Bartonella spp. comprises emergent and re-emergent fastidious Gram-negative bacteria with worldwide distribution. Cats are the main reservoir hosts for Bartonella henselae and dogs represent opportunistic hosts for the bacteria. Even though ticks may also play a role in transmission, their competence as vectors for Bartonella spp. has not been totally understood. Considering only a few studies had a focus on screening Bartonella in animals, humans and ectoparasites in Portugal, this study aimed to address the molecular occurrence of Bartonella sp. in 123 stray cats, 25 stray dogs, 30 humans from Lisbon and 236 questing ticks within the country. Using a qPCR targeting the nuoG gene, it was possible to detect Bartonella sp. DNA on 20.32% of cat samples (25/123). From these positive samples, 13 sequences were characterized as B. henselae, 11 as B. clarridgeiae and 1 presented co-infection with both species. The absolute quantification of nuoGBartonella DNA in sampled cats ranged from 2.78 × 10 to 1.03 × 105 copies/µL. The sampled dogs, humans and ticks were negative. These results showed that B. henselae and B. clarridgeiae are circulating in stray cats from Lisbon. Additional and more extended studies should be conducted to determine the impact of such infections on humans, particularly those in constant and direct contact with cats. Bartonella spp. encompasses small, fastidious, and facultative Gram-negative intracellular bacteria with a global distribution -3′ [® (IDT-Integrated DNA Technologies) encompassing the 83 bp B. henselae-nuoG gene fragment. The number of copies was determined by the following formula: Xg/μL DNA/[fragment length in bp × 660]) × 6.022 × 1023 × plasmid copies/μL.A quantitative PCR targeting a fragment of the NADH ubiquinone oxireductase subunit G (BHQ1]-3′  was carrnuoG-based qPCR gene were afterwards used in two cPCR assays. The citrate synthase gene (gltA) was targeted for amplification of a 767 bp fragment, using the primers 443F: 5′-GCTATFTCTGCATTCTATCA-3′ and 1210-R: 5′-GATCYTCAATCATTTCTTTCCA-3′ [ribC), using the primers BARTON-1: 5′-TAACCGATATTGGTTGTGTTGAAG-3′ and BARTON-2: 5′-TAAAGCTAGAAAGTCTGG CAACATAACG-3′ [gltA cPCR assay and cycling conditions consisted of an initial denaturation at 95 °C for 5 min, 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. Ultrapure water was used as a negative control. All assays were conducted in a T100™ Thermal Cycler . PCR products were analyzed by horizontal electrophoresis in 1.5% agarose gel stained with Green Safe Premium (NZYTech). Positive amplicons for both cPCR targeting the genes gltA and ribC were purified using the NZYGelpure kit (NZYTech) and Sanger sequenced at StabVida .Positive samples for the TTCCA-3′ . The 25 TAACG-3′ . ReactioE. coli competent cells (NZYTech) according to standard cloning protocols. Briefly, the purified amplified product was ligated into the plasmid pTZ57R/T. Chemically competent E. coli cells (NZYtech) were transformed, plated in LB agar (NZYtech) and incubated at 37 °C, overnight. Positive colonies were subcultured overnight on 3 mL of LB medium  at 37 °C and under 200 rpm of shaking. Finally, plasmid purification was carried out using the NZY Miniprep kit (NZYtech) following the manufacturer’s recommendations, and 10 µL of purified samples together with 3 µL of M3 forward primer were sent to StabVida , for sequencing. Sequences were manually trimmed and submitted to BLAST [B. henselae gltA, B. clarridgeiae gltA, B. henselae ribC and B. clarridgeiae ribC), and a ClustalW multiple alignment was performed in Bioedit for each group of gene sequences. The number of haplotypes (h) and the haplotype diversity (Hd) was evaluated using the software DNA Sequence Polymorphism (DnaSP) v.6.12.03 [p-distance model with 1000 bootstrap replications. For the phylogenetic trees’ construction, ClustalW multiple alignment was performed in Bioedit for each group of gene sequences (gltA and ribC), including representative sequences for each haplotype obtained in this study, together with other sequences selected from the GenBank (for gltA: Bartonella acomydis (AB444979.1), Bartonella alsatica (AF204273.1), Bartonella birtlesii (AF204272.1), Bartonella vinsonii subp. vinsonii (Z70015.1), Bartonella vinsonii subsp. arupensis (AF214557.1), Bartonella jaculi (AB444975.1), Bartonella rattaustraliani (EU111796.1), Bartonella japonica (AB242289.1), Bartonella coopersplainsensis (EU111803.1), Bartonella quintana (HQ014627.1), Bartonella koehlerae (AF176091.1), Bartonella henselae Human Australia (AJ439406.1), Bartonella henselae (L38987.1), Bartonella henselae Cat Flea Austria (MF374384.1), Bartonella henselae Cat Brazil (MH019304.1), Bartonella henselae Dog Chile (MG252490.1), Bartonella doshiae (AF207827.1), Bartonella rattimassiliensis (AY515124.1), Bartonella queenslandensis (EU111798.1), Bartonella elizabethae (Z70009.1), Bartonella schoenbuchii (AJ278184.1), Bartonella chomelii (AY254308.1), Bartonella capreoli (AF293392.1), Bartonella rochalimae (FN645459.1), Bartonella clarridgeiae Cat flea Chile (KY913636.1), Bartonella clarridgeiae Cat Brazil (MH019302.1), Bartonella clarridgeiae Cat Thailand (KX001761.1), Bartonella clarridgeiae (EU770616.1), Bartonella clarridgeiae strain 73 (FN645454.1), Bartonella baciliformis (DQ452947.1), Bartonella tamiae (DQ395177.1) and Brucella abortus (AE017223.1); and for ribC: Bartonella henselae Cat Brazil (HQ012583.1), Bartonella henselae fleas USA (AY953284.1), Bartonella henselae Cat Brazil (HM588661.1), Bartonella koehlerae (FJ832090.1), Bartonella quintana (AJ236917.1), Bartonella washoensis subsp. cynomysii (DQ825697.1), Bartonella heixiaziensis (KJ361664.1), Bartonella vinsonii subsp. arupensis (AY116631.1), Bartonella vinsonii subsp. vinsonii (AY116636.1), Bartonella vinsonii subsp. berkhoffii (AY116629.1), Bartonella alsatica (AY116630.1), Bartonella fuyuanensis (KJ361648.1), Bartonella doshiae (AY116627.1), Bartonella sp. Bat Kenya (HM363783.1), Bartonella bovis riboflavin synthase France (AY116637.1), Bartonella chomelii (AB290195.1), Bartonella capreoli (AB290194.1), Bartonella schoenbuchensis (AY116628.1), Bartonella bacilliformis (AJ236918.1), Bartonella clarridgeiae Cat China (EU571943.1), Bartonella clarridgeiae (AJ236916.1), Bartonella clarridgeiae Japan (AB292604.1), Bartonella clarridgeiae Cat Brazil (KR092386.1), Bartonella ancashensis (KP720649.1), Candidatus Bartonella ancashi Peru (KC886734.1), Brucella melitensis (CP008750.1)). No criteria regarding host or geographical localization were used to retrieve sequences from GenBank. Likelihood-mapping analyses were performed using the TREE-PUZZLE v5.3 program [Sequences were analyzed and manually cured using Bioedit Sequence Alignment Editor , in ordeto BLAST . Edited .6.12.03 . The hap.6.12.03  was usedlerae AF1091.1, BaBartonella spp. was not detected in ticks, dogs, and humans in the present study, but more studies need to be performed addressing this topic and putting emphasis on Bartonella spp. reservoirs and vectors. Regarding the dogs, surveys among endocarditis afflicted individuals can be carried out and similarly, studies conducted on humans should prioritize immune-compromised groups, as they are more prone to develop infections by Bartonella spp. About 20% of the cats sampled in Lisbon were positive for at least one Bartonella spp. and both B. henselae and B. clarridgeiae were identified in similar percentages. It is hoped that these results could bring attention to Bartonella sp. as an emerging pathogen in cats from Lisbon, and a potential threat for public health. As they are the main reservoir for B. henselae, preventive measurements should be implemented in order to avoid an outbreak of cat scratch disease in Lisbon.Bartonellosis is recognized as an emerging vector-borne disease that constitutes a threat to both animal and human health. Continuous and active surveillance to understand the epidemiological characteristics of bartonellosis is needed worldwide, including in Portugal.


Escherichia coli is a reservoir of antimicrobial resistance genes (ARGs). Here, we report the draft genome sequence of an E. coli strain (31HGR-CBG) that was isolated from a urine sample in Tamaulipas, Mexico. 31HGR-CBG harbors multiple ARGs, including blaCTX-M-15 and class 1 integron. This strain also carries multiple virulence genes. Escherichia coli represents a threat to public health (E. coli (ExPEC) are genetically diverse and complex  approval nor informed consent was required. 31HGR-CBG was grown on Trypticase soy agar and CHROMagar Orientation medium and incubated overnight at 37°C. Standard biochemical tests were also performed.https://www.bioinformatics.babraham.ac.uk/projects/fastqc) and Trim Galore! v0.6.6  were used to evaluate quality and to trim the raw reads, respectively. Assembly was performed using SPAdes v3.15.2 (https://github.com/ablab/quast). Contigs smaller than 900 bp were removed. Bacterial identification was confirmed by a BLASTN search (http://blast.ncbi.nlm.nih.gov) against the NCBI database and ribosomal multilocus sequence typing (rMLST)  at 37°C. DNA was extracted using the Wizard genomic DNA purification kit . DNA quantification was performed with the Qubit double-stranded DNA (dsDNA) HS assay kit in the Qubit 3.0 fluorometer . Libraries were constructed with the Nextera Flex library preparation kit and were sequenced using the MiniSeq sequencing system (150-bp paired-end reads). A total of 10,918,316 raw reads were generated. FastQC v0.11.3 (http://genomicepidemiology.org). ARGs were also predicted with the CARD v3.1.4 database using RGI v5.2.0 (Automatic annotation was performed by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.2. Multilocus sequence typing (MLST) was executed with PubMLST v1 . SerotypI v5.2.0 . Phages I v5.2.0 . DefaultN50 value of 91,860 bp, a GC content of 50.82%, and genome coverage of 220×. Annotation identified 4,939 genes, 4,850 coding sequences, and two CRISPR arrays. 31HGR-CBG belongs to sequence type 44 (ST44) and serotype 0101:H4.The genome was 4,981,584 bp in size and was assembled into 141 contigs, with an aadA5, aph(6)-Id, aac(6′)-Ib-cr, and aph(3″)-Ib], extended-spectrum β-lactams (blaCTX-M-15 and blaOXA-1), phenicol (catB3), macrolides [mph(A)], sulfonamides (sul1 and sul2), tetracycline [tet(B)], trimethoprim (dfrA17), and quaternary ammonium compounds (qacEΔ1). In addition, plasmid replicons IncFIA, IncFIB, and IncFII were detected.Multiple ARGs were identified, including genes conferring resistance to aminoglycosides [gyrA, parC, and parE were found, indicating fluoroquinolone resistance. Virulence-associated genes and four intact prophages were identified.Chromosomal mutations in JAKJKJ000000000. The reads were deposited in the Sequence Read Archive (SRA) under the accession number SRR15258840.This draft genome has been deposited in GenBank under the accession number


