C-X-C Motif Chemokine Receptor 4 (CXCR4) in CD4+ T cells in LBD. CSF protein levels of the CXCR4 ligand, C-X-C Motif Chemokine Ligand 12 (CXCL12) were associated with neuroaxonal damage in LBD. Furthermore, we observed clonal expansion and upregulated Interleukin 17A expression by CD4+ T cells stimulated with a phosphorylated α-synuclein epitope. Thus, CXCR4-CXCL12 signaling may represent a mechanistic target for inhibiting pathological interleukin-17-producing T cell trafficking in LBD.Recent studies indicate that the adaptive immune system plays a role in Lewy body dementia (LBD). However, the mechanism regulating T cell brain homing in LBD is unknown. Here, we observed T cells adjacent to Lewy bodies and dopaminergic neurons in post-mortem LBD brains. Single-cell RNA sequencing of cerebrospinal fluid (CSF) identified upregulated expression of  The immune system is implicated in the neurodegenerative process of Lewy body dementia. PDD is defined by changes in memory and behavior and afflicts patients in late stage Parkinson’s disease (PD) . The syme models –13 and ie models . Moreovee models , 5, 14. P = 8.6X10−5; P = 0.0031; P = 6.33X10−13) and DLB (P = 4.02X10−13) presented with lower cognitive scores than PD-NCI patients  and patients with clinical DLB and PD  . Montrea+ T cells in close proximity to neuronal processes labeled by the dopamine enzyme tyrosine hydroxylase (TH) in the substantia nigra of PDD and DLB brains + glutamatergic neurons in the hippocampal CA2 region , a kinase essential for cytokine signaling, and the T cell activation gene Cluster Of Differentiation 69 (CD69) (C-X-C Motif Chemokine Receptor 4 (CXCR4) was also highly upregulated in PD-DLB CD4+ T cells  , 16 of C subtype . Highly 9 (CD69) . The che T cells . Moreove T cells . Quantifn PD-DLB . Thus, e+ T cells in PD-DLB prompted us to determine whether clonally expanded (i.e. antigen-specific) cells were distinct in PD-DLB. To assess clonal expansion, we performed single-cell T cell receptor sequencing (scTCRseq) on the same CSF cells as above , a marker of pro-inflammatory IL-17-producing (Th17) memory CD4+ T cells  and PD-NCI (P = 7.67X10−10) subjects  = 31.697, P = 5.18X10−12); P = 1.00X10−4) and PD-NCI (P = 8.30X10−3) subjects  = 9.161, P = 0.0002); P = 0.071); s = 0.40; P = 0.023), and these correlations were lesser in healthy  and PD-NCI  subjects (ANCOVA (F (P = 0.031); We next sought to determine whether levels of CSF CXCL12 were associated with cognitive impairment in PD. We measured CXCL12 in a cohort of age- and sex-matched healthy (n=84) and PD (n=79) subjects . This re+ T cells of the peripheral immune system and CSF. We performed scRNAseq on peripheral blood mononuclear cells (PBMCs) of the same subjects we analyzed by CSF scRNAseq and focused our analysis on CD4+ T cells  and Actin Beta (ACTB)  , which ra (ACTB) . We also T cells .Interleukin 17A (IL17A) in cells stimulated with α-synuclein  from each subject (IL17A-expressing cells co-expressed CD4 and some clonotypes also expressed the Th17-associated cytokine gene Interleukin 22 (IL22; +IL-17A+ T cells in the PDD substantia nigra, which were adjacent to TH+IL-17A+ neurons (P = 0.007; IL17A RNA expression in the brain, yet the gene encoding the IL17A receptor, IL17RA, was highly expressed in the midbrain (Notably, we also detected higher expression of ynuclein . IL17A e17 cells . To detepatients . We then subject . IL17A-e22 IL22; . We conf neurons . We alsomidbrain , suggestmidbrain . Finallymidbrain .IL17A, a pro-inflammatory cytokine involved in autoimmune diseases (In conclusion, these results implicate Th17 cell involvement in the degeneration of neurons in LBD. Notably, CXCR4 regulates cell migration , and antdiseases . In animdiseases , 32. Mordiseases . Thus, odiseases .Supplementary MaterialData S3Data S2Data S1Data S4


A prior study demonstrated suboptimal antibiotic prescribing in the emergency department (ED) at 77.4% for uncomplicated lower respiratory tract infections (LRTI), urinary tract infections (UTI), and acute bacterial skin and skin structure infections (ABSSSI). This study measured the effect of indication-based antibiotic order sentences (AOS) on prescribing.IRB-approved quasi-experiment of adults prescribed antibiotics in ED for uncomplicated LRTI, UTI, or ABSSSI from January - June 2019 (pre-group) or September - December 2021 (post-group). Exclusion: hospital admission, immunocompromised, active cancer, or prophylactic antibiotics. AOS are lean process, electronic discharge prescriptions retrievable by name or indication; AOS implementation occurred July 2021. Optimal prescribing was defined as the correct antibiotic selection, dose, and duration per local and national guidelines. Seven-day endpoints: antibiotic escalation, ED or hospital readmission, any outpatient contact, and reported adverse drug event (ADE). Descriptive and bivariate statistics performed. Variables considered for multivariable logistic regression had p< 0.2 or plausible association with optimal prescribing.294 patients included: 147-pre and 147-post. Patient characteristics are in Table 1. Overall optimal prescribing improved from 12 (8.2%) to 34 (23.1%) (p< 0.001). Breakdown of optimal prescribing in pre- and post-groups: selection 90 (61.2%) vs 117 (79.6%) (p< 0.001), dose 99 (67.3%) vs 115 (78.2%) (p=0.036), duration 38 (25.9%) vs 50 (34%) (p=0.126). After adjustment, AOS were independently associated with optimal prescribing (Table 2). Secondary endpoints: antibiotic escalation 10 (6.8%) vs 7 (4.8%) (p=0.662), hospital or ED readmission 12 (8.2%) vs 10 (6.8%) (p=0.658), outpatient contact 31 (21.1%) vs 28 (19%) (p=0.662), and ADE 2 (1.4%) vs 4 (2.7%) (p=0.684). Post-hoc analysis showed suboptimal uptake of AOS by ED prescribers.AOS is an efficient and promising antimicrobial stewardship strategy; provider re-education is needed to increase AOS uptake.All Authors: No reported disclosures.


Nirmatrelvir coadministered with ritonavir (nirmatrelvir/r) is a COVID-19 treatment. This study evaluated nirmatrelvir/r in nonhospitalized, symptomatic adults with COVID-19 at high risk of progressing to severe disease. We report secondary efficacy endpoints associated with COVID-19─related medical visits, including hospitalization details and oxygen support, as of the primary completion data cutoff .In this phase 2/3 double-blind, interventional study, adults with confirmed SARS-CoV-2 and symptom onset ≤ 5 days (d) were randomized 1:1 to receive nirmatrelvir/r 300 mg/100 mg or placebo (PBO) orally every 12 hours for 5 d. COVID-19─related medical visits were collected through Day 28. Oxygen support for COVID-19 and details of COVID-19─related hospitalization, including duration, intensive care unit (ICU) status, and mechanical ventilation, were assessed.Table 1). In addition to fewer hospitalizations being reported with nirmatrelvir/r (n=8 [0.8%]) vs PBO (n=65 [6.2%]), pts receiving nirmatrelvir/r had fewer hospitalized d (Table 2), with mean durations of 9.6  d with nirmatrelvir/r and 11.2  d with PBO in hospitalized pts. No pts in the nirmatrelvir/r group and 9 pts (0.9%) in PBO group were admitted to the ICU. No pts in the nirmatrelvir/r group received mechanical ventilation vs 3 pts in the PBO group. Fewer other COVID-19─related nonhospital medical visits were reported with nirmatrelvir/r vs PBO (Table 3). In the full analysis set, fewer pts required oxygen therapy for COVID-19 with nirmatrelvir/r (n=9/1120 [0.8%]) vs PBO (n=54/1126 [4.8%]).Of the 2246 patients (pts) enrolled globally from Jul to Dec2021, 2085  started treatment and met criteria for the modified intent-to-treat population . Fewer overall COVID-19-related medical visits were reported with nirmatrelvir/r vs PBO  vs pts receiving PBO. Clinical Trial: NCT04960202.Jennifer Hammond, PhD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Heidi Leister-Tebbe, BSN, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Annie Gardner, MPH, MSPT, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Paula Abreu, PhD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Weihang Bao, PhD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Wayne Wisemandle, MA, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds Wajeeha Ansari, MPH, Pfizer Inc.: Stocks/Bonds Rienk Pypstra, MD, MBA, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds James M Rusnak, MD, PhD, Pfizer Inc: Employee|Pfizer Inc: Stocks/Bonds.


Correction: BMC Musculoskelet Disord 24, 49 (2023)10.1186/s12891-023-06160-zhttp://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=emed8&NEWS=N&AN=2009254927)” instead of Chan et al. 2013.Following publication of the original article , the autFurthermore, the word "Specific" in Secondary outcomes is without the "S" in Table 1.The original article  has been


Urinary tract infections (UTI) are the second most prevalent microbial infection, impacting 150 million people globally every year. Traditionally, urine culture is considered the gold standard for UTI pathogen detection. However, molecular techniques, such as real time multiplex PCR tests targeting multiple pathogens, are becoming the alternative diagnostic tools for rapid detection of pathogens with the potential to provide quick diagnosis and enable targeted treatment. Due to the serious economic and healthcare utilization burden UTIs pose, early pathogen detection with a rapid turn-around-time to results has the potential to be instrumental for improving patient care and outcomes.A total of 300 deidentified patient samples that were previously tested via urine culture  were subjected to real time PCR molecular testing employing the nanofluidic Open Array ® platform . Statistical analyses were performed using R version 3.6.0.Among 300 urine specimens studied, culture detected pathogens in 183 patient samples (61%), and 117 samples were deemed negative. Culture and PCR results demonstrated an overall agreement of 75.3% (n=226) with 59% positive (n=177) and 16.3% negative (n=49). Results for 24.7% samples (n=74) were discordant. A total of 2% (n=6) were culture positive and PCR negative, and 22.7% (n=68) were culture negative and PCR positive. Agreement between PCR and culture positive results was 0.97, 95%CI  (177/183). Among the PCR positive samples (n=245), 32.7% were poly-microbial (n=80) and 67.3% were mono-microbial (n=165). In comparison, among culture positive samples (n=183), 33.3% (n=61) were polymicrobial and 65.6% (n=120) monomicrobial.Our study demonstrates that multiplex real time PCR-based detection of UTI bacterial pathogens has positive agreement with the traditional urine culture method. PCR based testing has the potential to deliver quick pathogen identification to enable targeted treatment options and medication adjustments. Further studies will correlate semi-quantitative cfu/mL culture values with semi-quantitative copies/mL PCR values to further refine result reporting and test performance.Pallavi Upadhyay, PhD, HealthTrackRx: Stocks/Bonds Fahida Surar, B.S., HealthTrackRx: Salaried Employee Geun Kim, M.S., HealthTrackRx: Salaried Employee Jay Reddy, PhD, HealthTrackRx: Stocks/Bonds Barbara D. Alexander, MD, Astellas: Advisor/Consultant|HealthtrackRx: Advisor/Consultant|HealthtrackRx: Grant/Research Support|Scynexis: Grant/Research Support|UpToDate: Advisor/Consultant Kimberly Hanson, MD, MHS, FIDSA, HealthTrackRx: Advisor/Consultant|HealthTrackRx: Clinical Advisory Board Member Vijay Singh, PhD, HealthTrackRx: Stocks/Bonds.


Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017–2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-β-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 β-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)—ST11, ST258, and ST15 and five main Escherichia coli clones—ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented.The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of  Klebsiella pneumoniae and hospital-acquired infections [Enterobacterales species [K. pneumoniae, Escherichia coli, and Enterobacter cloacae complex have been globally reported [β-lactams are commonly used antimicrobials due to their safe profile and broad-spectrum activity ,2,3,4,5.fections . In the  species ,4,7. The species . These b species ,4,5,6. Ireported ,4,6. In reported . In addireported .The gastrointestinal tract can be an important reservoir of ESBL-/carbapenemase-producing enterobacteria in hospital settings  leading Fecal carriage of ESBL/carbapenemase producers has been widely reported all over the world ,10. DiffThe aim of this study was to evaluate the prevalence of β-lactamase production and clonal relatedness of fecal ESBL- and carbapenemase-producing enterobacteria, collected from hospitalized patients .TMKPC . Bacterial isolates were identified using routine biochemical identification and were confirmed by VITEK  or Phoenix . Hsp60 sequencing [Enterobacter spp. and Klebsiella oxytoca isolates.The study was conducted in six hospitals—University Multiprofile Hospital for active treatment (UMHAT), Varna; UMHAT, Plovdiv; UMHAT, Pleven, and three hospitals in Sofia during the period of December 2017–June 2019. The fecal samples were obtained from patients  during the routine diagnostic process and were additionally inoculated on selective MacConkey agar with 1 mg/L cefotaxime and on Chromagarquencing  was usedhttp://www.eucast.org/clinical_breakpoints/), last accessed on 10 August 2022. The following antibiotics were tested: amoxicillin/clavulanic acid 30 µg (AMC), ceftazidime 10 µg (CAZ), cefotaxime 5 µg (CTX), cefepime 30 µg (FEP), cefoxitin 30 µg (FOX), imipenem 10 µg (IMP), meropenem 10 µg (MEM), piperacillin/tazobactam 36 µg (PIP/TAZ), chloramphenicol 30 µg (CHL), gentamicin 10 µg (GEN), amikacin 30 µg (AMK), tobramycin 10 µg (TOB), trimethoprim/sulfamethoxazole 25 µg (SXT), ciprofloxacin 5 µg (CIP), levofloxacin 5 µg (LVX), fosfomycin 200 µg (FOS)(for E. coli isolates), and tigecycline 15 µg (TIG for E. coli isolates). Susceptibility to tigecycline in Klebsiella isolates was determined by the broth microdilution method . Susceptibility to colistin was initially tested with modified SuperPolymyxin medium [K. pneumoniae and E. coli that grew on this screening medium were tested with the broth microdilution method .Antimicrobial susceptibility testing was performed using the disk diffusion method on Müller-Hinton II agar and the microdilution broth method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (Version 10). The susceptibility testing was carried out according to the clinical breakpoints of the EUCAST (Version 10) , a phenotypic confirmation of carbapenemase production was performed by the KPC, MBL, and OXA-48 Kit .Presumptive ESBL production was detected with the double-disk synergy method . PotentiE. coli K12:W3110 RifR lac/-/(1.2 × 107 CFU/mL). After overnight incubation at 37 °C, growth of the indicator strain on the gel localized the β-lactamase band by which the β-lactam had been inactivated.Production and number of β-lactamases were detected and analyzed by analytical isoelectric focusing (IEF) according to the method of Mathew  with modblaVIM, blaIMP, blaKPC, blaNDM, blaOXA-48), AmpC , and ESBLs  as previously described [blaSHV, blaCTX-M-1-group, blaCTX-M-9-group, blaCMY,blaDHA,blaKPC, and blaNDM. [All isolates were screened for the presence of carbapenemase-encoding genes , last accessed on 30 July 2022. A clone was defined as isolates showing 80% similarity.Clonal relatedness was investigated by ERIC PCR and Multilocus Sequence Typing (MLST). For Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, ERIC-1 and -2a primers were used as described previously . GeneticK. pneumoniae MLST typing and Achtman scheme was applied for E. coli. For E. coli isolates, the assignment to allelic numbers and sequence types (STs) was performed according to the MLST database (https://bigsdb.web.pasteur.fr/ecoli/ecoli.html), last accessed on 30 July 2022. Detection of specific O25b-ST131 clone was performed with allele-specific PCR for pabB gene as previously described [Pasteur scheme was used for K. pneumoniae isolates, protocols and assignment to allelic numbers and sequence-types (STs) were carried out as described in the MLST database , last accessed on 30 July 2022. A clonal complex was defined as a group of two or more independent isolates that shared six identical alleles.For E. cloacae complex isolates, primers, protocols, and assignment to allelic numbers and sequence-types (STs) were carried out as described in the MLST database (https://pubmlst.org/organisms/enterobacter-cloacae), last accessed on 30 July 2022.For E. coli isolates as previously described [Phylotyping was applied for escribed .E. coli (n = 103); K. pneumoniae (n = 65); Klebsiella oxytoca (n = 8); E. cloacae complex (n = 16); Morganella morganii (n = 3); and Citrobacter freundii complex (n = 10).A total of 205 enterobacterial isolates resistant to cefotaxime were isolated from fecal samples, collected from 580 patients (35.3%) as follows: 61 cefotaxime-resistant isolates from 158 studied patients (38.6%) from University Hospital (UH)-Varna; 25 cefotaxime-resistant isolates from 71 patients (35.2%) from UH-Pleven; 58 cefotaxime-resistant isolates from 102 patients (56.9%) from UH-Plovdiv, and 61 cefotaxime-resistant isolates from 249 patients (24.5%) from three hospitals in Sofia. The isolates were identified as Hsp60 sequencing identified 15 E. cloacae complex isolates as Enterobacter hormaechei, subdividing them into three subspecies—E. hormaechei spp. hoffmannii (n = 3); E. hormaechei spp. steigerwaltii (n = 10); and E. hormaechei spp. xiangfangensis (n = 2). One isolate was identified as Enterobacter kobei. Two isolates, initially identified as K. oxytoca, were reidentified by hsp60 sequencing as Klebsiella michiganensis.Klebsiella isolates to tigecycline was 93%. For E. coli, 17% and 3% resistance to tigecycline and fosfomycin, respectively, were found. Only five isolates of Klebsiella spp. (2.4%) were colistin nonsusceptible. The results are shown in K. pneumoniae isolates were determined as pandrug-resistant according to the criteria of Magiorakos et al. [The nonsusceptibility (resistance or intermediate susceptibility) rates in the collection of isolates were as follows: amoxicillin/clavulanic acid 93%, ceftazidime 86%, cefotaxime 100%, cefoxitin 35%, cefepime 95%, piperacillin/tazobactam 72%, imipenem 7%, meropenem 7%, tobramycin 68%, gentamicin 56%, amikacin 50%, trimethoprim/sulfamethoxazole 52%, ciprofloxacin 68%, levofloxacin 63%, and chloramphenicol 22%. The nonsusceptibility rates in the s et al. .E. cloacae complex and three C. freundii complex isolates were determined as possible inducible AmpC hyperproducers, demonstrating antagonism between amoxicillin/clavulanic acid and cefotaxime or ceftazidime.Disk diffusion synergy method (DDST) confirmed 171 isolates  as ESBL producers. Four K. pneumoniae isolates, suggesting class B carbapenemase production (zone around the disk with EDTA is ≥5 mm). Three isolates (two K. pneumoniae and one E. coli) demonstrated increased zones of inhibition by the disk containing meropenem and phenylboronic acid, suggesting class A (KPC) carbapenemase activity.The phenotypic test with meropenem and meropenem/EDTA disks was positive in 11 carbapenem-resistant blaSHV-1 gene was detected (which suggests possible SHV-1 hyperproduction). Three cefotaxime-resistant isolates, susceptible to carbapenems and with positive DDST did not produce a positive PCR reaction with any of the used ESBL group-specific primers.PCR and sequencing revealed the presence of genes, encoding ESBL in 167 (28.8%) isolates, obtained from 580 patients. In one isolate, only p < 0.0001). The ESBL producers among the isolates of E. coli and K. pneumoniae were 56.2% (94/167) and 34.1% (57/167), respectively.The prevalence of ESBL producers in Sofia hospitals was significantly lower  than in the other locations, 35.6% (118 ESBL producers/331 patients) (blaCTX-M-15 in 41% (84/205) of the isolates, blaCTX-M-3 in 24% (49/205), blaCTX-M-27 in 11% (22/205), and 8 isolates (3.9%) displayed blaCTX-M-14. Only one isolate was positive for blaCTX-M-9 and one for blaSHV-12 , except one isolate that showed blaCTX-M-14, were produced by E. coli strains. In contrast, blaCTX-M-3 was the prevailing ESBL among K. pneumoniae and K. michiganensis isolates. One K. pneumoniae isolate showed mixed sequences , GGT is a codon for glycine (specific for blaCTX-M-15)). All K. pneumoniae isolates were blaSHV positive. Of them, 29 representative isolates were sequenced, and blaSHV-1 (n = 21) and blaSHV-11 (n = 8) were identified. In 71 isolates, blaTEM was found and, later, 10 of them were confirmed by sequencing as blaTEM-1.Among the 205 investigated cefotaxime-resistant isolates, we observed solely laSHV-12 . blaCTX-K. pneumoniae, were carbapenemase producers. Thirteen of them coproduced ESBL or/and plasmid AmpC (K. pneumoniae isolates had blaNDM-1 together with blaCMY-4 and blaCTX-M-3/-15. Two K. pneumoniae and one E. coli isolates had blaKPC-2.Fourteen isolates (2.4%) from 580 patients, mainly mid AmpC . Ten K. E. coli  isolates showed two types of plasmid AmpC, blaDHA-1, n = 5 or blaCMY-2, n = 2. Thirteen isolates (2.2%) were AmpC hyperproducers (inducible or constitutive) (Seven itutive) .Thus, 188 isolates (32.4%) were found to carry ESBL (28.8%) or carbapenemases (2.4%) or plasmid AmpC genes (1.2%).Klebsiella spp. isolates, 24 representative isolates were subjected to IEF   exhibited two bands—pI8.4 and pI8.8 (The isoelectric focusing was carried out in representative isolates according to the species and detected β-lactamase genes. Of 73 solates) . We confnd pI8.8   and did not show cefotaxime hydrolyzing bands corresponding to TEM and SHV ESBL β-lactamases. Three isolates with unidentified enzyme type gave bands with pI8.0 (two isolates) and pI9.2 (one isolate). They were without CTX hydrolytic activity.E. hormaechi, C. freundii complex and M. morganii isolates, 5 isolates were tested, and a single cefotaxime hydrolyzing band was detected, pIs corresponded with sequenced