobic bacteria were incubated aerobically at 37 ◦ C.
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2.2. Antibacterial susceptibility assays
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Antibiotic disks of metronidazole (4 g), imipenem (10 g), doxycycline (30 g) and gentamicin (500 g) were purchased from bioMérieux. In vitro susceptibility testing of these antibiotics against aerobic and anaerobic bacteria was performed by the disk diffusion method [9]. Minimum inhibitory concentrations (MICs) of metronidazole were determined only for anaerobic bacteria by the Etest method using Etest strips with metronidazole concentrations of 0.016–256 g (bioMérieux) and MICs of gentamicin were determined only for aerobic bacteria using Etest strips with gentamicin concentrations of 0.016–256 g (bioMérieux) incubated under an aerobic atmosphere. The aerobic culture of anaerobic and aerobic bacteria was performed using Schaedler agar medium (Sigma-Aldrich, Saint-Quentin-Fallavier, France) supplemented with 1 mg/mL ascorbic acid (VWR International SAS, Fontenay-sous-Bois, France) and 0.1 mg/mL glutathione (Sigma-Aldrich); the pH was adjusted to 7.2 by addition of 10 M KOH (Sigma-Aldrich). Then, a bacterial suspension containing 107 CFU/mL of an exponentially growing culture of each of the tested strains was homogeneously spread on a Petri dish containing fresh culture medium. One disk of each of the three antibiotics tested was then deposited on each Petri dish and the dishes were incubated at 37 ◦ C under an aerobic atmosphere. The bacterial growth was observed after 24 h of incubation for aerobic bacteria and after 72 h for anaerobic bacteria. Subculture of the anaerobic bacteria in Schaedler agar medium under an anaerobic atmosphere was performed as a control. Control cultures without antimicrobial agents were incubated in parallel to provide a baseline for the growth of all bacteria. A non-inoculated culture medium was included as a negative control. All of the growing cultures were verified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) to check for contamination [6,10]. Growth was assessed by direct observation after 24 h or 72 h of incubation and in parallel using SirSCANTM (i2A, Pérols, France). All of the experiments were performed in triplicate.
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3. Results
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As a positive control, the antibacterial activity of metronidazole against anaerobic bacteria was confirmed anaerobically by
the disk diffusion method, and diameters of growth inhibition of 36.5 ± 0.3 mm for B. fragilis, 35.7 ± 0.5 mm for B. thetaiotaomicron, 37 ± 0.2 mm for F. magna, 37.2 ± 0.7 mm for F. nucleatum and 36.4 ± 0.1 mm for P. micra were observed (Table 1). In parallel, MICs of 0.25 g/mL for B. fragilis, 1 g/mL for B. thetaiotaomicron, 0.19 g/mL for F. magna, 0.25 g/mL for F. nucleatum and 0.125 g/mL for P. micra were observed. Using the disk diffusion method, growth of anaerobic bacteria was inhibited by metronidazole, with mean inhibition diameters of 36 ± 0.5 mm for B. fragilis, 37.6 ± 0.9 mm for B. thetaiotaomicron, 37.6 ± 0.4 mm for F. magna, 37.4 ± 0.15 mm for F. nucleatum and 37.6 ± 0.1 mm for P. micra after 72 h of incubation (Fig. 1). By Etest, MICs of 0.25 g/mL for B. fragilis, 1 g/mL for B. thetaiotaomicron, 0.19 g/mL for F. magna, 0.25 g/mL for F. nucleatum and 0.125 g/mL for P. micra were observed. No growth inhibition by metronidazole was observed for aerobic bacteria (Table 1). Growth of all of the anaerobic bacteria cultured aerobically using antioxidants was inhibited by doxycycline after 72 h of incubation at 37 ◦ C, with mean inhibition diameters of 37.6 ± 0.8 mm for F. magna, 37.6 ± 0.1 mm for F. nucleatum, 37.6 ± 0.5 mm for B. thetaiotaomicron, 42.6 ± 0.3 mm for B. fragilis and 32.6 ± 0.15 mm for P. micra. The disk diffusion method using imipenem disks inhibited the growth of anaerobic bacteria after 72 h of incubation, with mean inhibition diameters of 42 ± 0.9 mm for F. magna, 37.6 ± 0.3 mm for F. nucleatum, 37.8 ± 0.8 mm for B. thetaiotaomicron, 42.4 ± 2.5 mm for B. fragilis and 41.7 ± 1.7 mm for P. micra. Growth of aerobic bacteria was inhibited by imipenem and doxycycline, and MICs in the range of previously published results were observed (Table 1). The aerobic bacteria tested here remained insensitive to metronidazole after 24 h of incubation despite their growth on the same culture medium. Using the Etest method, growth of all aerobic bacteria cultured here was inhibited by gentamicin after 24 h of incubation at 37 ◦ C, and