In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy.

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. In this study, we describe the Pseudomonas phage Motto, a T1-like phage that can clear P. aeruginosa infections in an animal model and also exhibits biofilm-degrading properties. The phage has a substantial anti-biofilm activity against strong biofilm-producing isolates (n = 10), with at least a twofold reduction within 24 h. To demonstrate the safety of using phage Motto, cytotoxicity studies were conducted with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in Caenorhabditis elegans, with a 90% survival rate when treated with the phage at a multiplicity of infection of 10.

Green Synthesis of Zinc Oxide Nanoparticles from Althaea officinalis Flower Extract Coated with Chitosan for Potential Healing Effects on Diabetic Wounds by Inhibiting TNF-α and IL-6/IL-1ß Signaling Pathways.

Background: Diabetes Mellitus is a multisystem chronic pandemic, wound inflammation, and healing are still major issues for diabetic patients who may suffer from ulcers, gangrene, and other wounds from uncontrolled chronic hyperglycemia. Marshmallows or Althaea officinalis (A.O.) contain bioactive compounds such as flavonoids and phenolics that support wound healing via antioxidant, anti-inflammatory, and antibacterial properties. Our study aimed to develop a combination of eco-friendly formulations of green synthesis of ZnO-NPs by Althaea officinalis extract and further incorporate them into 2% chitosan (CS) gel. Method and Results: First, develop eco-friendly green Zinc Oxide Nanoparticles (ZnO-NPs) and incorporate them into a 2% chitosan (CS) gel. In-vitro study performed by UV-visible spectrum analysis showed a sharp peak at 390 nm, and Energy-dispersive X-ray (EDX) spectrometry showed a peak of zinc and oxygen. Besides, Fourier transforms infrared (FTIR) was used to qualitatively validate biosynthesized ZnO-NPs, and transmission electron microscope (TEM) showed spherical nanoparticles with mean sizes of 76 nm and Zeta potential +30mV. The antibacterial potential of A.O.-ZnO-NPs-Cs was examined by the diffusion agar method against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Based on the zone of inhibition and minimal inhibitory indices (MIC). In addition, an in-silico study investigated the binding affinity of A.O. major components to the expected biological targets that may aid wound healing. Althaea Officinalis, A.O-ZnO-NPs group showed reduced downregulation of IL-6, IL-1ß, and TNF-α and increased IL-10 levels compared to the control group signaling pathway expression levels confirming the improved anti-inflammatory effect of the self-assembly method. In-vivo study and histopathological analysis revealed the superiority of the nanoparticles in reducing signs of inflammation and wound incision in rat models. Conclusion: These biocompatible green zinc oxide nanoparticles, by using Althaea Officinalis chitosan gel ensure an excellent new therapeutic approach for quickening diabetic wound healing.

The Potentiation Activity of Azithromycin in Combination with Colistin or Levofloxacin Against Pseudomonas aeruginosa Biofilm Infection.

Objective: Pseudomonas aeruginosa (PA) often displays drug resistance and biofilm-mediated adaptability. Here, we aimed to evaluate the antibiofilm efficacy of azithromycin-based combination regimens. Methods: Minimum inhibitory concentrations (MICs), minimal biofilm eradication concentrations (MBECs), and MBEC-combination of azithromycin, colistin, amikacin, and levofloxacin to bioluminescent strain PAO1 and carbapenem-resistant PAO1 (CRPAO1) were assessed. An animal biofilm infection model was established and detected using a live animal bio-photonic imaging system. Results: In vitro, PAO1 and CRPAO1 were susceptible to colistin, amikacin, and levofloxacin, while they were unsusceptible to azithromycin. The combinations based on azithromycin have no synergistic effect on biofilm in vitro. In vivo, azithromycin plus colistin or levofloxacin could shorten the PAO1 biofilm eradication time, which totally eradicates the biofilm in all mice on the 8th or 6th day, while monotherapy only eradicate biofilm in 70% or 80% mice on the 8th day. For CRPAO1 biofilm, only azithromycin-colistin combination and colistin monotherapy eradicated the bacteria in 60% and 40% of mice at the 6th day. Conclusion: Azithromycin-based combinations containing levofloxacin or colistin had no synergistic effect in vitro, and they are promising for clinical applications due to the good synergistic activity against PAO1 biofilms in vivo.