Spinirta Jin & Zhang, 2020 (Araneae: Corinnidae) from Jiangxi Province, China are described here: S.sanxiandiansp. nov. (♂♀) and S.sishuishansp. nov. (♂). Detailed descriptions and photographs of the new species are provided.Two new species of  Corinnidae Karsch, 1880 has increased greatly in the past ten years . Measurements were taken with the AxioVision software (SE64 ver. 4.8.3) and are given in millimetres. Terminology of the male and female copulatory organs follows Specimens were examined using a Jiangnan SZ 6100 stereomicroscope with a Zoom Microscope System. Both male palps and female copulatory organs were detached and examined in 80% ethanol, using an Olympus CX43 compound microscope with a KUY NICE CCD camera. All specimens treated in this work are deposited in the Animal Specimen Museum, Life Science of College, Jinggangshan University . The abbreviations used in the text and figures are:ALE anterior lateral eye;AME anterior median eye;At atrium;CD copulatory duct;CO copulatory opening;CS cone-shaped spines;d dorsal;E embolus;EA embolic apophysis;FD fertilization duct;GA glandular appendage;MOA median ocular area;p prolateral;PLE posterior lateral eye;PME posterior median eye;PTA prolateral tibial apophysis;r retrolateral;RTA retrolateral tibial apophysis;Sp spermatheca;St subtegulum;v ventral;VTA ventral tibial apophysis.Taxon classificationAnimaliaAraneaeCorinnidaeGenusJin & Zhang, 2020921B6204-15E0-54B8-B299-06A67E4FBE76Spinirtajinyunshanensis Jin & Zhang, 2020. Type locality: Chongqing.Spinirta species, S.wuyishanensis Zhou, 2022 was recorded from Jiangxi Province in southeast China. It is worth mentioning that the female remains unknown.The genus includes 11 species, all of which are distributed in southern and southwest of China (WSC 2022). Currently, most of them are known only from females (three species) or males (four species) (WSC 2022). Most of China’s nine species are recorded from southwestern China . Only onTaxon classificationAnimaliaAraneaeCorinnidaeLiusp. nov.E398719E-F60C-5419-B24C-A02AFD3BF57Ehttps://zoobank.org/6CEBCD02-A0B5-452B-A1AD-E0F681296427Holotype: 1 ♂, China: Jiangxi Province, Ji’an City, Qingyuan District, Donggu Town, Dawu Mountain, 26°40'48.69"N, 115°25'7.79"E, 1031 m, 25.X.2020, K. Liu et al. leg. (Cor-04). Paratype: 2 ♀, 13.XI.2021, K. Liu et al. leg., other data same as holotype (Cor-03 and Cor-05).The specific name is derived from the type locality, Sanxiandian Temple in Dawu Mountain; noun in apposition.Spinirtasparsula Jin & Zhang, 2020  with a curved posterior part (vs. straight in S.sparsula) and the ear-shaped retrolateral tibial apophysis (RTA) without protruded base (vs. digitiform with a kidney shaped protruded base in S.sparsula). It also resembles S.sishuishan sp. nov. in having a thumb-like ventral tibial apophysis (VTA), a thick horn-like prolateral tibial apophysis (PTA) and a curved sperm duct (SD), but can be separated from it by the ear-shaped retrolateral tibial apophysis (RTA) (vs. shield-shaped in S.sishuishan sp. nov.), the anterior part of the tegulum with a broad lateral apophysis (vs. absent in S.sishuishan sp. nov.) and the sharp embolic apophysis in retrolateral view (vs. relatively blunt in S.sishuishan sp. nov.)  , the shield copulatory openings (CO) (vs. round in S.qizimeiensis), and the copulatory ducts (CD) extending from the anteromedial to the posterolateral part of the epigyne .The male of this new species is similar to that of cf. Fig. . The femMale. Habitus as in Fig. AME 0.32, ALE 0.31, PME 0.2, PLE 0.27, AME-AME 0.15, AME-ALE 0.07, PME-PME 0.3, PME-PLE 0.36, AME-PME 0.25, AME-PLE 0.47, ALE-ALE 0.87, PLE-PLE 1.45, ALE-PLE 0.2. MOA 0.74 long, front width 0.77, back width 0.72. Chelicera with three promarginal and five retromarginal teeth  thumb-like in ventral view. Retrolateral tibial apophysis (RTA) ear-shaped, nearly as long as tibial length, ventral surface with two lines of short cone-shaped spines (CS). Prolateral tibial apophysis (PTA) thick horn-like, strongly sclerotised, nearly as long as 1/3 of tibia. Tegulum with strongly sclerotized apex. Subtegulum (St) with many wrinkles on posterior surface. Sperm duct (SD) S-shaped in posterior part. Embolus (E) short, with thick base, forming a C-shape with short spine-like embolic apophysis (EA), nearly 3× longer than embolic apophysis.Palp as in Fig. Female. Habitus as in Fig. AME 0.28, ALE 0.26, PME 0.19, PLE 0.24, AME-AME 0.16, AME-ALE 0.08, PME-PME 0.27, PME-PLE 0.31, AME-PME 0.26, AME-PLE 0.4, ALE-ALE 0.83, PLE-PLE 1.3, ALE-PLE 0.18. MOA 0.72 long, front width 0.66, back width 0.66. Abdomen: 5.55 long, 3.85 wide. Leg measurements: I 13.57 ; II 12.5 ; III 11.2 ; IV 14.77 ; spination  large, shield, covers equal or less than half of epigynal plate, anteromedially located. Copulatory openings (CO) very large, oval, located at anterolateral atrium. Copulatory ducts (CD) very broad, anteriorly touching, posteriorly slightly separated. Glandular appendages (GA) short, located at dorsal part of copulatory ducts, extending beyond medial part of copulatory ducts, directed anteriorly. Spermathecae (Sp) relatively broad, separated by 1/2 width of copulatory ducts. Fertilisation ducts (FD) directed anteriorly, shorter than spermathecal width.Epigyne as in Figs Spinirtasanxiandian sp. nov. may be the result of the influence of their development factors.The female specimens of this new species occur exactly in the same sites explored by the authors. They are identified as the same species based on appearance and epigyne. Variability was observed in the epigyne Fig. , which mKnown only from the type locality, Jiangxi Province, China Fig. .Taxon classificationAnimaliaAraneaeCorinnidaeLiusp. nov.0FA58825-87CA-51AD-843C-D1560BD78095https://zoobank.org/F06E9311-8FE0-4858-B870-22E9F59F3262Holotype: 1 ♂, China: Jiangxi Province, Ganzhou City, Chongyi County, Sishui Mountain, near parking lot, 25°27'11.73"N, 113°55'30.04"E, 965 m, 2.X.2020, K. Liu et al. leg. (Cor-02).The specific name, derived from the type locality, is a noun in apposition.S.sanxiandian sp. nov. by the shield retrolateral tibial apophysis (RTA) (vs. ear-shaped), the anterior part of the tegulum lacking lateral apophysis (vs. present in S.sanxiandian sp. nov.) and the relatively blunt embolic apophysis (EA) in retrolateral view (vs. sharp in S.sanxiandian sp. nov.)  thumb-like in ventral view. Retrolateral tibial apophysis (RTA) shield in retrolateral view, nearly as long as tibial length, ventral surface with four lines of short cone-shaped spines (CS). Prolateral tibial apophysis (PTA) thick horn-like, strongly sclerotised, nearly as long as 1/3 of tibia. Tegulum with strongly sclerotized apex. Subtegulum (St) with many wrinkles on posterolateral tegulum. Sperm duct (SD) S-shaped in posterior part. Embolus (E) spine-like, with thick base, forming a C-shape with short blunt embolic apophysis (EA), nearly 4× longer than embolic apophysis.Palp as in Fig. Female. Unknown.Known only from the type locality, Jiangxi Province, China Fig. .


This study aims to investigate the equivalence between Cambridge Neuropsychological Test Automated Battery (CANTAB) and Montreal Cognitive Assessment, Changsha version (MoCA-CS) as cognition measures for Chinese stroke survivors. Sixteen stroke survivors were recruited from stroke center in Shanghai, China. Participants completed Paired Associates Learning (PAL) task in CANTAB, MoCA-CS and questionnaire about demo-social characteristics. Pearson correlation and hierarchical regression were used for equivalence analysis. CANTAB-PAL task performances（PAL First Attempt Memory Score, PALFAMS, PAL Total Attempts 2 Patterns, PALTA2）were significantly associated with score of MoCA-CS and delayed recall (β=0.528, p=0.014; β=0.198, p=0.043; β= -3.885，p=0.017; β= -1.600,p=0.026, respectively）after controlling for age, gender and education. The results suggest CANTAB-PAL is a reliable tool for measuring Chinese stroke survivors’ cognitive function. Future studies are needed to establish the equivalence of CANTAB in multi-tasks in larger sample of Chinese stroke survivors.


Escherichia coli (ETEC) strain harboring genes encoding colonization surface antigen 13 (CS13) and a heat-labile toxin. The ESEI_597 strain was isolated from an 8-month-old child living in Korogocho, Kenya, in 2013.Here, we report the draft genome of ESEI_597, an enterotoxigenic  Escherichia coli (ETEC) strain ESEI_597 was isolated in 2013 from a stool sample from an 8-month-old child residing in Korogocho, an informal settlement northeast of Nairobi, Kenya. A pea-sized fecal sample was emulsified in 5 mL buffered peptone water . Approximately 10 μL of the overnight broth inoculum was plated on MacConkey agar (Oxoid), and a biochemically identified colony was subcultured in Mueller-Hinton agar (Oxoid) before being stocked at −80°C in tryptone soy broth (Oxoid) with 15% glycerol. All incubations were performed at 37°C for 18 to 24 h. Labile toxin (LT) and colonization surface antigen 13 (CS13) conventional uniplex PCR screening was performed using previously described primers (https://www.well.ox.ac.uk/ogc) and preprocessed using an automated protocol developed by the Modernising Medical Microbiology (MMM) Oxford Group. Read trimming to remove remnant adaptor sequences was performed using BBDuk, part of the BBTools package (www.ncbi.nlm.nih.gov/sra), with an automated step for removal of contaminant reads. The remaining reads were mapped (using Stampy v1.0.23) to an E. coli reference genome (GenBank accession AE014075.1) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), with all per-base-sequence quality flagged as passed.Enterotoxigenic 14075.1) . The quaN50 value of 140,019 bp, a G+C content of 50.71%, and a total length of 4,937,264 bp. ABRicate v1.0.1 (https://github.com/tseemann/abricate) was used to detect acquired antimicrobial resistance (AMR) genes with ResFinder v2.1 (https://github.com/tseemann/mlst) and the PubMLST database (https://pubmlst.org)  . Automataat operon, etpBAC operon, eatA, and tleA-like autotransporter are all absent. The cexE gene is present but truncated, with an LS-BSR value of 0.8893 (cshABCDEFGH) is 7,923 bp long, and LT in ESEI_597 has been assigned to allele 29.ESEI_597 belongs to sequence type 155 (ST155) (Achtman scheme) ; plasmidf 0.8893 . The CS1The Kenya Medical Research Institute (KEMRI)/National Ethics Review Committee approved all procedures (protocol number 2507).E. coli ESEI_597 has been deposited in GenBank under the accession number JALDSW000000000. Other features of the sequence data are provided in The draft genome of