MICs of 2 g/mL for E. coli, 3 g/mL for K. pneumoniae, 3 g/mL for P. aeruginosa, 3 g/mL for S. aureus and 2 g/mL for S. epidermidis were observed. All of the anaerobic bacteria tested remained insensitive to gentamicin after 72 h of incubation despite their growth aerobically. The positive control culture of anaerobic bacteria that was incubated anaerobically at 37 ◦ C in Schaedler agar medium without metronidazole grew as expected after 72 h of incubation; likewise, the positive control culture of aerobic bacteria incubated aerobically grew as expected after 24 h of incubation. The negative controls introduced in all of the manipulations remained negative. All of the growing cultures were verified by MALDI-TOF/MS, confirming the absence of contamination.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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Fig. 1. Antimicrobial activity of metronidazole, doxycycline, imipenem and gentamicin against anaerobic bacteria. (1) Finegoldia magna; (2) Bacteroides thetaiotaomicron; (3) Parvimonas micra; (4) Bacteroides fragilis; and (5) Fusobacterium nucleatum. (a) Metronidazole Etest in Schaedler agar medium with antioxidants grown aerobically; (b) aerobic activity of three antibiotics in Schaedler agar medium with antioxidants; (c) activity of three antibiotics in Schaedler agar medium without antioxidants grown in an anaerobic condition as activity controls; and (d) gentamicin Etest in Schaedler agar medium with antioxidants grown aerobically.
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4. Discussion It was recently shown that anaerobes could grow easily in an aerobic atmosphere using antioxidants [5,11]. It was possible to
grow six anaerobe species, including Fusobacterium necrophorum and Ruminococcus gnavus [5]. Therefore, in this study we tested the activity of antimicrobial agents known for their activity in anaerobic conditions. Thus, in this work and for the first time, we tested the
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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antimicrobial susceptibility of the five aerobic and five anaerobic bacteria most frequently isolated in the clinical microbiology laboratory of Timone Hospital [6]. The results demonstrate, for the first time, that it is possible to cultivate aerobic and anaerobic bacteria in the same culture condition and to determine their antibiograms aerobically. Initially, after La Scola et al. succeeded in the culture of anaerobes aerobically, we tested the susceptibility of anaerobes to metronidazole in this particular culture condition. Growth of anaerobes was inhibited by metronidazole, with observed results in the range previously described [2]. To verify that the result was not a bias by our culture medium, we tested the susceptibility of aerobic bacteria in this same condition, and we used doxycycline and imipenem, which are known to be effective against the chosen strains, as the activity control [12]. We observed that the aerobic bacteria tested here were susceptible to doxycycline and imipenem as expected but remained resistant to metronidazole. In the same perspective, we tested the sensitivity of anaerobic bacteria to gentamicin when they were cultured aerobically and observed that the anaerobic bacteria remained insensitive to gentamicin despite their growth aerobically. The results obtained from these experiments demonstrate that the anaerobic bacteria tested here are highly sensitive to metronidazole and insensitive to gentamicin and that there is no relationship with the culture medium used for their culture or the atmosphere of the incubation (Table 1). Despite the fact that metronidazole is considered oxygensensitive, it is widely and effectively used for the treatment of bloodstream infections, which account for 1–17% of positive blood cultures [13–15]. Therefore, based on the results presented here, as proof of concept, we can state that the sensitivity to metronidazole or gentamicin is in no way related to the culture condition or to the atmosphere used to incubate the bacterial culture; however, the results demonstrate a real sensitivity of the micro-organism. Our culture medium using antioxidants will facilitate not only the culture of strictly anaerobic bacterial species but also antibiotic susceptibility testing both for anaerobic and aerobic bacteria in routine bacteriology laboratories and an evaluation of the susceptibility of ‘anaerobes’ to other antibiotics. Funding
This work was funded by the Méditerranée Infection Fondation, Q4 Aix-Marseille Université (Marseille, France).