Persistence and evolution of Pseudomonas aeruginosa following initiation of highly effective modulator therapy in cystic fibrosis.

Today, more than 90% of people with cystic fibrosis (pwCF) are eligible for the highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy called elexacaftor/tezacaftor/ivacaftor (ETI) and its use is widespread. Given the drastic respiratory symptom improvement experienced by many post-ETI, clinical studies are already underway to reduce the number of respiratory therapies, including antibiotic regimens, that pwCF historically relied on to combat lung disease progression. Early studies suggest that bacterial burden in the lungs is reduced post-ETI, yet it is unknown how chronic Pseudomonas aeruginosa populations are impacted by ETI. We found that pwCF remain infected throughout their upper and lower respiratory tract with their same strain of P. aeruginosa post-ETI, and these strains continue to evolve in response to the newly CFTR-corrected airway. Our work underscores the continued importance of CF airway microbiology in the new era of highly effective CFTR modulator therapy. IMPORTANCE: The highly effective cystic fibrosis transmembrane conductance regulator modulator therapy Elexakaftor/Tezacaftor/Ivacaftor (ETI) has changed cystic fibrosis (CF) disease for many people with cystic fibrosis. While respiratory symptoms are improved by ETI, we found that people with CF remain infected with Pseudomonas aeruginosa. How these persistent and evolving bacterial populations will impact the clinical manifestations of CF in the coming years remains to be seen, but the role and potentially changing face of infection in CF should not be discounted in the era of highly effective modulator therapy.

The microenvironment in antibiotic susceptibility testing.

Antibiotic susceptibility testing (AST) by agar diffusion has been repeatedly standardized and, in most cases, gives results which predict clinical success when antibiotic treatment is based on such results. The formation of the inhibition zone is due to a transition from planktonic to biofilm mode of growth. The kinetics of the interaction of antibiotics with bacteria is similar during AST by agar diffusion and during administration of antibiotics to the patients. However, the Mueller-Hinton agar (MHA) recommended for AST agar diffusion test is fundamentally different from the composition of the interstitial fluid in the human body where the infections take place and human cells do not thrive in MH media. Use of RPMI 1640 medium designed for growth of eucaryotic cells for AST of Pseudomonas aeruginosa against azithromycin results in lower minimal inhibitory concentration, compared to results obtained by MHA. The reason is that the RPMI 1640 medium increases uptake and reduces efflux of azithromycin compared to MHA. During treatment of cystic fibrosis patients with azithromycin, mutational resistance occur which is not detected by AST with MHA. Whether this is the case with other antibiotics and bacteria is not known but it is of clinical importance to be studied.

Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modeling and whole genome sequencing.

Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modeling (MBM) and genomic studies. Two CF multidrug-resistant strains were investigated in a 168h CBR (n=2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2=3h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts, and whole genome sequencing were completed. Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. The combination of aztreonam and tobramycin was required to suppress regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilized for future investigations of this promising inhaled combination.

Antagonism between neuropeptides and monoamines in a distributed circuit for pathogen avoidance.

Pathogenic infection elicits behaviors that promote recovery and survival of the host. After exposure to the pathogenic bacterium Pseudomonas aeruginosa PA14, the nematode Caenorhabditis elegans modifies its sensory preferences to avoid the pathogen. Here, we identify antagonistic neuromodulators that shape this acquired avoidance behavior. Using an unbiased cell-directed neuropeptide screen, we show that AVK neurons upregulate and release RF/RYamide FLP-1 neuropeptides during infection to drive pathogen avoidance. Manipulations that increase or decrease AVK activity accelerate or delay pathogen avoidance, respectively, implicating AVK in the dynamics of avoidance behavior. FLP-1 neuropeptides drive pathogen avoidance through the G protein-coupled receptor DMSR-7, as well as other receptors. DMSR-7 in turn acts in multiple neurons, including tyraminergic/octopaminergic neurons that receive convergent avoidance signals from the cytokine DAF-7/transforming growth factor ß. Neuromodulators shape pathogen avoidance through multiple mechanisms and targets, in agreement with the distributed neuromodulatory connectome of C. elegans.