enzymes , ST11 , and ST37 being the dominant types. In addition, ST1198, ST280, ST34, ST15, ST258, ST17, ST253, and ST449 were also found. Six isolates demonstrated unique profiles . K. oxytoca and K. michiganensis isolates had unique ERIC profiles.Epidemiological typing revealed 18 ERIC clusters  among 65profiles . The isohospital . ST11 isE. coli isolates, 40 ERIC types were identified , ST38 , ST155 , ST405 , ST1196, and ST88 .Among entified . A totalE. coli (n = 43) was detected in five centers and was associated with the production of CTX-M-27 , CTX-M-15 , CTX-M-3 , CTX-M-14 , and KPC-2 carbapenemase, found in a single isolate ; D, n = 20 ; A, n = 12; and B1, n = 20. The association between MLST types and phylotypes is shown in The following E. cloacae isolates (n = 16) demonstrated 12 ERIC clusters and 6 ST types. The most common ST was ST90, associated with CTX-M-15 production (oduction .n = 580) was similar to that in Portuguese hospitals (24%) [E. coli and K. pneumoniae isolates  in Bulgaria in 2019, which was the highest in Europe and showed a stable trend during the last 5 years , last accessed on 10 August 2022. We also found increased nonsusceptibility rates in the tested ESBL fecal isolates to aminoglycosides (50%-68%) and quinolones (63–68%) in comparison to the rates found in 2015 (21%-57% for aminoglycosides and 15–27% for quinolones) [This study reveals a moderate rate of fecal colonization with ESBL producers 28.8%) among Bulgarian patients during the period 2017–2019. The rate of third-generation cephalosporin-resistant isolates (mostly due to production of ESBL/carbapenemase/plasmid AmpC) was high (35.3%), but lower than the rate observed in a pilot study on hospital fecal carriage in Varna city, Bulgaria in 2015 (42.5%) . A possils (24%)  and highls (24%) . The obs.8% amongnolones) . A similnolones) .E. coli (56.2% versus 69%) and an increased proportion of ESBL K. pneumoniae isolates (34.1% versus 20.4%) were found [E. coli have been reported in many studies [In comparison with our previous study in 2015, a decreased rate of ESBL-producing re found . Similarre found . The pre studies ,26,27,28E. coli, as well as in all other species except Klebsiella spp. CTX-M-15-producing isolates have been reported worldwide [Klebsiella isolates. Similar to CTX-M-15, CTX-M-3 was also widely distributed among enteric strains [E. coli isolates. CTX-M-9 and CTX-M-14 intestinal producers have been reported in Portugal, Spain, China, and Bulgaria [E. coli isolates. In Bulgaria, DHA-1 enzyme was identified for the first time in 2019 in an E. cloacae complex isolate obtained from a blood sample [The main β-lactamase type in our study was CTX-M-15 41%). It was the predominant ESBL in 1%. It waorldwide ,8,9. CTX strains ,26,27,28Bulgaria ,26. CTX-d sample .K. pneumoniae isolates and in a single E. coli isolate. All carbapenem-producing isolates were obtained from hospitalized patients in Sofia. Interestingly, the frequency of ESBL producers (19.7%) was not high among these patients. The coproduction of NDM-1, CTX-M-15/-3, and CMY-4 enzymes in nine K. pneumoniae isolates is a possible explanation for the high resistance rates, identified in the NDM-1 producing isolates, two of them being pandrug-resistant.An important finding in the current study is the detection of carbapenemase-producing isolates. Although the detection rate was low (2.4%), it is an indicator that the enteric tract may act as a reservoir for these problematic bacteria. The carbapenem resistance in the present collection of fecal isolates was associated with NDM-1 and KPC-2, detected in The rates of carbapenemase producers in Europe have steadily increased during the last years in both clinical and intestinal isolates ,33,34,35n = 17, 26%) among K. pneumoniae fecal isolates. Taking the second place, ST11 was represented in 17% of K. pneumoniae isolates, all obtained from patients in Sofia hospitals. Most NDM-1 carbapenemase-producers belonged to this clone, including two panresistant and the colistin-resistant isolates. This result is in concordance with our previous study from 2019 that detected a high level of ST11 blaNDM-1/blaCTX-M-15 or-3/blaCMY-4 positive isolates in the same hospitals [blaNDM-1/blaCTX-M-15/3/blaCMY-4 positive isolates have also been detected in the Czech Republic [Another important finding in the present study is the identification of the high-risk clones ST11, ST258, and ST15 , is another high-risk clone. It is also distributed worldwide and associated with KPC-2 production [ST258 oduction ,30,33,41oduction . ST11 anoduction ,40,42,43K. pneumoniae clone which has commonly been associated with the production of ESBLs, mainly CTX-M-15 [ST15 is another high-risk CTX-M-15 ,7,44, buCTX-M-15 . In the CTX-M-15 .K. pneumoniae clones found in this study were ST37 and ST17. These clones showed a sustained persistence in Bulgaria. These ST have been already identified in clinical and fecal isolates, associated with CTX-M-15 and KPC-2 production [Other intestinal multidrug-resistant oduction ,45. ST37oduction .In the present study, ST353 was presented by a high number of isolates (19%), mainly obtained from patients hospitalized in Plovdiv University Hospital(A). ST353 isolates were associated mainly with CTX-M-3 production. This ST type has been rarely reported. In studies from China and Colombia, authors reported ST353 isolates, producing KPC and OXA-48 ,47. ST11E. coli isolates with ST131 being the predominant, found in 41.7%. The second most common ST was ST38 (10%). ST405, ST95, and ST69 were presented by single isolates only. The isolates from these high-risk clones represented 57% (n = 59) of all the studied E. coli isolates. All of them belonged to the B2 or D phylogroups. This is an important finding as these phylogroups are associated with a prolonged fecal carriage [Five high-risk clones were observed among carriage . This win = 43) were representatives of phylogroup B2 and 60% (n = 26) belonged to the O25b:H4-ST131 clone. Our study showed that the O25b:H4-ST131 clone was the major clone among fecal E. coli isolates, harboring different ESBL genes, mainly blaCTX-M-15, but also blaCTX-M-3 and blaCTX-M-14, which is in concordance with other reports [E. coli isolates were detected in all centers except the Sofia hospital 1 (D), where NDM-1-producing ST11 K. pneumoniae was observed. In addition, the frequency of ST131 isolates in the present study was higher (42%) than the rate (35%) detected in 2015 [Almost all ST131 isolates  and the plasmid AmpC enzyme DHA-1. This type has also been reported as a high-risk clone, causing both nosocomial and community-acquired infections, mainly urinary tract infections [In our study, fections . The detfections .E. coli. Recently, it has been found that representatives of ST69 clone carry an intact locus of enterocytes effacement (LEE), coding second bacterial type III secretion systems involved in the pathogenesis of Gram-negative infections [E. coli were identified as ST405 (D phylogroup). This clone was previously detected in Bulgaria as a carrier of NDM-1 carbapenemases [E. coli ST405 is an emerging urosepsis pathogen, reported to carry blaCTX-M, blaNDM, and a number of virulent genes comparable with O25b:H4-ST131 [ST69 is an interesting lineage from the D phylogroup, identified in two isolates of fections . In our enemases . E. coliH4-ST131 .E. coli isolates belonging to B1 and A phylogroups, which have been reported as commensal gut bacteria [In addition to the isolates that represent highly virulent clones, we also detected bacteria ; the obsEnterobacter isolates, ST90 producing CTX-M-15 was detected as the dominant clone. Although no high-risk clones have been defined in Enterobacter spp. so far, ST90 isolates, producing different carbapenemases, were detected in many countries: VIM-1 in Greece, IMP-4 in Canada and UK, and NDM-1 in Romania [E. cloacae complex can be a candidate for an international high-risk clone.In the group of  Romania . Polish  Romania . So, we The fecal colonization with ESBL and/or carbapenemase producers from high-risk international clones, associated with significant virulence and invasive potential and multidrug or pandrug resistance, can be an important reservoir not only for difficult to treat nosocomial infections, but also for wide dissemination in the community. Given that this study was performed before the COVID-19 pandemic, we can assume that the increased antibiotic usage during the last three years has further worsened the situation.E. coli and 17 K. pneumoniae) were members of high risk clones (37%).Twenty six percent of the K. pneumoniae isolates were representatives of high-risk clones such as ST11, ST258 and ST15 K. pneumoniae isolates. A very worrying finding was the detection of two ST11 pandrug resistant isolates, coproducing NDM-1, CMY-4, and CTX-M-15. Among E. coli, five high risk clones (57% of E. coli isolates) were found . The investigated isolates were producers of a wide range of β-lactamases—CTX-M-15, CTX-M-3, CTX-M-27 (reported for the first time in Bulgaria), CTX-M-14, NDM-1, KPC-2, and plasmid AmpC DHA-1 and CMY-2 enzymes. The detected high frequency of ESBL/carbapenemases-producing enteric bacteria before COVID-19 and the dramatically increased selective pressure during the pandemic period will negatively impact the antimicrobial resistance in clinically significant bacterial species. Further studies should closely monitor the future trends. The routine screening for colonization with MDR bacteria at hospital admission and during the hospital stay, especially in high-risk departments, as well as strict infection control measures should be widely implemented in Bulgarian hospitals to limit the further dissemination of problematic multidrug-resistant bacteria.In conclusion, a high rate of fecal colonization (32.3%) with ESBL/carbapenemase/plasmid AmpC producers among patients in Bulgarian hospitals was found. A high rate of ESBL producers (28.8%) was detected with a relatively low rate of carbapenemase producers (2.4%). Seventy six isolates (59