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Competing interests The culture medium reported here is the subject of a pending patent. All authors declare no competing interests. Ethical approval Not required. References [1] Martin WJ, Gardner M, Washington JA. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob Agents Chemother 1972;1:148–58. [2] Lofmark S, Edlund C, Nord CE. Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis 2010;50:S16–23. [3] Ralph ED, Kirby WM. Unique bactericidal action of metronidazole against Bacteroides fragilis and Clostridium perfringens. Antimicrob Agents Chemother 1975;8:409–14. [4] Tally FP, Sutter VL, Finegold SM. Metronidazole versus anaerobes In vitro data and initial clinical observations. Calif Med 1972;117:22–6. [5] La Scola B, Khelaifia S, Lagier JC, Raoult D. Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report. Eur J Clin Microbiol Infect Dis 2014;33:1781–3. [6] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, et al. Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrixassisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 2013;51:2182–94. [7] Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 2012;18:1185–93. [8] Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 2015;28:237–64. [9] Knapp CC, Ludwig MD, Washington JA. In vitro activity of metronidazole against Helicobacter pylori as determined by agar dilution and agar diffusion. Antimicrob Agents Chemother 1991;35:1230–1. [10] Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009;49:543–51. [11] Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D. Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev 2015;28:208–36. [12] Hecht DW. Anaerobes: antibiotic resistance, clinical significance, and the role of susceptibility testing. Anaerobe 2006;12:115–21. [13] Brook I. The role of anaerobic bacteria in bacteremia. Anaerobe 2010;16:183–9. [14] La Scola B, Fournier PE, Raoult D. Burden of emerging anaerobes in the MALDITOF and 16S rRNA gene sequencing era. Anaerobe 2011;17:106–12. [15] Barreau M, Pagnier I, La Scola B. Improving the identification of anaerobes in the clinical microbiology laboratory through MALDI-TOF mass spectrometry. Anaerobe 2013;22:123–5.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
1
Short Communication
2
The aerobic activity of metronidazole against anaerobic bacteria Q1
3
Niokhor Dione, Saber Khelaifia, Jean-Christophe Lagier, Didier Raoult ∗ Aix-Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, 13005 Marseille, France
4 5
6 18
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10
Article history: Received 24 December 2014 Accepted 29 December 2014
11
17
Keywords: Metronidazole Antioxidants Aerobic susceptibility testing In vitro activity MALDI-TOF/MS
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1. Introduction
12 13 14 15 16
Recently, the aerobic growth of strictly anaerobic bacteria was demonstrated using antioxidants. Metronidazole is frequently used to treat infections caused by anaerobic bacteria; however, to date its antibacterial activity was only tested in anaerobic conditions. Here we aerobically tested using antioxidants the in vitro activities of metronidazole, gentamicin, doxycycline and imipenem against 10 common anaerobic and aerobic bacteria. In vitro susceptibility testing was performed by the disk diffusion method, and minimum inhibitory concentrations (MICs) were determined by Etest. Aerobic culture of the bacteria was performed at 37 ◦ C using Schaedler agar medium supplemented with 1 mg/mL ascorbic acid and 0.1 mg/mL glutathione; the pH was adjusted to 7.2 by 10 M KOH. Growth of anaerobic bacteria cultured aerobically using antioxidants was inhibited by metronidazole after 72 h of incubation at 37 ◦ C, with a mean inhibition diameter of 37.76 mm and an MIC of 1 g/mL; however, strains remained non-sensitive to gentamicin. No growth inhibition of aerobic bacteria was observed after 24 h of incubation at 37 ◦ C with metronidazole; however, inhibition was observed with doxycycline and imipenem used as controls. These results indicate that bacterial sensitivity to metronidazole is not related to the oxygen tension but is a result of the sensitivity of the micro-organism. In future, both culture and antibiotic susceptibility testing of strictly anaerobic bacteria will be performed in an aerobic atmosphere using antioxidants in clinical microbiology laboratories. © 2015 Published by Elsevier B.V.