M. avium complex (MAC) strains in a chronic mouse infection model either as monotherapy or in combination with standard of care .Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. These studies evaluated oral doses (PO) of EBO against 5 M. avium 2285R evaluated EBO at 1, 10, 30, 100, 300 and 500 mg/kg PO once daily (QD) compared to 250 mg/kg CLR PO QD. C57BL/6 mice were infected with a pulmonary aerosol of 1x1011 CFU. Treatment was administered for 56 days starting on day 28 post-infection. The bacterial burden (CFU) in lungs was evaluated on days 1, 28 and 84 post-infection by plating serial dilutions of homogenates on Middlebrook 7H11 charcoal agar plates. An additional 4 strains of MAC were evaluated with EBO doses of 100, 200, 300 or 400 mg/kg QD compared with the SOC therapy for MAC  QD and SOC plus EBO 200mg/kg QD. Oral exposures of EBO were determined in a group of uninfected mice (Table 1).A pilot chronic efficacy study against M. avium 2285R, a biofilm-forming strain, EBO at all doses tested was significantly better than CLR dosed at 250 mg/kg , and no CFU were detected on agar plates containing EBO (16 mg/L). In subsequent studies, SOC was compared to EBO in 4 additional MAC strains . Efficacy of EBO monotherapy was better than SOC against M. avium ATCC 700898, while it was as good as SOC with M. intracellulare 1956, M. intracellulareDNA00055, and M. intracellulare DNA00111 with CFU reductions ranging from 2 - 4.8 log10 compared to day 28 controls. In all four strains tested, 200 mg/kg EBO, which approximates the human oral equivalent dose of 500 mg, combined with SOC increased bacterial killing from 1.4 - 3.0 log10 CFU compared to SOC alone resulting in total lung CFU reductions of 4.6 - 5.6 log10.In a study with M. avium 2285R at day 84. EBO demonstrated potent in vivo efficacy against 5 MAC strains and significantly improved efficacy when combined with SOC, supporting further clinical development for EBO.In this chronic mouse lung infection model, no EBO resistance development was detected with Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn Shoen, PhD, AN2 Therapeutics: Grant/Research Support Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds.


Health care workers (HCWs) possess a potential risk to acquire and spread various infections. This study was planned to assess the immune status of HCWs in pediatric departments of two tertiary care hospitals in Northern India.In this cross-sectional study, HCW’s (Indians), working in pediatric departments of these hospitals, over 6-months period (July18-Dec18) were enrolled after taking written consent and their 5-ml venous blood sample was collected. Ethical clearance was obtained from Institute Ethics committee, before enrolling subjects. Serum were tested for antibodies against diphtheria-toxin (DT), pertussis-toxin (PT), measles, mumps, rubella, varicella, hepatitis-B (HbsAb) and hepatitis-A using commercial IgG (quantitative) ELISA kits.A total of 160 HCW’s (M:F=77:83), having mean age 30.6±7.8 years, were enrolled. Out of them 106 (66.3%) were resident doctors, 31 (19.4%) nursing-staff, 18 (11.3%) faculty members, 3 (1.9%) research-staff and 2 (1.3%) paramedical-staff. In our study, antibodies (IgG) against DT were between 0.01 to 0.1 IU/ml in 78.1% (120/160); requiring a Tdap booster; while 7 out of 125 had titers < 0.01 IU/ml, which needed 3-doses of primary vaccination. Antibodies (IgG) against PT < 30 IU/ml were seen in 60.6% (97/160), which were considered as seronegative, as per kit recommendations.A total of 3% (5/160), 13.1% (21/160), 10% (16/160), 17.5% (28/160) had titers < 12 IU/ml for IgG antibodies against measles, mumps, rubella and varicella respectively; were considered unprotected. A total of 25% (40/160) had Anti-HBs antibody titers < 20 mIU/ml; which were low, therefore were advised to take one booster dose of Hep-B vaccine. A total of 15.6% (25/160) had IgG antibodies against hepatitis-A < 10 mIU/ml; were unprotected.Alarming proportions of pediatric-HCWs had low antibody titres against DT (78.1%) and PT (60.6%), necessitating a dose of Tdap. A total of 10%, 17.5% and 15.6% lacked protective antibodies against rubella, varicella and hepatitis-A. A quarter of screened population had low anti-Hbs titres, requiring boosting of immunity. Our study emphasizes the urgent need for improving immunization status of pediatric HCW’s; as they continue to remain susceptible to various vaccine preventable infectious diseases.All Authors: No reported disclosures.


Correction: BMC Cancer 22, 689 (2022)https://doi.org/10.1186/s12885-022-09740-9The results of colony formation assay which have been used in Fig. 4C(c) and Fig. 4D(c) were accidentally used again in Fig. The western blot result annotations "Ctrl "and "MIR137HG" were missed in Fig. Following publication of the original article , the autThe correct Fig.


Scientific Reports, 10.1038/s41598-023-28540-0, published online 28 January 2023Correction to: The original version of this Article contained an error in Reference 37,et al. Inclusive Wealth Report 2022 Executive Summary. .37. Barbier, E. B. now reads:. UNEP. Inclusive Wealth Report 2022 Executive Summary. .37The original Article has been corrected.


