Salmonella have changed in recent years as multidrug-resistant (MDR) strains have become prevalent among various serovars. The recent expansion of MDR Salmonella enterica serovar Indiana sequence type 17 (ST17) poses an increasing threat to global public health, as 24.3% (61/251) of S. Indiana isolates in this study exhibited resistance to three clinically important antimicrobial agents: fluoroquinolones (ciprofloxacin), extended-spectrum β-lactams , and macrolides (azithromycin). Both the evolutionary histories and antimicrobial resistance (AMR) profiles of this serovar remain to be described. Bioinformatic analysis revealed multiple lineages have coexisted and spread throughout China. Specifically, emergence of a predominant lineage appears to be associated with accumulated various substitutions in the chromosomal quinolone resistance-determining regions (GyrA S83F D87N and ParC T57S S80R) (141 [56.2%]), as well as acquisition of an extended-spectrum β-lactamase (ESBL)-producing IncHI2 plasmid that has subsequently undergone extensive rearrangement and an IncX1 plasmid that contains mph(A), conferring resistance to azithromycin. Several other evolutionary events influencing the trajectory of this drug-resistant serovar were also identified, including sporadic acquisitions of blaCTX-M-carrying plasmids, along with chromosomal integration of blaCTX-M within subclusters. Most human isolates reside in clusters containing isolates from animals, mainly from chickens, indicating the close relationship of human isolates with those from food animals. These data demonstrate that MDR S. Indiana ST17 is already widespread and capable of acquiring resistance traits against the clinical important antimicrobial agents, suggesting it should be considered a high-risk global MDR pathogen. The complexity of its evolutionary history has implications for AMR surveillance, epidemiological analysis, and control of emerging clinical lineages.The genetic features of foodborne IMPORTANCE The emergence and worldwide spread of AMR Salmonella constitute great public health concerns. S. enterica serovar Indiana is a typical MDR serovar characterized by sporadic reports. However, comprehensive population genomics studies have not been performed on this serovar. This study provides a detailed and comprehensive insight into the rapid evolution of AMR in this important Salmonella serovar in the past 15 years in eight provinces of China. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, the persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance determinants that occur at all genetic levels . There are different mechanisms of antimicrobial resistance in S. enterica serovar Indiana from those of other serovars. This study sheds light on the formation of MDR S. enterica serovar Indiana with chickens as its potential reservoirs and paves the way to curb its further expansion among food animals. Salmonella enterica (NTS) remains one of the important foodborne pathogens globally -resistant Salmonella spp. were listed by the WHO in 2017 as priority pathogens for which new antimicrobials were urgently needed , or changes in the food chain. Some clonal lineages of MDR Salmonella have shaped the epidemiology of the disease at a global level, as in the case for sequence type 34 (ST34) S. enterica serovar 4,[5],12:i:−, ST313 S.enterica serovar Typhimurium, and ST198 S.enterica serovar Kentucky , extended-spectrum β-lactams , and macrolides (azithromycin [AZM]) approved by the FDA in the United States to treat infections caused by Salmonella (S. Indiana isolates can express resistance to carbapenems or colistin (CT), the last-resort antimicrobials, suggesting that these bacteria have now become a serious challenge for public health  of DNA gyrase gene (gyrA) and DNA topoisomerase IV gene (parC) conferred FQs resistance, along with plasmid-mediated quinolone resistance (PMQR) genes [oqxAB and aac(6′)-Ib-cr]. In addition, diverse blaCTX-M genes mapped on mobile genetic elements (MGEs) were reported earlier and found to be integrated into the chromosome (as in the case of blaCTX-M-55) (Recent studies investigated the genetic basis underpinning resistance to FQs and extended-spectrum β-lactamases (ESBLs) in TX-M-55) , 23. HowS. Indiana isolates collected from human and food-related samples in eight provinces of China during 2006 to 2017. Using phenotypic susceptibility data and whole-genome sequencing (WGS) analysis, we determined the prevalence and mechanisms of resistance and identified potential drivers of variation among the AMR profiles described within different lineages.Here, we report on the genomic epidemiology and AMR features of 251 S. Indiana isolates from eight provinces in China, 120 isolates were cultured from clinical samples  taken during 2007 to 2017 and 131 isolates from food-related samples taken during 2006 to 2016 (see in silico multilocus sequence typing (MLST) as S. Indiana ST17 after whole-genome analysis.Out of the 251 confirmed 10.1128/msystems.00253-22.4TABLE S1Salmonella Indiana isolates. Download Table S1, PDF file, 0.01 MB.Source and basic information of Copyright © 2022 Du et al.2022Du et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. Indiana isolates (P < 0.05) (P < 0.05).Testing of the susceptibility of 251 isolates  to 13 anisolates , with 21isolates . The res < 0.05) . Further10.1128/msystems.00253-22.5TABLE S2S. Indiana isolates against ciprofloxacin. Download Table S2, DOCX file, 0.02 MB.MIC value related to the PMQR gene and substitution mutations in QRDR of 251 Copyright © 2022 Du et al.2022Du et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n = 11) had S83F and D87N amino acid substitutions in GyrA along with two ParC amino acid substitutions. Furthermore, five PMQR genes were detected, including aac(6′)-Ib-cr (n = 146), oqxAB (n = 68), qnrS1 (n = 2), qnrD (n = 1), and qepA (n = 1). PMQR genes aac(6′)-Ib-cr and oqxAB coexisted in 54 isolates  and were only detected with 4 amino acid substitutions in GyrA and ParC, with MICs of ciprofloxacin ranging from 8 to 256 mg/L (qnrS1 and oqxAB coexisted only in two food-related isolates. Of the 97 isolates possessing amino acid substitutions in GyrA (S83F and D87G) and ParC (T57S and S80R), the detection rate (47.5% [57/120]) in human-derived isolates was higher than that identified in food-related isolates (30.5% [40/131]) (P < 0.01). However, of the 141 isolates possessing amino acid substitutions in GyrA (S83F and D87N) and ParC (T57S and S80R), the detection rate in human isolates (47.5% [57/120]) was lower than that in food-related isolates (64.1% [84/131]) (P < 0.01).The genomes of 251 isolates were screened for known genetic determinants of AMR, including mobile resistance genes and mutations within QRDRs . For mut256 mg/L , while q10.1128/msystems.00253-22.6TABLE S3S. enterica serovar Indiana isolates. Download Table S3, DOCX file, 0.02 MB.Comparisons of the resistance genes of human and food-related Copyright © 2022 Du et al.2022Du et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the blaCTX-M subtypes were identified among the 138 ESBL-producing isolates  distributed in different provinces, including blaCTX-M-65 , blaCTX-M-14 , blaCTX-M-55 , blaCTX-M-15 , blaCTX-M-27 , and blaCTX-M-123 . The three dominant subtypes blaCTX-M-65/55/14 were detected in human and food-related isolates, while blaCTX-M-15/123 was found only in human isolates. Moreover, the prevalence rate of blaCTX-M-65 among isolates from ≤1-year-old patients (42.6% [20/47]) was significantly higher than that among isolates from other patients (11.1% [8/72]) (P < 0.01), which is in line with the phenotypic characteristics of isolates from these two patient groups. In general, most of the blaCTX-M variants have been detected in humans and chickens.Six mph(A) gene, which is highly associated with azithromycin resistance, had the highest detection rate of 33.5% . The mph(E) (n = 7), msr(E) (n = 7), erm(42) (n = 1), and erm(B) (n = 1) genes were also detected.For macrolides, the 26). The pairwise co-occurrence matrix of AMR genes is shown in arr-3 (conferring resistance to rifamycin), catB3 (conferring resistance to chloramphenicols), aac(6′)-Ib-cr (conferring resistance to aminoglycosides), and blaOXA-1 (conferring resistance to penicillins), which co-occurred in 58.2% of genomes (146/251); the combination of arr-3, catB3, aac(6′)-Ib-cr, and blaOXA-1 occurred with sul1 (sulfonamides) in 50.6% (127/251), aac(3)-Iva (aminoglycosides)/aph(4)-Ia (aminoglycosides)/tet(A) (tetracycline) in 45.8% (115/251), floR (chloramphenicols) in 43.4% (109/251), and sul2 (sulfonamides) in 36.7% (92/251) of genomes. These co-occurring genes were formed into resistance regions comprising IS26 and tet(A), sul1, arr-3, catB3, blaOXA-1, aac(6′)-Ib-cr, aac(3)-Iva, aph(4)-Ia, and sul2 located in ps15D023-IncHI2, ps12177-CTX, pIndS104-CTX, and ps11011-CTX (aph(3″)-Ib, aph(6)-Id, and aadA5 co-occurred in 19.1% (48/251) of genomes; the combination of aph(3″)-Ib, aph(6)-Id, and aadA5 occurred with oqxAB in 14.7% (37/251), fosA in 12.0% (30/251), blaTEM-1 in 11.5% (29/251), and blaCTX-M-65 in 9.2% (23/251) of genomes. These were caused by the resistance region IS26-aph(3″)-Ib-aph(6)-Id-sul2-IS26 co-occurring with other resistance genes. Salmonella genomic island 1 (SGI1), the widely reported original chromosomal integron containing an antibiotic resistance gene cluster and identified in several S. enterica serovars . Download FIG S2, TIF file, 0.9 MB.Sequence comparison of IncHI2 plasmids in Copyright © 2022 Du et al.2022Du et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the blaCTX-M-positive isolates, 90 (65.2%) could be classified by the locations of blaCTX-M in the genome from the fragmented short-read assemblies. blaCTX-M-14 in 14 isolates (42.4% [14/33]) and blaCTX-M-55 from 9 isolates (30.0% [9/30]) were located on the chromosome. blaCTX-M-15 from 9 isolates (100% [9/9]) and blaCTX-M-65 from 58 isolates (96.7% [58/60]) were carried by plasmids. In addition, 76 blaCTX-M-positive isolates were from humans and 62 were food related, accounting for 63% (76/120) and 47% (62/131) of the two groups, respectively. Although the positive ratio of blaCTX-M was significant higher in human isolates (P < 0.05), among the six subtypes, only the positive ratio of blaCTX-M-15 was significant higher in human isolates .Among the 138 blaCTX-M among representative nonclonal isolates from different sources, genome sequences of five isolates  were successfully completed. The IndS102 and s15D023 isolates separately carried blaCTX-M-14 and blaCTX-M-55 on their chromosomes within different genetic contexts. Another three isolates carried blaCTX-M on IncHI2 plasmids ranging from 200 to 322 kbp and sharing similar core structures  with the specific genetic context IS26-ISEcp1-blaCTX-M-65-IS903B located in an MDR region . BLASTn