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Metronidazole and its derivatives are known for their antiparasitic, bactericidal and archaeacidal activity against anaerobic bacteria and methanogenic archaea [1]. These antibiotics are mainly active in anaerobic or microaerophilic conditions [2]; however, their mechanism of action has not yet been determined accurately. A study suggests that after crossing the cell wall by passive transport, metronidazole and its derivatives are converted by the bacterial or archaeal intracellular enzymes to a nitro free radical [3]. To the best of our knowledge, the in vitro activity of metronidazole and its derivatives has only been tested in anaerobic or microaerophilic conditions [4]. However, La Scola et al. recently demonstrated that it was possible to cultivate anaerobic bacteria in aerobic conditions using antioxidants [5]; therefore, we questioned the aerobic activity of metronidazole against anaerobic bacteria in this culture condition. In this work, we tested aerobically, as a proof
∗ Corresponding author. Present address: Aix-Marseille Université, URMITE, UM63 CNRS 7278, IRD 198, INSERM 1095, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France. Tel.: +33 4 91 32 43 75; fax: +33 4 91 38 77 72. E-mail address: [email protected] (D. Raoult).
of concept, the in vitro activity of metronidazole against anaerobic and aerobic bacteria cultured from human microbiota.
36 37
2. Materials and methods
38
2.1. Bacterial strains
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In this study, the five most frequent anaerobic and aerobic bacteria isolated in the clinical microbiology laboratory of Timone Hospital (Marseille, France) were tested for their sensitivity to metronidazole [6]. The anaerobic bacteria tested were Bacteroides thetaiotaomicron CSUR P766, Bacteroides fragilis CSUR P863, Finegoldia magna CSUR P588, Fusobacterium nucleatum CSUR P1151 and Parvimonas micra CSUR P875. The aerobic bacteria tested were Escherichia coli CSUR P729, Staphylococcus aureus CSUR P749, Staphylococcus epidermidis CSUR P748, Klebsiella pneumoniae CSUR P931 and Pseudomonas aeruginosa CSUR P1003. All of the strains tested were isolated in the laboratory as part of a culturomics study as previously described [7,8]. Aerobic and anaerobic bacterial strains were routinely subcultured using 5% sheep blood agar medium (bioMérieux, Marcy-l’Étoile, France). Anaerobic bacteria were incubated under an anaerobic atmosphere using a gas pack
http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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Table 1 Diameter of zones of growth inhibition of the antibiotics used, and minimum inhibitory concentrations (MICs) of metronidazole and gentamicin. Strain
Inhibition diameter (mm) Doxycycline
Imipenem
MIC (g/mL) Metronidazole
Metronidazole a
Aerobic bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis
14.6 15.2 15.4 21.7 37
Anaerobic bacteria Bacteroides thetaiotaomicron Bacteroides fragilis Finegoldia magna Fusobacterium nucleatum Parvimonas micra
37.6 ± 0.5 42.6 ± 0.3 37.6 ± 0.8 37.6 ± 0.1 32.6 ± 0.15
b
Gentamicin
Positive control
R-medium
Positive control
R-medium
26.9 26.3 20.2 37 36.9
R R R R R
R R R R R
R R R R R
R R R R R
2 3 3 3 2
37.8 ± 0.8 42.4 ± 2.5 42 ± 0.9 37.6 ± 0.3 41.7 ± 1.7
35.7 ± 0.5 36.5 ± 0.3 37 ± 0.2 37.2 ± 0.7 36.4 ± 0.1
37.6 ± 0.9 36 ± 0.5 37.6 ± 0.4 37.4 ± 0.15 37.6 ± 0.1
1 0.25 0.19 0.25 0.125
1 0.25 0.19 0.25 0.125
R R R R R
R, Resistant (no inhibition). a The positive control was incubated anaerobically for anaerobic bacteria and aerobically for aerobic bacteria. b R-medium was incubated aerobically for aerobic and anaerobic bacteria.
