Actinomycin D reduces virulence factors and biofilms against Aeromonas hydrophila.

AIMS: Aeromonas hydrophila, a Gram-negative bacterium, is ubiquitously found in many aquatic habitats, causing septicemia in humans and fishes. Attributed to abuse or misuse of conventional antimicrobial drug usage, antimicrobial resistance is at an alarming rise. There is an available alternative strategy to bacterial resistance to antimicrobials, which is inhibition of virulence and pathogenicity employing quorum sensing inhibitors (QSIs). Hence, actinomycin D's effectiveness against A. hydrophila SHAe 115 as a QSI was investigated in decreasing virulence factors and preventing biofilm formation. METHODS AND RESULTS: Actinomycin D, belongs to the QSI combating Pseudomonas aeruginosa PAO1 originally isolated from an entophytic actinomycete (Streptomyces cyaneochromogenes RC1) in Areca catechu L. In the present work, further investigations were carried out to assess the effect of actinomycin D at subminimal inhibitory concentrations (sub-MICs), QS-regulated virulence factors, and biofilm inhibition strategies. Intrinsic properties encompassing inhibition of the production of protease and hemolysin and subsequent activities on biofilm formation and eradication of mature biofilm were established along with weakened swimming and swarming motilities in A. hydrophila SHAe 115. In the Tenebrio molitor survival assay, actinomycin D effectively reduced the virulence and pathogenicity of A. hydrophila, resulting in elimination of mortality. However, the hydrolysate of actinomycin D, 2-hydroxy-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid (HDPD), had lost the QSI activity in A. hydrophila. CONCLUSIONS: Actinomycin D was proved as a viable QSI in lessening A. hydrophila's the virulence and pathogenicity, as evident from our research findings.

1H-Pyrrole-2,5-dicarboxylic acid, a quorum sensing inhibitor from one endophytic fungus in Areca catechu L., acts as antibiotic accelerant against Pseudomonas aeruginosa.

Pseudomonas aeruginosa has already been stipulated as a "critical" pathogen, emphasizing the urgent need for researching and developing novel antibacterial agents due to multidrug resistance. Bacterial biofilm formation facilitates cystic fibrosis development and restricts the antibacterial potential of many current antibiotics. The capacity of P. aeruginosa to form biofilms and resist antibiotics is closely correlated with quorum sensing (QS). Bacterial QS is being contemplated as a promising target for developing novel antibacterial agents. QS inhibitors are a promising strategy for treating chronic infections. This study reported that the active compound PT22 (1H-pyrrole-2,5-dicarboxylic acid) isolated from Perenniporia tephropora FF2, one endophytic fungus from Areca catechu L., presents QS inhibitory activity against P. aeruginosa. Combined with gentamycin or piperacillin, PT22 functions as a novel antibiotic accelerant against P. aeruginosa. PT22 (0.50 mg/mL, 0.75 mg/mL, and 1.00 mg/mL) reduces the production of QS-related virulence factors, such as pyocyanin and rhamnolipid, and inhibits biofilm formation of P. aeruginosa PAO1 instead of affecting its growth. The architectural disruption of the biofilms was confirmed by visualization through scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Real-time quantitative PCR (RT-qPCR) indicated that PT22 significantly attenuated the expression of QS-related genes followed by docking analysis of molecules against QS activator proteins. PT22 dramatically increased the survival rate of Galleria mellonella. PT22 combined with gentamycin or piperacillin presents significant inhibition of biofilm formation and eradication of mature biofilm compared to monotherapy, which was also confirmed by visualization through SEM and CLSM. After being treated with PT22 combined with gentamycin or piperacillin, the survival rates of G. mellonella were significantly increased compared to those of monotherapy. PT22 significantly enhanced the susceptibility of gentamycin and piperacillin against P. aeruginosa PAO1. Our results suggest that PT22 from P. tephropora FF2 as a potent QS inhibitor is a candidate antibiotic accelerant to combat the antibiotic resistance of P. aeruginosa.

Actinomycin D: a novel Pseudomonas aeruginosa quorum sensing inhibitor from the endophyte Streptomyces cyaneochromogenes RC1.

The infections caused by Pseudomonas aeruginosa are difficult to treat due to its multidrug resistance. A promising strategy for controlling P. aeruginosa infection is targeting the quorum sensing (QS) system. Actinomycin D isolated from the metabolite of endophyte Streptomyces cyaneochromogenes RC1 exhibited good anti-QS activity against P. aeruginosa PAO1. Actinomycin D (50, 100, and 200 µg/mL) significantly inhibited the motility as well as reduced the production of multiple virulence factors including pyocyanin, protease, rhamnolipid, and siderophores. The images of confocal laser scanning microscopy and scanning electron microscopy revealed that the treatment of actinomycin D resulted in a looser and flatter biofilm structure. Real-time quantitative PCR analysis showed that the expression of QS-related genes lasI, rhlI, rhlR, pqsR, pslA, and pilA were downregulated dramatically. The production of QS signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone were also decreased by actinomycin D. These findings suggest that actinomycin D, a potent in vitro anti-virulence agent, is a promising candidate to treat P. aeruginosa infection by interfering with the QS systems.

Lidocaine pretreatment up-regulates aquaporin-5 expression in primary alveolar epithelium type II cells injured by lipopolysaccharides.

