Analysis of xanthyletin and secondary metabolites from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 against Pythium insidiosum.

BACKGROUND: Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. RESULTS: Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. CONCLUSIONS: Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.

Evaluation for the clinical diagnosis of Pythium insidiosum using a single-tube nested PCR.

Pythiosis is a rare infectious disease caused by Pythium insidiosum, which typically occurs in tropical and subtropical regions. The high mortality rate may be in consequence of the lack of diagnosis. The objective of this study was to evaluate reliability of a new single-tube nested PCR for detection of P. insidiosum DNA. A total of 78 clinical isolates of various fungi and bacteria, 106 clinical specimens and 80 simulated positive blood samples were tested. The developed primer pairs CPL6-CPR8 and YTL1-YTR1 are located on 18S subunit of the rRNA gene of P. insidiosum. The specificity, negative and positive predictive values were 100, 100 and 87.5 %, respectively, as compared with direct microscopy and cultivation. The detection limit of the single-tube nested PCR was 21 zoospores corresponding to 2.7 pg of the DNA. The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum.

Evaluation of antifungal effect and toxicity of xanthyletin and two bacterial metabolites against Thai isolates of Pythium insidiosum.

Pythiosis is a harmful disease caused by Pythium insidiosum, an aquatic oomycete. Therapeutic protocols based on antifungal drugs are often ineffective because the cytoplasmic membrane of P. insidiosum does not contain ergosterol. Therefore, the treatment of pythiosis is still challenging, particularly making use of natural products and secondary metabolites from bacteria. In this study, xanthyletin and substances obtained from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 exhibited anti-P. insidiosum activity and, moreover, xanthyletin was non-toxic against human cell lines. The hyphae of P. insidiosum treated with these three substances exhibited lysis holes on a rough surface and release of anamorphic material. Therefore, xanthyletin could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.

Coumarinoids from the fruits of Micromelum falcatum.

Four new compounds, microminutin B (1), microminutin C (2), micromarinate (3), and secomicromelin (4) as well as 17 known compounds were isolated from the fruits of Micromelum falcatum. All compounds were evaluated for antifungal activity against Pythium insidiosum using disc diffusion assay. P. insidiosum is a fungus-like microorganism for which antifungal agents now available are not effective. The results show that four compounds including secomicromelin (4), 7-methoxy-8-(4'-methyl-3'-furanyl)coumarin (10), micromarin B (17), and isomicromelin (19) could inhibit the mycelia growth of P. insidiosum.

Detection of diketopiperazine and pyrrolnitrin, compounds with anti-Pythium insidiosum activity, in a Pseudomonas stutzeri environmental strain.

AIMS: Screening of bacterial flora for strains producing metabolites with inhibitory effects on the human pathogenic oomycete Pythium insidiosum. Separation and characterization of extracts from Pseudomonas stutzeri with anti-Pythium inhibitory activity. Search for genes with anti-Pythium effect within the genome of P. stutzeri. METHODS: A total of 88 bacterial strains were isolated from water resources in northeastern Thailand. Two screening methods were used to establish their inhibitory effects on P. insidiosum. One strain, P. stutzeri ST1302 was randomly chosen, and the extract with anti-P. insidiosum activity was fractionated and subfractionated using liquid column chromatography and purified by thin layer chromatography. The chemical structure of purified fractions was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance and mass spectrometry. Further, search for genes involved in the anti-Pythium activity (phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin and pyrrolnitrin) was undertaken in this P. stutzeri strain using primers described in the literature. RESULTS: Anti-P. insidiosum activity was detected in 16 isolates (18.2%). In P. stutzeri ST1302, a subfraction labeled PYK7 exhibited strong activity against this oomycete. It was assigned to the diketopiperazines as cyclo(D-Pro-L-Val). In the search for genes, one gene region was successfully amplified. This corresponded to pyrrolnitrin. The results suggest the possibility of using the related metabolites against P. insidiosum. This is the first report on the inhibitory effects of P. stutzeri against this oomycete. The results may contribute to the development of antimicrobial drugs/probiotics against pythiosis.

Clauraila E from the roots of Clausena harmandiana and antifungal activity against Pythium insidiosum.

A new carbazole alkaloid named clauraila E (1) together with 8 known compounds were isolated from the methanol extract of the roots of Clausena harmandiana. All compounds were evaluated for antifungal activity against Pythium insidiosum using disc diffusion assay. Pythium insidiosum is a fungus-like microorganism, for which antifungals available now are not effective. It was found that compounds 3, 6, 7 and 9 could inhibit the mycelia growth of P. insidiosum. The results show convincingly that they may be lead to compounds for the development of probiotic or novel antifungal drugs.

New lignan esters from Alyxia schlechteri and antifungal activity against Pythium insidiosum.

Three new lignan esters, alyterinates A-C (1-3), as well as 10 known compounds were isolated from the roots of Alyxia schlechteri. Antifungal activity against Pythium insidiosum of all lignan derivatives was evaluated using disk diffusion assay. P. insidiosum is not a true fungus since its cell walls do not contain ergosterol as usual fungi, so the antifungals available now are not effective. From activity testing, it was found that compounds 3, 4 and 5 could inhibit the mycelia growth of P. insidiosum.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.