In Vitro Susceptibility of Pythium insidiosum Isolates to Aminoglycoside Antibiotics and Tigecycline

Abstract

This study evaluated the in vitro activity of aminoglycoside antibiotics and tigecycline against Pythium insidiosum. The susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M38-A2. MIC values for gentamicin, neomycin, paromomycin, and streptomycin ranged from 32 to 64 mg/liter, and the minimal fungicidal concentration (MFC) ranged from 32 to 128 mg/liter, which are incompatible with safe concentrations of these drugs in plasma in vivo. Tigecycline showed the lowest MIC (0.25 to 2 mg/liter) and MFC (1 to 8 mg/liter) range values. The in vitro susceptibility observed to tigecycline makes this drug a good option in future tests in vitro and in vivo for the management of pythiosis.

TEXT

Pythiosis is a chronic disease that affects humans, mammals, and birds and is caused by the aquatic oomycete Pythium insidiosum. It occurs in tropical, subtropical, and temperate regions, and there have been no reports of direct transmission among animals or between animals and humans. Cutaneous, vascular, ocular, and systemic forms (humans) and subcutaneous and gastrointestinal forms (horses, dogs, cattle, cats, and sheep) are the clinical presentations most commonly observed (3).

Pythiosis progresses rapidly and can become life threatening if it is not treated in the early stages. The absence of ergosterol in the cell wall of this oomycete restrains the treatment of pythiosis with antifungal therapy due to the fact that most antifungal drugs act by inhibiting the synthesis of ergosterol. Radical surgery and immunotherapy are among the best therapeutic options, but the improved cure rates are directly associated with early diagnosis of pythiosis (14).

Members of the genus Pythium are known to be susceptible to some antibiotics of the tetracycline, macrolide, aminoglycoside, and amphenicol classes (5, 6, 8, 12). McMeekin and Mendoza (9) reported that streptomycin may have a stimulatory or inhibitory effect on P. insidiosum depending on the culture conditions, and Loreto et al. (7) described an in vitro inhibitory activity of minocycline against P. insidiosum. In this context, this study aims to evaluate the in vitro susceptibility of the aminoglycosides gentamicin, neomycin, paromomycin, and streptomycin and the minocycline-derived tigecycline against isolates of P. insidiosum.

Twenty-three clinical Brazilian isolates of P. insidiosum from equine pythiosis cases and the ATCC 58.637 reference strain were tested in this study. The identities of the isolates were confirmed by a PCR-based assay (4).

Gentamicin, neomycin, paromomycin, and streptomycin were obtained from Sigma Chemical Co. (St. Louis, MO). Tigecycline (Tygacil; Pfizer) was purchased commercially. The drugs were dissolved in dimethyl sulfoxide to obtain stock solutions (12,800 mg/liter) and stored at −70°C.

At present, there is no standardized susceptibility testing method for oomycetes. Because P. insidiosum produces hyphae and based on the results of previous in vitro and in vivo studies (7, 11), the susceptibility test was performed according to the CLSI M38-A2 protocol for filamentous fungi (2, 11). Briefly, working solutions were prepared by diluting the stock solutions in RPMI 1640 broth, pH 7.0, in the same way as recommended for antifungal agents. The zoospores of P. insidiosum do not produce turbidity as the conidia of filamentous fungi do. Thus, the final inoculum concentrations (2 × 10³ to 3 × 10³ zoospores/ml—the same density needed for testing dermatophyte molds) were obtained by the zoosporogenesis induction technique as previously described (15), which allows the production of abundant mycelium after 24 h of incubation at 37°C. The MICs were determined by visual observation and represent inhibition of 100% of mycelium growth after 24 h of incubation at 37°C. The minimum fungicidal concentrations (MFCs) were determined by transferring all the volume of the wells without mycelium growth to tubes containing 1 ml of Sabouraud dextrose broth and incubating for up to 96 h at 37°C. All experiments were performed in triplicate.

The in vitro susceptibility of 24 P. insidiosum isolates against gentamicin, neomycin, paromomycin, streptomycin, and tigecycline are listed in Table 1. For gentamicin, neomycin, paromomycin, and streptomycin, the MIC ranged from 32 to 64 mg/liter (the geometric mean [GM] ranged from 49.3 to 55.3 mg/liter), and the MFC ranged from 32 to 128 mg/liter (the GM ranged from 62.1 to 73.9 mg/liter). Tigecycline showed the lowest MIC (0.25 to 2 mg/liter; GM, 0.9 mg/liter) and MFC (1 to 8 mg/liter; GM, 2.4 mg/liter) ranges. The results indicated that 64 mg/liter of gentamicin, neomycin, paromomycin, or streptomycin was the concentration able to inhibit the growth (inhibitory and fungicidal effect) of at least 62.5% of all isolates. For tigecycline, a concentration of 1 mg/liter was able to inhibit the growth of 70.8% of the isolates, and 2 mg/liter showed a fungicidal effect in 50% of isolates.