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system (bioMérieux) at 37 ◦ C. Aerobic bacteria were incubated aerobically at 37 ◦ C.
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2.2. Antibacterial susceptibility assays
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Antibiotic disks of metronidazole (4 g), imipenem (10 g), doxycycline (30 g) and gentamicin (500 g) were purchased from bioMérieux. In vitro susceptibility testing of these antibiotics against aerobic and anaerobic bacteria was performed by the disk diffusion method [9]. Minimum inhibitory concentrations (MICs) of metronidazole were determined only for anaerobic bacteria by the Etest method using Etest strips with metronidazole concentrations of 0.016–256 g (bioMérieux) and MICs of gentamicin were determined only for aerobic bacteria using Etest strips with gentamicin concentrations of 0.016–256 g (bioMérieux) incubated under an aerobic atmosphere. The aerobic culture of anaerobic and aerobic bacteria was performed using Schaedler agar medium (Sigma-Aldrich, Saint-Quentin-Fallavier, France) supplemented with 1 mg/mL ascorbic acid (VWR International SAS, Fontenay-sous-Bois, France) and 0.1 mg/mL glutathione (Sigma-Aldrich); the pH was adjusted to 7.2 by addition of 10 M KOH (Sigma-Aldrich). Then, a bacterial suspension containing 107 CFU/mL of an exponentially growing culture of each of the tested strains was homogeneously spread on a Petri dish containing fresh culture medium. One disk of each of the three antibiotics tested was then deposited on each Petri dish and the dishes were incubated at 37 ◦ C under an aerobic atmosphere. The bacterial growth was observed after 24 h of incubation for aerobic bacteria and after 72 h for anaerobic bacteria. Subculture of the anaerobic bacteria in Schaedler agar medium under an anaerobic atmosphere was performed as a control. Control cultures without antimicrobial agents were incubated in parallel to provide a baseline for the growth of all bacteria. A non-inoculated culture medium was included as a negative control. All of the growing cultures were verified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) to check for contamination [6,10]. Growth was assessed by direct observation after 24 h or 72 h of incubation and in parallel using SirSCANTM (i2A, Pérols, France). All of the experiments were performed in triplicate.
