EDTA inhibits Rhizopus oryzae (Rhizopus arrhizus) growth in vitro in blood of diabetic and non-diabetic individuals - is it an option for combined therapy with antifungal agents in the future?

Grace Salazar, Esaú López Jácome, Dora Corzo León, Rafael Valdez Vázquez, Ana Patricia Rodriguez Zulueta, Rafael Franco Cendejas, Jesus Resendiz-Sanchez

Abstract: 

Background: The antimicrobial effect of EDTA has been described previously on bacteria, Candida and, Aspergillus species. The iron chelating therapy seems as a promising option in combination with antifungal agents for the treatment of mucormycosis in diabetic individuals. However, the role of EDTA has not been described previously in R. oryzae.

Material/methods: We recruited 95 individuals, 65 with diabetes and 30 without diabetes. Exclusion criteria were previous infection, antibiotic use, chronic kidney disease, neutropenia or malignant disease. From each individual, 2 blood samples were obtained, one in a tube with EDTA (Vacutainer® BD K2) and the other one in a tube without reagents (Vacutainer® BD). Each sample was mechanically lysed and freezing. Using a clinical isolate strain well characterized of R. oryzae we prepared the inoculum according to CLSI M38A recommendations and incubated in EDTA and nonEDTA tubes for each individual. Fungal growth (number of conidia/mm3 , and millimeters), budding conidia and hyphae production were evaluated at 3, 6, 12, 24 hours of incubation. Neubauer chamber and growth on SDA (Sabouraud agar) were used to perform these measures. The results were analysed with Wilcoxon matched-pair signed-rank and Mann-Whitney U test, p value ≤ 0.05 was considered significant.

Results: The amount of conidia/mm3 did not change in samples with EDTA after 24 hrs of incubation (0 hrs= 265 vs 24hrs= 240; p=0.36). On the other hand, in the non-EDTA samples, the number of conidias decreased (0 hrs= 267 vs 24 hrs= 20; p< 0.001) due to the increasing percent of budding conidia (EDTA 3 hrs= 1,9 vs non-EDTA 3 hrs= 30,2; p< 0.001 and EDTA 6 hrs= 4,8 vs non-EDTA 6 hrs= 38; p< 0.001). We did not observe hyphae production in microscopic analysis after 24 hrs in EDTA group compared with non-EDTA group (p=0.001). Macroscopic growth on SDA at 24 hrs was greater in the non-EDTA group compared with EDTA group (non-EDTA= all the samples grew up at a maximum of 85 mm vs EDTA group= median 40mm {0-72 mm}, p=0.001). No differences were found in the effect of EDTA between groups with and without diabetes.

Conclusions: Our findings show that EDTA inhibits the hyphae formation in R. oryzae in diabetics and non-diabetic individuals including those with glycaemic levels uncontrolled. The use of EDTA could have future applications in mucormycosis treatment but further studies are needed.

2016

Poster: 

AttachmentSize
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abstract No: 

#4713

Full conference title: 

26th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 26th (2016)