Table 1.

MIC and MFC results for aminoglycosides and tigecycline against 24 Pythium insidiosum isolates^a

Antibiotic and parameter	No. of isolates (%) with the following MIC or MFC (mg/liter):										MIC or MFC range (mg/liter)	GM (mg/liter)
	0.25	0.5	1	2	4	8	16	32	64	128
Paromomycin
MIC	0	0	0	0	0	0	0	9 (7.5)	15 (62.5)	0	32–64	49.3
MFC	0	0	0	0	0	0	0	2 (8.3)	15 (62.5)	7 (29.2)	32–128	73.9
Gentamicin
MIC	0	0	0	0	0	0	0	5 (20.8)	19 (79.2)	0	32–64	55.3
MFC	0	0	0	0	0	0	0	2 (8.3)	17 (70.8)	4 (16.7)	32–128	69.7
Neomycin
MIC	0	0	0	0	0	0	0	5 (20.8)	19 (79.2)	0	32–64	55.3
MFC	0	0	0	0	0	0	0	2 (8.3)	15 (62.5)	7 (29.2)	32–128	73.9
Streptomycin
MIC	0	0	0	0	0	0	0	8 (33.3)	16 (66.7)	0	32–64	50.7
MFC	0	0	0	0	0	0	0	3 (12.5)	17 (70.8)	3 (12.5)	32–128	62.1
Tigecycline
MIC	2 (8.3)	2 (8.3)	17 (70.8)	3 (12.5)	0	0	0	0	0	0	0.25–2	0.9
MFC	0	0	10 (41.7)	2 (8.3)	7 (29.2)	5 (20.8)	0	0	0	0	1–8	2.4

^aMFC, minimum fungicidal concentration; GM, geometric mean.

Currently, there is no “gold standard” treatment for the management of pythiosis. Therapeutic success is directly related to rapid diagnosis followed by treatment with surgical removal of the affected area, immunotherapy and/or antimicrobial therapy. Salipante et al. (13) reported an above-the-knee amputation in a case of advanced human pythiosis even after the patient had received extensive surgical debridement and treatment with liposomal amphotericin B, posaconazole, micafungin, terbinafine, and minocycline, which reinforces the challenge in the treatment of pythiosis.

McMeekin and Mendoza (9) reported that cultures of P. insidiosum in Sabouraud broth supplemented with streptomycin (100 mg/liter or 200 mg/liter) have a variable level of inhibition or stimulation of growth. In our study, using zoospores of P. insidiosum as the inoculum, we observed the need for concentrations between 32 and 128 mg/liter of the aminoglycoside antibiotics tested as necessary for the growth inhibition of P. insidiosum. Taken together, these results demonstrate that P. insidiosum, depending on in vitro culture conditions and drug concentration, can be inhibited by aminoglycoside antibiotics. However, serum levels of these compounds are a bottleneck for the clinical treatment of pythiosis. The therapeutic serum levels of this class of antibiotics are generally below 30 mg/liter, and the accumulation of higher doses can induce toxicity (1).

Tigecycline has an extensive distribution into tissues, a half-life ranging from 37 to 67 h, and a peak serum concentration after a 1-h infusion ranging from 0.109 mg/liter after a single dose of 12.5 mg to 2.817 mg/liter after a dose of 300 mg (10). These pharmacological properties, together with the results of our study, where concentrations of ≤2 mg/liter inhibited P. insidiosum isolates in vitro, suggest that tigecycline is a drug that should be considered for further P. insidiosum in vitro and in vivo assays alone and in combination with other drugs.

In conclusion, P. insidiosum demonstrated a dose-dependent in vitro inhibition by aminoglycoside antibiotics and tigecycline. Comparing the pharmacokinetic and pharmacodynamic profiles described for aminoglycosides and tigecycline (1, 10) and the data obtained in this study, MICs of aminoglycosides against P. insidiosum are incompatible with safe plasma concentrations of these drugs in vivo but compatible with the effective plasma concentrations achieved by tigecycline. The evaluation of the susceptibility profile of tigecycline against P. insidiosum based on drug combination, in vivo experimental pythiosis, and with geographically and genetically diverse P. insidiosum strains could elucidate the potential of this drug in the treatment of pythiosis.