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Bacterial blight (causal agent Pseudomonas syringae complex, Psc) is an endemic and economically important disease of northern highbush blueberry production in Canada and the Pacific Northwest of the USA. To date, there is no comprehensive survey of the disease in the region and detailed characterization of associated pathogens from Pacific western Canada. Therefore, we did comprehensive disease survey and characterization of associated pseudomonads population using pathogen morphology, biochemical tests, and molecular characterization. We isolated 380 strains of pseudomonads from symptomatic plants from 32 research and commercial fields in 10 diverse geographic locations in British Columbia. We used P . syringae specific (Psy) primers and identified 197 Psy-PCR positive isolates out of 380. We further sequenced Psy-PCR positive isolates of pseudomonads using four housekeeping genes and identified four phylogenomic species: P. syringae (40%), Pseudomonas avellanae (29%), Pseudomonas viridiflava (20%), and phylogenomic species A (7%). P . avellanae and P. viridiflava are new phylogenomic species of Psc causing bacterial blight in highbush blueberry. We found some patterns among geographical locations and highbush blueberry varieties in the frequency distribution of isolates of these phylogenomic species. Genetic fingerprinting with rep-PCR assays identified a very high genetic diversity of pseudomonads populations among geographical locations, varieties, and phylogenomic species. Biochemical characterization (LOPAT- levan, oxidase, pectolytic activity, arginine dihydrolase, and tobacco hypersensitivity) revealed that the vast majority of isolates were Pseudomonas Group Ia. Findings of this study provide insight into the population biology of pseudomonads infecting highbush blueberry, provide information for disease diagnosis, and exploit disease management options, including identifying sources of disease resistance. Key points • High prevalence of bacterial blight caused by P. syringae complex (Psc) in highbush blueberry in Pacific western Canada • We report two new phylogenomic species of Psc, P. viridiflava and P. avellanae, that cause bacterial blight and canker disease in highbush blueberry • The genetic diversity of the population of Psc was very high

The aim of this study is to evaluate opportunistic pathogenic bacteria of the genus Pseudomonas in anthropogenically impacted bathing waters, primarily focusing on bathing ponds. The findings include the detection of these bacteria, their susceptibility to selected antibiotics, and the determination of the Exotoxin A (exoA) gene using PCR method. P. aeruginosa was present in most samples, albeit in low concentrations (1–14 CFU/100 mL). The presence of P. otitidis , which is associated with ear infection, in this type of bathing water, was not rare (up to 90 CFU/100 mL). This species would not be detected by the standard methods, including tests on acetamid medium, used for P. aeruginosa in water. The isolated strains of P. otitidis lack the exoA gene and exhibited higher resistance to meropenem compared to P. aeruginosa .

Bacterial species exhibit biogeographical patterns like those observed in larger organisms. The distribution of bacterial species is driven by environmental selection through abiotic and biotic factors as well dispersal limitations. We asked whether interference competition, a biotic factor, could explain variability in habitat use by Pseudomonas species in the human home. To answer this question, we screened almost 8000 directional, pairwise interactions between 89 Pseudomonas strains including members of the Pseudomonas aeruginosa (n = 29), Pseudomonas fluorescens (n = 21), and Pseudomonas putida (n = 39) species groups for the presence of killing. This diverse set of Pseudomonas strains includes those isolated from several different habitats within the home environment and includes combinations of strains that were isolated from different spatial scales. The use of this strain set not only allowed us to analyze the commonality and phylogenetic scale of interference competition within the genus Pseudomonas but also allowed us to investigate the influence of spatial scale on this trait. Overall, the probability of killing was found to decrease with increasing phylogenetic distance, making it unlikely that interference competition accounts for previously observed differential habitat use among Pseudomonas species and species groups. Strikingly, conspecific P. aeruginosa killing accounted for the vast majority of the observed killing, and this killing was found to differ across the habitat type and spatial scale of the strains’ isolation. These data suggest that interference competition likely plays a large role in the within-species dynamics of P. aeruginosa but not other household Pseudomonas species.

Abstract Rationale To identify pathogens that require different treatments in community-acquired pneumonia (CAP), we propose an acronym, “PES” (P  seudomonas aeruginosa, E  nterobacteriaceae extended-spectrum β-lactamase–positive, and methicillin-resistant S  taphylococcus aureus). Objectives To compare the clinical characteristics and outcomes between patients with CAP caused by PES versus other pathogens, and to identify the risk factors associated with infection caused by PES. Methods We conducted an observational prospective study evaluating only immunocompetent patients with CAP and an established etiological diagnosis. We included patients from nursing homes. We computed a score to identify patients at risk of PES pathogens. Measurement and Main Results: Of the 4,549 patients evaluated, we analyzed 1,597 who presented an etiological diagnosis. Pneumonia caused by PES was identified in 94 (6%) patients, with 108 PES pathogens isolated (n = 72 P. aeruginosa, n = 15 E  nterobacteriaceae extended-spectrum β-lactamase positive, and n = 21 methicillin-resistant S  taphylococcus aureus). These patients were older (P = 0.001), had received prior antibiotic treatment more frequently (P < 0.001), and frequently presented with acute renal failure (P = 0.004). PES pathogens were independently associated with increased risk of 30-day mortality (adjusted odds ratio = 2.51; 95% confidence interval = 1.20–5.25; P = 0.015). The area under the curve for the score we computed was 0.759 (95% confidence interval, 0.713–0.806; P < 0.001). Conclusions PES pathogens are responsible for a small proportion of CAP, resulting in high mortality. These pathogens require a different antibiotic treatment, and identification of specific risk factors could help to identify these microbial etiologies.

