In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum

Abstract

We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC₉₀ between 2 and 32 μg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.

TEXT

Pythium insidiosum is the main oomycete pathogen associated with severe diseases in humans and animals, particularly horses and dogs. In humans, clinical presentations include ocular, cutaneous∕subcutaneous, vascular, and disseminated pythiosis. While underlying diseases, such as thalassemia and hemoglobinopathy syndrome, have been observed in cutaneous, vascular, and disseminated forms of disease, ocular pythiosis can affect otherwise healthy individuals. Moreover, no underlying diseases have been observed with animal pythiosis, for which most cases are associated with pyogranulomatous disease in subcutaneous tissue or in a gastrointestinal form (1, 2).

Treating pythiosis remains a challenge because this microorganism produces hyaline hyphae similar to those of true fungi but is unable to synthesize ergosterol, which is the direct or indirect main target of most antifungal drugs. Consequently, there are no definitive treatment protocols for this disease, and despite some contradictory results, P. insidiosum can be considered intrinsically resistant to most antifungal drugs (2–4). The most commonly used effective clinical management of pythiosis is aggressive surgical resection, such as amputation, and surgical debridement of skin lesions. Unfortunately, surgical interventions are not always possible, and a high rate of recurrence has been observed. Additionally, immunotherapy cure rates can reach approximately 55% and 80% in humans and horses, respectively (2, 3, 5). Independent of the use of single or combination therapies, the best cure rates are associated with rapid diagnosis and early treatment (1, 2). Regardless of the therapy of choice, little attention has been given to the specific topical treatment of pythiosis lesions.

Considering the high tissue concentrations achieved by azithromycin and terbinafine and the above context, this study aimed to assess the in vitro activity for combinations of azithromycin or terbinafine (systemic therapeutic options) and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate (topical therapeutic options).

We evaluated the susceptibility of 20 isolates of P. insidiosum from equine pythiosis cases and the reference strain ATCC 58637. Clinical isolates were previously identified by a nested PCR assay (6). The drugs (final concentrations tested in the wells) azithromycin (0.06 to 32 μg∕ml), benzalkonium chloride (0.25 to 32 μg∕ml), alkyltrimethylammonium bromide (cetrimide) (0.5 to 32 μg∕ml), cetylpyridinium chloride (1 to 64 μg∕ml), mupirocin (0.5 to 32 μg∕ml), potassium permanganate (1 to 64 μg∕ml), terbinafine hydrochloride (1 to 64 μg∕ml), and triclosan (8 to 0.125 μg∕ml) were purchased as standard powders from Sigma-Aldrich (St. Louis, MO). Broth microdilution susceptibility tests were performed following the CLSI M38-A2 protocol (7) as previously described (8, 9). The MICs were determined by visual observation and represented the inhibition of 100% of mycelium growth after 48 h of incubation at 37°C.

The interactions between azithromycin or terbinafine with benzalkonium, cetrimide, cetylpyridinium, mupirocin, potassium permanganate, and triclosan were evaluated using a checkerboard test (10). The lowest fractional inhibitory concentration index (FICI) was determined from the nonturbid wells along with the turbidity-nonturbidity growth interface after 48 h of incubation at 37°C and interpreted as follows: FICI ≤ 0.5, synergism; FICI > 0.05 to ≤4, indifference; FICI > 4, antagonism.

The results of the in vitro susceptibility and combination tests are shown in Table 1. The growth of P. insidiosum isolates was not inhibited by potassium permanganate alone (MIC > 64 μg∕ml). MIC values (geometric means) (in μg∕ml) ranged from 2 to 16 (4.68) for azithromycin and 4 to 32 (14.56) for terbinafine. Topical antimicrobial drug MICs (in µg/ml) ranged from 0.5 to 2 (1.25) for triclosan, 1 to 8 (2.49) for mupirocin, 2 to 8 (3.53) for cetylpyridinium, 4 to 8 (5.31) for benzalkonium, and 4 to 16 (7.75) for cetrimide.

TABLE 1.

In vitro activities of azithromycin and terbinafine, alone or in combination with topical antimicrobial drugs, against 21 Pythium insidiosum isolates