Use of systemic biofertilizers in sugarcane results in highly reproducible increments in yield and quality of harvests.

The utilization of a novel (systemic) biofertilizer containing Pseudomonas fluorescens, Azospirillum brasilense, and Bacillus subtilis and possessing the technology to facilitate the entry of bacteria through the stomata, was evaluated at three localities in Mexico (Potrero Nuevo, Veracruz; Ameca, Jalisco; and Champotón, Campeche) in two sugarcane varieties (NCO-310 and Mex 57-473) at different time scales. Inoculation of the systemic biofertilizer was imposed over the local agricultural management of the sugarcane; chemical fertilization of the experimental parcels at Potrero Nuevo was done using 70-20-20 and 120-80-80 at Ameca and Champotón. Three doses of the biofertilizer per hectare were applied during the annual productive cycle of sugarcane at each site; one year at Potrero Nuevo and Champotón; and six years at Ameca. The annual sugarcane yield was evaluated at each site. Additionally, sugar quality (°Brix or sucrose content) was evaluated at the three localities, while different variables of stalk performance were also measured at Ameca and Champotón. Our data provide evidence that this systemic biofertilizer consistently and reliably increased the sugarcane yield at all localities during the time of evaluation, ranging from 73.7 tons ha-1 at Potrero Nuevo (2.5 times increase; P < 0.05) and 77.7 tons ha-1 at Ameca (1.9 times increase; P < 0.05) to 23.8 tons ha-1 at Champotón (1.4 times increase; P < 0.05). This increase in sugarcane biomass was related to increased tillering rather than increased stalk height or diameter. This novel biological product improved the sugarcane quality in terms of °Brix (P < 0.05, 2.6° difference) and sucrose content (P < 0.5, 0.7% difference).

Effect of Elexacaftor/Tezacaftor/Ivacaftor on Pseudomonas aeruginosa Acquisition and Chronic Infection at a Single Pediatric Cystic Fibrosis Care Center.

OBJECTIVES: As cystic fibrosis (CF) lung disease progresses, the airways become infected with opportunistic pathogens, such as Pseudomonas aeruginosa (PA). In October 2019, the US Food and Drug Administration approved elexacaftor/tezacaftor/ivacaftor (ETI), a highly effective modulator therapy (HEMT), for individuals 12 years and older with 1 copy of the F508del cystic fibrosis transmembrane conductance regulator (CFTR) mutation. ETI increases the amount of and function of CFTR in the respiratory epithelium, improving mucociliary clearance and reducing static airway mucus, a major trigger for chronic infection and inflammation. METHODS: A retrospective analysis of inhaled tobramycin (iTOB) prescriptions between January 1, 2016, and December 31, 2021, was performed. This captured data before and after ETI approval at Children's Mercy Kansas City (CMKC). The number of individuals with new PA acquisition and individuals considered -chronically infected was analyzed. RESULTS: The number of eradication prescriptions declined in 2020 and 2021, with 15 (7%) and 12 (5%) -individuals prescribed therapy for those years, respectively. A similar pattern was observed for -prescriptions for chronic infection. A reduction was seen in 2020 and 2021, with 28 (13%) and 20 (9%) individuals -prescribed therapy for the respective years. CONCLUSIONS: The CMKC experienced a decrease in the number of courses of iTOB prescribed during the last 6 years. The reasons for this are likely multifactorial and may include the implementation of standardized PA surveillance and eradication protocols, the effect of HEMT on mucociliary clearance and airway microbiology, and the poorly understood effects of the SARS-CoV-2 pandemic on the epidemiology of respiratory infections.