HFE p.C282Y/p.C282Y are incompletely characterized.Screening program participants with iron overload (IO) phenotypes without HFE p.C282Y and p.H63D genotyping.We studied white participants who had IO phenotypes without p.C282Y/p.C282Y in post-screening clinical examinations (CE). We defined IO phenotypes as a) elevated serum ferritin (SF) and transferrin saturation (TS) at screening and CE, and b) absence of IO treatment, anemia, transfusion >10 units, alcohol intake >30 g/d, hepatitis B or C, and pregnancy. We defined IO-related disease as elevated alanine or aspartate aminotransferase (ALT/AST) or swelling/tenderness of 2nd/3rd metacarpophalangeal (MCP) joints. All participants had HFE genotypes were 12.9 (p.C282Y/p.H63D), 3.0 (p.H63D/p.H63D), 1.9 (p.C282Y/wt), 0.9 (p.H63D/wt), and 0.5 (wt/wt) compared to 42,640 white screening participants without IO phenotypes or p.C282Y/p.C282Y. Regression on SF revealed positive associations: MCV ; swelling/tenderness of MCP joints ; and p.H63D/wt . IO-related disease  occurred in 19 participants . Median MCV was higher in participants with IO-related disease . Logistic regression on IO-related disease revealed a significant association with diabetes ).There were 32 men and 26 women (mean age 54±16 y). Median food/supplemental iron intakes were 14.3/0.0 mg/d. Relative risks of HFE p.C282Y/p.C282Y, relative risks of HFE genotypes p.C282Y/p.H63D, p.H63D/p.H63D, and p.C282Y/wt were significantly higher than in 42,640 white screening participants with neither IO phenotypes nor p.C282Y/p.C282Y. SF was significantly associated with MCV, swelling/tenderness of 2nd/3rd MCP joints, and p.H63D/wt. IO-related disease was significantly associated with MCV and diabetes.In the present 58 screening program participants who had IO phenotypes without  HFE gene  [HFE alleles p.C282Y  and p.H63D  were discovered in referred white patients with hemochromatosis and iron overload (IO) in 1996 [The HFE alleles [HJV, chromosome 1q21.1), transferrin receptor 2 , hepcidin antimicrobial peptide , and solute carrier family 40 member 1 (ferroportin)  [Rare types of hemochromatosis, usually diagnosed in referred patients, are associated with novel  alleles  or deletHFE genotype and sex [Some population screening program participants who have elevated SF and elevated TS without p.C282Y/p.C282Y also have IO phenotypes due to hemochromatosis, although these participants have been incompletely characterized. A primary report of the Hemochromatosis and Iron Overload Screening (HEIRS) Study displayed separate initial screening TS and SF data of participants from a racially diverse population segregated only for  and sex . Those d and sex .HFE p.C282Y/p.C282Y who participated in the HEIRS Study post-screening clinical examinations (CE) [HFE genotypes other than p.C282Y/p.C282Y and identified variables significantly associated with IO phenotypes and IO-related disease. We discuss our observations in the context of previous reports.To learn more, we studied non-Hispanic whites who had IO phenotypes without ons (CE) , 8, 9. Gons (CE) , 8, 9. Wons (CE) . We compThe National Heart, Lung, and Blood Institute/National Human Genome Research Institute HEIRS Study evaluated the prevalence, genetic, and environmental determinants, and potential clinical, personal, and societal impacts of hemochromatosis and IO in a multiethnic, primary care-based sample of 101,168 adults enrolled during the interval 2001–2003 at four Field Centers in the US and one in Canada . The StuHFE p.C282Y and p.H63D allele-specific genotyping [The HEIRS Study recruited participants from a health maintenance organization, diagnostic blood collection centers, and public and private primary care offices in ambulatory clinics associated with five Field Centers . Ninety-notyping .HFE p.C282Y homozygotes (regardless of screening SF and TS), to all participants whose screening SF and TS exceeded study thresholds, regardless of HFE genotype, and to selected participants who had normal screening SF and TS and HFE wt/wt, defined as absence of p.C282Y and p.H63D. Median interval between screening and CE participation was 8 months [Invitations to participate in a post-screening CE were extended to all 8 months .HFE genotype. Each participant completed a questionnaire addressing medical history and medications [At CE, eligible participants were informed of their screening SF, TS, and ications  and a Unications . An HEIRications .HFE genotype based on p.C282Y and p.H63D allele-specific analyses [All testing was performed at the HEIRS Study Central Laboratory . At CE, a morning blood sample was obtained after an overnight fast to measure SF and TS , to confirm Elevated SF and TS were defined as SF >300 μg/L and TS >50% (men) and SF > 200 μg/L and TS >45% (women). Reference ranges for hemoglobin were 133–177 g/L (men) and 117–157 g/L (women) and for mean corpuscular volume (MCV) were 78–105 fL (men) and 78–106 fL (women) [Reflex testing for hepatitis B surface antigen and hepatitis C antibody was performed in all participants with elevated ALT or AST . Participants with elevated ALT or AST and positivity for hepatitis B surface antigen were defined to have viral hepatitis B . ParticiObtaining liver specimens by biopsy, estimating liver iron content using imaging techniques, and performing therapeutic phlebotomy were beyond the scope of the HEIRS Study.We defined IO phenotypes as the following: a) elevated SF and elevated TS at both screening and CE, and b) absence of IO treatment, anemia, erythrocyte transfusion >10 units, alcohol intake >30 g/d, hepatitis B or C, and pregnancy. We defined IO-related disease in participants with IO phenotypes according to modified criteria of Allen et al. : elevateHFE p.C282Y/p.C282Y who had IO phenotypes, who fasted overnight ≥8 h before CE, and whose data analyzed in this study were complete.We analyzed observations on all self-identified non-Hispanic white CE participants without Macrocytosis was defined as MCV greater than the respective sex-specific upper reference limits. Microcytosis was defined as MCV ≤77 fL.2.Estimates of dietary and supplemental iron and daily alcohol intake were taken from the Multi-Ethnic Dietary Questionnaire . We defiSelf-reported diabetes reported at screening was confirmed with medication reviews at CE . We defiPhysicians who performed physical examinations at CE recorded these manifestations as present or absent .The dataset for analyses consisted of complete observations on 58 CE participants with IO phenotypes. Age, SF, TS, hemoglobin, and MCV are expressed to the nearest integer. Descriptive data are displayed as enumerations, percentages, proportions, means (± 1 SD), or medians (range). Analyses of continuous data using d’Agostino’s and Shapiro-Wilk tests revealed that age and BMI values were normally distributed and thus these values are displayed as mean ± 1 SD and were compared using Student’s t tests (two-tailed). Continuous variables that were not normally distributed are displayed as medians (range) and were compared using Mann-Whitney U tests (unpaired data) or Wilcoxon’s signed-ranks test (paired data). Percentages were compared using Fisher’s exact test (two-tailed). The significance of the difference between two independent proportions was calculated using the z-ratio and two-tailed values of p. We compared screening and CE SF and TS data using Pearson’s correlation coefficient.HFE genotypes other than p.C282Y/p.C282Y. We performed forward stepwise regression on SF at CE using a) all independent variables available at CE (except TS) and b) the first independent variable with p ≥0.05 as the stopping rule. TS was not used as an independent variable because its positive correlation with SF at CE was significant. We display standardized coefficients β for each significant predictor variable in this regression.We computed relative risks of We performed logistic regression on IO-related disease using all available independent variables  for which values of p in univariate comparisons were ≤0.1500. We calculated odds ratios ) for significant independent variables.® , GB-Stat® , and GraphPad Prism 8® .We defined values of p <0.05 to be significant. Analyses were performed with Excel 2000HFE genotypes [HFE p.C282Y/p.C282Y [Screening observations in 43,453 non-Hispanic white participants  were evaluable for SF and TS levels and enotypes . Both sc/p.C282Y . ElevateHFE genotypes p.C282Y/p.H63D, p.H63D/p.H63D, and p.C282Y/wt in the present 58 CE participants with IO phenotypes were significantly greater than corresponding percentages in 42,640 non-Hispanic white screening participants without IO phenotypes (Percentages of enotypes . Relativenotypes . Aggregaenotypes .Detailed characteristics of 58 participants are displayed in HFE p.C282Y/p.H63D occurred in 18 participants (31.0%) and p.H63D/p.H63D in four participants (6.9%). Each of the genotypes p.C282Y/wt, p.H63D/wt, and wt/wt occurred in 12 participants. Two men and two women had SF >1000 μg/L . Macrocytosis was detected in one woman and microcytosis was detected in another woman. Median estimated daily iron intakes from food and iron supplements were 14.3 mg and 0.0 mg, respectively. Elevated ALT or AST occurred in 18 participants (31.0%). Swelling/tenderness of the 2nd/3rd MCP joints was observed in one participant (1.7%). Diabetes occurred in four participants (6.9%). 000 μg/L .HFE p.H63D/wt . This regression accounted for 32.0% of the variance of SF (regression ANOVA p = 0.0001).Forward stepwise regression on SF at CE using other CE observations (except TS) as independent variables revealed three positive associations: MCV ; swelling/tenderness of 2nd/3rd MCP joints ; and These data are displayed in There were 15 abstainers (25.9%) and 43 non-abstainers (74.1%). The difference between median MCV values of abstainers and non-abstainers was not significant (94 fL (86–97) vs. 96 fL (77–107), respectively; p = 0.1369). All nine of 58 participants (15.5%) with MCV >100 fL were non-abstainers, although proportions of abstainers and non-abstainers with MCV >100 fL did not differ significantly .HFE p.C282Y/p.C282Y who also reported that they had not received treatment for IO. Our study design excluded non-Hispanic white CE participants who may have had elevated SF and elevated TS due to types of anemia that increase iron absorption, a history of erythrocyte transfusion >10 units, viral hepatitis B or C, or increased alcohol intake. Elevated SF without elevated TS is common in patients with obesity [SLC40A1 alleles [We evaluated observations in 58 non-Hispanic white participants in the HEIRS Study CE who had both elevated SF and elevated TS defined as IO phenotypes without  obesity , metabol obesity , diabete obesity , non-alc obesity , 19, inf obesity , maligna obesity –22, "cla alleles , heredit alleles , and ben alleles . Thus, oHFE genotypes p.C282Y/p.H63D, p.H63D/p.H63D, and p.C282Y/wt in the present 58 CE participants with IO phenotypes were significantly greater than corresponding percentages in 42,640 non-Hispanic white screening participants without IO phenotypes. SF was significantly associated with MCV, swelling/tenderness of the 2nd/3rd MCP joints, and p.H63D/wt. IO-related disease was significantly associated with MCV and diabetes. IO and IO-related disease were not significantly associated with estimated intake of dietary or supplemental iron. Taken together, we infer that heritable factors in addition to common HFE genotypes other than p.C282Y/p.C282Y or acquired factors we did not study contribute to or account for IO phenotypes, IO-related disease, and other potential iron-related consequences of hemochromatosis in the present participants.Percentages of HFE p.C282Y/p.H63D was increased in the present participants, although we observed no significant association of IO-related disease with p.C282Y/p.H63D. Participants in an Australian population screening program with IO phenotypes and p.C282Y/p.H63D also had a low risk of IO-related disease  [Relative risk of 5 women) . In refe5 women) . In an a5 women) . In a me5 women) .GNPAT) p.D519G (rs11558492) occurred with greater prevalence in French and Australian subjects with HFE p.C282Y/p.H63D with higher SF than those with lower SF [CYBRD1) promoter polymorphism rs884409 was significantly higher in French patients with p.C282Y/p.H63D with higher SF than those with lower SF [HJV p.N196K  or HAMP -72C>T [HAMP p.G71D  did not modify SF and TS phenotypes [HFE iron-associated alleles probably account in part for the variable penetrance of p.C282Y/p.H63D phenotypes.Glyceronephosphate O-acyltransferase  [HFE coding region allele [HAMP Met50del IVS2+1(-G) and HAMP p.G71D, respectively [Relative risk of .9, 5.8) . In a me.9, 5.8) . Compounn allele , occurren allele , 36. Refn allele , 38. In ectively .HFE p.H63D/wt was increased in the present participants. In an analysis of 14 case-control studies of hemochromatosis, the pooled odds ratio of p.H63D/wt as a contributor to hemochromatosis IO was 1.9  [HFE alleles, e.g., p.I105T (c.314T>C) [HFE allele, e.g., p.E168Q (c.502G>C), in cis with p.H63D [Relative risk of .5, 2.5) . Rarely,.314T>C) , or withh p.H63D . In a meh p.H63D . It is pHFE p.C282Y/p.C282Y [Estimated median daily intake of dietary iron in the present participants is similar to that in adults in the U.S. not selected for IO phenotypes . SF in t/p.C282Y .HFE p.C282Y/p.C282Y were significantly higher than those of control subjects [HFE genotype [MCV was significantly associated with SF and IO-related disease in this study, after adjustment for other variables. In two previous reports, mean MCV values in referred hemochromatosis patients without subjects , 45. Incgenotype . In one genotype . By desigenotype ) and thagenotype ). We obsgenotype  and manygenotype , occurreHFE genotypes were p.H63D (n = 2) and p.H63D/p.H63D and wt/wt (one each). In 20,306 Canadian HEIRS Study participants linked to the Ontario Death Registry 9 years after screening, all-cause survival was significantly decreased in the 34 participants with screening SF >1000 μg/L and HFE genotypes other than p.C282Y/p.C282Y  [Four of the present 58 participants  had SF >1000 μg/L. Their 7 wt/wt) .HFE genotypes other than p.C282Y/p.C282Y in adults with IO phenotypes have meaningful clinical correlates. Some adults with IO phenotypes without p.C282Y/p.C282Y have hepatic steatosis [HFE hemochromatosis, or non-HFE "modifier" mutations. Guidelines for hemochromatosis management promulgated by expert groups in the U.S. [HFE genotyping. It is also recommended that first-degree relatives of persons diagnosed to have HFE hemochromatosis undergo TS/SF phenotyping and HFE genotyping [HFE alleles has disease-identification value other than to reveal IO risk.teatosis , 51 or cteatosis , 52, othteatosis . Adults the U.S.  and Eurothe U.S.  recommennotyping , 54. TheHFE p.C282Y/p.C282Y and unlikely to have other disorders that sometimes present with IO phenotype mimics. Another strength is the availability of screening and CE data sufficient to demonstrate a) significant differences in proportions and relative risks of HFE genotypes other than p.C282Y/p.C282Y and b) significant associations of IO phenotypes and IO-related disease with other potential iron-related consequences of hemochromatosis. An uncertainty of this study is that we may have excluded participants with "classic" SLC40A1 (ferroportin) hemochromatosis in whom SF but not TS is typically elevated [A strength of this study is that our selection criteria included only participants with IO phenotypes who were likely to have hemochromatosis in the absence of elevated .HFE genotype risk associated with IO phenotype because participants invited to the CE (other than those with HFE p.C282Y/p.C282Y or wt/wt) were selected on the basis of having both screening SF and screening TS values above HEIRS Study thresholds, not on the basis of HFE genotype [HFE p.C282Y and p.H63D. The HEIRS Study performed analyses to detect specific HFE alleles other than p.C282Y and p.H63D and specific HJV, TFR2, HAMP, SLC40A1 alleles in a small proportion of screening participants [In this study, we assessed genotype . The HEIicipants . None oficipants .HFE genotypes p.C282Y/p.H63D, p.H63D/p.H63D, and p.C282Y/wt were increased in screening program participants who had IO phenotypes without p.C282Y/p.C282Y. SF was significantly associated with MCV, swelling/tenderness of 2nd/3rd MCP joints, and p.H63D/wt. IO-related disease was significantly associated with MCV and diabetes. Novel HFE alleles, deleterious alleles of HJV, TFR2, HAMP, or SLC40A1, iron-related "modifier" alleles, or acquired factors could contribute to IO phenotypes and IO-related disease in some of the present participants.We conclude that proportions and relative risks of S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Although convenient single-tablet antiretroviral regimens have been developed to treat HIV in recent years, some patients have continued to take a multi-tablet treatment, nevirapine extended-release (NVP XR) plus two nucleoside reverse transcriptase inhibitors (NRTIs) due to its excellent safety profile. This observational study examined the demographic and clinical characteristics of HIV-positive patients who switched to dolutegravir plus lamivudine (DTG/3TC) from NVP XR plus 2 NRTIs, following discontinuation of NVP XR from the Canadian market.Virally suppressed (< 50 cps/mL) HIV-positive adults ≥18 years who switched from NVP XR plus 2 NRTIs to DTG/3TC between 20 August 2019 and 30 April 2020 were retrospectively identified from Electronic Medical Records housed at Spectrum Health in British Columbia, Canada. Baseline demographic and clinical characteristics were summarized using descriptive statistics at the date of first DTC/3TC prescription. Virologic control, CD4 cell count, weight-related changes and exploratory characteristics related to metabolic syndrome were summarized at baseline and at 12 ± 6-months post-switch using descriptive statistics. Reasons for treatment discontinuation were also captured.Sixty-nine patients were identified . Mean length of use (±SD) of NVP XR was 4.6 ± 2.6 years. Sixty-three (91.3%) persisted on DTG/3TC at the 12-month timepoint post-switch with 61 (96.8%) virally suppressed < 50 cps/mL and all 63 (100%) virally suppressed < 200 cps/mL. Among persistent patients with CD4 cell counts available, mean CD4 cell count (±SD) remained stable, increasing slightly from 724.4 ± 238.4 to 740.3 ± 240.6 cells/uL. All six (8.7%) patients who discontinued DTG/3TC, discontinued due to tolerability and not effectiveness reasons.Our findings are the first to examine real-world use of single-tablet, 2-drug DTC/3TC among patients who switched from multi-tablet, 3-drug NVP XR plus 2 NRTIs in Canada. The majority persisted on DTG/3TC and remained virally suppressed at the < 50 cps/mL level 12-months post-switch. This, coupled with excellent tolerability, demonstrates the effectiveness of DTG/3TC in maintaining viral suppression among a unique and stable group of older men.Joss de Wet, MBChB CCFP FCFP, Gilead: Advisor/Consultant|Gilead: Board Member|Gilead: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Board Member|ViiV: Grant/Research Support Joann K. Ban, PharmD, MSc, GlaxoSmithKline Canada Inc.: Employee Gustavo Verdier, BSc, BPharm, MBA, GlaxoSmithKline: Stocks/Bonds|ViiV Healthcare ULC: Salary Juejing Ling, MSc, GSK: Employee of IQVIA , a company that receives consulting fees from GSK. Andrean Bunko, MPH, GlaxoSmithKline Inc.: GSK contracted with IQVIA for this and other projects. I am acting as an author in capacity of my employment with IQVIA. Michael McKimm, BSc, ViiV Healthcare: employee.