analysis demonstrated pIndS104-CTX was most similar (99.99% identity at 100% coverage) to a locus on the chromosome of Chinese blaCTX-M-65-positve S. Indiana SI43, obtained from a spiral shell in China in 2010 , indicating pIndS104-CTX may have the ability to recombine with the chromosome entirely or evolve from ancestor clones like SI43. ps11011-CTX was 263,731 bp in size and possessed 783 predicted coding sequences. ps11011-CTX showed high similarity to ps12177-CTX , originating from a human sample from China in 2006, and also to Escherichiacoli plasmids pE648CTX-M-65  and pECJS-B60-267 , as well as Klebsiellapneumoniae plasmid pHNHF1_NDM-99 . ps12177-CTX was 200,106 bp in size (48.61% GC content), and it encoded an MDR region different from that described in ps11011-CTX , which might have resulted from the truncation of ISEcp1 by IS26.Although genetic contexts of  by IS26 . ISEcp1 mph(A) was positive in the chicken isolate IndS104 and was located on plasmid pIndS104_3_29k, a typical IncX1 plasmid of size 29,056 bp. It was found to be homologous  to six similar plasmids that ranged from 34,764 to 222,492 bp among Salmonella spp. and E. coli, as well as a chromosomal locus in a human S. Indiana isolate SI111 (CP050764) (mph(A)-bearing IncX1 plasmids were hypothetically mobilizable and could move into the chromosome via insertion sequences such as IS26. The typical IS26-mphR(A)-mrx-mph(A)-IS26 transposition unit was embedded in the IncX1 plasmid and other MDR plasmids such as IncHI2 (S. Indiana SI111 chromosome demonstrated a variant of mph(A)-bearing IncX1 plasmid recombined into the SI85 chromosome by IS26 to generate the chromosomal IncX1 segment in SI111 (26 in the transmission of mph(A) among plasmids and chromosomes. Detailed analysis of mph(A)-bearing contigs in the 84 mph(A)-positive isolates showed that the core structure mphR(A)-mrx-mph(A) (n = 84) and seven additional different core structures were prevalent among these isolates (mphR(A)-mrx-mph(A) were not identified because of short fragmented assembled contigs based on Illumina short-read data.Among the five isolates  with complete genome sequences, P050764) , which is IncHI2 . Linear in SI111 . This hiisolates . However10.1128/msystems.00253-22.3FIG S3mph(A)-harboring S. Indiana isolates. Seven different genetic structures are listed above. The numbers of isolates of the seven types are 1, 1, 6, 1, 11, 63, and 1, respectively. Boxes or arrows represent the ORFs. Red arrows represent the mph(A) gene. Blue arrows indicate mobile elements. mphR(A) and mrx are shown by green arrows. Light gray arrows represent the resistance gene and hypothetical protein. A triangle represents the truncated gene. Download FIG S3, TIF file, 1.3 MB.Genetic environment comparison of 84 Copyright © 2022 Du et al.2022Du et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. Indiana emerged within the study period and were transmitted to those enrolled provinces. All 251 isolates harbored the mutations on gyrA and parC associated with the FQ resistance. According to core-genome-based phylogenies, we divided them into six lineages : lineage 1 included seven isolates with D87G in GyrA and T57S in ParC, lineage 2 included two isolates with S83F in GyrA and T57S in ParC, and lineage 3 included four isolates with S83F in GyrA and T57S and S80R in ParC. The other three later lineages with 4- amino acid substitutions were the dominant groups. Lineage 4 (n = 49) and lineage 5 (n = 48) had S83F and D87G in GyrA and T57S and S80R in ParC, while lineage 6 (n = 141) had S83F and D87N in GyrA and T57S and S80R in ParC, which was the most common lineage (141/251 [56.2%]). Moreover, in the early stages of quinolone resistance evolution, most of the isolates with double-amino-acid substitutions in GyrA (D87G [lineage 1] or D87F [lineage 2]) and ParC (T57S) were susceptible to ciprofloxacin, except in two isolates that also carried PMQR genes (oqxAB and qnrS1), and the isolates from lineage 3 with three amino acid substitutions in GyrA (S83F) and ParC (T57S and S80R) exhibited low MIC values of ciprofloxacin , 76.7% (23/30), and 70% (42/60) of the isolates harboring each genotype, respectively (mph(A) appeared most frequently in lineage 6 (88.1% [74/84]), followed by lineage 5 (9.5% [8/84]), and only occurred once in lineage 2 and lineage 3, respectively. Furthermore, co-occurrence of mph(A) with diverse ESBL genes was observed . Both IncN and IncX1 replicons were dominantly distributed in lineage 6, with prevalence of 75.3% (55/73) and 98.5% (64/65). Based on the complete genome sequences of IndS104, mph(A) was located in an IncX1 plasmid. In addition, among the 251 isolates, mph(A) and IncX1 were both detected in 55 isolates belonging to lineage 6, and both were negative in 157 isolates. The distribution of mph(A) and IncX1 displayed a significant correlation with a calculated coefficient of 0.64 . The isolates carrying IncX1 plasmids might be more likely to carry mph(A) .Based on the 2,904 core genome single nucleotide polymorphisms (SNPs) obtained from 251 genomes, we performed phylogenic analysis and displayed the phylogenetic relationships of sequenced isolates . Multipllineages , which ifloxacin . In contfloxacin . Of noteectively . MoreoveSalmonella have changed significantly in recent years as MDR Salmonella strains have become prevalent among various serovars, such as S. 1,4,[5],12:i:−, S. Kentucky, and S. enterica serovar London  -Ib-cr was the dominant genotype, with the carriage rate increasing from 20% in 2006 to 65% in 2017  of the ciprofloxacin-resistant Salmonella strains of various serovars, the oqxAB and aac(6′)-Ib-cr combination was the predominant one in our study, which was located in the IncHI2 plasmid. This gene combination was also located in a nonconjugative plasmid, pCFSA244-1, mainly transmitted among S. Typhimurium isolates, and qnrS2-aac(6′)-lb-cr-oqxAB elements were also found on the chromosome in S. enterica serovar Derby (oqxAB-aac(6′)-Ib-cr-bearing IncHI2 plasmid found in S. Indiana was mobilizable by conjugation  . Furtherservoirs . Since 2servoirs . Our who E. coli . Recent  E. coli . It has  strains . This mijugation . The hig studies . This mi studies .S. Indiana, contributing to their cefotaxime resistance phenotypes   variant of blaCTX-M-15, blaCTX-M-55, possessing enhanced cephalosporin-hydrolyzing activity, was both prevalent in both isolates from humans and those from food-related samples, particularly in lineage 6, indicating potential higher adaptability of blaCTX-M-55. IncHI2 plasmids were typical MDR plasmids positive for blaCTX-M, mcr-1, class 1 integrons, and oqxAB, similar to those found in other Enterobacteriaceae  . Most blaCTX-M-55 genes in S. Indiana are located on the chromosome, which is different from its plasmid origin in other serovars, such as S.enterica serovars Enteritidis, Goldcoast, Typhimurium, and Choleraesuis  . FurtherE. coli) , 41. Furplasmids . The blaosporins . The occeraesuis –46.S. Indiana isolates harboring the plasmid-borne mph(A), mph(E), erm(42), and erm(B) genes. Moreover, in the present study, 24% of isolates were coresistant to azithromycin, ciprofloxacin, and cefotaxime, which is considerably higher than the levels reported in a Chinese national surveillance study (0.2%) from 2005 to 2011 (mph(A) and mph(E), raising concern about the spread of resistance among bacteria (mph(A)-positive isolates were mostly grouped into lineages 5 and 6 coexisting with diverse ESBL genes. This feature is highly related to the IncX plasmid, which has played an important role in the spreading of resistance genes, such as blaNDM (IncX3) and mcr-1 (IncX4 and IncX2) (–Macrolide (azithromycin) resistance was determined in 36.3% 91/251) of  to 2011  and a Ch to 2011 . In the bacteria . In our d IncX2) –51. More of  to 2 IncX2) –.Salmonella. The high prevalence of IncHI2 plasmids in S. Indiana is similar to that reported for S. Typhimurium  and food  collected previously by the National Health Commission Key Laboratory of Food Safety Risk Assessment from four provinces in China during 2006 to 2016. In total, isolates from eight provinces were included. Clinical fecal samples were enriched in selenite brilliant green sulfa enrichment broth for 18 h at 37 ± 1°C, and anatomical site samples  were enriched on blood agar plates for 18 h at 37 ± 1°C. Clinical sample isolates were purified as described previously , along with amplification of the invA gene by PCR. All isolates were identified to the serogroup level and then serotyped by slide agglutination with commercial Salmonella antisera  following the Kauffmann-White scheme at the National Health Commission Key Laboratory of Food Safety Risk Assessment in Beijing, China.A total of 251 confirmed eviously . S. enteuidebook . The isoS. Indiana isolates was performed by the agar dilution method and interpreted according to 2018 Clinical and Laboratory Standards Institute (CLSI) guidelines (https://eucast.org/). The antimicrobial susceptibilities of the following antimicrobials were assessed: ampicillin (AMP), cefotaxime (CTX), cefotaxime-clavulanic acid (CTX-CLA), chloramphenicol (CHL), ciprofloxacin (CIP), nalidixic acid (NAL), gentamicin (GEN), streptomycin (STR), imipenem (IPM), meropenem (MEM), tetracycline (TET), azithromycin (AZM), sulfonamide (SUL), and colistin (CT). The MICs were calculated. Multidrug resistance was defined as resistance to three or more classes of antimicrobials. Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as the quality control strains.Antimicrobial susceptibility testing (AST) of the idelines  and the in silico multilocus sequence typing (MLST) scheme was used to subtype the isolates using BLASTn and seven housekeeping genes: aroC, dnaN, hemD, hisD, purE, sucA, and thrA  and then sequenced using an Illumina HiSeq 2500 platform  to generate 150-bp paired-end reads from a library with an average insert size of 500 bp. Raw reads were filtered to remove low-quality reads by fastQC  and then62–and thrA .blaCTX-M were determined after whole-genome sequence analysis. blaCTX-M-containing contigs were examined for plasmid Inc types using PlasmidFinder  was used as the reference in phylogenetic analysis. Illumina reads were mapped to the reference genome using Bowtie 2 v2.2.8, with single nucleotide polymorphisms (SNPs) identified using Samtools v1.9, and the data were combined as described previously . Multiple alignments of core genomes identified from the pairwise alignments with S. Indiana D90 were used as the input for Gubbins to detect and remove recombination sites (The complete genome sequence of eviously . The higon sites . Phylogeon sites  and seleon sites . The conon sites . The phyon sites .PRJNA850394.The genome sequences in this study were deposited into the National Center for Biotechnology Information database under BioProject accession no.