Abstract Rationale Chronic infection with Pseudomonas aeruginosa is associated with an increased exacerbation frequency, a more rapid decline in lung function, and increased mortality in patients with bronchiectasis. Objectives To perform a randomized placebo-controlled study assessing the efficacy and safety of inhaled colistin in patients with bronchiectasis and chronic P. aeruginosa infection. Methods Patients with bronchiectasis and chronic P. aeruginosa infection were enrolled within 21 days of completing a course of antipseudomonal antibiotics for an exacerbation. Participants were randomized to receive colistin (1 million IU; n = 73) or placebo (0.45% saline; n = 71) via the I-neb twice a day, for up to 6 months. Measurements and Main Results The primary endpoint was time to exacerbation. Secondary endpoints included time to exacerbation based on adherence recorded by the I-neb, P. aeruginosa bacterial density, quality of life, and safety parameters. All analyses were on the intention-to-treat population. Median time (25% quartile) to exacerbation was 165 (42) versus 111 (52) days in the colistin and placebo groups, respectively (P = 0.11). In adherent patients (adherence quartiles 2–4), the median time to exacerbation was 168 (65) versus 103 (37) days in the colistin and placebo groups, respectively (P = 0.038). P. aeruginosa density was reduced after 4 (P = 0.001) and 12 weeks (P = 0.008) and the St. George’s Respiratory Questionnaire total score was improved after 26 weeks (P = 0.006) in the colistin versus placebo patients, respectively. There were no safety concerns. Conclusions Although the primary endpoint was not reached, this study shows that inhaled colistin is a safe and effective treatment in adherent patients with bronchiectasis and chronic P. aeruginosa infection. Clinical trial registered with http://www.isrctn.org/ (ISRCTN49790596)

Background The effect of dual systemic antibiotic therapy against Pseudomonas aeruginosa in patients with pre-existing lung disease is unknown. To assess whether dual systemic antibiotics against P. aeruginosa in outpatients with COPD, non-cystic fibrosis (non-CF) bronchiectasis, or asthma can improve outcomes. Methods Multicenter, randomised, open-label trial conducted at seven respiratory outpatient clinics in Denmark. Outpatients with COPD, non-CF bronchiectasis, or asthma with a current P. aeruginosa -positive lower respiratory tract culture (clinical routine samples obtained based on symptoms of exacerbation not requiring hospitalisation), regardless of prior P. aeruginosa- status, no current need for hospitalisation, and at least two moderate or one hospitalisation-requiring exacerbation within the last year were eligible. Patients were assigned 1:1 to 14 days of dual systemic anti-pseudomonal antibiotics or no antibiotic treatment. Primary outcome was time to prednisolone or antibiotic-requiring exacerbation or death from day 20 to day 365. Results The trial was stopped prematurely based in lack of recruitment during the COVID-19 pandemic, this decision was endorsed by the Data and Safety Monitoring Board. Forty-nine outpatients were included in the study. There was a reduction in risk of the primary outcome in the antibiotic group compared to the control group (HR 0.51 (95%CI 0.27–0.96), p  = 0.037). The incidence of admissions with exacerbation within one year was 1.1 (95%CI 0.6–1.7) in the dual antibiotic group vs. 2.9 (95%CI 1.3–4.5) in the control group, p  = 0.037. Conclusions Use of dual systemic antibiotics for 14 days against P. aeruginosa in outpatients with chronic lung diseases and no judged need for hospitalisation, improved clinical outcomes markedly. The main limitation was the premature closure of the trial. Trial Registration ClinicalTrials.gov, NCT03262142, registration date 2017–08-25.

The continuously increasing carbapenem resistance within and poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS). A total of 78 , 30 were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark. A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with (42.7%), (23.3%) and (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in (76.3%). Among , the major sequence types were st307 and st11, while among and , the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in , the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for , detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for , carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%). The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among isolates, and the former performed better than CDT among .

