Microbiology - Current Issue

While Pseudomonas aeruginosa ATCC 27853 is a widely used reference strain with previously characterized prophage regions, our use of one of the latest prophage prediction tools, PHASTEST, helped reveal a critical misclassification in its genome. Using this tool, we initially identified six prophage regions, with four classified as intact; however, in-depth analysis demonstrated that one of these predicted intact prophages was, in fact, a functional pyocin-encoding region. Specifically, the region spanning 679,586–698,056 bp, initially annotated as an intact prophage, was definitively re-identified as a region harbouring an R1-type pyocin. The most recent literature regarding prophages in P. aeruginosa ATCC 27853 classifies the region spanning 683,173–696,044 bp as a prophage. This region falls entirely within the genomic region we describe and reclassify, further emphasizing the importance of the reclassification performed in this study. The identified R1-type pyocin was induced using mitomycin C, processed via tangential flow filtration, and its bactericidal activity was confirmed against a clinical P. aeruginosa isolate via spot-test killing assays and absorbance-based assays. Transmission electron microscopy revealed R-type pyocin particles averaging 133 nm in length. This misidentification of a pyocin as a prophage critically underscores the inherent limitations of current bioinformatic tools in accurately distinguishing between these distinct phage-derived elements, thereby highlighting the urgent need for more refined annotation methodologies. Accurate identification of such elements is essential, as they may influence experimental outcomes and provide new insights into bacterial defence mechanisms.

Reusable medical devices are reprocessed between uses, including cleaning and, as necessary, disinfection or sterilization. Healthcare-associated infections have been attributed to reusable medical devices and linked to inadequate reprocessing, which can result in residual soil on the device, insufficient disinfection, microbial resistance to used disinfectants and biofilm-related contamination. These factors can lead to microbial proliferation on and biofouling of reusable medical devices, increasing the risk of patient infection. While there are FDA-recognized standards for cleaning validation (including artificial test soils), there is a lack of standards or guidance documents available to advise on determining whether biofilm has been adequately cleaned off reusable devices after reprocessing. Additionally, relatively few studies report reproducible models of biofilm formation on medical devices or device materials; such models are necessary to begin the identification of the microbial biofilm burden present before and after reprocessing. Moreover, appropriate analytes to quantify that biofilm burden, and the endpoints of those analytes after reprocessing, need to be determined. The study described herein utilized a drip flow reactor (DFR) to develop single-species biofilms of two Gram-negative (Pseudomonas aeruginosa and Klebsiella pneumoniae) and two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacterial species that are prone to contaminate medical devices as biofilms. Biofilm was extracted at early and late biofilm stages and then tested for several analytes, including protein, ATP, endotoxin, peptidoglycan and total organic carbon. The levels of these analytes were compared to c.f.u. and metabolic activity to qualitatively compare analyte levels with biofilm burden. The results presented demonstrate that the DFR can be used to model biofilm formation of several medically relevant micro-organisms on stainless steel. Furthermore, the analytical data obtained with this study indicate that the analytes used can be a good starting point for informing the selection of endpoints in future studies that evaluate the efficacy of cleaning and disinfection within the context of biofilm reduction.