Difficult-to-treat (DTR) Pseudomonas aeruginosa harboring Verona-Integron metallo-ß-lactamase (blaVIM): infection management and molecular analysis.

Pseudomonas aeruginosa harboring Verona Integron-encoded metallo-ß-lactamase enzymes (VIM-CRPA) have been associated with infection outbreaks in several parts of the world. In the US, however, VIM-CRPA remain rare. Starting in December 2018, we identified a cluster of cases in our institution. Herein, we present our epidemiological investigation and strategies to control/manage these challenging infections. This study was conducted in a large academic healthcare system in Miami, FL, between December 2018 and January 2022. Patients were prospectively identified via rapid molecular diagnostics when cultures revealed carbapenem-resistant P. aeruginosa. Alerts were received in real time by the antimicrobial stewardship program and infection prevention teams. Upon alert recognition, a series of interventions were performed as a coordinated effort. A retrospective chart review was conducted to collect patient demographics, antimicrobial therapy, and clinical outcomes. Thirty-nine VIM-CRPA isolates led to infection in 21 patients. The majority were male (76.2%); the median age was 52 years. The majority were mechanically ventilated (n = 15/21; 71.4%); 47.6% (n = 10/21) received renal replacement therapy at the time of index culture. Respiratory (n = 20/39; 51.3%) or bloodstream (n = 13/39; 33.3%) were the most common sources. Most infections (n = 23/37; 62.2%) were treated with an aztreonam-avibactam regimen. Six patients (28.6%) expired within 30 days of index VIM-CRPA infection. Fourteen isolates were selected for whole genome sequencing. Most of them belonged to ST111 (12/14), and they all carried blaVIM-2 chromosomally. This report describes the clinical experience treating serious VIM-CRPA infections with either aztreonam-ceftazidime/avibactam or cefiderocol in combination with other agents. The importance of implementing infection prevention strategies to curb VIM-CRPA outbreaks is also demonstrated.

Development of an enzyme-linked phage receptor-binding protein assay (ELPRA) based on a novel biorecognition molecule- receptor-binding protein Gp130 of Pseudomonas aeruginosa bacteriophage Henu5.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818-0.991), and the specificity was 0.905 (0.682-0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples.

Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave.

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

Evidence for intracellular Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a significant cause of global morbidity and mortality. Although it is often regarded as an extracellular pathogen toward human cells, numerous investigations report its ability to survive and replicate within host cells, and additional studies demonstrate specific mechanisms enabling it to adopt an intracellular lifestyle. This ability of P. aeruginosa remains less well-investigated than that of other intracellular bacteria, although it is currently gaining attention. If intracellular bacteria are not killed after entering host cells, they may instead receive protection from immune recognition and experience reduced exposure to antibiotic therapy, among additional potential advantages shared with other facultative intracellular pathogens. For this review, we compiled studies that observe intracellular P. aeruginosa across strains, cell types, and experimental systems in vitro, as well as contextualize these findings with the few studies that report similar observations in vivo. We also seek to address key findings that drove the perception that P. aeruginosa remains extracellular in order to reconcile what is currently understood about intracellular pathogenesis and highlight open questions regarding its contribution to disease.

Local control: a hub-based model for the c-di-GMP network.

The genome of Pseudomonas fluorescens encodes >50 proteins predicted to play a role in bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-mediated biofilm formation. We built a network representation of protein-protein interactions and extracted key information via multidimensional scaling (i.e., principal component analysis) of node centrality measures, which measure features of proteins in a network. Proteins of different domain types (diguanylate cyclase, dual domain, phosphodiesterase, PilZ) exhibit unique network behavior and can be accurately classified by their network centrality values (i.e., roles in the network). The predictive power of protein-protein interactions in biofilm formation indicates the possibility of localized pools of c-di-GMP. A regression model showed a statistically significant impact of protein-protein interactions on the extent of biofilm formation in various environments. These results highlight the importance of a localized c-di-GMP signaling, extend our understanding of signaling by this second messenger beyond the current "Bow-tie Model," support a newly proposed "Hub Model," and suggest future avenues of investigation.

