In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals

Fonseca, Anelise O.S.; Pereira, Daniela Isabel Brayer; Filho, F. S. Maia; Osório, L.G.; Maroneze, Beatriz Persici; Valente, Júlia de Souza Silveira; Pötter, Luciana; Meireles, Mário Carlos Araújo

doi:10.1093/jac/dku021

Cited by 18 publications

(27 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, in our hands, generation of zoospores from Thai strains is difficult and labor-intensive and did not provide enough zoospores for the susceptibility testing. Likewise, hyphal suspensions, prepared as described by other investigators (15,30,31) were not reproducibly uniform in our hands (data not shown). As an alternative, we have used the method of Brown and coworkers (32), starting with agar plugs of active hyphal growth and measuring radial growth of the hyphae.…”

Section: Discussionmentioning

confidence: 41%

“…insidiosum, respectively. To completely inhibit the growth of 50% of the isolates tested required at least 16 mg/liter of itraconazole and 4 mg/liter of terbinafine in the studies by Argenta et al (34) and at least 64 mg/liter of itraconazole and 8 mg/liter of terbinafine in those by Fonseca et al (30), while our group found that more than 128 mg/liter of both drugs was required. The different inhibitory effects of the two drugs against Py.…”

Section: Discussionmentioning

confidence: 58%

“…insidiosum. In our preliminary work to develop such a test, we considered using zoospores or hyphal suspensions as inocula (29,30). However, in our hands, generation of zoospores from Thai strains is difficult and labor-intensive and did not provide enough zoospores for the susceptibility testing.…”

Section: Discussionmentioning

confidence: 99%

“…insidiosum (30), we have chosen to use Sabouraud dextrose (SD) agar for these assays. Since a particular medium could interfere with antifungal drug activity, this may be a concern.…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

Lerksuthirat

Sangcakul

Lohnoo

et al. 2017

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum. However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches. It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum. We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum. In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

show abstract

Section: Discussionmentioning

confidence: 41%

Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

“…insidiosum (30), we have chosen to use Sabouraud dextrose (SD) agar for these assays. Since a particular medium could interfere with antifungal drug activity, this may be a concern.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

Lerksuthirat

Sangcakul

Lohnoo

et al. 2017

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…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 g/ml for mupirocin and tobramycin and from 256 to 0.125 g/ml for azithromycin, clarithromycin, clindamycin, chloramphenicol, doxycycline, erythromycin, florfenicol, fluconazole, fusidic acid, lincomycin, linezolid, minocycline, roxithromycin, terbinafine, tetracycline, tigecycline, and tilmicosin.…”

mentioning

confidence: 99%

New Insights into the In Vitro Susceptibility of Pythium insidiosum

Loreto

Tondolo

Pilotto

et al. 2014

Antimicrob Agents Chemother

View full text Add to dashboard Cite

bWe 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. P ythiosis 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 g/ml for mupirocin and tobramycin and from 256 to 0.125 g/ml for azithromycin, clarithromycin, clindamycin, chloramphenicol, doxycycline, erythromycin, florfenicol, fluconazole, fusidic acid, lincomycin, linezolid, minocycline, roxithromycin, terbinafine, tetracycline, tigecycline, and tilmicosin. The final conce...

show abstract

In Vitro Susceptibility of Pythium insidiosum to Melaleuca alternifolia, Mentha piperita and Origanum vulgare Essential Oils Combinations

et al. 2016

View full text Add to dashboard Cite

Pythium insidiosum is the etiologic agent of pythiosis, a severe and emerging disease that affects mammals. Failure of conventional antifungal therapies is partially justified by the absence of ergosterol in the plasma membrane of this oomycete. Despite research advancement, the treatment of pythiosis has not been not fully established. The present study investigated the in vitro susceptibility profile of Brazilian isolates of P. insidiosum (n = 20) against Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils, and their combinations. Susceptibility tests were performed according to CLSI M38-A2 protocol, and combinations were evaluated by the microdilution cherkerboard method. All tested essential oils showed antimicrobial activity against P. insidiosum, and the greatest activity of O. vulgare was highlighted. Synergistic and/or indifferent effect was observed for all combinations evaluated, especially the M. piperita and O. vulgare combination, which showed 65 % synergism. This is the first study to report in vitro combinations of essential oils against P. insidiosum indicating the susceptibility of this oomycete to M. alternifolia, M. piperita and O. vulgare essential oils, as well as their combinations.

show abstract

In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals

Abstract: We conclude that the use of hyphal inocula of P. insidiosum for in vitro susceptibility tests could be a suitable method for evaluating antimicrobial susceptibility, particularly when it is not possible to obtain a standardized zoospore inoculum.

Cited by 18 publications

References 17 publications

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance

New Insights into the In Vitro Susceptibility of Pythium insidiosum

In Vitro Susceptibility of Pythium insidiosum to Melaleuca alternifolia, Mentha piperita and Origanum vulgare Essential Oils Combinations

Contact Info

Product

Resources

About