Background: Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell–cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed. Objective: the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma. Methods and Materials: we determined exosome quantity, size distribution, marker expression, and downstream application. Results: among the precipitation methods, we found the size of particles and concentrations with 10–20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10–20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum. Conclusions: together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications.

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand’s dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%), C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

XenorhabdusandPhotorhabduscan produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity ofXenorhabdusandPhotorhabdusagainst 15 antibiotic-resistant bacteria strains.Photorhabdusextracts had strong inhibitory the growth of Methicillin-resistantStaphylococcus aureus(MRSA) by disk diffusion. TheP.akhurstii ssubsp.akhurstii(bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between eitherP.akhurstiisubsp.akhurstii(bNN141.3_TH) orP.akhurstiisubsp.akhurstii(bNN168.5_TH) orP.hainanensis(bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay forP.akhurstiisubsp.akhurstii(bNN168.5_TH) extract againstS.aureusstrain PB36 significantly decreased cell viability from 10⁵CFU/ml to 10³CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treatedS.aureusstrain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.

A promising means of rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co-producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co-β-lactamase-producing Enterobacteriaceae are endemic.

Acne vulgaris is the most common dermatological problem among adolescents and young adults. Acne-associated pathogens, including Cutibacterium acnes and Staphylococcus epidermidis, and inflammation are among predominant etiologies of acne. Drugs targeting pathogens and inflammation response are an effective avenue to treat acne vulgaris. Rice bran has high pharmacological value, but its therapeutic potential for the treatment of acne remains unexplored. This study, therefore, aimed to purify and identify an anti-acne component from the ethanolic extract of Thai black rice (Oryza sativa cv. Neow Dam 3) bran and to investigate its underlying mechanisms of action. Protocatechuic acid (PA) was purified by bio-guided extraction and identified by a nuclear magnetic resonance. The antibacterial action was elucidated by minimum inhibitory concentration (MIC) determination and electron microscopy. PA cytotoxicity in HaCaT keratinocytes was assessed by an MTT assay, expression of inflammatory-associated genes and proteins by RT-qPCR, western blotting, and ELISA, and the nuclear translocation of NF-κB by immunofluorescence. The MIC of PA against C. acnes DMST 14,916 and S. epidermidis ATCC 12,228 and DMST 14,932 was 2048 µg/mL, which was similar to ferulic acid. Scanning and transmission electron microscopy demonstrated C. acnes and S. epidermidis treated with PA had unambiguous membrane damage and abnormal morphology. C. acnes lysate induced the expression of IL-6, IL-8, IL-1β, iNOS, TNF-α, and COX-2 genes, increased phosphorylated NF-κB, IL-6, and TNF-α protein levels, and caused translocation of NF-κB into the nucleus, while noncytotoxic levels of PA suppressed these effects. In summary, PA had anti-acne activity by inhibiting acne-associated pathogens, possibly through cell membrane damage, and suppressed inflammation induced by C. acnes via the NF-κB pathway suggesting that it may have therapeutic use for treating acne in the future.