Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Molecular detection of Pythium insidiosum from soil in Thai agricultural areas.

Pythium insidiosum is an aquatic fungus-like organism in the kingdom Stramenopila that causes pythiosis in both humans and animals. Human pythiosis occurs in ocular, localized granulomatous subcutaneous and systemic or vascular forms. Individuals whose occupations involve exposure to aquatic habitats have an elevated risk of contracting pythiosis. Previously, we reported the first successful isolation of Pythium insidiosum from aquatic environmental samples by culture including confirmation using molecular methods. In this study, we show that P. insidiosum inhabitats moist soil environments in agricultural areas. A total of 303 soil samples were collected from 25 irrigation sources in the areas nearby the recorded home addresses of pythiosis patients residing in northern provinces of Thailand. P. insidiosum DNA was identified directly from each soil extract by using a nested PCR assay and subsequent phylogenetic analysis of the ribosomal intragenic spacer region. P. insidiosum DNA could be detected from 16 of the 25 soil sources (64%). Conventional culture methods were also performed, however all samples exhibited negative culture results. We conclude that both irrigation water and soil are the natural reservoirs of P. insidiosum. In endemic areas, the exposure to these environmental reservoirs should be considered a risk factor for hosts susceptible to pythiosis.

Antioxidative and immunogenic properties of catalase-peroxidase protein in Penicillium marneffei.

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.