Bacteria and archaea are central to the production, consumption, and remineralization of dissolved and particulate organic matter and contribute critically to carbon delivery, nutrient availability, and energy transformations in the deep ocean. To explore environmentally relevant genomic traits of sinking-particle-associated versus free-living microbes, we compared habitat-specific metagenome-assembled genomes recovered throughout the water column in the North Pacific Subtropical Gyre. The genomic traits of sinking-particle-associated versus free-living prokaryotes were compositionally, functionally, and phylogenetically distinct. Substrate-specific transporters and extracellular peptidases and carbohydrate-active enzymes were more enriched and diverse in particle-associated microbes at all depths than in free-living counterparts. These data indicate specific roles for particle-attached microbes in particle substrate hydrolysis, uptake, and remineralization. Shallow-water particle-associated microbes had elevated genomic GC content and proteome nitrogen content and reduced proteome carbon content in comparison to abyssal particle-associated microbes. An inverse trend was observed for their sympatric free-living counterparts. These different properties of attached microbes are postulated to arise in part due to elevated organic and inorganic nitrogen availability inside sinking particles. Particle-attached microbes also were enriched in genes for environmental sensing via two-component regulatory systems, and cell-cell interactions via extracellular secretion systems, reflecting their surface-adapted lifestyles. Finally, particle-attached bacteria had greater predicted maximal growth efficiencies than free-living bacterioplankton at all depths. All of these particle-associated specific genomic and proteomic features appear to be driven by microhabitat-specific elevated nutrient and energy availability as well as surface-associated competitive and synergistic ecological interactions. Although some of these characteristics have been previously postulated or observed individually, we report them together here in aggregate via direct comparisons of cooccurring free-living and sinking-particle-attached microbial genomes from the open ocean. Thes10–16–in situ by divers  and FL microbes found throughout the water column in the oligotrophic Pacific Ocean, we leveraged time series metagenomic samples recovered at Station ALOHA in the North Pacific Subtropical Gyre, generating a total of 407 mid- to high-quality MAGs from both sample types. FL microbes (captured on 0.2-μm-pore-size filters) were collected from surface waters to a depth of 4,000 m at Station ALOHA, and metagenomes were generated, assembled, and analyzed –45. In p–To expand the genomic data from sympatric SPA and FL prokaryote communities throughout the water column, metagenomic data sets were prepared from time series samples collected from the surface to 4,000 m during Station ALOHA time series efforts and expeditions –47. Thes–43–10.1128/mbio.01569-22.6TABLE S1Table S1, XLSX file, 0.03 MB.Sample descriptions and metadata of metagenomes. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The FL and PA MAGs were classified into five sample types . FL MAGs10.1128/mbio.01569-22.7TABLE S2Table S2, XLSX file, 0.04 MB.Checkm statistics, taxonomic classification, and sample designation of the MAGs. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria, including Alphaproteobacteria, Gammaproteobacteria, Planctomycetota, Chloroflexota, Bacteroidota, and Verrucomicrobiota. Ten other bacterial phyla were each represented by only a single MAG. This included the poorly characterized phylum UBP17, formerly known as Cloacimonetes (WWE1), of which only a few MAGs have been included in the genome taxonomy database to date  FL and PA MAGs shared some taxa at higher classification levels, only nine genera were found in both the FL and PA data sets. These included MAGs closely related to Alcanivorax, Prochlorococcus_A, Thalassarchaeum, Henriciella, Idiomarina, Nitrosopumilus, Nitrosopelagicus, Roseibacillus_B, and Bythopirellula  identifiers (IDs) and clustered into 49,146 orthologous protein families. Nonmetric multidimensional scaling (NMDS) profiles based on the presence of KO IDs  and orth10.1128/mbio.01569-22.1FIG S1FIG S1, PDF file, 1.1 MB.Nonmetric multidimensional scaling (NMDS) plots of the recovered MAGs based on gene annotations. (A) NMDS plot generated based on the presence/absence of KEGG Orthology (KO) annotations. (B) NMDS plot generated based on the presence/absence of orthologous protein families. Shapes indicate the sample type designation of the MAG. Color indicates the taxonomic classification of the MAG. PA and FL designations are as described in the legend to Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P value < 0.05)  , and S4. < 0.05) . For FL 10.1128/mbio.01569-22.2FIG S2q value of <0.05 for Fisher’s exact test. Download FIG S2, PDF file, 0.5 MB.Venn diagram of significantly enriched genes. (A and B) Venn diagrams of significantly enriched genes based on KEGG Orthology (KO) annotations and orthologous protein family. Significantly enriched KOs and orthologous gene families were determined using Scoary with a Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01569-22.8TABLE S3Table S3, XLSX file, 0.4 MB.Scoary enrichment of KO IDs (A) and orthologous protein families (B) based on sample types. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01569-22.9TABLE S4Table S4, XLSX file, 0.1 MB.Count tables of transporter genes (A), peptidase genes (B), CAZyme genes (C), and extracellular secretion system genes (D) included in MAGs. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The most enriched KO IDs identified by Scoary were significantly associated with the PA_Shallow MAGs A and B. Previous studies have shown that transporters of carbohydrates and energy sources are ubiquitous in heterotrophic prokaryotic communities throughout the open water column, from the surface to abyssal depths, and are crucial for organic matter transformation . To inve10.1128/mbio.01569-22.3FIG S3FIG S3, PDF file, 0.1 MB.Counts of MAGs encoding substrate-specific transporters. Genes encoding putative transporters were grouped based on their substrate specificity. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01569-22.4FIG S4FIG S4, PDF file, 0.1 MB.Counts of MAGs encoding substrate-specific CAZymes. Genes encoding putative CAZymes were annotated against dbCAN (3.0) with default parameters  and filtCopyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A greater proportion of MAGs encoding mannopine transporters were identified in the FL samples than in the PA samples (~6.2% versus ~1.2%) . The subGiven the higher prevalence and diversity of transporters in SPA MAGs, POC solubilization capabilities of both SPA and FL MAGs were investigated. Extracellular hydrolytic enzymes that include peptidases and carbohydrate-active enzyme (CAZymes), key components in particulate organic carbon hydrolysis, were examined in terms of their abundance, diversity, and function B and C.The SPA MAGs possessed a significantly higher count of genes encoding peptidases and CAZymes per genome than the FL MAGs  and B. ASince polysaccharides constitute a large fraction of DOC  and POC 59–l-rhamnohydrolase (GH106), α-l-rhamnosidase (GH106), alginate lyase (PL6 and -14), endo-α-1,5-l-arabinanase (GH43), β-l-arabinofuranosidase (GH146), chitinase (GH18), and β-porphyranase (GH16). These CAZymes hydrolyze polysaccharides such as alginate, porphyran, ulvan, and xylan, which are structural components of algae, and chitin, a highly abundant polysaccharide in marine environments. These CAZymes also hydrolyze an assortment of polysaccharides, releasing sugars such as rhamnose, xylan, and arabinose, which are found enriched in spring phytoplankton blooms , was found to be enriched in FL MAGs in comparison to PA MAGs . This en63–n blooms .l-fucosidase (GH29 and GH151), β-glucosidase , cellulase , cellobionic acid phosphorylase (GH94), α-N-acetylgalactosaminidase (GH109 and -114), β-galactosidase (GH2 and -42), and α-galactosidase . The enrichment of these CAZymes in SPA MAGs also correlates well with the spike in multiple sugars detected during summer phytoplankton blooms, such as mannan, fucose, glucose, galactosamine, and galactose , β-mannosidase (GH2), α-alactose .N-acetylhexosaminidase , which is involved in the hydrolysis of N-acetylated oligo/polysaccharides such as chitooligosaccharides and chitin, which are prevalent and abundant in marine crustaceans. MAGs containing genes for β-N-acetylhexosaminidase were present only within the Gammaproteobacteria and include the orders Arenicellales, Burkholderiales, Enterobacterales, Nitrosococcales, Pseudomonadales, UBA11654, and Xanthomonadales. Lastly, genes encoding laminarin endo-1,3-β-d-glucosidase (GH16 and -81) were found to be enriched in 25 SPA MAGs, which degrades laminarin, a polysaccharide abundant in microalgae such as diatoms  enable rapid transcriptional responses to environmental variations, including light, temperature, and nutrient availability . TCSs arExamination of the SPA and FL MAGs for genes encoding TCSs showed significantly higher representation of histidine kinases  in PA MAGs than in FL MAGs . In addiExtracellular protein secretion systems (ESSs) are widespread among bacteria and archaea and are central components in fimbria- and pilus-associated attachment and adhesion, nutrient transport, predation, cell-cell interactions, surface colonization, and pathogenicity in diverse environmental contexts and settings –74. The –10.1128/mbio.01569-22.5FIG S5FIG S5, PDF file, 0.1 MB.Frequency of different extracellular secretion systems in particle-associated versus free-living bacterial genomes in the water column. Genes encoding bacterial secretion systems were predicted using MacSysFinder with the TSScan reference database (v1.0rc1). Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Prior work has suggested that deep-sea bacteria may have larger genomes than bacteria in the epipelagic zone , 76, andPA microbial communities are often viewed as “hot spots” for microbial activity in comparison to their FL counterparts, based on their larger cell size and cell densities, higher enzyme activities, and overall rates of heterotrophic microbial metabolism , 12, 13.77–80–in situ inorganic nitrogen availability and depth for FL bacterioplankton at Station ALOHA  had significantly larger genome sizes on average than FL microbes  . In termon ALOHA  44). No. Noin sion ALOHA . As for on ALOHA . These gon ALOHA .Recently, codon usage bias CUB) metrics in genes encoding ribosomal proteins have been used to estimate the maximum growth rates (minimum doubling times) of microorganisms in both laboratory and environmental settings –87. We t–UB metric10.1128/mbio.01569-22.10TABLE S5Table S5, XLSX file, 0.03 MB.Estimated growth efficiencies based on MAG codon usage biases. Download Copyright © 2022 Leu et al.2022Leu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Prochlorococcus as well as motile heterotrophic copiotrophs like Idiomarina. These results further illustrate the distinctive microhabitat-specific phylogenetic, physiological, and ecological divergence between SPA and FL microbial communities  and energy , 61, 67.Previous metagenomic and metaproteomic studies of 0.2-μm-filtered samples throughout the water column reported that total peptidases and CAZymes decreased from epipelagic (0 to 200 m) to bathypelagic  microbial communities, while the percentage of extracellular enzymes and their diversity increased with depth . In our Given their particle-associated lifestyles, we postulated that SPA microbial genomes are also enriched in genes that promote environmental sensing and response, motility, attachment, colonization, and cell-cell interactions. Consistent with this hypothesis, both the shallow- and deep-water SPA microbes contained greater proportions of genes associated with two-component regulatory systems, extracellular secretion systems, and flagella than did FL microbes from the same depth horizons. The genomic distributions of TCSs support prior observations with respect to general metabolic types  and suggPrior gene-centric studies have demonstrated that genome size, GC content, and genome and proteome nitrogen content tend to positively correlate with environmental inorganic nitrogen availability among FL bacterioplankton populations , 81, 82.3−, NO2−) to reduce the energetic costs of nitrogen acquisition. Our data further suggest that as particles sink and nitrogen depletion ensues, SPA microbial assemblages undergo dynamic compositional shifts in response to the more nutrient-depleted particle microenvironments found at greater depths.SPA microbes, however, showed an opposite trend with respect to DNA GC content and proteome N-ARSC with increasing depth. Strikingly, shallow-water SPA microbes collected at 150 m had higher DNA GC and proteome nitrogen contents than did deeper, 4,000-m SPA microbes. The higher genome and proteome nitrogen content in shallow- versus deep-water SPA microbes is consistent with recent studies of the elemental composition and energy content (in Joules per unit mass of organic carbon)  of sinkiin situ maximum growth rates (minimum doubling times) of prokaryote environmental settings  at 37°C for 30 min. Subsequently, 50 μL of a proteinase K solution (0.8 mg mL−1) was added, followed by the addition of 50 μL of 10% SDS. Samples were incubated at 55°C for 2 h. Final DNA purification was robotically performed using a Chemagen MSM I instrument with the CMG-1037 DNA saliva kit .Metagenomic sequence data from FL samples see –46. Brie–Sequencing libraries were prepared and sequencing for all the above samples was performed as described previously , 45. Brihttps://sourceforge.net/projects/bbmap/) in two passes. The first pass used parameters “ktrim=r k = 23 mink = 11 hdist = 1 tbo tpe tbo tpe” for Illumina sequencing adapters, and the second pass used parameters “k = 27 hdist = 1 qtrim=rl trimq = 17 cardinality=t mingc = 0.05 maxgc = 0.95” to remove phiX, low-quality bases, and sequences with unrealistically high or low GC. Additional low-quality bases and sequences were removed with Trimmomatic 0.38 (parameters: LEADING:10 TRAILING:10 MINLEN:100) (https://github.com/lh3/seqtk). The cleaned reads from each sample were assembled using SPAdes 3.11.1   . Unpaire,99,127) .https://github.com/wwood/CoverM). For each assembled metagenome, metagenome-assembled genomes were recovered using MetaBAT1 v0.32.5 (https://github.com/wwood/finishm).Mapping of quality reads was performed using CoverM v0.4.0 with default parameters  were reclassified as PA_Shallow (see Methods)  , since tMethods) .Sixty-one 4,000-m PA_Deep MAGs were reannotated as PA_Shallow based on their photic zone-dependent gene representation, well documented physiologies and lifestyles , and/or depth profile read mapping densities. These included the following MAGs: DT-Alcanivorax-1, DT-Alteromonas-1, DT-Bdellovibrionales-2, DT-Bdellovibrionales-3, DT-Caenarcaniphilales-1, DT-Chromohalobacter-1, DT-Cognatishimia-1, DT-Coraliomargarita-1, DT-Dinoroseobacter-1, DT-Ekhidna-1, DT-Epibacterium_A-1, DT-Erythrobacter-1, DT-Erythrobacter-2, DT-Flavobacteriaceae-1, DT-Flavobacteriaceae-3, DT-Flavobacteriaceae-4, DT-Flavobacteriales-4, DT-Gilvibacter-1, DT-Halieaceae-1, DT-Halioglobus-1, DT-Halomonas-1, DT-Halomonas-3, DT-Henriciella-1, DT-Henriciella-2, DT-Henriciella-3, DT-Hyphomicrobiaceae-1, DT-Idiomarina-1, DT-Idiomarina-3, DT-Ilumatobacteraceae-1, DT-Legionellales-1, DT-Micavibrionaceae-1, DT-Mycoplasmatales-1, DT-Mycoplasmatales-2, DT-Oleispira-1, DT-Oligoflexales-1, DT-Parvularculaceae-1, DT-Phaeodactylibacter-1, DT-Phycisphaerales-1, DT-Pseudoalteromonas-1, DT-Pseudobacteriovorax-1, DT-Pseudomonadales-2, DT-Psychroserpens-1, DT-Psychrosphaera-1, DT-Rhizobiales-4, DT-Rhodobacteraceae-1, DT-Rhodobacteraceae-2, DT-Rhodobacteraceae-3, DT-Richelia-1, DT-Rickettsiaceae-1, DT-Rickettsiales-1, DT-Rivularia-1, DT-Salinicola-1, DT-Saprospiraceae-1, DT-Saprospiraceae-2, DT-Shewanella-1, DT-Simkaniaceae-1, DT-Verrucomicrobiales-1, DT-Vibrio-1, DT-Winogradskyella-1, DT-Winogradskyella-2, DT-Xanthomonadales-1, and DT-Crocosphaera.https://github.com/Ecogenomics/GTDBTk). Briefly, marker genes were identified in each genome, aligned, concatenated, and classified with pplacer to identify the maximum-likelihood placement of each genome’s concatenated protein alignment in the GTDB-Tk reference tree. GTDB-Tk classifies each genome based on its placement in the reference tree, its relative evolutionary distance, and FastANI distance.Classification of the MAGs was determined using GTDB-Tk  v.1.3.0 https://github.com/bbuchfink/diamond.git) against UniRef100 (accessed September 2019)  , clusterer 2019) , and Pfaer 2019)  and TIGRer 2019) . Genes wer 2019) .https://github.com/JessAwBryant/gene-characteristics.Amino acid sequences from the dereplicated genome set were used to calculate N-ARSC and C-ARSC values using custom scripts from Mende et al.  and avaiGenes were annotated against dbCAN (3.0) with default parameters  and filt−10. SignalP v5.0  domain annotations , 110. HiGenes encoding the bacterial secretion systems were predicted using MacSysFinder  and the https://www.protocols.io/view/viral-sequence-identification-sop-with-virsorter2-5qpvoyqebg4o/v3). Prophage genes were annotated using DRAM-v.py v1.2.4  ordination analysis with the Jaccard distance matrix using the metaMDS function in the Vegan v2.6-2 package in R , 119.To calculate the relative abundance, reads from each metagenomic data set were mapped to the dereplicated MAGs using CoverM v0.3.1 with the “contig” command, a cutoff of 95% minimum identity, and a minimum aligned read length of 75% of each read. Coverage of each contig was calculated with the CoverM “trimmed_mean” option, and the coverage for each MAG was calculated as the average of all contig coverages, weighted by their length. The relative abundance of each MAG in each metagenomic data set was calculated as its coverage divided by total reads in the sample multiplied by 100,000,000.The presence/absence of each KO ID or orthologous family in each genome was used as input for the program Scoary  to identP values that were affected by multiple testing were corrected for false discovery using the Benjamini-Hochberg procedure.The mean statistical significance of different proteins of interests and genomic characteristic metrics between sample types were determined through one-way analysis of variance (ANOVA), followed by the Tukey test using the R package multicompview v0.1-8 with the Tukey honestly significant different (HSD) function. All https://github.com/dparks1134/CompareM).Average amino acid identity (AAI) between the genomes was calculated using orthologous genes identified through reciprocal best BLAST hits by use of compareM v0.0.5 (http://bioinformatics.psb.ugent.be/webtools/Venn/). Figures were further refined using Adobe Illustrator.Figures were generated using pheatmap  and ggplPRJNA482655, and assembled MAGs are available under NCBI BioSample no. SAMN14675689 to SAMN14675809. Metagenomic reads produced from DNA extracted from shallow 150-m sediment trap samples (collected in 2015) (PRJNA358725. For Station ALOHA 0.2-μm-pore-size filter-collected metagenomes/MAGs, read sequence data and FL MAGs are available at NCBI SRA under BioProject no. PRJNA352737, and assemblies can be found under BioSample no. SAMN12604809.All data were deposited in the NCBI SRA archive as follows. Deep 4,000-m-trap metagenomes and MAGs are deposited under NCBI BioProject no.


Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an encochleated oral formulation of amphotericin B which has been shown to be safe and effective against murine aspergillosis and murine cryptococcal meningoencephalitis. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM.Rhizopus delemar 99-880 or M. circinelloides f. jenssenii DI15-131. Treatment with placebo (diluent control), oral MAT2203  or LAMB , began 16 h post infection. Survival (n=10-20/group from 1/2 experiments) through Day +21 and tissue fungal burden of lungs or brain (n=10/group) euthanized on Day +4 post infection  served as a primary and secondary endpoint, respectively.ICR mice were immunosuppressed with cyclophosphamide and cortisone acetate on days -2, -3 and +8, relative to infection with intratracheally instilled Rhizopus delemar infection, doses of MAT2203 5,15 mg/kg qd or 7.5 mg/kg bid, significantly prolonged median survival time (MST) and enhanced overall survival vs. placebo-treated mice . Importantly, MAT2203 treatments were as effective as LAMB . For mice infected with M. circinelloides, MAT2203 at 15 mg/kg, qd significantly prolonged MST and enhanced overall survival vs. placebo-treated mice . In both infection models MAT2203 treatment of 15 mg/kg or LAMB resulted in significant ∼1.0-1.5 log reduction and ∼2.0-2.2 log reduction in lung and brain fungal burden vs. placebo, respectively (Wilcoxon Rank Sum).For in vivo efficacy in treating R. delemar or M. circinelloides pulmonary infection in immunosuppressed mice, which was equivalent to the LAMB current standard of care. Continued investigation and development of MAT2203 as a novel, and oral formulation of amphotericin antifungal agent against mucormycosis is warranted.MAT2203 demonstrated Theresa Matkovits, PhD, Matinas BioPharma: Employee Jenel Cobb, PhD, Matinas BioPharma: Employee Raphael J. Mannino, PhD, Matinas BioPharma: Employee Ashraf S. Ibrahim, PhD, Matinas BioPharma: Advisor/Consultant|Matinas BioPharma: Grant/Research Support|SFunga: Grant/Research Support.