Based on the pharmacokinetic-pharmacodynamic (PK-PD) breakpoints, cefepime-zidebactam inhibited 98.5% of Enterobacterales and 98.9% of Pseudomonas aeruginosa isolates, respectively. Against carbapenem-resistant and difficult-to-treat resistant Gram-negative bacilli, cefepime-zidebactam demonstrated better activity against Enterobacterales  and P. aeruginosa . Among the 379 carbapenem-resistant Enterobacterales isolates, the most common carbapenemase genes detected were blaKPC-2 (64.1%) and blaNDM (30.9%). Cefepime-zidebactam showed an MIC90 of ≤2 mg/L for 98.8% of blaKPC-positive isolates and 89.7% of blaNDM-positive isolates. Ceftazidime-avibactam also showed efficient in vitro activity against Enterobacterales (93.6%) and P. aeruginosa (87.7%). Ceftazidime-avibactam was active against 97.5% of blaKPC-positive isolates and 100% of blaOXA-232-positive isolates. Cefepime-zidebactam inhibited 97.3% of Acinetobacter baumannii isolates with an MIC50/90 of 16/32 mg/L. Our study systematically evaluated the in vitro activities of these new BLBLIs against a variety of Gram-negative bacilli, provided preclinical data for the approval of these BLBLIs in China, and supported cefepime-zidebactam and ceftazidime-avibactam as potential efficient therapies for infections caused by carbapenem-resistant Enterobacterales (CRE), carbapenem-resistant P. aeruginosa (CRPA), and DTR isolates.Novel β-lactam–β-lactamase inhibitor combinations (BLBLIs) are in clinical development for the treatment of infections caused by carbapenem-resistant and difficult-to-treat resistant (DTR)  Gram-negative bacilli. This study evaluated the IMPORTANCEEnterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii are the most common Gram-negative bacilli to cause nosocomial infections throughout the world. Due to their large public health and societal implications, carbapenem-resistant A. baumannii (CRAB), carbapenem-resistant P. aeruginosa (CRPA), and carbapenem-resistant and third-generation-cephalosporin-resistant Enterobacteriaceae were regarded by the World Health Organization (WHO) as a global priority for investment in new drugs in 2017. The present study showed the potent in vitro activity of these novel BLBLIs and other comparators against Gram-negative bacillus isolates, including carbapenem-resistant or difficult-to-treat resistant phenotypes. Polymyxins, tigecycline, and ceftazidime-avibactam (except for blaNDM-positive isolates) were available for the treatment of infections caused by CRE isolates. Currently, cefepime-zidebactam and other BLBLIs have not yet been approved for use in China. Here, our study aimed to evaluate the in vitro activities of BLBLIs against Gram-negative bacillus isolates, especially CRE, before clinical use. Carbapenem-resistant Gram-negative bacilli have rapidly increased worldwide in the last decade, which is related to the emergence and prevalence of plasmid-mediated extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases among these isolates, conferring resistance to β-lactam antibiotics, and make difficulties in empirical treatment for clinicians  were carbapenem-resistant Enterobacterales (CRE), including K. pneumoniae , E. coli , and Enterobacter cloacae .Among the tested P. aeruginosa (CRPA) and carbapenem-resistant A. baumannii (CRAB), respectively, and 11.9%  of Enterobacterales isolates and 8.6% (65/756) of P. aeruginosa isolates were difficult-to-treat resistant (DTR) isolates.For glucose-nonfermenting bacteria, 228/756 (30.2%) and 471/630 (74.8%) were carbapenem-resistant in vitro activities of cefepime-zidebactam, ceftazidime-avibactam, cefepime-tazobactam, ceftolozane-tazobactam, and other comparator agents against 4,042 clinical isolates are summarized in Tables 1Enterobacterales isolates  with an MIC50/90 of 0.06/1 mg/L. A total of 98.5% of isolates were inhibited at the provisional cefepime-zidebactam pharmacokinetic-pharmacodynamic (PK-PD) breakpoint (≤8 mg/L), with 24 E. coli, 4 K. pneumoniae, 7 Proteus rettgeri, 3 P. mirabilis, 1 E. cloacae, and 1 Serratia marcescens isolates showing MICs of ≥16 mg/L among the various genera of Enterobacterales. Besides cefepime-zidebactam, ceftazidime-avibactam was also active against all Enterobacterales clinical isolates with an MIC50/90 of 0.25/4 mg/L. Among 171 ceftazidime-avibactam-resistant isolates, cefepime-zidebactam showed an MIC of 8 mg/L or lower against 84.1% of the tested isolates (data not shown). Apart from cefepime-zidebactam and ceftazidime-avibactam, tigecycline (96.5% susceptible) and amikacin (90.4% susceptible) also displayed potent activity against Enterobacterales. The rate of susceptibility to cefepime-tazobactam was 85.8%, similar to those for polymyxin B (81.4% susceptible) and meropenem (85.6% susceptible), which showed good activity against the tested isolates. More than 60% of the Enterobacterales isolates were susceptible to ceftolozane-tazobactam (74.2% susceptible), imipenem (74.6% susceptible), piperacillin-tazobactam (75.5% susceptible), cefoperazone-sulbactam (69.8% susceptible), and ceftazidime (60.5% susceptible). The following other comparator agents showed limited activity: cefepime (55.3% susceptible), ceftriaxone (46.1% susceptible), aztreonam (54.7% susceptible), ciprofloxacin (42.1% susceptible), levofloxacin (48.1% susceptible), and trimethoprim-sulfamethoxazole (53.7% susceptible) (The eptible) .P. aeruginosa were highly inhibited by cefepime-zidebactam with an MIC50/90 of 2/8 mg/L at a PK-PD breakpoint of ≤32 mg/L (98.9% susceptible). The rate of susceptibility of P. aeruginosa to cefepime-zidebactam was similar to or slightly higher than those for ceftazidime-avibactam (87.7% susceptible), ceftolozane-tazobactam (90.2% susceptible), polymyxin B (95.6% susceptible), and amikacin (95.4% susceptible)  . The rat50/90 value of cefepime-zidebactam against 630 A. baumannii isolates was 16/32 mg/L , blaNDM-positive (n = 117), blaIMP-positive (n = 8), blaOXA-232-positive (n = 7), blaVIM-positive (n = 1), as well as carbapenemase-negative (n = 3) isolates  and polymyxin B (98.2% versus 96.9%), whereas the rates of susceptibility were 76.3% for ceftolozane-tazobactam and 68% for ceftazidime-avibactam as the most active comparators. Except for imipenem and meropenem, CRPA isolates were moderately resistant to other β-lactams, aztreonam, and fluoroquinolones, with susceptibility rates of 30% to 50%.90 values of >32 mg/L.For CRAB, cefepime-zidebactam, tigecycline, and polymyxin B showed high susceptibility rates of 96.6%, 88.5%, and 96.6%, respectively, and amikacin and trimethoprim-sulfamethoxazole showed limited activity, with susceptibility rates of 18.7% and 23.1%, respectively. The MICs of other agents were higher, with MICEnterobacterales isolates with an MIC50/90 of 1/4 mg/L at ≤8 mg/L, and 74.7% of DTR Enterobacterales isolates were susceptible to ceftazidime-avibactam with an MIC50/90 of 2/>64 mg/L. Only tigecycline (95.9% susceptible) and polymyxin B (92.4% susceptible) displayed greater in vitro activity than cefepime-zidebactam and ceftazidime-avibactam against all DTR Enterobacterales isolates  demonstrated greater in vitro activity than the above-described agents against DTR P. aeruginosa isolates  were blaNDM-5 positive, 12.4% (47/379) were blaNDM-1 positive, 2.1% (8/379) were blaIMP positive, 1.8% (7/379) were blaOXA-232 positive, and 0.3% (1/379) were blaVIM positive, respectively. Additionally, blaKPC-2 was mainly detected in K. pneumoniae , S. marcescens , Citrobacter freundii , and Morganella morganii . The highest prevalences of blaNDM-5 were 82.5% (33/40) in E. coli and 55.6% (5/9) in Klebsiella aerogenes isolates. blaNDM-1 was the predominant type of carbapenemase gene among E. cloacae  and P. rettgeri  isolates.In this study, 99.2% (376/379) of the CRE isolates had a single carbapenemase gene, and only 3 isolates were negative for all five common carbapenemase genes . Among tP. aeruginosa, and A. baumannii, which has substantially increased morbidity and mortality rates and caused nosocomial outbreaks , European Committee on Antimicrobial Susceptibility Testing (EUCAST), or U.S. Food and Drug Administration (FDA) clinical breakpoints of cefepime-zidebactam, so according to its PK-PD breakpoint (≤32 mg/L), P. aeruginosa had a rate of susceptibility to cefepime-zidebactam of 99.6%, whereas it was 98.9% in our study. The potent activity of cefepime-zidebactam against CRE, P. aeruginosa, and A. baumannii isolates harboring carbapenemase genes has been previously reported. In another study of a worldwide surveillance program, Sader et al. (n = 153) had cefepime-zidebactam MICs of ≤8 mg/L, similar to the results of this study (98.5%).In this study, 98.5% of ≤32 mg/L , respect≤32 mg/L , the autr et al.  reportedE. coli, 0.6% to 1.0% for Enterobacter spp., 0.6% to 3.0% for Klebsiella spp., and 8.4% for P. aeruginosa (data not shown). In our study, we observed slightly higher DTR rates of 1.2% for E. coli, 0.04% to 0.5% for Enterobacter spp., 9.3% for Klebsiella spp., and 8.6% for P. aeruginosa. The differences in DTR rates between the 2 studies may reflect the characteristics of the strains among different regions and different specimen sources. Kadri et al. reported that mortality was significantly higher for DTR than for carbapenem-resistant, extended-spectrum-cephalosporin-resistant, or fluoroquinolone-resistant infections  (96% to 98.2%) and DTR isolates (96.9% to 97.2%).The DTR phenotype, a novel category in the study of Gram-negative bacteremia, focuses on treatment-limiting resistance to all first-line agents. The DTR phenotype was defined as an isolate that tests not susceptible (intermediate or resistant) to all β-lactam categories, including carbapenems and fluoroquinolones, and it was demonstrated that isolates that were not susceptible to first-line agents were associated with increased patient mortality and clinical failure. Karlowsky et al.  studied fections . The in blaKPC- or blaOXA-48-positive isolates. blaKPC-positive isolates showed a low rate of resistance to ceftazidime-avibactam (2.5%), but the majority (87.5% to 100%) of blaNDM-positive isolates were resistant to ceftazidime-avibactam. The major resistance mechanisms that confer reduced susceptibility to ceftazidime-avibactam are as follows: the production of metallo-β-lactamases (MBLs) such as NDM, VIM, or IMP; blaKPC variants; and the transposition of KPC with porin deficiency  and P. aeruginosa  isolates producing important β-lactamases, including MBLs (except for ceftazidime-avibactam), KPCs, and OXA-232, for which treatment agents are limited. The results from this study support the use of cefepime-zidebactam and ceftazidime-avibactam as potential therapies for infections caused by CRE, CRPA, and DTR isolates.We studied a recent nationwide collection of Gram-negative bacilli and observed that new BLBLIs, especially cefepime-zidebactam and ceftazidime-avibactam, demonstrated potent The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University (no. 2019-460).Klebsiella pneumoniae (n = 979), Escherichia coli (n = 900), P. aeruginosa (n = 756), A. baumannii (n = 630), Enterobacter cloacae (n = 172), Proteus mirabilis (n = 119), Serratia marcescens (n = 118), K. aerogenes (n = 103), Morganella morganii (n = 89), Citrobacter freundii (n = 84), Proteus vulgaris (n = 51), Proteus rettgeri (n = 29), and Klebsiella oxytoca (n = 12). Among the tested clinical isolates, 23.6% of the isolates were isolated from patients in the intensive care unit, followed by outpatient and emergency departments (18.5%), urology surgery (6.7%), respiratory medicine (5.6%), neurosurgery departments (4.2%), and other departments. A total of 33.9% of the tested isolates were isolated from sputum, followed by urine (22.5%), blood , secreta (7.7%), bronchoalveolar lavage fluid (3.9%), pus (2.8%), wound (2.7%), abdominal fluid (2.0%), bile (1.7%), shunt fluid (1.3%), drain (1.2%), and other sources (8.2%). Species identification was performed at each participating site and confirmed by the central laboratory using matrix-assisted laser desorption ionization–time of flight mass spectrometry . Quality control was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines using E. coli ATCC 25922 and ATCC 35218, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 for antimicrobial susceptibility testing.The China Antimicrobial Surveillance Network (CHINET) is a multicenter bacterial resistance surveillance program in operation since 2005 in China. In 2019, 46 hospitals in 28 provinces or cities collected up to 4,042 nonduplicate, clinically significant Gram-negative isolates from CHINET, including Enterobacterales . Cefepime-tazobactam MICs were interpreted using provisional breakpoints of ≤16 mg/L for Enterobacterales and P. aeruginosa based on PK-PD studies  in the study. Quality control and test results were interpreted according to 2021 CLSI breakpoints . Difficult-to-treat resistance phenotypes were defined by testing resistance to all tested β-lactams (including carbapenems and β-lactamase inhibitor combinations) and fluoroquinolones .Enterobacterales (CRE) isolates were selected for analysis of carbapenemase. The five most common carbapenemase genes  were confirmed for all of the CRE isolates by PCR with specific primers and DNA sequencing, as described previously (Carbapenem-resistant eviously .