Stenotrophomonas maltophilia with a 44,659-bp genome. This phage is closely related to Stenotrophomonas phage SM171, sharing 92% overall nucleotide identity as determined by BLASTn, and it shares 14 similar proteins  with some Pseudomonas phages from the genus Beetrevirus.Phage Suso is a temperate siphophage of  Stenotrophomonas maltophilia is emerging as a multidrug-resistant respiratory pathogen  and trimmed with FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). The trimmed reads  were used for assembly using SPAdes v3.5.0 . Annotation of the phage genome was performed using the Center for Phage Technology (CPT) Galaxy-Apollo platform  , in Wadsworth, TX, in September 2019, using reads 87,54 reads xy-pub) 79. Gene cxy-pub) 7 and Metaxy-pub) 7. tRNA gexy-pub) 7 and tRNAxy-pub) 7. Gene fuxy-pub) 7, TMHMM vxy-pub) 7, HHPred xy-pub) 7, and Sigxy-pub) 7. Genome-xy-pub) 7. All sofStenotrophomonas phage SM171 (MZ611865), sharing 92% overall nucleotide identity as determined by BLASTn. Besides this phage, Suso shares 14 similar proteins  with some Pseudomonas phages from the genus Beetrevirus (taxid 2560098), such as phages B3 (GenBank accession number NC_006548), JBD67 (GenBank accession number NC_042135), and vB_Pae_BR141c (GenBank accession number MK511065).Phage Suso has a siphophage morphology  possessiMZ326866. The associated BioProject, SRA, and BioSample accession numbers are PRJNA222858, SRR14095259, and SAMN18509666, respectively.The Suso sequence was deposited in GenBank with accession number


Mindfulness (being present in the moment without judgement) has been linked to greater caregiver emotional health. Recent mindfulness-based interventions report improved coping skills, mood, and reduced stress in dementia caregivers. In this cross-sectional study of 141 ADRD caregivers, we assessed whether the relationship between caregiver mindfulness and caregiver experience varies by caregiver gender, relationship to patient (spouse-vs-child), etiology (AD-vs-LBD), or stage (MCI-vs-dementia). A stratified univariate analytic approach was used. Four mindfulness parameters  were used: global score (GS), decentering (F1), positive (F2), and negative emotional regulation (F3). Outcomes included positive and negative appraisals of caregiving (PANAC), preparedness, care confidence, and depression. GS was linked to positive outcomes in male (rPANAC+=0.32/p=0.005), spouse caregivers (rPANAC+)=0.32/p=0.006 ) of ADRD patients regardless of etiology  and stage . Inverse relationships were observed with negative outcomes in male (rPANAC-=-0.46/p=0.002 and rdepression=-0.41/p=0.005), spouse caregivers (rPANAC-=-0.25/p=0.035 and rdepression=-0.30/p=0.009) of AD patients (rPANAC-=-0.25/p=0.043 and rdepression=-0.33/p=0.009) in early stages (rdepression=-0.41/p=0.001). F2 contributed to most relationships, with F3 and F1 significant in some but not all caregiver groups. Specifically, male spouse caregivers of AD patients regardless of stage may benefit from full-scope (F1-F3) programs while those of LBD patients from programs focused on improving emotional regulation (F2-F3). Wives of AD and LBD patients may in turn benefit from programs to improve positive emotional regulation (F2). Findings suggest that tailoring mindfulness-based interventions to specific caregiver groups may be effective in improving caregiver experience and mood.


Rationale: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives: Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods: Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p = 0.0397) and NETosis (p = 0.0332), and impaired phagocytosis (p = 0.0036) associated with impaired ROS generation (p < 0.0001). The percentage of CD54+ neutrophils (p < 0.001) was significantly increased, while surface expression of CD11b (p = 0.0014) and PD-L1 (p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p = 0.0396) and impaired DNAse activity (p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p = 0.0129). Conclusions: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity. Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) virus, was declared a global pandemic by The World Health Organization (WHO) on 11 March 2020 [Dysregulated virus induced host-immune responses are thought to be the primary cause of severe COVID-19 . NeutropAdvanced age is a recognised risk factor for severe COVID-19, including the development of ARDS ,7,8. AgeEmerging studies suggest that neutrophils are implicated in the pathogenesis of severe COVID-19 and every reporting study has described cell dysfunction (or inferred cell dysfunction through transcriptomics), which could contribute to tissue damage and secondary infection ,25,26,27These are important considerations. As neutrophil functions change with age, age matched controls are important in identifying pathological differences. Although approximately 12% of hospitalized COVID-19 patients require ICU support, the majority of hospital admissions and deaths occur on non-ICU wards ; understWe hypothesised that neutrophils from COVID-19 patients would exhibit diverse altered effector functions and changes to phenotype, with the degree of dysfunction associated with adverse patient outcomes.This study aimed to perform, for the first time, a comprehensive assessment of ex vivo neutrophil phenotypes and functions in a statistically powered cohort of hospitalized, non-ICU SARS-CoV2 infected patients compared to aged-matched controls and patients with non-COVID community acquired pneumoniae (CAP) and to investigate relationships with clinical outcomes.Recruitment is summarized in 2: FiO2 ratio (SF), converted to PaO2: FiO2 (PF) (SF = 57-0.61PF).COVID-19 patients were recruited within 48 h of hospital admission due to pneumonitis/pneumonia related to COVID-19. All patients had a positive COVID-19 PCR swab. No patients received novel treatments or were part of a COVID clinical medicinal trial on recruitment to this study. Exclusion criteria is listed in CAP patients were recruited within 48 h of hospital admission due to non-COVID-19 pneumonia. All patients had a negative COVID-19 swab. Exclusion criteria is listed in the Age matched controls (AMC) were either recruited from patients attending pre-booked face-to-face outpatient appointments or from hospital staff. AMC had no evidence of acute illness, including COVID-19, within the last two weeks, as assessed by a respiratory physician, and met the other exclusion criteria.S. pneumoniae and NETosis by the release of cell-free (cf) DNA  DNA . Plasma t-test or ANOVA. A Mann-Whitney U test for unpaired data, a Wilcoxon test for paired data, or a Kruskall-Wallis test was used to analyse non-normally distributed data. Data are presented throughout as median (IQR), with each n number representing a separate study participant. Significance was defined at p < 0.05. There were no corrections for multiple comparisons, but exact p values are given. A power calculation performed on isolated neutrophil NETosis data  suggested that 18 participants were required in each group . A Kolmogorov-Smirnov Test was used to determine data distribution. Normally distributed data were analysed using a student’s 41 COVID-19 patients (mean age 71.5 years), two healthy AMC (mean age 70 years) and 26 CAP patients (mean age 67.5 years) were included in the study. Demographics are provided in p = 0.0332) and CAP  AMC vs. 40.63 (115.2) COVID-19, S. pneumoniae was significantly decreased in COVID-19 patients compared to AMC (Median fluorescence intensity (MFI): 8.0 (4.2) AMC vs. 6.6 (2.6) COVID-19, p = 0.0366) and CAP (MFI 9.8 (4.8), p = 0.0052, p = 0.3257, p = 0.9228, p = 0.6726, Neutrophil phagocytosis of p = 0.0091), and CAP patients (MFI: 285 (211) baseline vs. 357 (260) 30 min, p = 0.038), but not in COVID-19 patients (MFI: 44.1 (35) baseline vs. 65.3 (60) 30 min, p = 0.134, p < 0.0001, Cytoplasmic (c)ROS, and nuclear/mitochondrial (n/m)ROS were measured in resting neutrophils and following phagocytosis. Compared to unstimulated cells, cROS levels were elevated after phagocytosis in both AMC (MFI: 46.8 (28) baseline vs. 64.9 (51) 30 min, p < 0.0001), but not in COVID-19 patients (MFI: 18.9 (12) baseline vs. 21.2 (12), p = 0.0329) or CAP patients (MFI: 22.2 (21) baseline vs. 26.7 (17), p = 0.989, p < 0.0001) and CAP neutrophils  baseline vs. 32.0 (38), p = 0.0394, p = 0.0118, A larger increase in the level of cfDNA was detected in supernatants obtained from COVID-19 patient neutrophils post-PMA treatment compared to AMC (fold change in absorbance of neutrophils stimulated with PMA vs. vehicle control: 1.29 (0.32) AMC vs. 1.53 (1.66) COVID-19, p = 0.0396, p = 0.0186, p = 0.0322).At hospital admission, COVID-19 patients presented with higher concentrations of plasma cfDNA compared to AMC (621 ng/mL (1324) AMC vs. 1071 ng/mL (856) COVID-19, To determine whether neutrophils, via NETosis, were a source of the cfDNA, plasma samples were screened for the presence of CitH3, a protein that decorates the DNA backbone of NETs . Westernp < 0.0001, vs. 77.45% (34) CAP, p < 0.0001, Using NETs as a substrate, serum DNase activity was lower in COVID-19 patients at the time of hospital admission when compared to both AMCs and CAP patients (% degradation: 88.4% (93) AMC vs. 12.8% (49) COVID-19, To determine whether changes observed in neutrophil function were associated with phenotype, expression of key surface molecules were investigated by flow cytometry. This was compared to both AMC and CAP patients. A table of percentage receptor expression and MFI is shown in p = 0.0014, p = 0.0026, p = 0.046; MFI:1701 (1268) CAP, p < 0.0001). Compared to AMC, both the percentage of neutrophils expressing the activation marker CD11b (82% (15) AMC vs. 68% (44) COVID-19, p < 0.0001, vs. 7% (11) CAP, p < 0.0001, The percentage of neutrophils expressing CD54, a marker of reverse transmigration, was elevated in COVID-19 patients compared to both AMC and CAP patients (26% (37) AMC vs. 71% (21) COVID-19, p < 0.006, vs. 524 (264) and CAP, p < 0.0001, The percentage of neutrophils expressing CD66b, CD62L, CD10, CXCR2, CXCR4, CD11c and PD-L1 did not differ between AMC and COVID-19 patients see . The surThere was no association of neutrophil phenotypic marker expression with 4C severity score, or in survivors and non-survivors.p = 0.0322, p = 0.0273, p = 0.0420, On day three to five follow-up, relative to baseline readings, there was a decrease in CXCR2 expression: (MFI: 1960 (1601) day one vs. 1126 (1262), day three to five, p = 0.0322, p = 0.0273, p = 0.0420, On day three to five follow-up, relative to baseline readings, there was a decrease in CXCR2 expression: (MFI: 1960 (1601) day 1 vs. 1126 (1262), day three to five, p = 0.0296, CAP p = 0.0106, p < 0.0001, CAP p = 0.0032, p < 0.0001, CAP p < 0.0001, p < 0.0001, Compared to AMC, both COVID-19 and CAP patients showed elevated levels of IL-6 , p = 0.0156, Pre-incubation of COVID-19 neutrophils from cohort 2, with Pi3k delta inhibitor CAL101 and gamma inhibitor AS252434 significantly decreased PMA induced NETosis (PMA RFU 247 (228.5) vs. delta 207.5 (242.5), We present novel findings of COVID-associated neutrophil dysfunction across all main effector functions. In summary, compared to AMC and patients with CAP, systemic neutrophils from patients hospitalized with moderate severity COVID-19 demonstrated increased migration, impaired anti-microbial responses including reduced phagocytosis and nuclear/mitochondrial ROS generation after phagocytosis. Later/end phase neutrophil responses were increased, namely ex vivo NETosis with evidence of increased systemic NETosis, coupled with reduced DNase activity, which was also elevated in CAP. We also show an altered but distinct neutrophil phenotype, not compatible with a purely activated, immature, senescent or anti-inflammatory phenotype as described before (results summarised in Individually, as described in other studies, these changes to effector function could compromise aspects of the host defence. Collectively, these changes represent a clear mechanism for significant tissue damage. Poor phagocytosis would impede pathogen clearance, increasing the likelihood of secondary infection and amplifying inflammation. NETosis is implicated in tissue damage and thrombotic events in several disease settings ,38. The S. pneumoniae, the most common bacteria implicated in secondary infection in COVID-19 [Secondary infection in COVID-19 is associated with increased severity of lung disease and poorer outcomes ,41. ImpaCOVID-19 , alongsiCOVID-19 , may conElevated NETosis ,24,44 anThe collective pattern of neutrophil dysfunction in COVID-19 speaks of alterations to mechanosensing within these cells. Elevated migration and impaired phagocytosis could both be linked to reduced pseudopod extrusion, which is known to increase migratory speed . FurtherWe observed an altered neutrophil phenotype in moderate COVID-19, not compatible with previously described populations, and not in keeping with our AMC or CAP control cohorts. COVID-19 neutrophils expressed decreased levels of the activation marker CD11b and a lack of CD62L shedding, which has previously been observed in sepsis , alongsi+ CXCR2- neutrophils, confirming a report of reduced CXCR2+ neutrophils in ICU COVID-19 patients [Finally, we observed that COVID-19 neutrophils expressed elevated CD54, a marker of reverse transmigration, whereby neutrophils migrate from the tissues back into the circulation. These cells are capable of high levels of oxidative burst , which mpatients .The majority of COVID-19 hospitalisations and deaths occur in non-ICU wards , making Our data suggests a distinct cellular response in moderate COVID-19 which contributes to on-going immune mediated harm, but which may be modifiable using a targeted therapy, such as PI3Kδ or PI3K γ inhibitors administered at this crucial point in disease progression.® to isolate neutrophils, and the authors combined data from multiple blood samples taken over eleven days of hospitalization. As we show changes in neutrophil phenotype and function over the three to five time course of our study, we suggest that combining time points obscures the complex changes occurring in this short-lived cell population. We also used opsonized S. pneumoniae for phagocytosis studies which may be phagocytosed by different mechanisms to S. aureus bioparticles [Our data complements studies which showed increased systemic NETosis in COVID-19 ,68, and articles , confounMore recently, Loyer et al., demonstrated the increased expression of CD11b and ROS production with decreased CD62L expression in a cohort of COVID-19 patients treated in the ICU in comparison with CAP and healthy controls . The autThis study was limited due to the safety measures required when handling biological fluids for a new infectious disease. All experiments were carried out within a BSL2 hood and methods were chosen based on tolerance to inactivation/fixation with 4% PFA. Our patients did not include an ICU group; however, mild-moderate disease affects a larger proportion of overall COVID-19 patients, and we believe it is this point in the patient pathway which holds most potential for successful intervention. Our AMC group highlights changes in COVID-19 and our CAP group highlights differences between disease types.Our study shows that moderate COVID-19 is associated with alterations in neutrophil phenotype, increased migratory capacity and NETosis, and impaired antimicrobial function, which contributes to the severity of COVID-19. Elevated NETosis in the lung is associated with disease severity, and elevated systemic NET production is likely to contribute to inflammation, which may drive ARDS associated damage and thrombosis. Targeting neutrophils and their downstream effectors may be beneficial in the treatment of COVID-19.


