Abstract
We have determined the in vitro activity of several antibacterial and antifungal drugs against Pythium insidiosum using broth microdilution (BMD), disk diffusion, and Etest methods. The largest zones of inhibition (disk diffusion) and the lowest BMD and Etest MICs were observed for azithromycin, clarithromycin, linezolid, mupirocin, doxycycline, minocycline, and tigecycline. The in vitro activities observed suggest that antibacterials, which act by inhibiting protein synthesis, are promising candidate therapies for the treatment of pythiosis.
TEXT
Pythiosis is a life-threatening and chronic pyogranulomatous disease caused by the fungus-like pathogen Pythium insidiosum, the main oomycete species capable of infecting humans and other animals (1, 2). Although there have been a few reports of clinical cures of cases of pythiosis with antifungal therapy (3, 4), the data from the literature on the clinical management of pythiosis patients with treatment by antifungals indicate that such therapy has been largely ineffective (2, 5, 6). The genus Pythium is unable to synthesize ergosterol, the active target of most antifungals, which partly explains why this class of drugs has been ineffective (2).
Studies on the in vitro susceptibility of the clinical isolates of P. insidiosum to antifungal drugs have shown divergent results, and there are no international protocols approved for evaluating the in vitro susceptibility of P. insidiosum. Interestingly, previous studies have shown that P. insidiosum is quite sensitive to antibacterials belonging to the classes of macrolides, tetracyclines, and glycylcyclines (7, 8) and that its combination with antifungal drugs can result in synergistic interactions in vitro (9). However, the effects of antibacterial agents that inhibit protein synthesis on P. insidiosum have not been studied extensively. Thus, the objective of this study was to compare the in vitro susceptibilities of P. insidiosum to a number of antibacterial and antifungal drugs using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth microdilution, CLSI M51-A disk diffusion, and Etest methods.
We evaluated the susceptibility of 25 Brazilian P. insidiosum strains isolated from equine pythiosis lesions. All of the isolates were identified using a PCR-based assay according to Botton et al. (10). The reference strains included P. insidiosum CBS 101555 from an equine pythiosis case and P. insidiosum CBS 119452 and Pythium aphanidermatum CBS 128995 from human pythiosis cases.
The broth microdilution (BMD) reference assay was carried out following the CLSI M38-A2 guidelines (11), as previously described (12, 13). The final concentrations of the antimicrobial agents tested in the wells ranged from 1,024 to 0.5 μg/ml for mupirocin and tobramycin and from 256 to 0.125 μg/ml for azithromycin, clarithromycin, clindamycin, chloramphenicol, doxycycline, erythromycin, florfenicol, fluconazole, fusidic acid, lincomycin, linezolid, minocycline, roxithromycin, terbinafine, tetracycline, tigecycline, and tilmicosin. The final concentrations for amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole ranged from 32 to 0.015 μg/ml. All standard antimicrobial powders were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MIC of each agent was determined by visual observation and represents the inhibition of 100% of mycelium growth after 24 h and 48 h of incubation at 37°C.
Susceptibilities of the isolates to antibacterial and antifungal drugs were also determined by the Etest (AB bioMérieux, Solna, Sweden) method according to the manufacturer's recommendations and by the disk diffusion reference (CLSI M51-A) assay (14). A P. insidiosum inoculum at a concentration of 3 to 5 × 104 zoospores/ml was set as the optimal inoculum size for the Etest and disk diffusion assay. Briefly, the entire surface of each 100-mm-diameter nonsupplemented Mueller-Hinton (MH) agar (pH 7.3 ± 0.1) (Hi-Media Laboratories, India) plate was inoculated with 200 μl of the inoculum suspension and spread with a Drigalsky spreader. Excess surface moisture was removed with a sterile pipette. Etest strips (listed in Table 1) and the disks (Sensifar, São Paulo, Brazil) containing 2 μg of clindamycin and lincomycin; 5 μg of mupirocin; 10 μg of gentamicin, neomycin, streptomycin, and tobramycin; 15 μg of azithromycin, clarithromycin, erythromycin, roxithromycin, tigecycline, and tilmicosin; and 30 μg of chloramphenicol, doxycycline, florfenicol, linezolid, minocycline, and tetracycline were placed on the surface of each plate of inoculated agar and incubated for 24 to 48 h at 37°C, after which the zone diameters for disk diffusion testing and the MICs for the Etest strips were recorded. Susceptibility endpoints were read at the intersection of the scale of the strip with the first completely clear ellipse (Etest) and clear inhibition zone (disk diffusion). Off-scale MICs were transformed to the next highest dilution matching the scale for calculating the geometric mean and the essential agreement. The MIC pairs obtained using the BMD and Etest methods were evaluated to either be in essential agreement or show nonsubstantial or substantial differences (15). CLSI and Etest MIC90s (MICs at which 90% of isolates were inhibited) were also determined.