Deutereulophus Schulz, D.felixsp. nov. and D.daguisiensissp. nov., are described from China. A key to species of Deutereulophus known from China is provided.Two new species of  Deutereulophus Schulz is one of several small genera of the tribe Eulophini, subfamily Eulophinae (Hymenoptera: Eulophidae). Species of the genus are distributed in all zoogeographic regions, except for the Afrotropical region. Currently Deutereulophus contains 23 valid species (D.froudei (Girault), D.interruptus Zhu & Huang, D.marginatus Zhu & Huang, and D.tennysoni (Girault)  , 2002b.Deutereulophus (as Entedonomorpha Girault) was subdivided into two species groups by tennysoni-group and provides a key to all species occurring in China.The genus All specimens were collected by sweeping or yellow-pan trapping, and were dissected and mounted in Canada balsam on slides following the method of Terminology follows the F1–3 flagellomeres 1–3;MV marginal vein;OOL minimum distance between a posterior ocellus and corresponding eye margin;PMV postmarginal vein;POL minimum distance between posterior ocelli;SMV submarginal vein;STV stigmal vein.NEFU), Harbin, China.All type material is deposited in the insect collections at Northeast Forestry University : SMV = 36; MV = 36; PMV = 12; STV = 16.ing Fig.  2.1× as Metasoma .felix, meaning lucky.Named after the Latin adjective Deutereulophusmarginatus, and can be separated using the key given above.The new species is similar to Taxon classificationAnimaliaHymenopteraEulophidae08C5DC71-9949-5063-A04A-0D42C97F314Ahttps://zoobank.org/02F37BE0-42A2-4F4D-B908-7AF3845F0280Holotype, ♀ , China, Hubei Province, Suizhou City, Daguisi National Forest Park, 12. VI. 2012, Guo-Hao Zu and Jiang Liu, by sweeping. Paratypes: 1♀ [on slide], same data as holotype.Head and mesosoma black. Vertex with scattered pits. Antennal scrobes smooth. Antenna yellowish-white with F3 dark brown, clava dark brown with apex yellowish-white. Female funicle 3-segmented, clava 4-segmented. Legs yellowish-white with procoxa and profemur dark brown. Face with raised reticulation. Metasoma dark brown. Mesoscutum strongly reticulate with large meshes. Sublateral grooves on mesoscutellum converging and meeting posteriorly. Propodeum with a raised triangular cup-shaped area anteromedially, median carina split and diverging posteriorly.Female. Length 2.4 mm, fore wing length 1.7 mm. Head and mesosoma black. Eyes gray. Ocelli pale yellow. Antenna yellowish-white with F3 dark brown, clava dark brown with apex yellowish-white. Mandibles dark brown. Petiole black. Metasoma dark brown. Legs yellowish-white with procoxa and profemur dark brown. Wings hyaline with veins brown.Head : SMV = 29; MV = 32; PMV = 12; STV = 10.ing Fig.  2.4× as Metasoma .Named after the type locality, the Daguisi National Forest Park in Hubei Province.D.malabarensis Narendran, but can be separated from it by the following combination of characters: antenna yellowish-white with F3 dark brown, clava dark brown with apex yellowish-white ; sublateral grooves on mesoscutellum converging and meeting posteriorly ; propodeum with a raised triangular cup-shaped area anteromedially .The new species is similar to


Among current e-cigarette users, 14.5% reported that the brand they usually used was Puff Bar, followed by Vuse (12.5%), Hyde (5.5%), and SMOK (4.0%). Approximately one fifth (21.8%) of current e-cigarette users reported “some other brand” as their usual brand.https://stacks.cdc.gov/view/cdc/121630). Among current users of flavored pods or cartridges, the reported flavor types used were fruit (58.4%); menthol (53.9%); candy, desserts, or other sweets (30.3%); and mint (27.6%). Among current users of flavored tanks or mod systems, the reported flavor types used were fruit (69.6%); candy, desserts, or other sweets (47.7%); mint (40.1%); and menthol (35.2%).Among current e-cigarette users overall, 84.9% used flavored e-cigarettes; of these, the reported flavor types, in descending order of use, were fruit (69.1%); candy, desserts, or other sweets (38.3%); mint (29.4%); and menthol (26.6%). A similar pattern was observed among current users of flavored disposable e-cigarettes: fruit (75.2%); candy, desserts, or other sweets (40.4%); mint (29.6%); and menthol (16.7%) (Supplementary Table, In 2022, 2.55 million U.S. middle and high school students currently used e-cigarettes. Most reported using flavored products, and, among those students, approximately seven of 10 used fruit flavors. Disposable products were the most commonly reported device type. Further, among middle and high school students who used e-cigarettes, approximately four in 10 reported frequent use, and approximately one in four reported daily use. The use of tobacco products in any form, including e-cigarettes, by middle and high school students is unsafe. Sustained implementation of comprehensive tobacco prevention and control strategies at the national, state, and local levels,


We present a comprehensive data set that describes an anaerobic microbial consortium native to polychlorinated biphenyl (PCB)-contaminated sediments. Obtained from sediment microcosms incubated for 200 days, the data set includes 4 metagenomes, 4 metatranscriptomes (in duplicate), and 62 metagenome-assembled genomes and captures microbial community interactions, structure, and function relevant to anaerobic PCB biodegradation. P < 0.0001).Polychlorinated biphenyl (PCB)-contaminated sediments threaten human and ecological health but often harbor PCB-transforming bacteria that help detoxify sediments –3. We es–After 200 days of incubation, DNA was extracted from single slurry samples (2 mL) with a modified DNeasy PowerWater Sterivex kit protocol , and RNAMethanosarcina barkeri (GenBank accession number NZ_CP009530.1) and Methanobacterium subterraneum (GenBank accession number NZ_CP017768.1) were added to further deplete methanogenic archaeal rRNA sequences. RNA was sequenced on a single Illumina NovaSeq 6000 flow cell lane (2 × 150-bp paired-end reads).High-throughput DNA and RNA sequencing (4 metagenomes and 4 metatranscriptomes in duplicate) was performed at the Iowa Institute of Human Genetics (IIHG) . Indexed DNA libraries, prepared with the KAPA HyperPrep kit  using sheared DNA , were pooled and sequenced on separate lanes of an S Prime NovaSeq 6000 flow cell (2 × 150-bp paired-end reads). RNA libraries were indexed and rRNA depleted with the stranded total RNA preparation with Ribo-Zero Plus kit . To improve mRNA sequencing efficiency, supplemental rRNA probes developed from Metagenome sequencing yielded 97,279,994 (E2_1), 105,614,978 (E2_2), 102,655,932 (F4_1), and 103,672, 591 (F4_2) raw reads from each bottle. Metatranscriptome sequencing (RNA-seq) yielded 50,403,972 (E2_1 metatranscriptome-A), 60,356,358 (E2_1-B), 49,353,924 (E2_2-A), 45,023,913 (E2_2-B), 54,860,340 (F4_1-A), 47,603,523 (F4_1-B), 55,440,491 (F4_2-B), and 71,530,818 (F4_2-C) raw reads. After trimming and filtering of unassembled sequence reads with Trimmomatic (v0.39) .Dehalococcoides mccartyi) (Metagenome-assembled genomes (MAGs) were obtained by removing low-abundance k-mers from quality-filtered reads with khmer (v3.0.0) , coassem12–ccartyi) , which wccartyi) , is expePRJNA743546. Metagenomic data are available under SRA accession numbers SRX11347095 to SRX11347098. Metatranscriptomic data are available under accession numbers SRX14430540 to SRX14430547. PCB data are available at Iowa Research Online (https://www.doi.org/10.25820/data.006156).Raw data files (24 fastq files) and MAGs are available under BioProject accession number


Actinomyces oris strain K20 was isolated from oral apical lesions. Here, we report the complete circular genome sequence of this strain, obtained by means of hybrid assembly using two next-generation sequencing datasets. The strain has a 3.1-Mb genome with 2,636 coding sequences. Actinomyces are ubiquitous in soil as well as human and animal microbiota  using NanoFilt v.2.3.0 , following the manufacturer’s standard protocol. Paired-end (2 × 156-bp) reads were obtained using a MiSeq instrument (Illumina). The raw sequencing data  were processed using the FASTQ preprocessing program fastp v.0.19.5  (The strain was cultured overnight in heart infusion broth (BD Difco) at 37°C under aerobic conditions. Genomic DNA (gDNA) was extracted from the culture medium of this strain using the MagAttract high-molecular-weight (HMW) DNA kit . The obtained gDNA was subjected to long- and short-read sequencing. Long-read sequencing was performed using the GridION X5 sequencing platform ; 1.0 μg unfragmented gDNA was used for library construction using a ligation sequencing kit SQK-LSK109; ONT). The prepared library was applied to an R9.4.1 flow cell . The base calling of long-read sequences using Dogfish v.0.9.6-3 generated 114,245 reads (1.1 Gb), with an ; ONT. Thde novo genome assembly, the remaining long- and short-read data were processed using the Unicycler v.0.4.4 pipeline (https://dfast.ddbj.nig.ac.jp/), predicted 2,636 coding sequences, 9 rRNA genes, and 52 tRNA genes.For complete pipeline . Assemblpipeline . BlobToopipeline  was usedpipeline  until nopipeline , provideA. oris strain K20 has been deposited at DDBJ/EMBL/GenBank under the accession number AP025590. The associated BioProject and BioSample accession numbers are PRJDB13022 and SAMD00442649, respectively. The SRA accession numbers are DRR351395 (Illumina) and DRR351396 (Nanopore).The complete genome sequence of