Pseudomonas aeruginosa ( P. aeruginosa ) is an important zoonotic pathogen. It is also the primary causative agent of systemic infections in the endangered Moschus berezovskii . The emergence of multidrug-resistant strains of P. aeruginosa has made these infections increasingly difficult to control, and bacteriophages are considered important alternatives or adjuncts to antibiotic therapy. This study isolated P. aeruginosa strains that induce suppurative infections in Moschus berezovskii from a farm in Shaanxi Province, China. The bacteriophages vB_PaeP_FMD5 (FMD5) and vB_PaeM_H24-1 (H24-1) were isolated using these bacteria as hosts. The safety and practicality of the two phages were analyzed through methods such as biological characteristic assessment, whole genome sequencing analysis, and animal experiments. FMD5 is classified within the Podoviridae family, whereas H24-1 belongs to Myxoviridae. Biological characterization revealed that both FMD5 and H24-1 exhibit tolerance to temperature, pH, chloroform, and Ultraviolet(UV) exposure. The optimal multiplicity of infection (OMOI) for FMD5 and H24-1 were 0.01 and 0.1, respectively, and the burst sizes from the one-step growth curve were 200 PFU/cell and 150 PFU/cell, respectively. In vitro inhibitory assays demonstrated that FMD5, H24-1, and their cocktail exerted a favorable inhibitory effect for up to 11 hours. Whole genome sequencing confirmed that both phages possess double-stranded DNA genomes, with FMD5 having a length of 72,254 bp and a G+C content of 55.16%, containing 91 ORFs(Open Reading Frame)​, whereas H24-1 has a genome length of 66,281 bp, a G+C content of 56.26%, and encompasses 94 ORFs. No drug-resistance genes, virulence factors, or lysogenic genes were identified in either phage. Phylogenetic analysis of conserved genes revealed that FMD5 is closely related to the previously published Pseudomonas phage LP14 (LP14), while H24-1 is closely related to the previously published Pseudomonas phage vB_PaeM_LS1(LS1), but both are newly discovered bacteriophages. In a mouse model of bacteremia treated with bacteriophages, both individual phages and the cocktail exhibited favorable therapeutic effects. The two novel bacteriophages isolated in this study exhibit efficient and stable characteristics. They demonstrate sound therapeutic effects against bacteremia in mice caused by multidrug-resistant P. aeruginosa , suggesting their great potential as alternatives or adjuncts to antibiotic therapy for treating infection.

A novel lytic bacteriophage, PSA-KC1, was isolated from wastewater. In this study, the whole genome of the bacteriophage PSA-KC1 was analyzed, and its lytic properties were assessed. PSA-KC1 has a linear double-stranded DNA genome with a total length of 43,237 base pairs and a GC content of 53.6%. In total, 65 genes were predicted, 46 of which were assigned functions as structural proteins involved in genome replication, packaging or phage lysis. PSA-KC1 belongs to the genus Septimatrevirus under the Caudoviricetes class. The aim of this study was to investigate the efficacy of the lytic bacteriophage PSA-KC1 and compare it with that of the Pyophage phage cocktail on 25 multi drug resistant (MDR) Pseudomonas aeruginosa strains isolated from sputum samples of cystic fibrosis patients. Seventeen of these strains were susceptible (68%) to the PSA-KC1 lytic phage we isolated, whereas eight clinical strains were resistant. However, 22 (88%) of the P. aeruginosa strains were susceptible to the Pyophage cocktail, and three (12%) were resistant to the Phage cocktail. At the end of our study, a new lytic phage active against multidrug-resistant P. aeruginosa strains from CF patients was isolated, and its genome was characterized. Since the PSA-KC1 phage does not contain virulence factors, toxins or integrase genes, it can be expected to be a therapeutic candidate with the potential to be used safely in phage therapy.

The rise of infections caused by multidrug-resistant bacteria has driven the search for alternative therapies, with Pseudomonas aeruginosa representing a major target. Among these alternatives, the use of bacteriophages (phage therapy) employs these viruses that specifically infect bacteria to fight infections. Although phage efficacy has been demonstrated both in vitro and in vivo, few studies have directly correlated these two dimensions. This study aimed to evaluate, compare, and correlate in vitro and in vivo phage activity. For this, three strictly lytic bacteriophages (ph9027, ph1461, and ph3678) were isolated and tested against 15 clinical isolates and three reference strains of P. aeruginosa . Their in vitro activity was assessed through Spot-Test, Efficiency of Plating, Local Virulence, and Virulence Index assays, followed by in vivo assays in Galleria mellonella larvae and Balb/C mice in a peritonitis model with bacteremia induction. Virulence Index and animal treatments were performed using a phage cocktail (1:1:1 ratio, 1.0 × 10⁹ PFU/mL). In vitro assays revealed varying phage activity, with the Virulence Index showing the strongest correlation with in vivo results in G. mellonella (rs = 0.7648). Larval treatment achieved up to 100% survival, significantly different ( p  < 0.05) from bacteria-only controls. In mice, phage therapy reduced bacterial loads by up to 3 logs in blood, spleen, peritoneal lavage, liver, and lung ( p  < 0.05) in comparison with control groups. Therefore, the present study, in addition to reiterating the safety and therapeutic efficacy of phage therapy in animal models, presents evidence that Virulence Index techniques, associated with the survival results of G. mellonella larvae, can be excellent tools for directing preclinical assays due to the strong statistical correlation observed between these data and those obtained in mouse experiments.