Genetic dissection of the tissue-specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Metal-based nanoparticles: an alternative treatment for biofilm infection in hard-to-heal wounds.

Metal-based nanoparticles (MNPs) are promoted as effective compounds in the treatment of bacterial infections and as possible alternatives to antibiotics. These MNPs are known to affect a broad spectrum of microorganisms using a multitude of strategies, including the induction of reactive oxygen species and interaction with the inner structures of the bacterial cells. The aim of this review was to summarise the latest studies about the effect of metal-based nanoparticles on pathogenic bacterial biofilm formed in wounds, using the examples of Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Pseudomonas aeruginosa, as well as provide an overview of possible clinical applications.

First report of Pseudomonas cichorii causing bacterial leaf blight of pocketbook plant (Calceolaria hybrida) in Taiwan.

Pocketbook plants (Calceolaria spp.) are flowering ornamentals often grown as potted plants (Poesch 1937). In December 2022, leaf blight symptoms were observed on 2-mo-old plants of C. hybrida F1 'Dainty'. The disease was found in a nursery in Ren'ai Township, Nantou, and about 20% of the plants exhibited symptoms. Symptomatic plants had brown or gray necrotic lesions of different sizes and shapes, mostly around leaf margins. Lower leaf wilting was also observed (Fig. S1, A and B). Three plants were sampled. Leaf lesions were surface-disinfected with 75% ethanol and cut into smaller pieces in 10 mM MgCl2. After observing bacterial streaming under a microscope, the bacteria were streaked onto nutrient agar (NA). Following 2 days at 28°C, a type of round, creamy white colony predominated on all the plates. Three strains (Calc-A, Calc-B, and Calc-C) were obtained, one from each plant. The strains produced fluorescent pigments on King's B medium and were tested Gram-negative. The strains were characterized with the LOPAT scheme (Schaad et al. 2001). They did not exhibit activities of pectic enzymes, arginine dihydrolase and levan sucrase, but produced oxidase and induced the hypersensitive response in tobacco. DNA was extracted from the strains for PCR amplification of the 16S rDNA with primer pair 27f/1492r as described by Lane (1991). The 16S rDNA sequences were compared with entries in the GenBank database. The sequences obtained (GenBank accession no. OR824302) matched that of Pseudomonas cichorii MAFF 301158 (accession no. AB724288; 1,403/1,403 bp) and were 99% identical to that of DSM 50259T (accession no. CP074349; 1,391/1,405 bp). The strains were also tested with the species-specific primers hrp1a and hrp2a (Cottyn et al. 2011). The amplicons were sequenced and a BLASTn search showed that the sequences (accession no. OR827305) shared the highest identity (99.3%) with that of P. cichorii strain 83-1 (accession no. DQ168848; 848/854 bp) and were 97.3% identical to the sequence of DSM 50259T (accession no. CP074349; 831/854 bp). Calc-A was selected as a representative strain and deposited in the Bioresource Collection and Research Center, Taiwan (reference no. BCRC 81432). Koch's postulates were fulfilled by spray-inoculating a suspension of Calc-A on three 2-mo-old C. hybrida F1 'Dainty' plants. The inoculum was prepared by suspending NA-grown cells in 10 mM MgCl2 including 0.02% Silwet L-77 (OD600 = 0.3; 1.5 x 108 CFU/ml). For the controls, three plants were sprayed with bacteria-free solution. The plants were bagged throughout the experiment and kept in a growth chamber (14/10 h light/dark; 26/24°C day/night). Leaf blight and wilting symptoms developed on all leaves of the inoculated plants after 30 h, but not the controls (Fig. S1, C and D). The pathogen was reisolated from the treatment group, and colony PCR with hrp1a/hrp2a showed that the reisolated strain shared the same sequence with Calc-A to Calc-C. Repeating the inoculation assay produced consistent results. This is the first report of P. cichorii affecting Calceolaria in Taiwan. The bacterium has been reported infecting diverse crops in Taiwan, such as tomato and lettuce (Tsai et al. 2014). Expanding the understanding of the pathogen's potential hosts could help prevent its spread across important crops.