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As a positive control, the antibacterial activity of metronidazole against anaerobic bacteria was confirmed anaerobically by
the disk diffusion method, and diameters of growth inhibition of 36.5 ± 0.3 mm for B. fragilis, 35.7 ± 0.5 mm for B. thetaiotaomicron, 37 ± 0.2 mm for F. magna, 37.2 ± 0.7 mm for F. nucleatum and 36.4 ± 0.1 mm for P. micra were observed (Table 1). In parallel, MICs of 0.25 g/mL for B. fragilis, 1 g/mL for B. thetaiotaomicron, 0.19 g/mL for F. magna, 0.25 g/mL for F. nucleatum and 0.125 g/mL for P. micra were observed. Using the disk diffusion method, growth of anaerobic bacteria was inhibited by metronidazole, with mean inhibition diameters of 36 ± 0.5 mm for B. fragilis, 37.6 ± 0.9 mm for B. thetaiotaomicron, 37.6 ± 0.4 mm for F. magna, 37.4 ± 0.15 mm for F. nucleatum and 37.6 ± 0.1 mm for P. micra after 72 h of incubation (Fig. 1). By Etest, MICs of 0.25 g/mL for B. fragilis, 1 g/mL for B. thetaiotaomicron, 0.19 g/mL for F. magna, 0.25 g/mL for F. nucleatum and 0.125 g/mL for P. micra were observed. No growth inhibition by metronidazole was observed for aerobic bacteria (Table 1). Growth of all of the anaerobic bacteria cultured aerobically using antioxidants was inhibited by doxycycline after 72 h of incubation at 37 ◦ C, with mean inhibition diameters of 37.6 ± 0.8 mm for F. magna, 37.6 ± 0.1 mm for F. nucleatum, 37.6 ± 0.5 mm for B. thetaiotaomicron, 42.6 ± 0.3 mm for B. fragilis and 32.6 ± 0.15 mm for P. micra. The disk diffusion method using imipenem disks inhibited the growth of anaerobic bacteria after 72 h of incubation, with mean inhibition diameters of 42 ± 0.9 mm for F. magna, 37.6 ± 0.3 mm for F. nucleatum, 37.8 ± 0.8 mm for B. thetaiotaomicron, 42.4 ± 2.5 mm for B. fragilis and 41.7 ± 1.7 mm for P. micra. Growth of aerobic bacteria was inhibited by imipenem and doxycycline, and MICs in the range of previously published results were observed (Table 1). The aerobic bacteria tested here remained insensitive to metronidazole after 24 h of incubation despite their growth on the same culture medium. Using the Etest method, growth of all aerobic bacteria cultured here was inhibited by gentamicin after 24 h of incubation at 37 ◦ C, and MICs of 2 g/mL for E. coli, 3 g/mL for K. pneumoniae, 3 g/mL for P. aeruginosa, 3 g/mL for S. aureus and 2 g/mL for S. epidermidis were observed. All of the anaerobic bacteria tested remained insensitive to gentamicin after 72 h of incubation despite their growth aerobically. The positive control culture of anaerobic bacteria that was incubated anaerobically at 37 ◦ C in Schaedler agar medium without metronidazole grew as expected after 72 h of incubation; likewise, the positive control culture of aerobic bacteria incubated aerobically grew as expected after 24 h of incubation. The negative controls introduced in all of the manipulations remained negative. All of the growing cultures were verified by MALDI-TOF/MS, confirming the absence of contamination.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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Fig. 1. Antimicrobial activity of metronidazole, doxycycline, imipenem and gentamicin against anaerobic bacteria. (1) Finegoldia magna; (2) Bacteroides thetaiotaomicron; (3) Parvimonas micra; (4) Bacteroides fragilis; and (5) Fusobacterium nucleatum. (a) Metronidazole Etest in Schaedler agar medium with antioxidants grown aerobically; (b) aerobic activity of three antibiotics in Schaedler agar medium with antioxidants; (c) activity of three antibiotics in Schaedler agar medium without antioxidants grown in an anaerobic condition as activity controls; and (d) gentamicin Etest in Schaedler agar medium with antioxidants grown aerobically.