Vaccination strategies that provide enhanced immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are needed. We evaluated the safety and immunogenicity of a bivalent omicron containing vaccine, mRNA-1273.214 (50 µg), administered as a second booster dose in adult participants.In this ongoing phase 2/3 trial, 50 µg of the bivalent vaccine mRNA-1273.214  or 50 µg of the authorized mRNA-1273 were administered as second boosters in adults who previously received a 2 dose (100 µg) primary series and a first booster (50 µg) dose of mRNA-1273 (≥ 3 months prior). Primary objectives were safety and reactogenicity and immunogenicity 28 days post-booster dose.Figure). Safety and reactogenicity were similar for both vaccine groups.In participants with no prior SARS-CoV-2 infection who received booster doses of mRNA-1273.214 (n=334) or mRNA-1273 (n=260), neutralizing antibody (nAb) geometric mean titers ]) against omicron BA.1 were 2372.4 (2070.6−2718.2) and 1473.5 (1270.8−1708.4), respectively. The model-based GMT ratio (GMR [97.5% CI]) of mRNA-1273.214 compared to mRNA-1273 was 1.75 (1.49−2.04), meeting the pre-specified superiority criterion against omicron BA.1. The pre-specified criterion for non-inferiority against the ancestral SARS-CoV-2 strain was also met. Additionally, mRNA-1273.214 elicited higher GMTs (727.4 [632.8−836.1]) than mRNA-1273 (492.1 [431.1−561.9]) against omicron subvariants BA.4/BA.5 [GMR (95% CI) 1.69 [1.51−1.90])]. Binding antibody responses against alpha, beta, gamma, delta, and omicron were numerically higher in the mRNA-1273.214 group compared to mRNA-1273. mRNA-1273.214 GMTs were consistently higher across age (18-< 65 and ≥ 65 years) and pre-booster SARS-CoV-2 infection subgroups (The bivalent omicron containing mRNA-1273.214 elicited superior nAb responses against omicron 28 days post-immunization compared to mRNA-1273 regardless of age and prior SARS-CoV-2 infection; no new safety concerns were identified.Spyros Chalkias, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Stephen R. Walsh, MD, Janssen Vaccines: Grant/Research Support|Moderna, Inc.: Grant/Research Support|NIAID/NIH: Grant/Research Support|Sanofi Pasteur: Grant/Research Support Nichole McGhee, B.S., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Joanne Tomassini, Ph.D., Moderna, Inc.: Advisor/Consultant Xing Chen, Sc.D., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Ying Chang, M.S., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Andrea Sutherland, M.D., MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds David Montefiori, Ph.D., Moderna, Inc.: Grant/Research Support Bethany Girard, Ph.D., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Darin Edwards, Ph.D., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Jing Feng, M.S., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Honghong Zhou, Ph.D., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Lindsey R. Baden, MD, Moderna, Inc.: Grant/Research Support|NIAID: Grant/Research Support Jacqueline Miller, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Rituparna Das, M.D., Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds.


Piper species  has been studied, along with their antifungal and phytotoxic activities. These EOs contained β-bisabolene/nerolidol (Pc), β-bisabolene/δ-cadinene/caryophyllene (Pt), caryophyllene oxide (Pcs), bicyclogermacrene/10-epi-Elemol (Po), bicyclogermacrene/germacrene-D/apiol (Pd), caryophyllene/germacrene-D (Pa), germacrene-D (Pr), limonene/apiol (Ps), apiol (Psf), and apiol/bicyclogermacrene (Pm) as major components, and some are described here for the first time . A composition-based dendrogram of these Piper species showed four major groups . The spore germination effects  and phytotoxicity (Lolium perenne and Lactuca sativa) of these EOs were studied. Most of these Piper essential oils showed important activity against phytopathogenic fungi (except G1), especially against B. cinerea. Similarly, most of the essential oils were phytotoxic against L. perenne (except G1), with P. sancti-felicis (G4), P. casapiense (G2), and P. reticulatum (G2) being the most effective. Caryophyllene oxide, β-caryophyllene, β-pinene, limonene, α-humulene, and apiol were evaluated against B. cinerea, with the most effective compounds being β-pinene, apiol, and limonene. This work demonstrates the species-dependent potential of essential oils from Peruvian Piper species as fungicidal and herbicidal agents.The chemical composition of essential oils (EOs) from ten Peruvian  Diseases caused by plant pathogens significantly contribute to annual loss in crop yield worldwide . The appIn plants, essential oils (EOs) play a protective role against herbivores, phytopathogenic fungi, and weeds. Essential oils also represent a new class of crop protectants due to their volatility and low toxicity to the environment and have been proposed as an alternative to synthetic pesticides ,7,8,9. APiper is found in tropical and subtropical areas, and in America, there are approximately 700 species [The Piperaceae family has approximately eight genera and 3000 species . The gen species ,13, with species ,15. This species , includi species ,17,18, a species ,20, inse species ,21.Piper EOs are characterized by the presence of monoterpene hydrocarbons , oxygenated monoterpenoids , sesquiterpene hydrocarbons , oxygenated sesquiterpenoids -nerolidol, caryophyllene oxide, α-cadinol, epi-α-bisabolol), and phenylpropanoids , among others [Piper essential oils have been described as being insect antifeedant, acaricidal, nematicidal, and herbicidal agents [Piper essential oils are a promising source of new potential biopesticide ingredients.g others ,22,23,24l agents ,21. TherPiper species for their biopesticidal potential, ten species native to the Peruvian Amazonian region  have been extracted by hydrodistillation to study the chemical composition of their EOs by GC-MS, along with their fungicidal activity against phytopathogens  and their phytotoxic effects (against Lolium perenne and Lactuca sativa) to assess their potential applications in phytopathogen and/or weed control.As part of an ongoing project on the bioprospection of Peruvian Piper coruscans; β-bisabolene (40.2%), δ-cadinene (9.8%), caryophyllene (9.7%), germacrene-D (5.0%), and nerolidol (4.5%) for P. tuberculatum; caryophyllene oxide (10.2%), and caryophyllene (4.7%) for P. casapiense; bicyclogermacrene (7.9%), 10-epi-Elemol (7.3%), caryophyllene (6.3%), α-pinene (6.0%), β-pinene (5.1%), β-selinenol (4.9%), α-eudesmol (4.5%), and camphene (4.4%) for P. oblicuum; bicyclogermacrene (16.5%), germacrene-D (10.4%), apiol (8.9%), caryophyllene (6.8%), β-pinene (6.3%), α-cubebene (5.9%), and β-elemene (4.5%) for P. dumosum; caryophyllene (11.3%), germacrene-D (9.6%), α-humulene (6.6%), δ-cadinene (6.6%), and (-)-β-copaene (5.8%) for P. anonifolium; germacrene-D (12.6%), bicyclogermacrene (8.1%), δ-cadinene (6.0%), copaene (4.6%), and caryophyllene (4.5%) for P. reticulatum; limonene (38.5%), apiol (15.0%), caryophyllene oxide (8.4%), eudesma-3,7-(11)-diene and copaene (5.8%) for P. soledadense; apiol (76.1%), and caryophyllene (4.1%) for P. sancti-felicis and apiol (51.6%), bicyclogermacrene (9.0%), germacrene-D (6.7%), and myristicin (4.6%) for P. mituense.The chemical compositions of the essential oils are shown in P. sancti-felicis and P. mituense, which were characterized by phenylpropanoids, and P. soledadense, which was characterized by monoterpene hydrocarbons.The overall composition of these oils is shown in Piper species (P. coruscans (Pc) and P. tuberculatum (Pt), characterized by the presence of sesquiterpene hydrocarbons; (G2) P. casapiense (Pcs), P. obliquum (Po), P. dumosum (Pd), P. anonifolium (Pa), and P. reticulatum (Pr), characterized by sesquiterpenes; (G3) P. soledadense (Ps), with monoterpenes and sesquiterpenes; and (G4) P. sancti-felicis (Psf) and P. mituense (Pm), characterized by phenylpropanoids.A dendrogram based on the composition of the  species  showed fPiper essential oils against Aspergillus niger, Botrytis cinerea, and Alternaria alternate is shown in B. cinerea was the fungal species most susceptible to the action of Piper essential oils. The antifungal activity showed a pattern in accordance with the grouped EOs. P. coruscans and P. tuberculatum (G1) were not active. P. casapiense (G2), P. obliquum (G2), P. dumosum (G2), P. anonifolium (G2), and P. reticulatum (G2) were only active against B. cinerea with varying potencies, with P. obliquum and P. anonifolium being the most effective. P. soledadense (G3) inhibited the spore germination of A. niger and B. cinerea. P. sancti-felicis (G4) oil was active against all three fungal species with moderate effects, and P. mituense (G4) only acted on B. cinerea, probably due to the lower concentration in apiol of this oil.The antifungal activity (spore germination inhibition) of the Piper oils components tested against B. cinerea , β-pinene showed strong antifungal activity , followed by apiol  and limonene , with effective doses similar to the positive control  for apiol.Among the Lactuca sativa (dicotyledonous) and Lolium perenne (monocotyledonous) plants. The phytotoxic activity did not follow the grouping pattern observed for the antifungal effects. P. sancti-felicis (G4), P. casapiense (G2), P. mituense (G4), and P. reticulatum (G2), effectively inhibited germination, leaf and root growth of L. perenne , P. obliquum (G2), P. dumosum (G2), and P. soledadense (G3) inhibited leaf growth (>50%), followed by P. tuberculatum (G1) with a 50% inhibition.The essential oils were tested for phytotoxic effects on ne >50%, . P. anonP. sancti-felicis, P. casapiense, P. reticulatum, and P. mituense effectively inhibited the root growth of L. sativa (data not shown). P. soledadense reduced the root growth of L. sativa (data not shown). These results show strong selective herbicidal potential of the EOs tested against monocotyledonous plants.Piper essential oils present a wide variety of chemical compounds with important biological activities that may be of interest in agriculture, medicine, and food industries, among others. These oils play an important role in the defense of the plant against pests, and many studies have reported activities as insecticidal, antifeedants, phytotoxic and antifungal [tifungal ,26,27,28Piper species described here have been previously reported to show quantitative