We found that tricalcium phosphate-solubilizing bacteria, phytate-degrading bacteria, and gcd and bpp abundances were more abundant in silt plus clay  than in macroaggregate (250 to 2000 μm) and microaggregate (53 to 250 μm). Fertilization treatment and aggregate fractionation showed distinct effects on PSB number and P-cycling-related gene abundance. We found significantly negative correlation between gcd gene abundance and tricalcium phosphate-solubilizing bacterial number (Col-CaP) and dramatically positive correlation between bpp gene abundance and phytate-degrading bacterial number (Col-Phy). P fractions were responsible for PSB number and P-cycling-related gene abundance. The isolated Pseudomonas sp. strain PSB-2 and Arthrobacter sp. strain PSB-5 exhibited good performances for solubilizing tricalcium phosphate. The inoculation of Pseudomonas sp. PSB-2 could significantly enhance plant fresh weight, plant dry weight, and plant height. Our results emphasized distinct distribution characteristics of PSB and P-cycling-related genes in soil aggregates and deciphered a close linkage between PSB number and P-cycling-related gene abundance. Our findings might guide the isolation of PSB from agricultural soils and provide a candidate plant-growth-promoting bacterium for agro-ecosystems.Deciphering distribution patterns of phosphate-solubilizing bacteria (PSB) and phosphorus-cycling-related genes in soils is important to evaluate phosphorus (P) transformation. However, the linkage between PSB number and P-cycling-related gene abundance in soils, especially soil aggregates, remains largely unknown. Here, we estimated the numbers of PSB and abundances of P-cycling-related genes (i.e., IMPORTANCE Phosphate-solubilizing bacteria are responsible for inorganic P solubilization and organic P mineralization. Elucidating the linkage between phosphate-solubilizing bacterial number and P-cycling-related gene abundance is important to isolate plant-growth-promoting bacteria for agro-ecosystems. Our findings reveal differentiating strategies of phosphate-solubilizing bacteria in soil aggregates, and the deciphered P fractions show strong effects on distribution patterns of phosphate-solubilizing bacteria and P-cycling-related genes. Additionally, we isolated phosphate-solubilizing bacteria with good plant-growth-promoting ability. This study enriches our knowledge of P cycling in soil aggregates and might guide the production and management of farmland. Prenic acid . In contnic acid , 14. Phynic acid , 15. Phyin soils . ConsequAcinetobacter, Citrobacter, Massilia, and Pseudomonas) (21–Bacillus)   , 21–24, acillus) . For insosum L.) . Consequosum L.) , 25, 26.gcd and bpp), (ii) estimate abiotic  and biotic  factors on numbers of PSB, and (iii) decipher the performance of PSB for promoting plant growth. Considering that P availability affects the abundances of P-cycling-related genes  circulation , 27 and 3–13–ed genes , 30, we ed genes , 17, 31,5 to 8.77 × 108 CFU/g soil) was significantly higher in NPK  and N  fertilization treatments than that in CK (control without fertilizer), M (swine manure), and MN (combined swine manure and N fertilizer) fertilization treatments (P < 0.05). The phytate-degrading bacterial number (Col-Phy) (4.0 × 104 to 3.27 × 108 CFU/g soil) was remarkably higher in N and MN fertilization treatments than that in CK, M, and NPK fertilization treatments (P < 0.05). In five fertilization treatments, the Col-CaP and Col-Phy were basically slightly higher in silt+clay than that in macroaggregate and microaggregate  rather than aggregate fractionation  showed a significant effect on PSB number (Col-CaP and Col-Phy) (P < 0.05) but the opposite for MN fertilization treatment (P < 0.05). Col-CaP was slightly higher than Col-Phy in the same soil aggregate with N fertilization treatment (P > 0.05). These results indicated different distribution patterns of PSB in soil aggregates with different fertilization treatments.The numbers of PSB represented by CFU varied in different soil aggregates . The triCol-Phy) . Additiogcd gene (4.32 × 106 to 2.20 × 107 copies/g soil) was more abundant in M and MN fertilization treatments than in other fertilization treatments (P < 0.05), whereas the bpp gene (1.99 × 105 to 3.42 × 106 copies/g soil) showed no significant difference among five fertilization treatments (P > 0.05). Basically, gcd and bpp were more abundant in silt+clay than in macroaggregate and microaggregate with the same fertilization treatment  and aggregate fractionation  showed significant effects on abundances of P-cycling-related genes (gcd-harboring bacteria were significantly higher than that of bpp-harboring bacteria in the same soil aggregate with the same fertilization treatment (P < 0.01). These results reflected that fertilization treatment and aggregate fractionation might influence the distribution and abundance of gcd-harboring bacteria and bpp-harboring bacteria.The abundances of P-cycling-related genes varied in different soil aggregates . The gcdreatment . Consequed genes . In addigcd gene abundance was significantly negatively correlated with Col-CaP  (P < 0.05 or P < 0.01 or P < 0.001), whereas only OP was significantly correlated with Col-Phy (P < 0.05). Similarly, nonphosphorus nutrients  and P fractions  were significantly positively correlated with both gcd and bpp gene abundances (P < 0.05 or P < 0.01 or P < 0.001) (Linear regressions reflected that and bpp) . Nonphos< 0.001) . Accordi< 0.001) . Additiogcd abundance, bpp abundance, Col-CaP, and Col-Phy . Additionally, soil TC and P fractions, rather than gcd abundance, showed strong indirect and direct effects on Col-CaP, respectively  were identified as Bacillus, Pseudomonas, Massilia, Citrobacter, Arthrobacter, and Acinetobacter genera according to a phylogenetic tree based on 16S rRNA gene sequences  and soluble P levels of PSB-2 cultured in liquid National Botanical Research Institutes phosphate growth medium (NBRIP) media were significantly higher than that for PSB-5 at the same period , and growth tended to be stable after day 5 (P > 0.05). The soluble P levels of PSB-5 displayed a fast increase during 5 days and presented slight fluctuation after day 5. In contrast, soluble P levels of PSB-2 exhibited significantly fast increases during 8 days   affect abundances of P-cycling-related genes  –8, 34. Fcd genes . The abuicient P , 39, 40.icient P  and difficient P . Additioicient P . These ricient P , 43, 44,icient P , 14, 15.icient P . This phicient P .bpp gene abundance and phytate-degrading bacterial number, which is expected, and this might be because phytate is a relatively stable compound   , 16, 47.rganisms , 34, whirganisms . Organicsing CO2 , which fironment , 41, 50.ironment . Consequ studies , 42; to Pseudomonas sp. PSB-2 exhibited good performance for P-solubilizing, and the best P-solubilizing level was 59.98 mg/L. The solubilizing capacity for Ca3(PO4)2 by Pseudomonas sp. PSB-2 is higher than that for Pantoea dispersa Cav.cy3 (<50 mg/L)  (Serratia marcescens RP8 (974 mg/L)   but lowe93 mg/L)  and Serr74 mg/L) . PSB in 74 mg/L) , 14, 23,74 mg/L) . The Pser peanut , Enterobhick pea , Acinetothaliana , and Psegeranium . The inogeranium . Previougeranium , 57. Therogenase , 57. Futgcd and bpp) in soil aggregates under different fertilization treatments. We found strong linkages between gcd gene abundance and tricalcium phosphate-solubilizing bacterial number, as well as between bpp gene abundance and phytate-degrading bacterial number. The phosphate-solubilizing bacteria Pseudomonas sp. PSB-2 and Arthrobacter sp. PSB-5 showed good performances for inorganic phosphorus solubilization. The phosphate-solubilizing bacterium Pseudomonas sp. PSB-2 could enhance growth of Chinese cabbage, showing significant increases in plant fresh and dry weight as well as plant height. Our findings extend the knowledge of mechanisms for distribution patterns of phosphate-solubilizing bacteria and P-cycling-related genes in soil aggregates and might guide the isolation of phosphate-solubilizing bacteria. Future work will use multiple techniques  to investigate P-cycling-related bacterial abundance and community composition and optimize condition for P solubilization by PSB and decipher mechanisms for P solubilization at both the gene and protein levels.In conclusion, we found distinct distribution patterns of phosphate-solubilizing bacteria and P-cycling-related genes  and winter wheat (Triticum aestivum L.) rotation since it was built in 1978. Five fertilization treatments were used, including CK, N, NPK, M, and MN  for soil aggregates by using quantitative PCR (qPCR). Primer bppF (5′-GAC GCA GCC GAY GAY CCN GCN NTN TGG-3′) and primer bppR (5′-CAG GSC GCA NRT CAN CRT TRT T-3′) were employed to amplify the bpp gene 2 or sodium phytate solid medium and incubated at 30°C for 5 days. The NBRIP contained 10 g/L glucose, 5 g/L Ca3(PO4)2 or sodium phytate, 0.25 g/L MgSO4·7H2O, 5 g/L MgCl2·7H2O, 0.2 g/L KCl, 0.1 g/L (NH4)2SO4, 2 mL/L trace element solution, 0.2 g/L cycloheximide, and 18 g/L agar 2 were subcultured by picking and streaking five times to isolate pure colonies. We gained six strains  and identified them using simple 16S rRNA gene sequencing at Wuhan Qingke Innovation Biotechnology Co., Ltd. The universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used to amplify the 16S rRNA gene (Single colonies from the NBRIP containing CaRNA gene . The phy3(PO4)2 and incubated it at 30°C for 3 days.The isolated PSB  were incubated in both solid and liquid NBRIP to estimate their solubilization potentials for tricalcium phosphate . We inoc7 CFU/mL) (Exp group). Each treatment had five replicates. Chinese cabbage (Shanghai Qing) seeds were purchased from China National Seed Group, precultivated in sterile nutritious soils, and allowed to grow to about 10-cm length of sprouts . The original physicochemical properties and processing of experimental potted soils were described previously . Two pot sprouts . Each sporimetry .If not otherwise stated, we estimated significant differences by using the one-way analysis of variance. Permutational multivariate analysis of variance was conducted using the function adonis in the vegan package of R. Canonical analysis of principal coordinates was used to estimate effects of soil physicochemical factors on PSB number and gene abundance using the capscale function in the vegan package. A structural equation model was built to reflect potential linkage among soil physicochemical factors, gene abundance, and PSB number using IBM SPSS Amos v.21. For the structural equation model (SEM), the PC1 value of the first axis of the principal-component analysis, accounting for 99.89% of the total variation, was applied as a proxy for representing P components .