TABLE 1.
In vitro susceptibilities of 27 isolates of Pythium insidiosum and Pythium aphanidermatum CBS 128995 to antibacterials and antifungal agentsd
| Antimicrobial agent | Results with CLSI M38-A2 method: |
Results with Etest method: |
% EA ata: |
% of isolates showing any discrepancyb |
Zone diameter by disk diffusion techniquec (range [mean]) (mm) at: |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC90 (μg/ml) at: |
MIC (range [GM]) (μg/ml) at: |
MIC90 (μg/ml) at: |
MIC (range [GM]) (μg/ml) at: |
NSD at: |
SD at: |
|||||||||||
| 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | 24 h | 48 h | |
| Antibacterials | ||||||||||||||||
| Azithromycin | 8 | 16 | 1–8 (2.9) | 1–16 (3.9) | 4 | 4 | 0.03–4 (0.7) | 0.03–16 (1.0) | 78 | 75 | 18 | 18 | 4 | 7 | 14–40 (29.2) | 14–40 (27.7) |
| Chloramphenicol | 128 | 256 | 4->256 (23.1) | 4->256 (52.5) | 256 | >256 | 2->256 (25.6) | 8->256 (53.8) | 93 | 82 | 3.5 | 14 | 3.5 | 4 | NZ–40 (26.3) | NZ–40 (23.8) |
| Clarithromycin | 8 | 8 | 0.25–8 (1.8) | 0.25–8 (3.1) | 8 | 32 | 0.5–16 (2.4) | 0.5–32 (3.9) | 93 | 93 | 7 | 7 | 0 | 0 | 20–38 (28.3) | 12–36 (25.4) |
| Clindamycin | 64 | 256 | 4 -> 256 (16) | 4->256 (26.9) | 64 | 256 | 2–256 (7.6) | 2->256 (14.5) | 82 | 79 | 14 | 17 | 4 | 4 | NZ–21 (11.5) | NZ–21 (9.4) |
| Doxycycline | 8 | 16 | 1–8 (3.3) | 2–16 (6.4) | 4 | 16 | 1–8 (2.3) | 2–16 (5.8) | 100 | 100 | 0 | 0 | 0 | 0 | 22–38 (30.0) | 20–38 (29.4) |
| Erythromycin | 32 | 64 | 1–32 (7.7) | 2–64 (15.5) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ–34 (22.9) | NZ–34 (21.2) |
| Florfenicol | 128 | >256 | 8->256 (25.1) | 16->256 (50.2) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ–39 (28.6) | NZ–36 (25.6) |
| Gentamicin | ND | ND | ND | ND | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ | NZ |
| Fusidic acid | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
| Lincomycin | >256 | >256 | >256 | >256 | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ | NZ |
| Linezolid | 16 | 32 | 1–32 (5.6) | 4–32 (8.8) | 4 | 4 | 0.5–8 (1.7) | 0.5–8 (2.0) | 86 | 75 | 14 | 25 | 0 | 0 | 18–46 (31.5) | 18–40 (29.3) |
| Minocycline | 2 | 4 | 0.125–4 (0.9) | 0.25–4 (1.6) | 1 | 2 | 0.06–4 (0.2) | 0.06–4 (0.4) | 82 | 82 | 18 | 18 | 0 | 0 | 21–40 (31.9) | 20–40 (31.5) |
| Mupirocin | 16 | 32 | 2–32 (3.2) | 2–32 (6.9) | 2 | 4 | 0.125–2 (0.6) | 0.125–4 (1.0) | 75 | 79 | 21 | 18 | 4 | 3 | 20–32 (25.8) | 16–32 (23.6) |
| Neomycin | ND | ND | ND | ND | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ | NZ |
| Roxithromycin | 32 | 128 | 2–128 (9.7) | 4–128 (20.6) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | 10–34 (18.9) | NZ–30 (14.6) |
| Streptomycin | ND | ND | ND | ND | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ | NZ |