and qualitative chemical variations that can be attributed to environmental factors  [The essential oils from some of the umidity) ,29,30. AP. coruscans (Pc) and P. tuberculatum (Pt). P. coruscans EO had β-bisabolene (33.4%) and nerolidol (10.2%) as the main components, while this species collected in Ecuador contained β-caryophyllene (24.1–25.0%), α-humulene (11.6–12.0%), and caryophyllene oxide (9.3–10.9%) [P. tuberculatum studied here showed β-bisabolene (40.2%) as the main component followed by δ-cadinene (9.8%), caryophyllene (9.7%), germacrene-D (5.0%), nerolidol (4.5%), copaene (4.2%), and β-elemene (3.3%). This Piper species (Pt) EO has been previously reported for plants collected from different locations. Pt collected in Venezuela gave an EO with α-farnesene (6.2%), humulene epoxide II (6.0%), 2-pentadecanone (4.1%), β-eudesmol (4.4%), 2-tridecanone (4.3%), ledane (3.6%), -farnesylacetone (3.6%), and α-cadinol (2.9%) [E)-caryophyllene (30.1%) [E-cariofilene (7.1%), and trans-4-muurola(14)-5-diene (9.9%) [(G1) 3–10.9%) . The EO l (2.9%) , while E (30.1%)  or myrissabolene .4% and nP. casapiense (Pcs), P. obliquum (Po), P. dumosum (Pd), P. anonifolium (Pa), and P. reticulatum (Pr): P. casapiense (Pcs), is reported here for the first time. The EO from P. obliquum had bicyclogermacrene (7.9%), 10-epi-Elemol (7.3%), caryophyllene (6.3%), and α-pinene (6.0%). However, the Po essential oil of Ecuadorian origin contained safrole (45.9%), γ-terpinene (17.1%), and terpinolene (11.5%) [P. dumosum contained bicyclogermacrene (16.5%) and germacrene-D (10.4%). Similarly, the EO from Pd plants collected in the Brazilian Amazonia had bicyclogermacrene (16.2%), β-caryophyllene (15.9%), β-pinene (16.0%), and α-pinene (12.1%) [P. anonifolium studied here contained caryophyllene (11.3%), germacrene-D (9.6%), δ-cadinene (6.6%), α-humulene (6.6%), and neoalloocimene (5.5%), while a previously reported EO from the Brazilian Pará was composed of selin-11-en-4-β-α-ol (20.0%), β-selinene (12.7%), α-selinene (11.9%), and α-pinene (8.8%) [P. reticulatum studied here contained the phenylpropanoid apiol as the main component (15.0%) while β-elemene (24.6%) and β-caryophyllene (16.7%) were abundant in a Pr oil from the northern region of Brazil [(G2)  (11.5%) , while a (11.5%) . P. dumo (12.1%)  as the me (8.8%) . Furthere (8.8%) . The EO f Brazil .soledadense (Ps) is reported here for the first time.(G3) P. P. sancti-felicis (Psf) and P. mituense (Pm). The EO from P. sancti-felicis and P. mituense (Pm reported here for the first time) contained the phenylpropanoid apiol as the main component . δ-3-carene (35.3%) and limonene (27.1%) were the main components of the EO from P. sancti-felicis collected in Choco, Colombia [(G4) Colombia ,25.Piper have been reported to have a wide range of biological properties [P. tuberculatum -β-ocimene 14% and β-caryophyllene 32.1%) [P. anonifolium  showed strong antifungal activity against Cladosporium cladosporioides and C. sphaerospermum [The essential oils studied here have shown important activities against phytopathogenic fungi (G2-4). Essential oils from the genus operties ,14,21,40operties ,38,39. Se 32.1%) , and P. ospermum .B. cinerea , β-pinene, limonene, and apiol showed strong antifungal activity. Therefore, β-pinene could contribute to the activity of Po and Pd EOs, while apiol and limonene could explain the effect of Ps, Psf, and Pm oils. β-caryophyllene (inactive) could be a synergist.The composition-based grouping of the EOs overlapped with the antifungal activity, suggesting that the presence of bicyclogermacrene, 10-epi-Elemol, germacrene-D, caryophyllene, limonene, β-pinene, and/or apiol could be responsible for significant antifungal effects. Among the oil components tested against Rhizoctonia solani, Fusarium oxysporum, Penicillium digitatum and Asperigallus niger [Botrytis cinerea [Aspergillus flavus, A. niger, A. fumigatus, and A. parasiticus [Botryodiplodia theobromae and Colletotrichum acutatum [Fusarium solani [Rhizoctonia solani and Helminthosporium oryzae [B. cinerea spore germination.(S)-Limonene has reported antifungal activity against us niger . Apiol s cinerea , Aspergiasiticus , Botryodacutatum ,46. The acutatum . Caryophm solani  and carym oryzae , but in P. sancti-felicis, P. mituense, P. casapiense, P. reticulatum, P. anonifolium, P. obliquum, P. dumosum, and P. soledadense) were phytotoxic to the monocotyledonous Lolium perenne, and this activity did not overlap with the composition-based groups, suggesting a multi-component phytotoxic action. This is the first report on the phytotoxic effects of these species, except for an EO from P. sancti-felicis that was not active and did not contain apiol [Piper species, including P. hispidinervum rich in safrole [P. dilatatum rich in apiol, and P. divaricatum rich in eugenol and methyleugenol [Most of the essential oils tested here  [Brassica campestris and Raphanus sativus [Echinochloa crusgalli, Lolium perenne, Amaranthus retroflexus, and Digitaria sanguinalis [Among the main compounds present in the  species , apiol i plants) , and car sativus  and the guinalis .2SO4. All investigated Piper species contained essential oils that range from 0.078 to 1.26% based on dry weight  of the selected Piperaceae species were collected in Iquitos, Loreto Department, Peru in different seasons. The taxonomic identification was carried out at the Herbarium Amazonense of the National University of the Peruvian Amazon, Iquitos, Peru. A voucher for each species has been deposited in the herbarium. All the plants were permitted for collection . The EOs extraction was performed by hydrodistillation using the dried aerial parts of the plants. The EOs were separated by decantation and dried over anhydrous Nay weight .Essential oils were analyzed by gas chromatography (GC) on a Shimadzu 2010 and gas chromatography-mass spectrometry (GC-MS) equipped with a mass spectrometer Shimadzu GCMS-QP2010-Ultra Mass Detector . The carrier gas was helium. The capillary column was a Teknokroma TRB (95%) dimethyl (5%) dimethylpolysiloxane (30 m × 0.25 mm ID and 0.25 µm phase thickness). Working conditions were as follows: injector temperature, 300 °C; column temperature 70–290 °C, for 6 min, staying at 290 °C for 15 min, temperature of the transfer line connected to the mass spectrometer, 250 °C, and ionization source temperature 250 °C. The identification of compounds was performed with standard terpenes analyzed under the same conditions and by comparison of the mass spectra with those available in the library Wiley Mass Spectral Database , while relative area% has been used for quantification of all the peaks obtained in the chromatograms. The mass spectra and Kovats retention indexes obtained were compared with the literature reported ,54.Aspergillus niger, Alternaria alternata, and Botrytis cinerea came from the fungal collection of Instituto de Ciencias Agrarias-CSIC, Madrid, Spain where they are maintained. The antifungal activity of the essential oils was determined using a modified spore germination inhibition growth assay [5 cells/mL in NaCl 0.9% for A. niger and 1 × 107 cells/mL in distilled water for B. cinerea and A. alternate. Amphotericin B (5 µg/mL) was used as a positive control.The fungal species th assay . The essA. niger and 25 °C for B. cinerea). After the incubation process, 25 uL of an MTT (5 mg/mL) plus menadione (1 mM) solution in RMPIMOPS were added, the plates were incubated again for 3 h, the medium was removed, 200 µL of acidic isopropanol (95% isopropanol and 5% 1 M HCl) was added, and the plates were incubated for another 30 min. The absorbance was read at 490 nm in an Elisa reader. The IC50 values (the effective dose to give 50% inhibition) were calculated by a regression curve of % spore germination inhibition on log dose.The samples and spore suspensions (4 replicates) were placed on 96-well plates and incubated for 24 h .Lactuca sativa, and Lolium perenne seeds (40 seeds/test) in 12-well microplates, as described previously [L. sativa) or seven days (L. perenne), and the root length  was measured  at the end of the experiment. A nonparametric analysis of variance (ANOVA) was performed on root/leaf length data [These experiments were conducted with eviously . The essgth data ,21.https://www.statgraphics.com, accessed on 2 July 2022).The data were analyzed using STATGRAPHICS Centurion XIX  subjected to cluster analysis . The groups were chosen with a distance >2.Piper species as fungicidal and herbicidal agents based on their composition. A dendrogram based on the composition of the Piper species showed four groups: (G1) P. coruscans (Pc) and P. tuberculatum (Pt), characterized by the presence of sesquiteterpene hydrocarbons; (G2) P. casapiense (Pcs), P. obliquum (Po), P. dumosum (Pd), P. anonifolium (Pa) and P. reticulatum (Pr), characterized by sesquiterpenes; (G3) P. soledadense (Ps), with monoterpenes and sesquiterpenes; and (G4) P. sancti-felicis (Psf) and P. mituense (Pm), characterized by phenylpropanoids. The essential oils in G2-4 showed important activity against Botrytis cinereal and were phytotoxic against Lolium perenne.This work demonstrates the species-dependent potential of essential oils from Peruvian Considering the composition-based grouping of the EOs, we can conclude that the presence of bicyclogermacrene, 10-epi-Elemol, germacrene-D, caryophyllene, limonene, β-pinene, and/or apiol could be responsible for significant antifungal and herbicidal effects. β-pinene, apiol, and limonene showed antifungal activity, but not caryophyllene, suggesting that this compound could be a synergist.Piper germplasm bank and the domestication of selected species to grant a sustainable biomass source for the production of essential oils with biopesticidal activity.These findings have important implications for the development of a