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4. Discussion It was recently shown that anaerobes could grow easily in an aerobic atmosphere using antioxidants [5,11]. It was possible to
grow six anaerobe species, including Fusobacterium necrophorum and Ruminococcus gnavus [5]. Therefore, in this study we tested the activity of antimicrobial agents known for their activity in anaerobic conditions. Thus, in this work and for the first time, we tested the
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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antimicrobial susceptibility of the five aerobic and five anaerobic bacteria most frequently isolated in the clinical microbiology laboratory of Timone Hospital [6]. The results demonstrate, for the first time, that it is possible to cultivate aerobic and anaerobic bacteria in the same culture condition and to determine their antibiograms aerobically. Initially, after La Scola et al. succeeded in the culture of anaerobes aerobically, we tested the susceptibility of anaerobes to metronidazole in this particular culture condition. Growth of anaerobes was inhibited by metronidazole, with observed results in the range previously described [2]. To verify that the result was not a bias by our culture medium, we tested the susceptibility of aerobic bacteria in this same condition, and we used doxycycline and imipenem, which are known to be effective against the chosen strains, as the activity control [12]. We observed that the aerobic bacteria tested here were susceptible to doxycycline and imipenem as expected but remained resistant to metronidazole. In the same perspective, we tested the sensitivity of anaerobic bacteria to gentamicin when they were cultured aerobically and observed that the anaerobic bacteria remained insensitive to gentamicin despite their growth aerobically. The results obtained from these experiments demonstrate that the anaerobic bacteria tested here are highly sensitive to metronidazole and insensitive to gentamicin and that there is no relationship with the culture medium used for their culture or the atmosphere of the incubation (Table 1). Despite the fact that metronidazole is considered oxygensensitive, it is widely and effectively used for the treatment of bloodstream infections, which account for 1–17% of positive blood cultures [13–15]. Therefore, based on the results presented here, as proof of concept, we can state that the sensitivity to metronidazole or gentamicin is in no way related to the culture condition or to the atmosphere used to incubate the bacterial culture; however, the results demonstrate a real sensitivity of the micro-organism. Our culture medium using antioxidants will facilitate not only the culture of strictly anaerobic bacterial species but also antibiotic susceptibility testing both for anaerobic and aerobic bacteria in routine bacteriology laboratories and an evaluation of the susceptibility of ‘anaerobes’ to other antibiotics. Funding
This work was funded by the Méditerranée Infection Fondation, Q4 Aix-Marseille Université (Marseille, France).
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Competing interests The culture medium reported here is the subject of a pending patent. All authors declare no competing interests. Ethical approval Not required. References [1] Martin WJ, Gardner M, Washington JA. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens. Antimicrob Agents Chemother 1972;1:148–58. [2] Lofmark S, Edlund C, Nord CE. Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis 2010;50:S16–23. [3] Ralph ED, Kirby WM. Unique bactericidal action of metronidazole against Bacteroides fragilis and Clostridium perfringens. Antimicrob Agents Chemother 1975;8:409–14. [4] Tally FP, Sutter VL, Finegold SM. Metronidazole versus anaerobes In vitro data and initial clinical observations. Calif Med 1972;117:22–6. [5] La Scola B, Khelaifia S, Lagier JC, Raoult D. Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report. Eur J Clin Microbiol Infect Dis 2014;33:1781–3. [6] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, et al. Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrixassisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 2013;51:2182–94. [7] Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 2012;18:1185–93. [8] Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 2015;28:237–64. [9] Knapp CC, Ludwig MD, Washington JA. In vitro activity of metronidazole against Helicobacter pylori as determined by agar dilution and agar diffusion. Antimicrob Agents Chemother 1991;35:1230–1. [10] Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009;49:543–51. [11] Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D. Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev 2015;28:208–36. [12] Hecht DW. Anaerobes: antibiotic resistance, clinical significance, and the role of susceptibility testing. Anaerobe 2006;12:115–21. [13] Brook I. The role of anaerobic bacteria in bacteremia. Anaerobe 2010;16:183–9. [14] La Scola B, Fournier PE, Raoult D. Burden of emerging anaerobes in the MALDITOF and 16S rRNA gene sequencing era. Anaerobe 2011;17:106–12. [15] Barreau M, Pagnier I, La Scola B. Improving the identification of anaerobes in the clinical microbiology laboratory through MALDI-TOF mass spectrometry. Anaerobe 2013;22:123–5.
Please cite this article in press as: Dione N, et al. The aerobic activity of metronidazole against anaerobic bacteria. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.12.032
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