Mycobacterium smegmatis mc2155. McGee has a genome 156,008 bp long, containing 237 protein-coding genes, 31 tRNA genes, and 1 transfer-messenger RNA (tmRNA) gene. McGee shares high gene content similarity to phages in actinobacteriophage cluster C1.Mycobacteriophage McGee is a myovirus isolated from a wet soil sample collected at Manassas, VA, using  Mycobacterium smegmatis mc2 155 have been isolated and characterized, providing important insights into viral diversity and population structure . The soil sample was washed with 7H9 liquid medium, the wash filtered , and the filtrate plated in soft agar overlay containing Mycobacterium smegmatis mc2 155. McGee was purified with multiple rounds of plating. Negative-stain transmission electron microscopy revealed McGee to possess a Myoviridae morphology , and sequenced on an Illumina MiSeq instrument v3 reagents), resulting in 77,248 single-end 150-bp reads providing 73× coverage. The raw reads were assembled and assessed using the programs Newbler v2.9 ( reagentshttp://cobamide2.bio.pitt.edu/) (http://phages.wustl.edu/starterator), whereas gene functional assignments were determined using BLASTp searches against the NCBI nonredundant v2.13.0 (13Genes were identified and annotated using DNA Master v5.23.6 (tt.edu/) , GLIMMER v2.13.0  and acti v2.13.0 , HHpred  v2.13.0 , and Pha v2.13.0 . Aragorn v2.13.0  and tRNA v2.13.0  were use2.13.0 1315 and TM2.13.0 13, 17. AllA total of 269 putative genes were identified in McGee, including 237 protein-coding genes, 31 tRNAs, and 1 tmRNA. Among those that could be assigned a function, the virion structure and assembly genes include a major capsid protein (gp101), capsid decoration protein , tail sheath protein (gp129), tail assembly chaperones , tape measure protein (gp134), minor tail proteins , and baseplate wedge protein . The genes involved in McGee’s DNA metabolism include DNA helicase (gp188), thymidylate kinase (gp197), DnaC-like helicase loader (gp201), DnaB-like double-stranded DNA (dsDNA) helicase (gp202), DNA primase (gp204), DnaJ-like chaperonin (gp206), single-stranded DNA (ssDNA) binding protein (gp207), DnaE-like DNA polymerase III α (gp208), RF-1 peptide chain release factor (gp209), RecA-like DNA recombinase (gp210), Holliday junction resolvase (gp210), RusA-like resolvase (gp214), Ro-like RNA binding protein (gp233), and multiple proteins with a helix-turn-helix DNA binding domain . Genes involved in lysis, lysin A, holin, and lysin B are encoded by gp252, gp253, and gp254, respectively. The largest gene in McGee is a hypothetical protein gp98  and the smallest gp64 (75 bp long).https://phagesdb.org/), McGee was assigned to phage cluster C1 (Based on its gene content similarity (GCS) of at least 35% to phages in the Actinobacteriophage database (uster C1 , 18, 19.ON637764, while its raw reads have been deposited under Sequence Read Archive accession number SRX14483217.The complete genome sequence for McGee can be found at GenBank under accession number


