An Optimized/Scale Up-Ready Protocol for Extraction of Bacterially Produced dsRNA at Good Yield and Low Costs.

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

Genomic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Clinical Acinetobacter baumannii Isolates from Horses.

Acinetobacter (A.) baumannii is an opportunistic pathogen that causes severe infections in humans and animals, including horses. The occurrence of dominant international clones (ICs), frequent multidrug resistance, and the capability to form biofilms are considered major factors in the successful spread of A. baumannii in human and veterinary clinical environments. Since little is known about A. baumannii isolates from horses, we studied 78 equine A. baumannii isolates obtained from clinical samples between 2008 and 2020 for their antimicrobial resistance (AMR), clonal distribution, biofilm-associated genes (BAGs), and biofilm-forming capability. Based on whole-genome sequence analyses, ICs, multilocus (ML) and core-genome ML sequence types (STs), and AMR genes were determined. Antimicrobial susceptibility testing was performed by microbroth dilution. A crystal violet assay was used for biofilm quantification. Almost 37.2% of the isolates were assigned to IC1 (10.3%), IC2 (20.5%), and IC3 (6.4%). Overall, the isolates revealed high genomic diversity. We identified 51 different STs, including 22 novel STs (ST1723-ST1744), and 34 variants of the intrinsic oxacillinase (OXA), including 8 novel variants (OXA-970 to OXA-977). All isolates were resistant to ampicillin, amoxicillin/clavulanic acid, cephalexin, cefpodoxime, and nitrofurantoin. IC1-IC3 isolates were also resistant to gentamicin, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. All isolates were susceptible to imipenem. Thirty-one multidrug-resistant (MDR) isolates mainly accumulated in the IC1-IC3 groups. In general, these isolates showed less biofilm formation (IC1 = 25.0%, IC2 = 18.4%, IC3 = 15.0%) than the group of non-IC1-IC3 isolates (58.4%). Isolates belonging to the same ICs/STs revealed identical BAG patterns. BAG blp1 was absent in all isolates, whereas bfmR and pgaA were present in all isolates. At the level of the IC groups, the AMR status was negatively correlated with the isolates' ability to form a biofilm. A considerable portion of equine A. baumannii isolates revealed ICs/STs that are globally present in humans. Both an MDR phenotype and the capability to form biofilms might lead to therapeutic failures in equine medicine, particularly due to the limited availability of licensed drugs.