Acute respiratory distress syndrome (ARDS) or acute lung injury (ALI) is associated with decreased aquaporin-5 (AQP5) expression. Lipopolysaccharides (LPS) can decrease AQP5 expression. The effects and mechanisms of lidocaine pretreatment on primary alveolar epithelium type II (AEC II) cells injured by LPS were investigated. Primary AEC II cells were isolated from rats previously injured with LPS as an ALI model. The groups of cells were evaluated: 1) pretreated with lidocaine (2, 20, 200µg/ml) and/or Infliximab, an anti-TNF-α neutralizing antibody, 2) uninjured cells; 3) solvent pretreated injured cells and 4) untreated injured cells as controls. TNF-α levels were evaluated by ELISA. AQP5 expression was determined by mRNA and protein expression (q-PCR and western blot).The release of TNF-α was increased significantly in AEC II cells following LPS injury. The release of TNF-α was decreased by 33%-100% as a result of lidocaine pretreatment in a dose-dependent fashion. This decrease was accompanied by up-regulated AQP5 expression in LPS injured AEC II cells, and Infliximab can greatly block AQP5 expression in LPS injured AEC II cells pretreated with lidocaine. Lidocaine pretreatment (2-200µg/ml) of LPS injured AEC II cells results in a decrease in TNF-α release, then up-regulates AQP5 expression, which maybe involved in the mechanism of its effects on AEC II cells injured by LPS.

Characterization and chemical modification of PLN-1, an exopolysaccharide from Phomopsis liquidambari NJUSTb1.

Phomopsis liquidambari is a classical endophytic fungus with great application potential in ecology and agriculture; however, studies on its exopolysaccharides are lacking. Here, we aimed to evaluate the structure and bioactivity of PLN-1, an exopolysaccharide derived from the P. liquidambari NJUSTb1 strain. The structure was elucidated by chromatography/spectral methods and hydrolyzation. Immunomodulation, moisture absorption, and retention properties were investigated after sulfation and carboxymethylation modification. Results showed that PLN-1 contained a linear repeating unit of →[4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→4)-α-d-Glcp-(1→]n, with a molecular weight of 343 kDa. The degrees of substitution of sulfated polysaccharide (S-PLN-1) and carboxymethylated polysaccharide (C-PLN-1) were 1.228 and 0.903, respectively. S-PLN-1 showed stronger moisture absorption and retention properties than PLN (crude EPS), C-PLN1, and PLN-1. Furthermore, PLN, S-PLN-1, and C-PLN-1 stimulated the proliferation of RAW 264.7 cells with no cytotoxicity. The elucidation of PLN-1 in this study paves the way for future applications.

Comparing post-operative analgesic effects of varying doses of dexmedetomidine as an adjuvant to ropivacaine for ultrasound-guided dual transversus abdominis plane block following laparotomy for gynecologic malignancies.

The aim of the present study was to determine the analgesic effects of ropivacaine combined with different doses of dexmedetomidine for ultrasound-guided transversus abdominis plane (TAP) block immediately following laparotomy in patients with gynecologic malignancies. A further aim was to determine the appropriate clinical dose of dexmedetomidine as an adjuvant for ropivacaine. Patients with gynecologic malignancies scheduled for laparotomy were randomly assigned to group R (TAP block with 0.3% ropivacaine), group RD1 (TAP block with ropivacaine and 0.5 µg/kg dexmedetomidine), group RD2 (TAP block with ropivacaine and 1 µg/kg dexmedetomidine) and group RD3 (TAP block with ropivacaine and 2 µg/kg dexmedetomidine). TAP blocks were performed post-operatively. The four groups all received patient-controlled intravenous analgesia (PCIA) after the operation. The numerical rating scale (NRS) as well as the Ramsay sedation scale (RSS) scores, the first request time for PCIA bolus, oxycodone hydrochloride consumption, the plasma concentration of ropivacaine, the incidence of post-operative complications and adverse events, and patient satisfaction were recorded. Post-operative NRS scores at rest exhibited significant differences between the R group and all the RD groups at 24 h after surgery (P<0.05). Compared with the other groups, the NRS score in the RD3 group was decreased (P<0.05). The RSS scores were higher in all of the RD groups compared with those in the R group at 2 h (P<0.05) and were highest in the RD3 group compared with those in all other groups at 4 h (P<0.05). The first request time for PCIA was significantly longer in the RD3 group compared with that in the RD2, RD1 and R groups (510.47±102.67, 595.47±100.11, 682.43±104.46 and 776.42±143.91 min, respectively; P<0.05). Cumulative opioid consumption based on the number of PCIA bolus requested at 24 and 48 h post-operatively indicated that the total number of PCIA boluses was significantly lower in the RD groups compared with those in the R group at 24 and 48 h (P<0.05). The ropivacaine concentration did not differ among the four groups. There was no significant difference between groups with respect to post-operative nausea and vomiting, bradycardia and hypotension; however, all RD groups had a higher patient satisfaction than group R (P<0.05). Compared with that in the other groups, the duration of post-anesthesia care unit stay in group RD3 was relatively longer due to excessive sedation (P<0.05). In conclusion, TAP blockade using 0.5-2 µg/kg dexmedetomidine combined with 0.3% ropivacaine is a safe and effective treatment for analgesia in laparotomy procedures for gynecologic malignancies. The study was registered in the Chinese Clinical Trial Registry (CHICTR; www.chictr.org.cn) on January 15th, 2019 (registration no. ChiCTR1900020995).