Commiphora have been used in traditional medicines for centuries. More than 200 Commiphora species exhibit highly variable phytochemical compositions. A novel highly selective, sensitive, accurate HPLC-MS/MS method was developed and validated to quantify five characteristic phytosteroids and furanosesquiterpenoids, namely (E)-guggulsterone, (Z)-guggulsterone, curzerenone, furanoeudesma-1,3-diene, and myrrhone. The resulting contents and additionally GC analysis were used to classify and differentiate Commiphora oleogum resins of the species C. myrrha, C. erythraea, C. mukul, C. holtziana, C. confusa, and C. kua, as well as unspecified resins. Interestingly, a Commiphora sample from Ogaden, Ethiopia, comprised 446 ng/mg guggulsterones presumed to be unique to C. mukul from the Indian subcontinent. However, Commiphora from Ogaden differed considerably from C. mukul in respect to guggulsterones isomer’s ratio. Moreover, the cytotoxicity of Commiphora extracts, essential oils, botanical drugs containing Commiphora, and pure compounds against the epidermoid carcinoma A431, malignant melanoma RPMI-7951 and SK-MEL-28 cells was investigated in vitro. Thereby, especially C. mukul extract and C. myrrha essential oil exhibited high cytotoxicity against skin cancer cells with IC50 of 2.9–10.9 µg/mL, but were less toxic to normal keratinocytes. In summary, Commiphora oleogum resins and its phytochemicals warrant further investigation aiming at chemotaxonomical classification as well as application in skin cancer treatment.Oleogum resins of the genus  Commiphora Jacq. of the Burseraceae family are shrubs or small trees with thorny branches and aromatic oleogum resin exudates with characteristic odors  Engl. (syn. C. molmol Engl.) predominantly growing in Somalia Commiphormponents . In factCommiphora oleogum resin or extract, namely Myrrhinil-Intest® and Gugulipid® were investigated. Additionally, two botanical drugs containing ® is intended for use against gastrointestinal disorders, such as non-specific diarrhea, mild cramps, or flatulence. It contains chamomile flower extract (70 mg/pill), coffee charcoal (50 mg/pill), and powered C. molmol (syn. C. myrrha) oleogum resin (100 mg/pill) [C. myrrha analysis. However, Myrrhinil-Intest® contained with 0.456 µg/mg considerably less furanoeudesma-1,3-diene than the crude C. myrrha oleogum resin (87.7 µg/mg) (Myrrhinil-Intestmg/pill) . HPLC-MS7 µg/mg) a. ® is considered a hypolipidemic drug containing an ethyl acetate extract of C. mukul [Z)-guggulsterone is the dominant isomer over (E)-guggulsterone with guggulsterone(ER/Z) = 0.61, which corresponds to the natural guggulsterone proportion in C. mukul [GugulipidC. mukul . ChemicaC. mukul ,22.Commiphora oleogum resin extracts and essential oils against the epidermoid carcinoma cell line A431 and the malignant melanoma cells lines RPMI-7951 and SK-MEL-28 were compared. The cell lines selected are rather resistant to treatment with the standard chemotherapeutic drugs cisplatin and 5-fluorouracil  between 8.4 and 21.4 µg/mL. Furthermore, Commiphora extracts were toxic for RPMI-7951 and SK-MEL-28 with IC50 = 2.9–11.5 µg/mL and IC50 = 10.9–23.4 µg/mL, respectively, which is comparable or even higher (particularly for SK-MEL-28 cells) than the cytotoxicity of cisplatin and 5-fluorouracil. Interestingly, the extracts obtained from C. mukul exhibited the highest cytotoxicity against all cell lines investigated. Statistical analysis confirmed higher cytotoxic efficacy of a C. mukul extract compared to extracts from C. erythraea, C. holtziana, C. kua, and C. from Tarraxo  to treat various conditions including cancers [C. myrrha, C. erythraea, and C. holtziana essential oils was shown to exhibit antiproliferative, proapoptotic and cytotoxic effect on human lung adenocarcinoma cells in vitro and in cancer xenografts in mice [Likewise, essential oils of ll lines . Essential cells . In Chin cancers . Particu cancers . Curzere in mice .C. mukul extract and C. myrrha essential oil were additionally tested for their cytotoxicity against non-carcinogenous human keratinocytes (MBU-IM). Here, the C. mukul extract and C. myrrha essential oil were less toxic to normal human keratinocytes than to malignant melanoma cells indicating their selectivity towards cancer cells -guggulsterone have been shown to induce caspase-dependent apoptosis in prostate cancer cells at concentrations > 10 µM [E)-guggulsterone exhibited cytotoxic effect on all three cancer cells lines with an IC50 = 12.6 µg/mL for A431, an IC50 = 3.7 µg/mL for RPMI-7951, and an IC50 = 11.1 µg/mL for SK-MEL-28. Similarly, (Z)-guggulsterone was also cytotoxic with an IC50 = 18.9 µg/mL for A431, an IC50 = 6.4 µg/mL for RPMI-7951, and an IC50 = 10.7 µg/mL for SK-MEL-28 - and (Z)-guggulsterone with regard to their toxicity against skin cancer cell lines were investigated. The combination index (CI) [E)- and (Z)-guggulsterones exhibited slight to moderate antagonistic interactions at effective doses ED50, ED75, and ED90. The only moderate synergistic interaction was observed in RPMI-7951 at ED50 . In addition to guggulsterones, C. mukul contains a further steroid substance group, termed guggulsterols [Because dex (CI) ,43, a qu at ED50 . This inlsterols . HoweverE)-guggulsterone, (Z)-guggulsterone, curzerenone, and furanoeudesma-1,3-diene were purchased from Sigma-Aldrich  and myrrhone from ChemFaces . Commiphora oleogum resins were provided by field experts  and subsequently deposited at the Herbarium of the Botanical Garden of Ulm University, Institute of Systemic Botany and Ecology, Germany (voucher: ULM-24224). For more precise sample information please see Commiphora botanical drugs Myrrhinil-Intest® (Batch No. 9100816) and Gugulipid® (Batch No. 1353619) were from Repha GmbH  and Natural Organics Inc. , respectively.All solvents and chemicals were of analytical reagent grade. The solvents used for the extraction, sample preparation, and HPLC-MS/MS analysis were MeOH, acetic acid , and ultrapure water  coupled to an Arium Pro station . The compounds  and evaporated to dryness by using a rotary evaporator.For extraction, 600 mg of freshly grounded n-hexane. The solvent of the combined organic phases was evaporated at 60 °C (water bath) with nitrogen stream.Essential oils were obtained by hydrodistillation of oleogum resins as previously described . ShortlyThe HPLC-MS/MS experiments were carried out on an Agilent 1260 Infinity HPLC system  coupled with an AB API 2000 triple quadrupole mass spectrometer  using an electrospray ionization ion source (ESI) in positive ionization mode. Devices were controlled and data were processed by means of Analyst 1.6.1 software .The chromatographic separation was performed using an analytical reversed-phase HPLC column  with a precolumn . v/v) (eluent A) and methanol (eluent B), both acidified with 0.2% acetic acid. Initial conditions were 32% eluent A and 68% eluent B followed by a linear gradient to 95% eluent B over 6.8 min, then 95% eluent B until 11.8 min. Thereafter, a linear gradient to initial conditions until 12.0 min and reequilibration continued until 17.0 min. In order to stabilize the chromatographic system, the column was kept at a temperature of 28 °C. The injection volume was set to 10 µL.By means of Design of Experiments (DoE) a novel method for chromatographic separation of guggulsterones was developed, in which the flow rate, the starting concentration of eluent B, and the gradient slope were optimized. Thus, the flow rate was 600 µL/min, the mobile phase consisted of methanol/water  and m/z 97.1 (product ion) as quantifier for (E)-guggulsterone and (Z)-guggulsterone. Moreover, m/z 313.3 (precursor ion) and m/z 109.1 (product ion) were used as qualifier for both guggulsterones. The ions m/z 231.0/83.0 (quantifier) and m/z 231.0/149.1  were used for curzerenone, m/z 229.0/159.0 (quantifier) and m/z 229.0/187.2  for myrrhone, and m/z 215.0/119.1 (quantifier) and m/z 215.0/105.2  for furanoeudesma-1,3-diene. The optimized source parameters and MS tune parameter are listed in The MS/MS detection was performed in multiple reaction monitoring (MRM) mode with Commiphora oleogum resins were not mixed with Boswellia oleogum resin, all samples were additionally analyzed for ten boswellic and lupeolic acids namely, α-boswellic acid (α-BA), acetyl-α-boswellic acid (α-ABA), β-boswellic acid (β-BA), acetyl-β-boswellic acid (β-ABA), 11-keto-α-boswellic acid (α-KBA), 11-keto-β-boswellic acid (β-KBA), acetyl-11-keto-α-boswellic acid (α-AKBA), acetyl-11-keto-β-boswellic acid (β-AKBA), lupeolic acid (LA), and acetyl-lupeolic acid (ALA). The boswellic and lupeolic acid analysis by HPLC-MS/MS was carried out as previously described [To ensure that escribed ,24.Commiphora species by GC-FID and GC-MS was carried out as described previously in detail [n-alkanes and mass spectra comparison with NIST17 libraries and in-house libraries. Moreover, 2-methoxyisofuranogermacrene and dihydropyrocurzerenone were identified by comparison of retention indices and mass spectra with expert literature [Analysis of essential oils in oleogum resins of different n detail ,45. The terature ,46. ResuThe skin cancer cell lines, epidermoid carcinoma cells A431, malignant melanoma cells RPMI-7951 and SK-MEL-25, and normal human keratinocytes MBU-IM were from the German Collection of Microorganisms and Cell Cultures  and cultured as recommended. 50 values were determined using SigmaPlot 14.0 software . Cells were seeded into 96-well plates and treated 24 h later using Tecan D300e digital dispenser . After 72 h incubation period, cell viability was analyzed by addition of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt . Absorbance of the formed orange formazan dye was analyzed with an Infinite M1000 PRO Tecan plate reader (Tecan) at λ = 450 nm with a λ = 630 nm reference filter . For quaE)- and (Z)-guggulsterone in human skin cancer cell lines were carried out according to recommendations [Combination studies of cytotoxic effects of , SigmaPlot 14.0 software , and Valoo 2.10 software . All data were tested for normal distribution by the Anderson-Darling test and equality of variances by Levene’s test. Sample groups were compared by one-way analysis of variance (ANOVA) and post hoc by Dunett’s test. Comparison of two sample groups was carried out by Student’s Commiphora by HPLC-MS/MS has been developed and validated. Additionally, essential oils of the respective oleogum resins were analyzed by GC. The phytochemical profiles were used to classify Commiphora oleogum resins of the species C. myrrha, C. erythraea, C. mukul, C. holtziana, C. confusa, and C. kua as well as unspecified Commiphora resins. Hence, patterns in the phytochemical composition were discovered assisting a chemotaxonomical differentiation among different Commiphora species. Interestingly, a Commiphora oleogum resin from the Ogaden region in Ethiopia comprised guggulsterones, which are unique for C. mukul from the India subcontinent. Considering the guggulsterones isomer’s ratio and essential oil composition, Commiphora from Ogaden in Africa differs considerably from C. mukul suggesting that at least one African Commiphora species produces also phytosteroids such as guggulsterones. Moreover, the study provides evidence for cytotoxic efficacy of Commiphora extracts and essential oils against human epidermoid carcinoma and malignant melanoma cells in vitro. Here, especially C. mukul extract and C. myrrha essential oil exhibited the highest cytotoxicity against all three skin cancer cell lines investigated, but were less toxic to normal keratinocytes. Commiphora preparations and phytochemicals should be investigated more detailed regarding, for example, their systemic toxicity aiming at the development of new anticancer drugs.A highly, selective, and accurate method for the simultaneous determination of five phytosteroids and furanosesquiterpenoids in oleogum resins of the genus