| Synercid | ND | ND | ND | ND | >32 | >32 | 0.5->32 (5.8) | 0.5->32 (6.9) | NA | NA | NA | NA | NA | NA | ND | ND |
| Tetracycline | 16 | 32 | 1–32 (7.4) | 4–32 (16) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | 11–42 (27.4) | 10–38 (25.1) |
| Tigecycline | 4 | 4 | 0.25–4 (1.3) | 0.5–4 (2) | 1 | 1 | 0.03–4 (0.2) | 0.03–4 (0.3) | 68 | 64 | 32 | 32 | 0 | 4 | 23–40 (32.2) | 22–40 (33.4) |
| Tilmicosin | 128 | 128 | 4–128 (27.6) | 8–128 (42.8) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | NZ–28 (17.6) | NZ–28 (15.2) |
| Tobramycin | >1,024 | >1,024 | >1,024 | >1,024 | >1,024 | >1,024 | >1,024 | >1,024 | 100 | 100 | 0 | 0 | 0 | 0 | NZ | NZ |
| Antifungals | ||||||||||||||||
| Amphotericin B | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
| Anidulafungin | ND | ND | ND | ND | >32 | >32 | >32 | >32 | NA | NA | NA | NA | NA | NA | ND | ND |
| Caspofungin | >32 | >32 | 16->32 (47.5) | 16->32 (60.9) | >32 | >32 | 4->32 (39.0) | 4->32 (46.3) | 96 | 96 | 4 | 4 | 0 | 0 | ND | ND |
| Fluconazole | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
| Micafungin | ND | ND | ND | ND | >32 | >32 | >32 | >32 | NA | NA | NA | NA | NA | NA | ND | ND |
| Itraconazole | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
| Posaconazole | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
| Terbinafine | 16 | 32 | 2–16 (8.0) | 4–64 (13.3) | ND | ND | ND | ND | NA | NA | NA | NA | NA | NA | ND | ND |
| Voriconazole | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 100 | 100 | 0 | 0 | 0 | 0 | ND | ND |
Essential agreement (EA) (discrepancies of no more than ±2 2-fold dilutions) between the CLSI and Etest methods.
Nonsubstantial differences (NSD) (discrepancies of 3 or 4 2-fold dilutions) or substantial differences (SD) (discrepancies of >4 2-fold dilutions) with respect to the BMD method.
Not performed for P. insidiosum CBS 101555, P. insidiosum CBS 119452, or P. aphanidermatum CBS 128995 control strains.
Abbreviations: GM, geometric mean; ND, not determined; NA, not applicable; NZ, no zone.
Table 1 summarizes the in vitro susceptibility of the Pythium isolates determined using the BMD, disk diffusion, and Etest methods. Tigecycline, minocycline, azithromycin, and clarithromycin were considered the most effective drugs tested because they required the lowest concentrations (geometric mean MICs of <4 μg/ml) for the inhibition of the Pythium species. For all strains, the lowest MIC90 values (≤4 μg/ml after 48 h) were observed for tigecycline, minocycline, azithromycin, linezolid, and mupirocin using the BDM and the Etest methods. Lower essential agreements (64 to 86%) were observed for azithromycin, chloramphenicol, clindamycin, linezolid, minocycline, mupirocin, and tigecycline. Excellent essential agreements (93 to 100%) were observed for the other MIC comparisons.