Antimicrobial agenta	Individual drugs		Drug combinations			Interpretation (%)
	MIC90 (in µg/ml)	MIC range (GM)b (μg/ml)	Drugs	Associated MIC ranges (GMs) (μg/ml)	Mean FICI range (GM) (μg/ml)	Synergistic	Indifferent
Systemic drugs			AZT + BAC	0.25–4 (1.07) + 2–4 (2.57)	0.38–1 (0.75)	4.8	95.2
AZT	8	2–16 (4.68)	AZT + CET	1–8 (2.13) + 2–8 (3.53)	0.50–1 (0.91)	14.3	85.7
TRB	32	4–32 (14.56)	AZT + CPC	0.25–8 (1.25) + 1–4 (1.66)	0.50–1 (0.76)	10	90
Topical drugs			AZT + MUP	1–4 (2.06) + 0.25–2 (1.10)	0.50–1.5 (0.91)	19	81
BAC	8	4–8 (5.31)	AZT + TCS	0.06–8 (0.60) + 0.25–1 (0.53)	0.38–1 (0.62)	19	81
CET	16	4–16 (7.75)	TRB + BAC	0.25–8 (2.34) + 0.5–4 (1.82)	0.19–1 (0.52)	38.1	61.9
CPC	8	2–8 (3.53)	TRB + CET	0.125–8 (1.76) + 0.5–4 (1.94)	0.19–1 (0.38)	71.4	28.6
MUP	4	1–8 (2.49)	TRB + CPC	1–8 (2.27) + 1–4 (1.71)	0.5–1 (0.67)	4.8	95.2
Potassium permanganatec	>64	>64	TRB + MUP	0.06–8 (3.01) + 0.5–4 (1.0)	0.38–1 (0.64)	33.3	66.7
TCS	2	0.5–2 (1.25)	TRB + TCS	0.25–16 (4.13) + 0.25–1 (0.55)	0.50–1 (0.77)	19	81

^aAZT, azithromycin; BAC, benzalkonium; CET, cetrimide; CPC, cetylpyridinium; MUP, mupirocin; TCS, triclosan; TRB, terbinafine.

^bGM, geometric mean.

^c100% of indifferent interactions when combined with azithromycin or terbinafine.

The combinations of antimicrobial drugs showed a predominance of indifferent interactions (>80%) when azithromycin was associated with topical antimicrobials. Similarly, when terbinafine was combined with topical antimicrobials, there was a predominance of indifference (>60%), except for its combination with cetylpyridinium, for which a 71.4% predominance of synergistic interactions was observed. The interaction of azithromycin or terbinafine with potassium permanganate resulted in 100% indifferent interactions (data not shown). Antagonistic interactions were not observed.

In this study, we described systemic and topical antimicrobial drugs that individually inhibit the in vitro growth of P. insidiosum. Almost none of the evaluated antimicrobials has a very low MIC, and MIC values were relatively high for some of them, such as cetrimide and terbinafine. However, the use of these compounds is favored by the high concentration allowed by the topical mode of application. Additionally, although cetylpyridinium and triclosan are widely used as oral antiseptics, they also have potential for the treatment of skin infections (11, 12).

Previous studies demonstrated the in vitro and in vivo susceptibility of P. insidiosum to azithromycin, mupirocin, and terbinafine (8, 13–19) with in vitro results similar to those of our study. Although all other evaluated antimicrobials have a wide range of known antimicrobial activities against bacteria, some fungi, and parasites (20, 21), this study was the first to evaluate the antimicrobial activities of benzalkonium, cetrimide, cetylpyridinium, potassium permanganate, and triclosan against clinically isolated P. insidiosum based on broth microdilution susceptibility tests.

Toxicity data for triclosan from mammalian studies, including those of humans, were been reviewed by Rodricks et al. (12). Triclosan products are not expected to cause adverse health effects, and there has been little indication of toxicity or sensitization. Pharmaceutical formulations containing quaternary ammonium compounds (benzalkonium, cetrimide, and cetylpyridinium) are nontoxic when applied to skin or mucous membranes and are generally well tolerated (22). Azithromycin, mupirocin, and terbinafine have known pharmacology, are known to be safe, and are widely used to treat infections. Azithromycin (23) and terbinafine (24) are generally safe and well tolerated after oral administration, and most cases of toxicity are related to higher doses. Mupirocin is well tolerated with topical treatment, and adverse effects are observed in less than 2% of users (25). Potassium permanganate should always be thoroughly diluted in water (e.g., 1:10,000), as higher concentrations can cause necrosis of the skin (26).

It is important to note that the treatment of pythiosis is complex and, in most cases, requires surgical intervention, immunotherapy, and systemic antimicrobial chemotherapy. Therefore, the use of topical antimicrobials for lesions should be a complementary treatment to the main therapies. Larger in vitro and in vivo susceptibility studies are needed to elucidate the potential of the topical antimicrobials evaluated in this study for the treatment of pythiosis.

In conclusion, the in vitro susceptibility data reported in this study show that the growth of P. insidiosum clinical isolates was inhibited by all antimicrobials evaluated, with the exception of potassium permanganate. Although there was a predominance of indifferent interactions between the combinations of the evaluated antimicrobials, the absence of antagonism and the lack of information regarding the topical therapy of pythiosis suggest that the antimicrobials evaluated have potential as new supplementary topical therapeutic options for the treatment of pythiosis.