Effects of swine manure dilution with lagoon effluent on microbial communities and odor formation in pit recharge systems.

Pit recharge systems (PRS) control odor by managing organic solids in swine manure. However, there needs to be more understanding of PRS's effect on the microbiome composition and its impact on odor formation. A study was conducted to understand how recharge intervals used in PRS impact manure microbiome and odor formation. Bioreactors dynamically loaded simulated recharge intervals of 14, 10, and 4 days by diluting swine manure with lagoon effluent at varying ratios. Treatment ratios tested included 10:0 (control), 7:3 (typical Korean PRS), 5:5 (enhanced PRS #1), and 2:8 (enhanced PRS #2). Manure microbial membership, chemical concentrations, and odorant concentrations were used to identify the interactions between microbiota, manure, and odor. The initial microbial community structure was controlled by dilution ratio and manure barn source material. Firmicutes and Proteobacteria were the dominant microbial phyla in manure and lagoon effluent, respectively, and significantly decreased or increased with dilution. Key microbial species were Clostridium saudiense in manure and Pseudomonas caeni in lagoon effluent. Percentages of these species declined by 8.9% or increased by 17.6%, respectively, with each unit dilution. Microbial community composition was controlled by both treatment (i.e., manure dilution ratio and barn source material) and environmental factors (i.e., solids and pH). Microbiome composition was correlated with manure odor formation profiles, but this effect was inseparable from environmental factors, which explained over 75% of the variance in odor profiles. Consequently, monitoring solids and pH in recharge waters will significantly impact odor control in PRS.

Computational and experimental strategies for combating MBL P. aeruginosa (MBLPA) biofilms using phytochemicals: Targeting the quorum sensing network.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, ubiquitously found in nature and causative agent in many infections. Due to increased antibiotic resistance, there is a need to develop more robust antibacterial agents from natural sources. In this study, we worked on two metallo-ß-lactamase (MBL) producing Pseudomonas aeruginosa strains and targeted the Quorum Sensing mechanism (QS) of these bacteria to combat antibiotic resistance. Our study aimed at using phytochemicals which have been used since centuries in herbal medicine. We used fifteen commercially available phytochemicals and check their effects on biofilm formation, quorum sensing and inter-related mechanisms. Sub-inhibitory concentration of isoliquiritin inhibited biofilm formation 55 % in P8 at day 6 and 48 % in P6 at day 6; quorum sensing 83 % in P6 and 61 % in P8 whereas sub-inhibitory concentration of 6-gingerol suppressed biofilm formation by 48 % in P8 at day 6 and 44 % in P6 at day 6; quorum sensing 69 % in P6 and 48 % in P8, respectively. The results indicated isoliquiritin, epigallocatechin gallate, eugenol, luteolin and chrysin to be the potential candidates in inhibiting QS and related mechanisms. Isoliquiritin which was never been used before against biofilm and QS related studies, showed remarkable results and found to be more efficient in inhibiting QS than 6-gingerol -a known QS inhibitor. For examining the molecular interaction between phytochemicals and QS, In-silico molecular docking was performed between phytoligands and four QS proteins (Las I, Las R, RhlI and Rhl R). In-silico docking analysis revealed that isoliquiritin showed strong bond with amino acids (Trp34, Asp35, Asp35, Tyr105, Arg104, Val138, Thr140) present at the active site of RhlI with binding energy value of -8.4 kcal/mol as compared to that of 6-gingerol with Rhl1 (-7.3 kcal/mol). In conclusion, our study may help in controlling nosocomial infections caused by carbapenem-resistant metallo beta-lactamase P. aeruginosa (MBL-PA) by utilizing these phytochemicals in biofilms disruption and quorum sensing inhibition. Moreover their synergism with antibiotics may help in lowering the MIC of carbapenem antibiotics against such Multi-drug resistant strains.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.