Cytochrome P450 1B1 (CYP1B1) gene are a major cause of PCG. Current study was conducted to screen CYP1B1 gene in highly consanguineous PCG affected families from Pakistani population consistent with the autosomal recessive pattern of PCG inheritance.Primary congenital glaucoma (PCG) is a heterogeneous rare recessively inherited disorder prevalent in regions with high consanguinity. Disease phenotype is associated with increased intra ocular pressure and is a major cause of childhood blindness. Sequence variations in CYP1B1 gene was done for all enrolled families. In-silico analysis was performed to identify and predict the potential disease-causing variations.For this study, patients and controls  from 25 consanguineous families belonging to Punjab, Baluchistan and Khyber Pakhtunkhwa, Pakistan were recruited through ophthalmologists. DNA was isolated from collected blood samples. Genetic screening of Pathogenicity screening revealed sequence variants segregating with disease phenotype in homozygous or compound heterozygous form in eleven out of 25 analyzed families. We identified a total of sixteen disease causing variants among which five frameshift i.e., c.629dup (p.Gly211Argfs*13), c.287dup (p.Leu97Alafs*127), c.662dup (p.Arg222Profs*2), c.758_759insA  and c.789dup (p.Leu264Alafs*63), two silent c.1314G>A, c.771T>G and six missense variations c.457C>G (p.Arg153Gly), c.516C>A (p.Ser172Arg), c.722T>A , c.740T>A (p.Leu247Gln), c.1263T>A (p.Phe421Leu), and c.724G>C (p.Asp242His) are previously un reported. However two frameshift c.868dup (p.Arg290Profs*37), c.247del (p.Asp83Thrfs*12) and one missense variant c.732G>A (p.Met244Ile), is previously reported. Furthermore, six polymorphisms c.1347T>C, c.2244_2245insT, c.355G>T, c.1294G>C, c.1358A>G and c.142C>G were also identified. In the intronic region, a novel silent polymorphism i.e., g.35710_35711insT was found in homozygous state. All the newly detected disease-causing variants were negative in 96 ethnically matched controls.CYP1B1 gene. Genetic counseling was provided to families to refrain from practicing consanguinity and perform premarital screening as a PCG control measure in upcoming generations.Among twenty-five screened families, eight families  were segregating disease causing variants in recessive manner. Two families (PCG049 and PCG062) had compound heterozygosity. Our data confirms genetic heterogeneity of PCG in Pakistani population however we did not find molecular variants segregating with PCG in fifteen families in coding exons and intron-exon boundaries of  GLC3A [GLC3B [GLC3C [GLC3D [CYP1B1) at GLC3A and Latent Transforming growth factor-β-binding Protein-2 (LTBP2) at GLC3D have been reported to cause PCG [MYOC) [FOXC1) [TEK) [Glaucoma is characterized by impaired vision due to increased intraocular pressure, a primary risk factor for irreversible optic nerve damage. This disorder can be categorized according to etiology (primary glaucoma/secondary glaucoma), onset  and iridocorneal angle (open/close) . PrimaryGLC3A , GLC3B [A [GLC3B , GLC3C [B [GLC3C  and GLC3C [GLC3D  at positC [GLC3D . Among tause PCG . HoweverG [MYOC) , Forkhea [FOXC1) , and the1) [TEK)  have alsCYP1B1 gene has three exons out of which the last 2 codes for a 543 amino acid (a.a) protein [CYP1B1 in development of eye is uncertain however it is believed that due to mutations in this gene, generation of some important morphogens is affected leading to structural defects in trabecular meshwork and the aqueous humour outflow pathways [CYP1B1 gene including missense, small deletions, indels, gross deletions and regulatory mutations [CYP1B1 gene in PCG cases belonging to consanguineous Pakistani families, we enrolled and screened twenty-five families for CYP1B1 variants. Each family had at least one child affected with primary congenital glaucoma. protein . Cytochr protein . This me protein . Exact fpathways , 17, 18.utations . Studiesutations , 15, 20.http://haplopainter.sourceforge.net/about.html) [Based on clinical assessment provided by ophthalmologists, 25 diagnosed families of PCG were enrolled belonging to Khyber Pakhtunkhwa (KPK), Baluchistan and South Punjab. Clinical data, family history and blood samples of patients and each available family member was collected after informed written consent following the principles of world medical association of Helsinki . The stuut.html)  was usedet al., 2010 [Average 4ml of peripheral blood sample was taken from each participating individual and stored in 5ml EDTA (Ethylene Diamine Tetra Acetic acid) vacutainer. Extraction was performed using non-organic method of DNA extraction described by Kaul l., 2010 . To checet al., 2019 [et al., 2019 [https://www.mutationtaster.org/) [Amplification of coding regions and at least 50 base pairs of flanking non-coding regions was performed using primers reported previously by Afzal er.org/) . Furtherhttp://www.hgvs.org/) guidelines, Mutalyzer (2.0.35)  [https://varsome.com/) [https://www.genomnis.com/access-hsf) was used to determine pathogenicity due to disruption of splicing signals because of sequence variants.For significance of each variant and to check their nomenclature according to Human Genome Variation Society HGVS (zer.nl/)  was usedme.com/)  was usedhttp://genetics.bwh.harvard.edu/pph2/) [https://sift.bii.a-star.edu.sg/) [http://provean.jcvi.org/seq_submit.php) [https://folding.biofold.org/i-mutant/i-mutant2.0.html) [http://mupro.proteomics.ics.uci.edu/) [fwt-ΔGfmut) of protein structure that corresponds to stabilizing or destabilizing effect of variations. To check the conservation between different mammalian species, Clustal omega  [https://weblogo.berkeley.edu/) [https://www3.cmbi.umcn.nl/hope/) [PolyPhen-2 (Polymorphism Phenotyping v2) (u/pph2/) , SIFT (Sedu.sg/)  and PROVmit.php)  were use.0.html)  and MUprci.edu/)  softwareustalo/)  was usedey.edu/)  was usedl/hope/)  was usedIn this study, twenty-five PCG segregating consanguineous Pakistani families were enrolled. Among these families i.e., 09 belonged to Punjab province of Pakistan, 01 to Azad Kashmir whereas 12 and 03 belonged to Khyber Pakhtunkhwa and Baluchistan respectively. At the time of enrollment, seventeen families  had a single PCG affected individual, three families  had two, one family (PCG058) had three, three families  had four whereas family PCG050 had six affected members respectively .CYP1B1 coding regions and at least 50 base pairs of flanking noncoding region using DNA of each proband revealed thirteen novel disease-causing variations in coding regions according to mutation taster , (c.247del), (c.732G>A) and six reported polymorphisms (c.1347T>C), (c.1294G>C), (c.1358A>G), (c.2244_2245insT), (c.355G>T), (c.142C>G) were also found. Another polymorphism g.35710_35711insT was also found in intronic region in family PCG062 that has not been reported previously. Out of the novel disease-causing variants, five were frame shift variations c.629dup (p.Gly211Argfs*13), c.287dup (p.Leu97Alafs*127), c.662dup (p.Arg222Profs*2), c.758_759insA  and c.789dup (p.Leu264Alafs*63). Other novel disease-causing variants include six missense variants c.457C>G (p.Arg153Gly), c.516C>A (p.Ser172Arg), c.722T>A , c.740T>A (p. Leu247Gln), c.1263T>A (p.Phe421Leu), and c.724G>C (p.Asp242His) and two silent variations c.1314G>A and c.771T>G. In addition to disease causing variants, seven polymorphisms were also detected .In family PCG049, two disease causing variations were detected. A single nucleotide substitution i.e., c.457C>G was present in heterozygous condition, it changed arginine at position 153 to glycine and was deleterious according to PROVEAN and Polyphen-2 with a score of -5.21 and 1.00 respectively  Table 3Table 3. TTCGGC and Fas ESS site TGTTTC was broken. Two new ESS sites TTTTCG and GTTTTC were created for IIE and one TGTGTTTT for PESS (putative exonic splicing silencer).Insertion of thymine (T) in PCG050 in exon 2 at position 629–630  changed CYP1B1 gene  that replaced aspartic acid at position 83 to threonine shifting reading frame and creating stop codon after 12 residues . I-MutanCTGTGGTTTTTGTC>CTGTGGTTTTAGTC changing consensus value (CV) from 50.91 to 78.78. CV for newly created site showed that it is not a very strong site (strong site CV> 80). PCG063 had two unreported mutations, a silent heterozygous mutation c.771T>G and a frameshift homozygous mutation c.789dup , Nomascus leucogenys (XP_003262792.2), Pongo abelii (XP_009235654.1) and Pan troglodytes (XP_001167556.1) with high similarity index to Homo sapiens   was founCYP1B1 coded protein is reported to cause abnormal development of ocular structures resulting in impeded outflow of aqueous humor and PCG phenotype [CYP1B1 gene varies among different populations i.e.; p.Ser476Pro is 44% prevalent in India, p.Arg469Trp, p.Arg368His, p.Arg390His, p.Gly61Glu and p.Glu173Arg are 70% prevalent in Iran, p.Gly61Glu, p. Arg390His and p.Glu229Lys are 80–100% prevalent in Saudi Arabia however p.Arg330Phe and p.Arg390His are predominantly reported from China [High frequency i.e., 70–100% of consanguineous marriages  is the mhenotype , 36. Datom China . Founderom China . Previouom China , 38, 39;CYP1B1 analysis in 25 cases enrolled through various regions of Punjab, Baluchistan and Khyber Pakhtunkhwa, Pakistan revealed a total of seven frameshift, seven missense and two silent disease-causing variations. Among seven frameshift variations five are novel however two are previously reported in patients of different ethnicities. The variant c.868dup (p.Arg290Profs*37) (rs67543922) was initially identified in PCG affected Pakistani family by Sheikh et al., 2014 [et al., 2015 [CYP1B1 conducted on population of Sindh and Punjab province of Pakistan by Rashid et al., 2019 [In present study l., 2014  and then7) rs675422 was inl., 2019  reportedet al., 2009 [et al., 2018 [Second reported frame shift variant c.247del (p.Asp83Thrfs*12) was initially identified in a study conducted on Indian population by Tanwar l., 2009 . Accordil., 2009 . Five hol., 2018  have shol., 2018 .CYP1B1 gene. Previously compound heterozygosity has been reported in developmental glaucoma, [et al., 2021 reported that two heterozygous mutations c.1310C>T (p.P437L) and c.3G>A (p.M1I) are responsible for glaucoma in a Chinese family [et al., 2019 identified compound heterozygosity  in two consanguineous families of PCG belonging to different ethnic groups of Pakistan [CYP1B1 with heterozygous TEK alleles in PCG cases [CYP1B1, recessive inheritance pattern and previously reported allelic interactions of two un linked genes for PCG phenotype [Missense disease-causing variants found in family PCG049, 052, 060, 062 and 067 and two silent disease-causing variants found in family PCG062, 063 are also previously unreported , Table 3laucoma,  and primlaucoma, . Cai et e family . In anote family . Waryah Pakistan . FurtherCG cases . In preshenotype , 13, 48 CYP1B1 disease causing variations in PCG patients [LTBP2, TEK, MYOC, FOXC1 and regulatory effect of cis-acting elements, splicing elements or possible modifiers [Due to epigenetic modifications and different environmental factors incomplete penetrance and increased variability could be observed in manifestation of patients , 49. We odifiers , 40, 50.et al., 2019 [All single nucleotide polymorphisms (SNPs) identified in current study except one present in intronic region i.e., g.35710_35711insT in homozygous state are previously reported. Four reported SNPs i.e., rs1056836 (c.1294G>C), rs1800400 (c.1358A>G), rs1056827 (c.355G>T) and rs10012 (c.142C>G) showed amino acid change while two polymorphisms rs1056837 (c.1347T>C) and rs4646431 (c.2244_2245insT) were silent. Most prevalent polymorphism (45%) c.1347T>C in present study was also reported in other studies conducted on PCG cases from Pakistani population . Afzal eCYP1B1 gene to PCG pathophysiology in our population.In conclusion we identified thirteen previously unreported and three reported mutations as well as six SNPs (one novel) in PCG probands born to parents having consanguineous marriages highlighting the autosomal recessive pattern of disease. Proper genetic testing and counseling should be provided to people in high consanguinity areas to help ophthalmologists in disease management and treatment. Mass screening and additional studies are required to better understand the heterogeneous pattern and contribution of


Corona virus disease-19 (Covid-19) has significantly affected organ transplantation with concerns regarding severe infection and mortality. Data on Covid-19 in renal transplant recipients (RTRs) is scarce from Pakistan. The aim of this study is find out the factors effecting mortality among Covid-19 patients in renal transplant recipients from the largest transplant center of Pakistan.All RTRs >18 years, with positive severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) polymerase chain reaction (PCR) and diagnosed as severe disease, between April to December 2020 were retrospectively reviewed. The severe disease was defined as O2 saturation < 94% at room air on admission. Survivors and non- survivors were compared. Demographics, immunosuppression, comorbid conditions, clinical features, laboratory investigations and graft function were noted.A total of 95 RTRs had severe disease. There was no difference in mortality between age, gender and co-morbid conditions among survivors and non-survivors. Both groups received similar immunosuppressive regimen. Intensive care unit (ICU) admission [16.5% vs 68.8% p< 0.001 OR 11.17 95% CI (3.3-37.6)] and high D-dimers >1.5µg/ml (p=0.052) at the time of admission were significantly associated with mortality. There was no association of graft function with mortality. Treatment with methyl-prednisolone was found to be significantly associated with survival [83% vs 43% P=0.02 OR 0.15 95% CI (0.05-0.49)]. (Table 1) WHO grading of the disease is shown in figure 1, there was a 100% mortality among patients on mechanical ventilator.ICU admission and high D-dimers at the time of admission are the significant risk factors for mortality in patients with Covid-19 infection. There was no association of graft dysfunction with mortality. Steroids use has significantly improved survival in renal transplant recipients with severe Covid-19 infection.All Authors: No reported disclosures.