The growth of Pythium spp. was not inhibited by amphotericin B, anidulafungin (Etest), micafungin (Etest), itraconazole, posaconazole, or voriconazole (MICs, >32 μg/ml), fluconazole, fusidic acid, or lincomycin (MICs, >256 μg/ml), or tobramycin (MICs, >1,024 μg/ml). Inhibition zones were observed for all of the selected disks with the exception of lincomycin and the aminoglycoside antimicrobials. After 48 h, the highest ranges in halos (zone diameter range in mm [arithmetic mean in mm]) were observed for azithromycin (14 to 40 [27.7]), clarithromycin (12 to 36 [25.4]), linezolid (18 to 40 [29.3]), mupirocin (16 to 32 [23.6]), doxycycline (20 to 38 [29.4]), minocycline (20 to 40 [31.5]), tetracycline (10 to 38 [25.1]), and tigecycline (22 to 40 [33.3]).
In this study, we describe antibacterial drugs that inhibit the in vitro growth of P. insidiosum at concentrations 100 times lower than those observed for previously studied antifungal drugs. It is noteworthy that antibacterial drugs such as azithromycin, minocycline, and tigecycline can inhibit the growth of P. insidiosum isolates in vitro at concentrations of <1 μg/ml, while the MICs for amphotericin B, echinocandins, and triazole antifungals are >32 μg/ml.
Previous studies have determined the in vitro susceptibility of P. insidiosum to antifungal and antibacterial agents using three main assays. In the first of these assays, antimicrobial agents are added to the culture medium, and the changes in the radial growth (16) or the mycelium weight (17) are measured. The second assay, the broth macrodilution or microdilution technique, uses a hyphal suspension scraped from the culture medium, which is adjusted spectrophotometrically (13, 18). The third method, macrodilution (12, 19–21) or microdilution (7, 8, 22, 23) in broth, utilizes the CLSI M38-A2 protocols (11) and inocula with final concentrations of 2 to 3 × 103 zoospores/ml. Additionally, some studies have reported that P. insidiosum isolates were insensitive or resistant to all of the antimicrobials evaluated (24–27) or described the MICs obtained by in vitro susceptibility tests (3, 4, 28) without describing how the inocula were prepared or how the susceptibility test was performed. This diversity in susceptibility testing techniques likely correlates with the diversity of MICs observed for P. insidiosum (see Table S2 in the supplemental material). The phylogenetic diversity, especially among the clinical isolates from the Americas and Asia (29–31), may be another factor that is related to the different susceptibility profiles of P. insidiosum.
This study represents the first assessment of P. insidiosum susceptibility using the disk diffusion technique based on the CLSI M51-A method as well as the first comparison of antimicrobial susceptibility testing between the Etest and CLSI M38-A2 methods. The established pharmacology and safety of the antibacterials described in this work support their consideration as new therapeutic options for the treatment of pythiosis infections. However, it is important to note that in vivo experimental testing of the disease in animal models is needed to determine the actual therapeutic potential of these drugs and their associations with treatments by other antimicrobial agents before they can be used safely in the clinic.
In conclusion, the in vitro data reported here suggest that antibacterial drugs that act by inhibiting bacterial protein synthesis merit attention as new candidates for the treatment of pythiosis infections. Therefore, additional studies using geographically and genetically diverse P. insidiosum strains, in addition to in vitro and in vivo susceptibility studies with combinations of antimicrobials, are needed for a better understanding of the susceptibility of this species to the antibacterials described in this study.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES-AUX PE-PNPD 743/2012). E.S.L. is a Ph.D. fellow of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (PNPD-CAPES).
Footnotes
Published ahead of print 15 September 2014
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02680-13.
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