The aim of this study was to determine the sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) for PC-local recurrence and metastases on a per region basis.Despite increasing use for the detection of biochemically recurrent prostate cancer (rPC), the diagnostic accuracy of positron emission tomography/computed tomography (PET/CT) with [18F]PSMA-1007 PET/CT for rPC were retrospectively analysed. Six body regions were defined: prostate fossa, pelvic lymph nodes (LN), retroperitoneal LN, supradiaphragmatic LN, bones, and soft tissue. A region was counted positive if at least one PSMA-positive lesion suspicious for PC was observed. Confirmation of a true-positive PSMA-avid lesion was defined as positive by histopathology, fall in serum prostate-specific antigen (PSA) (> 50%) after targeted therapy or confirmatory further CT, MRI, PET/CT, or bone scan imaging. Regions where additional imaging was able to confirm the absence of suspicious PC lesions or regions outside exclusively targeted RT with serum PSA decline (> 50%) were counted as true-negative regions. SE, SP, PPV, and NPV were calculated for all six regions.One hundred seventy-seven consecutive patients undergoing [n = 6) was too small for an accurate statistical analysis.The overall PET-positivity rate was 91%. Conclusive follow-up for affirmation or refutation of a PSMA-positive lesion was available for 81/152 patients on a per region basis. In this subgroup, overall sensitivity, specificity, PPV, and NPV were 95% (CI: 0.90–0.98), 89% (CI: 0.83–0.93), 86% (0.80–0.90), and 96% (CI: 0.92–0.98), respectively. On a per region basis, PPV was 97% (CI: 0.83–0.99) for local recurrence, 93% (CI: 0.78–0.98) for pelvic LN, 87% (CI: 0.62–0.96) for retroperitoneal LN, 82% (CI: 0.52–0.95) for supradiaphragmatic LN, and 79% (0.65–0.89) for bone lesions. The number of solid organ metastases  sensitivity and NPV on a per region basis. However, overall PPV was limited (86%), particularly for bone lesions (79%), which are a potential diagnostic weaknesses when using this tracer.The known high PET-positivity rate of [The online version contains supplementary material available at 10.1007/s00259-022-05693-0. Radioligands to the prostate-specific membrane antigen (PSMA) have become the gold standard for the staging of primary prostate cancer (PC) –4 and re68Ga]Ga-PSMA-11, a large number of alternative radioligands have become available, including but not limited to [68Ga]Ga-PSMA-I&T; [68Ga]Ga-THP-PSMA; and [18F]-labelled PSMA-radiotracers such as [18F]-rhPSMA-7, [18F]-DCFPyL, [18F]-JK-PSMA-7, or [18F]PSMA-1007 [18F]-labelled PSMA-radiotracers have numerous advantages over 68Ga-labelled ligands: [18F] is cyclotron produced with a longer half live and a lower positron energy compared to [68Ga] (0.65 MeV vs. 1.90 MeV), leading to improved spatial resolution [18F]PSMA-1007 suggest improved detection rates especially in local relapses and pelvic lymph node metastases in proximity to the urinary tract [18F]PSMA-1007 might be of benefit [68Ga]Ga-PSMA-11 can be an alternative PSMA-1007 in rPC. Hitherto, studies report only preliminary observations in small cohorts (n = 40) [18F]PSMA-1007 in a cohort of men undergoing PSMA-PET/CT for rPC in a clinical setting on a per patient and per region basis using a composite standard of truth (CSOT) for verification or refutation of imaging findings.Despite widespread adoption, few studies report the diagnostic accuracy of PSMA-1007 PET/CT in the University Clinic for Nuclear Medicine, Inselspital, Bern. The period was chosen to enable a minimum follow-up period of 12 months. Inclusion criteria were biochemical recurrence of PC (rPC) according to the ASTRO/AUA Guideline as rising PSA value after a PSA-Nadir after definitive treatment  PSMA-1007 was produced as previously described [18F]PSMA-1007 solution was given by intravenous bolus injection .[escribed . The [1818F]PSMA-1007. They were investigated on either Biograph-mCT PET/CT  (n = 90) or Biograph-VISION 600 PET/CT  PET/CT (n = 96) scanner. The examination protocols and reconstruction algorithms used are previously published PSMA-1007 PET/CT, but where histopathology of the PSMA-avid lesion was negative, where the PSA after targeted RT did not decrease by 50% or where post therapy imaging were not confirmatory. Regions where no PSMA-positive lesion was detected with additional confirmatory imaging or regions outside exclusively targeted RT with PSA decline were counted as true-negative (TN) regions. Patients with no histopathology, no further treatment or imaging, and therefore no composite standard of truth were not included in the final analysis  following exclusively targeted radiotherapy of a PSMA-positive lesion suspicious for PC were true positive (TP). The absence of PSMA-positive legions in a region where additional imaging revealed the presence of PC or where histopathology showed a positive result were counted as false negative (FN). A false-positive (FP) region was counted where a PMSA-positive lesion was observed in 8FPSMA-10 Figures .Fig. 2Coα) for α = 0.05 PSMA-1007 in patients with rPC was 95.6% : 0.90–0.98).Of these 81 PSMA-PET/CTs in 73, at least one suspicious PSMA-avid lesion was detected, and therefore, the PET scan was rated as “positive”. This difference in PET-positivity rate for this subgroup to the overall positivity rate was not statistically significant . On a region basis, PSMA-positive lesions were detected in the prostatic fossa in 51%, in pelvic LN in 48%, in retroperitoneal LN in 23%, in supradiaphragmatic LN in 16%, in bones in 53%, and in other metastasis (soft tissue lesions) in 7%  and 0.89 (CI: 0.83–0.93), PPV and NPV were 0.86 (CI: 0.80–0.90) and 0.96 (CI: 0.92–0.98), and positive and negative likelihood ratio (LR+/LR-) were LR+: 8.71 (CI: 5.69–13.34) and LR−: 0.06 (CI: 0.03–0.12).n = 6) showed a PPV of 0.40 (0.12–0.77) and a NPV of 0.97 (CI: 0.86–0.99). Further details are given in Figure Sensitivity and specificity on a region basis were 0.94 (CI: 0.81–0.98) and 0.92 (CI: 0.64–0.99) for local recurrence, 0.93 (CI: 0.77–0.99) and 0.92 (CI: 0.0.73–0.99) for pelvic LN, 1.00 (CI: 0.77–1.00) and 0.94 (0.80–0.98) for retroperitoneal LN, 1.00 (CI: 0.70–1.00) and 0.94 (CI: 0.81–0.98) for supradiaphragmatic LN, 0.97 CI: 0.85–0.99) and 0.74 (CI: 0.57–0.85) for bone lesions, and 0.67 (CI: 0.21–0.94) and 0.92 (CI: 0.79–0.97) for soft tissue lesions. PPV and NPV on a region basis were 0.97 (CI: 0.83–0.99) and 0.86 (CI: 0.60–0.96) for local recurrence, 0.93 (CI: 0.78–0.98) and 0.92 (CI: 0.74–0.98) for pelvic LN, 0.87 (CI: 0.62–0.96) and 1.00 (CI: 0.89–1.00) for retroperitoneal LN, 0.82 (CI: 0.52–0.95) and 0.96 (CI: 0.81–0.99) for supradiaphragmatic LN, and 0.79 (CI: 0.65–0.89) and 0.96 (CI: 0.81–0.99) for bone lesions. The small number of soft tissue lesions (–0.99 andROC and corresponding area under the curves (AUC) were 0.70 ± 0.03 overall. For local recurrence, AUC was 0.76 ± 0.05, for pelvic LN 0.71 ± 0.09, for retroperitoneal LN 0.55 ± 0.17, and for supradiaphragmatic LN 0.51 ± 0.14. For bone lesions, the AUC was comparatively lower at 0.63 ± 0.06. Corresponding ROC curves are outlined in Figure 18F]PSMA-1007 was clinically first introduced in 2015 [18F]PSMA-1007, there are currently very few evidence-based recommendations supporting its use [18F]PSMA-1007 on a patient-level and a per region basis. In this retrospective analysis of 177 patients, we present the largest cohort of men undergoing [18F]PSMA-1007 for rPC with a confirmatory standard of follow-up PSMA-1007 PSMA-1007. No significant difference in the PET-positivity rate between the subgroup with CSOT and those without was observed (p = 0.21), implying no bias in the patients with follow-up data available.One strength of our study was the high rate of follow-up available for our patients (86%). Through scrutiny of clinical imaging reports, multi-disciplinary team meeting minutes, and clinical records, we are able to demonstrate the performance of this radiopharmaceutical under clinical conditions in a large cohort of men undergoing PSMA-PET/CT in an academic nuclear medicine centre. Nevertheless, in common to all studies with rPC, follow-up data which can be used to verify imaging findings is not available for all patients. For example, a watchful waiting strategy might be chosen by the patient and his treating physicians. Using a CSOT including histopathology, PSA decline (> 50%) after targeted RT, and further imaging after the PSMA-PET showing response to system therapies, we were able to confirm or refute PSMA-PET findings. With a high number of consecutively screened patients with rPC (n = 152) is not small, the relatively smaller number of supradiaphragmatic lymph nodes (n = 13) and solid organ metastases (n = 6) precluded an accurate analysis of diagnostic performance in these regions. Further studies, ideally of prospective design, are required to confirm these data. In order to reduce selection bias, patients were consecutively followed up. This heterogeneous cohort of patients at a variety of stages of rPC and prior treatment furnishes an overview of the radiotracer’s performance under routine clinical conditions. Further studies, for example, directed at early stages of recurrence should be performed, particularly since the PPV, which is dependent upon pre-test prevalence [We highlight several weaknesses with our study. Our overall cohort size was limited by the introduction of the radioligand in our centre in Oct 2019 and the requirement to collect 12 months’ of follow-up. Although our cohort of patients with follow-up Below is the link to the electronic supplementary material.


COVID-19-related physical isolation, fear and anxiety determined de novo mental illnesses, by potentially facilitating the emergence of Hikikomori traits .The present study aims at screening a cohort of university students for the Hikikomori traits and assessing a set of psychopathological determinants associated with Hikikomori, particularly boredom and loneliness dimensions.A cross-sectional web-based survey was carried out by administering Hikikomori Questionnaire (HQ-11), Italian Loneliness Scale (ILS), Multidimensional State Boredom Scale (MSBS), Depression Anxiety Stress Scale (DASS-21) and Toronto Alexithymia Scale (TAS-20).1,148 respondents  were recruited. 70.7% declared to have experienced psychological distress. HQ-11 average total score was 18.4±SD=7.5 with statistically significant higher values in the males (p=0.017) and amongst students studying Informatics, Mathematics/Physics/Chemistry, Science of Communication and Engineering. The HQ-11 positively correlated with ILS (r=0.609), MSBS (r=0.415), TAS-20 (r=0.482) and DASS-21 (r=0.434).This study represents the first screening of the Hikikomori phenomenon in Italian university students. Hikikomori traits appear to be particularly represented in the Italian youth population and should be carefully investigated in future studies.No significant relationships.