Small regulatory RNAs (sRNAs) act as key regulators in many bacterial signalling cascades. However, in P. aeruginosa, the sRNAs involved in quorum sensing (QS) regulation and their function are still largely unknown. Here, we explored how the prrH locus sRNA influences P. aeruginosa virulence in the context of the QS regulatory network. First, gain- and loss-of-function studies showed that PrrH affects pyocyanin, elastase and rhamnolipid production; biofilm formation; and swimming and swarming motility and impaired the viability of P. aeruginosa in human whole blood. Next, our investigation disclosed that LasI and PhzC/D were directly repressed by PrrH. In addition, RhlI, the key member of the rhl QS system, diminished the expression of PrrH and enhanced the expression of downstream genes. Bioinformatics analysis found two binding sites of RhlR, the transcription factor of the rhl system, on the promoter region of prrH. Further β-galactosidase reporter and qPCR assays confirmed that PrrH was transcriptionally repressed by RhlR. Collectively, our data identified a novel RhlI/R-PrrH-LasI/PhzC/PhzD regulatory circuitry that may contribute to P. aeruginosa pathogenesis. Our findings indicate that PrrH is a quorum regulatory RNA (Qrr) in P. aeruginosa and provide new insight into PrrH’s function.

No abstract

Background: Antimicrobial peptides (AMPs) have shown promise in the treatment of multi-resistant pathogens. It was therefore of interest to analyze the effects of the AMP LL-37 on the regulation of several virulence factors related to the quorum sensing (QS) system of Pseudomonas aeruginosa (P. aeruginosa) in vitro. Methods:The minimum inhibitory concentration (MIC) was evaluated by the micro broth dilution method. The expression of QS-related and QS-regulated virulence factor genes was also evaluated. Exotoxin A activity was measured with the nicotinamide adenine dinucleotide (NAD) (Coenzyme I) method; Elastase activity was detected with the elastin-Congo red (ECR) method; Pyocyanin detection was performed using the chloroform extraction method. The effects of LL-37 were assessed by measuring the expression changes of the virulence protein-encoding genes of the strains with quantitative polymerase chain reaction (PCR). Results:The MIC of LL-37 against both P. aeruginosa reference strain (ATCC 15692 PAO1) and PA-ΔlasI/ rhII was therefore determined to be 256 μg/mL. LL-37 at sub-minimum inhibitory concentrations (sub-MICs) had no significant effects on P. aeruginosa bacterial growth (P>0.05), but significantly downregulated the expression of all 3 virulence factors.Conclusions: Interestingly, this effect appeared to be dose-related. These findings suggest that LL-37 could be a potential candidate for QS inhibition against bacterial infection and may have significant clinical potential in the treatment of P. aeruginosa biofilms.

BackgroundMulticenter laboratory may apply both automated flow cytometer and microscopy for urinalysis. Automated flow cytometer such as Sysmex UF‐1000i evaluates particles with native urine without centrifugation and reports as “counts per μL.” Microscopic examination recommended as the reference method for urine sediment analysis reports results as “counts per HPF (or μL).” Moreover, some results from flow cytometer are needed to be checked visually under microscopy. Therefore, it is worth to establish the consistency of the results from these two methods.MethodsUrine specimens from 412 patients were examined with Sysmex UF‐1000i and manual microscopy using FAST‐READ disposable counting chambers. White blood cell (WBC) and red blood cell (RBC) counting results from UF‐1000i after transferred with the converting factor (0.297) we estimated were compared with that from microscopic examination. Method comparison was performed using Passing‐Bablok analysis.ResultsAfter transferred with the converting factor (0.297), cell counting results from UF‐1000i showed a good correlation with that derived by the reference method (R 2 was 0.868 for RBCs (P < 0.001), 0.882 for WBCs (P < 0.001)). Passing‐Bablok analysis showed no systematic difference (intercept estimate, −1 [95%CI, −7 to 3] and slightly proportional (slope estimate, 1.2 [95%CI, 1.0 to 1.7]) bias between concentrations of cells measured by manual microscopy and Sysmex UF‐1000i using the converting factor.ConclusionThe converting factor (0.297) helps to transfer “counts per μL (non‐centrifugal urine)” to “counts per μL (equal to centrifugal urine),” and to keep the urine particle analysis results of Sysmex UF‐1000i consistent with the results from the reference method.