However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmid-mediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids.The rapid dissemination of plasmid-mediated  Acinetobacter species is a heterogeneous group of opportunistic pathogens and easily acquires antibiotic resistance genes (ARGs) (tet(X) genes have been reported in at least 10 different Acinetobacter species, including Acinetobacter baumannii, Acinetobacter gandensis, Acinetobacter piscicola, Acinetobacter schindleri, Acinetobacter johnsonii, Acinetobacter indicus, Acinetobacter towneri, Acinetobacter lwoffii, Acinetobacter pseudolwoffii, and Acinetobacter variabilis (tet(X3) and tet(X6) genes were detected with carbapenem resistance gene blaNDM–1 in A. baumannii, A. indicus, A. schindleri, and A. lwoffii isolates, posing a serious public health threat  Gram-negative and Gram-positive pathogens . Howevers (ARGs) . To dateriabilis . Worrisoh threat .Acinetobacter spp. genome and plays an important role in the horizontal transmission of ARGs, such as blaNDM–1 and blaOXA–23 , a series of plasmid classification schemes were developed based on replication initiator protein, mobilization protein and plasmid size (tet(X)-positive Acinetobacter spp. plasmids since the first mobile plasmid-mediated tet(X3) gene in 2019, and their homology groups remained to be analyzed (tet(X)-carrying Acinetobacter spp. plasmids by genome-based replicon typing, followed by analyses of structural characteristics, transferability, and in vivo effect.The plasmid is a self-replicating component of laOXA–23 . With thmid size . As the mid size . Howeveranalyzed . Herein,tet(X)-positive Acinetobacter spp. strains in China (tet(X3)-positive Acinetobacter spp. YH16040 and tet(X3)- and tet(X6)-positive Acinetobacter spp. YH12068 from pig; tet(X3)-positive Acinetobacter spp. YH16056 from soil; tet(X3)-positive A. pseudolwoffii YH18001 from human; tet(X4)-positive A. indicus Q22-2, Q85-2, and Q278-1 from migratory bird. In addition, a newly isolated tet(X6)-positive A. baumannii YC103 by CHROMagar™ Acinetobacter plates  containing tigecycline (2 μg/mL) from duck in 2019 was also analyzed (tet(X)-harboring Acinetobacter spp. plasmids deposited at the NCBI database were collected by tblastn -positive Acinetobacter spp. isolates were sequenced by Oxford Nanopore , respectively. Combining the clean data with our previous Illumina HiSeq data , genome assembly was performed by Unicycler version 0.4.1 and corrected by Pilon version 1.12 (Genomic DNA of eight Seq data  as well ion 1.12 .tet(X)-harboring plasmids were annotated by Rapid Annotation using Subsystem Technology (RAST) version 2.0 -positive structures was conducted by Easyfig version 2.2.5 (All sion 2.0 . Plasmidsion 2.0 . Plasmidsion 2.0 . A maximsion 2.0 . Plasmidme group . Similaron 2.2.5 .tet(X)-mediated tigecycline resistance was evaluated by filter mating with rifampin-resistant Acinetobacter baylyi ADP1 and A. baumannii ATCC 19606 (tet(X) detection and PCR-based fingerprinting (Transferability of plasmid-borne CC 19606 . The putprinting . In paraprinting .tet(X3)-harboring GR31 plasmid pYH16040-1 was cured of Acinetobacter spp. YH16040 using sodium dodecyl sulfate (SDS) with adjustment (tet(X3) gene .The justment . An overX3) gene  and repl1 whereas eravacycline was uninterpreted with no breakpoint. Escherichia coli ATCC 25922 served as a quality control strain.Minimum inhibitory concentrations (MICs) of all strains were determined by broth microdilution and interpreted according to the Clinical and Laboratory Standards Institute guideline M100-Ed28 . The tesG. mellonella  were randomly grouped (16 per group), and then infected with GR31 plasmid-mediated tet(X3)-positive Acinetobacter spp. YH16040 or its plasmid-cured strain YH16040C  via the last left proleg. After incubation at 35°C for 2 h, the infected larvae were treated with tigecycline (2 μg/g) or phosphate-buffered saline (PBS) by injection into the last right proleg. Finally, the larvae were observed for survival rates per 24 h in the next 4 days. All in vivo experiments were performed in triplicate.As previously described , the heatet(X)-positive Acinetobacter spp. isolates successfully revealed four tet(X)-carrying plasmids. These included tet(X3)-positive plasmids pYH16040-1 (GenBank accession number: CP094542) from Acinetobacter spp. YH16040, pYH16056-1 (CP094546) from Acinetobacter spp. YH16056, and pYH12068-1 (CP094556) as well as a tet(X6)-positive plasmid pYH12068-2 (CP094557) from Acinetobacter spp. YH12068 (tet(X6)-positive chromosome cYC103 (CP054560) from A. baumannii YC103 was also obtained (tet(X)-positive strains A. pseudolwoffii YH18001, A. indicus Q278-1, A. indicus Q85-2, and A. indicus Q22-2 (tet(X)-harboring plasmids or chromosomes despite repeated attempts. By querying Nanopore raw data of A. pseudolwoffii YH18001 , A. indicus Q278-1 , A. indicus Q85-2 , and A. indicus Q22-2 , a repeated structure consisting of multiple copies of tet(X) genes was detected, respectively, which may lead to the failure of complete sequence assemblies.WGS analyses of eight  YH12068 . Meanwhiobtained . For theus Q22-2 , we failtet(X)-positive Acinetobacter spp. plasmids deposited at the NCBI database has been growing  subtypes located on plasmids (tet(X3) , tet(X5) , and tet(X6) , of which tet(X3) usually coexisted with tet(X6) . Besides tet(X) genes, large amounts of plasmid-mediated genes conferring resistance to tetracyclines, aminoglycosides, β-lactams, phenicols, sulfonamides, macrolides, lincosamides, and rifamycins were present (tet(X)-positive plasmids ranging from 42,489 to 332,451 bp were widely distributed in A. indicus , A. baumannii , A. towneri , A. schindleri , A. variabilis , A. pseudolwoffii , A. piscicola , Acinetobacter junii , Acinetobacter pittii , and unidentified Acinetobacter spp. strains -positive MDR Acinetobacter spp. plasmids.Since the first report in 2019 , the num n = 34; , includiplasmids , namely  present . Worrisotet(X)-positive Acinetobacter spp. plasmids carried replicase genes and all of them belonged to a Rep_3 superfamily (pfam01051). Therefore, plasmid classification was conducted based on nucleotide sequence alignment with already classified replicase genes (tet(X)-positive Rep_3 superfamily plasmids, a total of six homology groups were successfully identified, such as the dominant GR31 , GR26 , GR41 , and GR59 -positive plasmids pYH16040-1 (CP094542), pYH16056-1 (CP094546), and pYH12068-1 (CP094556) fell within the same group GR31, which has been sporadically detected with tet(X) genes in A. baumannii, A. indicus, A. schindleri, A. towneri, and other Acinetobacter spp. strains (tet(X6)-positive plasmid pYH12068-2 (CP094557) was unclassified due to the lack of replicase genes. In contrast, the replicase genes of tet(X3)- and tet(X6)-carrying plasmids pXMC5X702-tetX-145k (CP084302) and pYH12207-2 (CP048661) have < 75% nucleotide identities with existing homology groups GR1-GR59, and therefore were defined as novel groups GR60  and GR61 , respectively (tet(X)-positive Acinetobacter spp. plasmids.Replicon conserved domain analyses showed that 82.4% (28/34) of se genes . Among t strains . Howeverectively . We provAcinetobacter spp. isolates . In essence, the GR31 plasmids have been detected in 13 validly named Acinetobacter species, including A. baumannii , A. lwoffii , A. indicus , A. towneri , A. schindleri , and others  genes especially in the genera Acinetobacter.In order to evaluate the host range of plasmids belonging to GR26, GR31, GR41, GR59, GR60, and GR61, we conducted a blastn search against the NCBI database and confirmed they were mainly distributed in d others . The plaplasmids . Particuplasmids , but thetet(X)-carrying GR31 plasmids available in the NCBI database. A total of 23 plasmids belonging to GR31 were analyzed and they exhibited GC contents ranging from 39.1 to 46.1%. According to our WGS results, pYH16040-1 was 87,435 bp and harbored 94 putative open reading frames (ORFs), whereas pYH16056-1 was 98,709 bp consisting of 107 ORFs and pYH12068-1 was 100,866 bp consisting of 115 ORFs. Although they originated from different sources, pYH16056-1 and pYH12068-1 showed an average 76.5% nucleotide coverage and 99.8% nucleotide identity to pYH16040-1, with the encoding sequence insertion, deletion and rearrangement  and the accessory modules (tet(X)-positive GR31 plasmids owned a repB gene involved in plasmid replication, and parA and parB genes responsible for plasmid stability. However, only 21.7% (5/23) of them harbored plasmid transfer modules, including conjugal transfer (n = 3) and mobilization genes (n = 2). For accessory modules, they mainly consisted of ARGs  and heavy-metal resistance gene clusters .Predominantly, we compared the complete plasmid sequences of pYH16040-1, pYH16056-1, and pYH12068-1 with angement . Moreoveangement . Archety modules . Our restet(X) genes in Acinetobacter species, including tet(X3) (n = 28), tet(X6) (n = 12), and tet(X5) (n = 2), were further analyzed  and ISAba14 (n = 3). Similarly, the truncation by IS26 was found on GR60 (n = 2) and GR61 (n = 2) plasmids, one of which was also upstream truncated by ISAcsp12. Except the GR26 plasmid carrying a complete ISCR2-mediated transposition unit (n = 1), GR41 and GR59 plasmids were detected with truncation by IS3 (n = 1) and IS1006 (n = 1), respectively. In addition, our WGS results of A. pseudolwoffii YH18001 (JALHBG010000013), A. indicus Q278-1 (JALHBF010000002), A. indicus Q85-2 (JALHBE010000004), and A. indicus Q22-2 (JALHBD010000003) revealed the incomplete tet(X)-carrying structures were also related to ISCR2 (tet(X) variants and ISCR2 as well as truncation by other insertion sequences indicated the frequent recombination events.Genetic environments of all plasmid-mediated analyzed . The res process . For GR3to ISCR2 . The clotet(X3) genes were successfully transferred from Acinetobacter spp. YH16040  and YH16056  to the recipient A. baylyi ADP1 , but failed to A. baumannii ATCC 19606. In Acinetobacter spp. YH12068, the tet(X3)-carrying GR31 plasmid pYH12068-1 (CP094556) couldn’t be transferred, which may be explained by the lack of entire conjugative transfer regions (tet(X6)-positive unclassified plasmid pYH12068-2 (CP094557) carrying plasmid transfer modules was transferred from Acinetobacter spp. YH12068 to A. baylyi ADP1 with a similar efficiency mentioned above. MICs of all transconjugants against tetracyclines increased by at least 32-fold when compared with A. baylyi ADP1, including tetracycline (≥64 μg/mL), tigecycline (≥4 μg/mL), the newly FDA-approved eravacycline (≥2 μg/mL) and omadacycline  gene (CP040912) was also able to be transferred from human-derived A. baumannii to A. baumannii 5AB via electro-transformation, whereas the GR31 plasmid co-harboring tet(X3) and tet(X6) genes (CP044451) was from environmental A. indicus to A. baylyi ADP1 by natural transformation. The transferability of tet(X) genes between Acinetobacter species of human, animal and environment origins reminded us to consider the “One Health” approach to prevent mobile tigecycline resistance.Conjugation experiments showed the GR31 plasmid-mediated  regions . Meanwhitet(X3)-mediated tigecycline resistance from Acinetobacter spp. YH16040, YH16056, and YH12068 to A. baylyi ADP1, as well as florfenicol resistance (128 μg/mL) from Acinetobacter spp. YH16040 and YH12068 and gentamicin resistance (16 μg/mL) from Acinetobacter spp. YH16056, which was consistent with the result of plasmid-mediated ARG mining (tet(X)-mediated tigecycline resistance and blaNDM–1-mediated carbapenem resistance between Acinetobacter species (tet(X) genes, as previously described for resistance determinants mcr-1 and blaIMP–1  was transferred with G mining . Recentl species . With thblaIMP–1 .tet(X3) gene, a plasmid-cured strain YH16040C [tet(X3)-negative] was obtained by serially passaging of tet(X3)-positive Acinetobacter spp. YH16040. For tigecycline, a significant decrease in MIC was detected (1 μg/mL) when compared with the parental isolate YH16040 , eravacycline (0.06 μg/mL), omadacycline (0.125 μg/mL), florfenicol (8 μg/mL) and sulfamethoxazole-trimethoprim (20 μg/mL) also exhibited at least 16-fold decrease. The result further confirmed the elimination of GR31 plasmid pYH16040-1 (CP094542) that co-harbored resistance genes tet(X3), floR and sul2  gene was evaluated using a G. mellonella model. 96 h later of tigecycline treatment (2 μg/g), the larval mortality rate of Acinetobacter spp. YH16040C significantly decreased to 37.5% while Acinetobacter spp. YH16040 was 87.5%  gene compromised the clinical effectiveness of tigecycline. Notably, the infection by Acinetobacter spp. YH16040 and Acinetobacter spp. YH16040C under the treatment of PBS resulted in a greater larval mortality rate (100%) than PBS only (12.5%) after 48 h (p < 0.0001), whereas no significant difference was observed between Acinetobacter spp. YH16040 and Acinetobacter spp. YH16040C -positive Enterobacteriaceae bacteria have also been constructed, and therefore G. mellonella was available for in vivo functional analyses of plasmid-mediated tet(X) genes.Subsequently, the tet(X)-carrying MDR plasmids in Acinetobacter species. Our results classified the tet(X)-positive Acinetobacter spp. plasmids of Rep_3 superfamily into six homology groups, including two newly assigned GR60 and GR61. To the best of our knowledge, this study first revealed a dominant GR31 plasmid mediating the horizontal transfer of tigecycline resistance gene tet(X) across different Acinetobacter species and contributed to the failure of tigecycline treatment in vivo. Accordingly, more efforts are needed to monitor and prevent the plasmid-mediated tet(X)-positive Acinetobacter spp. strains.Taken together, the data presented in this study highlighted the diverse distribution of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/J-LH, Y-HL, and JS designed the study. CC, P-YH, C-YC, and QH performed the experiments. CC and P-YH analyzed the data. CC wrote the draft of the manuscript. All authors reviewed, revised, and approved the final report.


Azadirachta indica L. and Melia azedarach L., belonging to Meliaceae family, have been shown to have medicinal benefits and are extensively employed in traditional folk medicine. Herein, HPLC analysis of the ethyl acetate fraction of the total methanolic extract emphasized the enrichment of both A. indica L., and M. azedarach L. leaves extracts with phenolic and flavonoids composites, respectively. Besides, 4 limonoids and 2 flavonoids were isolated using column chromatography. By assessing the in vitro antiviral activities of both total leaves extracts against Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2), it was found that A. indica L. and M. azedarach L. have robust anti-SARS-CoV-2 activities at low half-maximal inhibitory concentrations (IC50) of 8.451 and 6.922 μg/mL, respectively. Due to the high safety of A. indica L. and M. azedarach L. extracts with half-maximal cytotoxic concentrations (CC50) of 446.2 and 351.4 μg/ml, respectively, both displayed extraordinary selectivity indices (SI>50). A. indica L. and M. azedarach L. leaves extracts could induce antibacterial activities against both Gram-negative and positive bacterial strains. The minimal inhibitory concentrations of A. indica L. and M. azedarach L. leaves extracts varied from 25 to 100 mg/mL within 30 min contact time towards the tested bacteria. Our findings confirm the broad-spectrum medicinal value of A. indica L. and M. azedarach L. leaves extracts. Finally, additional in vivo investigations are highly recommended to confirm the anti-COVID-19 and antimicrobial activities of both plant extracts.The leaves of  Drug resistance to infections in humans brought on by microorganisms including bacteria, viruses and fungi is a life-endangering phenomenon with considerable number of mortalities worldwide. Medicinal herbs were applied to produce a plenty of pharmaceuticals that are currently used to alleviate various health disorders or diseases. Numerous researches are performed to find out new antimicrobial treatment alternatives that really can prevent the spread of bacteria or eradicate them without endangering humans by conducting comprehensive screenings of medicinal herbs , 2.In December 2019, the SARS-CoV-2 virus has emerged as an unknown severe respiratory illness, namely corona virus illness 2019 (COVID-19) . Few weeAzadirachta indica L. (Neem) and Melia azedarach L. (China tree) are popular medicinal herbs that belong to the Meliaceae family. A. indica L, originated in India, is extensively scattered in all countries , 1H-NMR : 7.11 , 5.45 , 2.89 ,4.29 , 4.89, 4.95, 2.67 , 1.91, 1.64 , 7.39 , 6.21 , 6.93 , 1.70 , 2.15 , 1.10 . 13C-NMR : 158.2 (C-1), 126.4 (C-2), 206.1 (C-3), 41.3 (C-4), 50.4 (C-5), 69.8 (C-6), 76.9 (C-7), 43.7 (C-8), 40.2 (C-9), 41.3 (C-10), 16.1 (C-11), 28.3 (C-12), 45.7 (C-13), 72.8 (C-14), 57.5 (C-15),33.1 (C-16), 40.2 (C-17), 122.8 (C-20), 141.8 (C-21), 111.2 (C-22), 138.7(C-23), 21.51, 171.81 (OAC), 21.9 (C-methyls). From the formentioned spectral data, this compound was identified as 14,15-β-epoxynimonol + 612 for C35H48O9, 549 (100%), 449, 389 and 371. Additionally, 1H-NMR, and13C-NMR data were in agreement with previous studies + 694 for C40H54O10, 649 for ethoxyl group loss at C-12, then 449 for two tigloyl side chain loss, followed by 389(100%) due to acetyl group loss and subsequently water loss giving 371 which are in accordance to nimbolinin limonoids fragmentation pattern. The 1H and 13C NMR data were similar to  +, 552, 537 [M–H2O + H]+. The data were consistent with the molecular formula C35H54O5. The 1H-NMR and13C-NMR data confirmed this compound as 3-α-tigloylmelianol which was previously identified from M. azedarach fruits by [Compound 6: yellow amorphous powder, m.p. 164°C. EI-MS (m/z): 462 (M+) for C22H22O11, in addition to 300, 285, 257, 152, 137, 116. IR data, UV absorbance in different shift reagents and 13C-NMR data characterized this compound as chrysoeriol 7-O-glucoside. This compound has been isolated for first time in the present study. The structures of the isolated compounds were illustrated in Fig 1.50), the half maximal inhibitory concentration 50 (IC50) and selectivity index (SI)(CC50/IC50) against SARS-CoV-2 were individually evaluated (Table 2). Both leaves extracts of A. indica and M. azedarach showed high safety in Vero E6 cells with CC50 values of 446.2 μg/mL for A. indica and 351.4 μg/mL for M. azedarach, respectively. Additionally, both extracts exhibited promising inhibition of SARS-CoV-2 replication in Vero E6 cells at potent IC50 concentrations of 8.541 μg/mL for A. indica and 6.922 μg/mL for M. azedarach . Interestingly, both extracts exhibited extraordinary selectivity indices (SI) (>50) (Table 2). The determined SI values are meaningfully greater than those recommended for further assessment of the bioactive compounds (> 10) [A. indica and M. azedarach indicate the bioactive potential of A. indica and M. azedarach leaves extracts. Compared to the reference drug control remedesivir , both plant extracts showed comparable safety and antiviral activities with slightly lower selectivity indices. These data demonstrate that both extracts can be considered as promising candidates for further in vitro and in vivo studies against SARS-CoV-2 virus and potentially other viruses.To appraise the antiviral efficacy of leaves extracts against SARS-CoV-2 virus, the half maximal cytotoxic concentration (CCs (> 10) , 39. TheA. indica extracts against the novel SARS-CoV-2 virus [et al. [To our knowledge, there is a few previous studies that assessed the antiviral activity of -2 virus , 41. In  [et al.  concludeBorkotoky and Banerjee , isolatein vitro viral inhibitory activity of M. azedarach L. leaves extract against SARS-CoV-2 which is so far not studied. Nethmini et al. found that M. azedarach L. could turn off the replication of herpes simplex virus, coxsackie virus, enterovirus, influenza A virus, and bovine rhinovirus in both epithelial and macrophage cell lines [In this study, we explored the ll lines .in vitro anti-SARS-CoV-2 effect in this study for both plant leaves extracts, the inhibitory zone assay was done to assess the in vitro antibacterial properties of the two leaves extracts against E. coli, P. aeruginosa, S. aureus, L. monocytogenes, and E. faecalis using agar well-diffusion assay. The obtained results indicated that 50 μg/ml of A. indica L. and M. azedarach L. displayed high potency for inhibiting a broad spectrum of examined Gram-negative (GN) species (.In addition to the Table 3). Results revealed that the size of ZOI of vancomycin against the selected pathogens; E. coli, S. enterica, P. aeruginosa, S. aureus, L. monocytogenes, and E. faecalis using disc diffusion assay were ranged between 7 and 11 mm as mentioned in Table 3).Simultaneously, two reference antibiotics drugs vancomycin (30 μg/disc) and ciprofloxacin (5 μg/disc) were applied in this study as a positive control . ResultsThese results show the tendency that the GN bacterial species were more susceptible to all tested plant extracts than the GP species. Conversely, the width of ZOI around discs was lower than those around wells. Our data are in agreement with previous study , in whicA. indica L. and of M. azedarach L. extracts were examined to estimate MIC values to assess their antibacterial potential during various exposure times  to different bacterial strains. As shown in Figs 4, the extracts demonstrated a notable bactericidal effect against all the tested bacterial strains. The detected MIC values depend on the concentrations (mg/mL) and exposure time (min). The consequences of MICs exhibited by the extract of M. azedarach displayed that the perfect antibacterial effects against E. coli (MIC = 50 mg/mL within 30 min), S. enterica (MIC = 25 mg/mL within 30 min), P. aeruginosa (MIC = 25 mg/mL within 30 min), S. aureus (MIC = 75 mg/mL within 30 min), E. faecalis (MIC = 25 mg/mL within 30 min) and L. monocytogenes (MIC = 75 mg/mL within 30 min). The measured MIC values of A. indica plant extract were lower in GN than GP bacteria. The enhanced antibacterial activity against GN could be due to variation in active compounds in M. azedarach extract and their penetration ability through bacterial cell wall and membrane [The different concentrations (10–100 mg/mL) of membrane . This fiM. azedarach extract. The results represented in Fig 4 revealed significant antibacterial effects against all test bacteria. Also here, the calculated MIC values depended on the applied extract concentrations (mg/mL) and contact time (min). The results of MIC exhibited that the extract has clear bactericidal effect against E. coli (MIC = 50 mg/mL within 30 min), S. enterica (MIC = 25 mg/mL within 30 min), P. aeruginosa (MIC = 50 mg/mL within 30 min), S. aureus (MIC = 75 mg/mL within 30 min), E. faecalis (MIC = 75 mg/mL within 30 min) and L. monocytogenes (MIC = 100 mg/mL within 30 min). Interestingly, the results showed that the M. azedarach extract possess higher antibacterial activities against the tested GN bacteria in comparison to GP bacteria, which is matching with what we detected in case of A. indica plant extract except that MIC of Listeria monocytogenes was higher than S. aureus and E. faecalis .Using the same procedure and bacterial strains, we also assessed the antibacterial activity of Table 4. The results gained revealed that there was an exceptional positive correlation with strong significance between GP and GN species and the concentrations of all plant extracts. On the other hand, results perceived that a moderate correlation had been reported for E. coli.From statistical analysis results, the potential correlation with significance between various concentrations of particular extract towards all the targeted pathogenic bacterial strains was briefly reported in S. aureus, vancomycin-resistant enterococci, and Mycobacterium tuberculosis are widely documented as challenging hospital-associated infections to treat with known antibiotics. To this point, medicinal plants are recently applied to provide a supplementary treatment line against these bacterial infections [M. azadirachta L. and A. indica L. showed powerful antibacterial activities against various GP and GN species. The Meliaceae family has a substantial percentage of polyphenolic chemicals that could possess oxidative activities. A. indica L. phytochemicals have such a broad range of biological characteristics, especially antibacterial and cancer-fighting potential [M. azedarach L. towards numerous types of bacterial pathogens [As a public biohazard with millions of deaths every year, antibiotic resistance emerged recently due to the continuous prescription of improper antibiotics at non-lethal doses. Methicillin-resistant fections . M. azadotential . This bootential . Furtherathogens . Limonoiathogens .M. azedarach L. was established to be biocidal agent against P. aeruginosa, and E. coli, the lowest activity was documented in aqueous extract of M. azedarach against E. coli (8.5 mm) and S. aureus (8.2 mm). In previous study, E. coli showed the lowest sensitivity to methanolic extract (6 mm), and no activity was observed in 10, 20, 30 μg/ml of methanolic extract of M. azedarach L. [M. azedarach L. seed extracts efficiently manage illnesses caused on by both GP and GN species and reported that the that ethyl acetate fraction exhibited greatest suppression, followed by aqueous and methanol-based extracts, each of which reduced the proliferation of all the studied pathogenic strains. The petrol and benzene extracts displayed antimicrobial activity versus 15 pathogens, however, in comparison to the polarity extracts. The findings previously illustrate that seed extracts are powerful antibiotics that can effectively prohibit both disease-caused pathogens to human infections.The ethanolic extract of arach L. . PreviouA. indica L. and M. azedarach L. leaves were chemically characterized and were found to be rich in phenolic compounds and flavonoids. In vitro assessment of both extracts revealed that they have robust antiviral activity against SARS-CoV-2 as well as antibacterial effects against broader spectra of GN and GP bacteria. These data emphasize their broad-spectrum medicinal value and demand further in vitro and in vivo investigations as anti-SARS-CoV-2 candidate and antibacterial agents.