Colitis-associated cancer (CAC) is a subtype of inflammatory bowel disease (IBD)-associated colorectal cancer. Huoxiang Zhengqi (HXZQ) is a classical Chinese herbal medicine and has been used to treat intestinal disorders, however, anti-CAC effects and underlying mechanisms of HXZQ have not been reported. An azoxymethane/dextran sulfate sodium-induced CAC mice model was used to investigate the anti-CAC effect of HXZQ. HXZQ significantly reduced colonic inflammation, suppressed the size and number of tumors, and reduced the levels of pro-inflammatory cytokines  and oxidative stress markers , and increased the levels of anti-inflammatory cytokines (IL-10 and IL-27) in CAC mice. Intestinal microbiota and serum metabolomics analyses indicated that HXZQ altered the gut microbial composition and the abundance of 29 serum metabolites in CAC mice. Additionally, HXZQ activated the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling pathway and increased the levels of antioxidants such as catalase (CAT), heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductases-1 (NQO-1), and superoxide dismutase-1 (SOD-1). HXZQ inhibited the activation of the nuclear factor kappa-B (NF-κB) signaling pathway and decreased the expression of NLR family pyrin domain containing 3 (NLRP3) by inhibiting the phosphorylation of inhibitor of nuclear factor kappa-B (IκB), inhibitor of nuclear factor kappa-B kinase (IKK), and NF-κB. In conclusion, HXZQ alleviated CAC in mice by modulating the intestinal microbiota and metabolism, activating Nrf2-mediated antioxidant response, and inhibiting NF-κB-mediated NLRP3 inflammasome activation against inflammation. The present data provide a reference for the use of HXZQ as a therapeutic or combination agent for clinical CAC treatment. Colitis-associated cancer (CAC) is a subtype of inflammatory bowel disease (IBD)-associated colorectal cancer. IBD includes Crohn’s disease (CD) and ulcerative colitis (UC) . InflammAtractylodes, regulates oxidative stress through the Nrf2 pathway and alleviates 2,4,6-trinitrobenzenesulfonic acid-induced acute colitis by influencing the composition of intestinal microbiota  that does not express BF enterotoxin to prevent DSS-induced colitis and prevent the formation of polyps in the model of CAC production, which affects the homeostasis of the intestinal microbiota . ROS canel of CAC.Prescriptions of Peaceful Benevolent Dispensary for approximately 900 years and has been used to treat intestinal disorders (Huoxiang Zhengqi (HXZQ) is a classic Chinese herbal medicine  that wasisorders . Recent isorders . Norfloxisorders . HoweverThe azoxymethane (AOM)/DSS-induced CAC mouse model mimics the pathology of CAC in humans and has been used for screening agents with anti-CAC properties and revealing the underlying mechanisms . In the Thirty-two 6-week-old male C57BL/6 mice (SCXK(Liao)2020–0,001, Liaoning Changsheng Biotechnology Co., Ltd. Benxi, China) were housed under specific pathogen-free conditions at the appropriate temperature (22 ± 2°C) and humidity (50% ± 10%) and kept on a 12/12 h light/dark cycle. The mice had unrestricted access to food and water. The experimental protocol complied with the ARRIVE guidelines and was approved by the Institution Animal Ethics Committee of Jilin University (SY202104007).n = 8) (serving as the model group) or 0.45 or 1.35 g/kg HXZQ  (serving as the HXZQ-treated groups) daily for 6 weeks. Another eight mice were intraperitoneally injected with normal saline on the first day, received normal drinking water for the entire experimental period, and received normal saline orally daily from the fifth to 10th week (serving as the control [Ctrl] group)  on the first day, and their drinking water was changed to 2% DSS  at the second, fifth, and eighth week. From the fifth week and beyond, the mice were randomly divided into three groups and orally received normal saline (] group) . Six houThe fixed colon, liver, spleen, and kidney tissues were paraffin-embedded, sectioned, and dewaxed in xylene for 40 min, anhydrous ethanol for 10 min, and 75% ethanol for 5 min, and then washed with running water. The sections were stained with hematoxylin and eosin (H&E) and treated sequentially with ethanol and xylene for dehydration. The sealed sections were observed and analyzed using an ECLIPSE E100 upright optical microscope  .Paraffin slides of colorectal tumors were subjected to antigen retrieval after de-paraffinization and blocked with 3% bovine serum albumin. The slides were incubated overnight at 4°C with primary antibody for nuclear factor kappa-B factor kappa-B (NF-κB) p65  in a wetn = 4) were used for 16S rRNA analysis of the gut microbiota. Nucleic acids were extracted from the contents of each cecum, and PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene was performed. PCR products were quantified and 2 × 250 bp double-end sequencing was performed. Sequencing was performed at Shanghai Personal Biotechnology Co. Ltd. .The cecum contents obtained from Ctrl, Model, and HXZQ-treated mice (https://www.ncbi.nlm.nih.gov/sra/PRJNA860221). These analyses were performed as previously described (The 16S rRNA sequencing results were clustered into amplicon sequence variants (ASVs) using DADA2 with 100% similarity. Based on the ASV abundance data, a flower plot was generated, and alpha diversity indices and the weighted UniFrac distance matrix were calculated. Abundance data from each group of microbiota were used to generate a species composition heatmap and for LDA effect size (LEfse) analysis. Microbial functions were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. The sequences of bacteria were uploaded to the NCBI Sequence Read Archive with accession number PRJNA860221  for vortex mixing. The supernatant obtained by centrifugation of the mixture was vacuum dried to a solid state. Before the analysis, the samples were dissolved in an aqueous solution of acetonitrile . Samples were separated on an ultra-high-performance liquid chromatography system  equipped with a ACQUITY UPLC BEH Amide 1.7 μm, 2.1 mm × 100 mm column  and were analyzed using a quadrupole-time of flight mass spectrometer . The separation conditions were identical to those reported in previous studies .p-value <0.05 as filtering criteria were used to draw heatmaps and correlation plots. The abundance data of the microbiota and metabolites with significant changes were jointly analyzed to obtain association heatmaps. These analyses were performed as described in our previous study , and score plots were plotted. The abundance of signature metabolites screened with OPLS-DA VIP >1 and us study .The collected tumor tissues were homogenized separately in phosphate buffer solution (PBS). Protein concentrations were determined using a Pierce™ BCA Protein Assay Kit . IL-1α, IL-1β, IL-6, IL-17A, IL-23, IL-27 and TNF-α in tumor tissues were detected using the LEGENDplex™ Mouse Inflammation Panel (13-plex) with a V-bottom plate . Enzyme-linked immunosorbent assay (ELISA) kits  were used to measure IL-10 (#MM-0176M1), IL-21 (#MM-0688M1), granulocyte-macrophage colony-stimulating factor (GM-CSF) (#MM-0185M1), malondialdehyde (MDA) (#MM-0897M1), and ROS (#MM-43700M1) levels in tumors.Tumor tissues were lysed at low temperatures in Radioimmunoprecipitation assay (RIPA) Buffer  supplemented with 1% Protease and Phosphatase Inhibitor Cocktail . Cytoplasmic and nuclear proteins in tumors were isolated by NE-PER Nuclear and Cytoplasmic Extraction Kit . Total protein in each sample was measured using the Pierce™ BCA Protein Assay Kit . The protein samples were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to PVDF membranes, and the membranes were blocked with western fast blocking solution  for 10 min at 25°C. The membranes were incubated with primary antibodies  for 12 hp-value less than 0.05 was considered statistically significant.All values are presented as mean ± standard error of the mean (S.E.M.) Biochemical indices were compared between Ctrl and Model using Student’s t-test. Comparisons among Model, 0.45 g/kg HXZQ, and 1.35 g/kg HXZQ were performed with one-way analysis of variance (ANOVA) followed by a post hoc multiple comparisons (Dunnett) test using BONC DSS Statistics 25 software . A p < 0.05) (HXZQ remarkably reduced the number and size of colonic tumors in CAC mice without influencing their body weight ( < 0.05) . Compare < 0.05) . HXZQ fa < 0.05) .Candidatus_Arthromitus, Turicibacter, Dorea, Acinetobacter, Clostridium, and Desulfovibrio, which were suppressed by HXZQ. Compared to the model group, HXZQ resulted in an increased abundance of 25 genera  (p < 0.001)  . The abu LDA >2) . Compara< 0.001) .p < 0.05) (The score plots of OPLS-DA showed significant differences in metabolite levels between the model and HXZQ-treated mice . The lev < 0.05) .Phascolarctobacterium, Prevotella, Helicobacter, and Butyricimonas (p < 0.05) (Candidatus_Arthromitus and p-75-a5 (p < 0.05) (Serum metabolite concentrations were significantly correlated with changes in colony abundance. Indoxyl sulfate was negatively correlated with  < 0.05) . L-Gluta < 0.05) .p < 0.01) (p < 0.01) (p < 0.001) (p < 0.01) (p < 0.001) (p < 0.01) (p < 0.05) (p < 0.05) (p < 0.001) (p < 0.05) (p < 0.001) (p < 0.01) (Inflammation severity tended to correlate positively with CAC development. HXZQ do < 0.01)  and ROS  < 0.01) , and red< 0.001) , IL-1β ( < 0.01) , IL-6 (>< 0.001) , IL-17A  < 0.01) , IL-21 ( < 0.05) , IL-23 ( < 0.05) , TNF-α (< 0.001) , GM-CSF  < 0.05) . Corresp< 0.001)  and IL-1 < 0.01) .p < 0.05), catalase (CAT) (>400%) (p < 0.001), heme oxygenase-1 (HO-1) (>400%) (p < 0.05), NAD(P)H quinone oxidoreductases-1 (NQO-1) (>470%) (p < 0.001), and superoxide dismutase-1 (SOD-1) (>220%) (p < 0.01) in colorectal tissues (p < 0.05), p-NF-κB (>52%) (p < 0.05), phosphorylated inhibitor of nuclear factor kappa-B kinase (p-IKK) (>33%) (p < 0.001), IL-1β (>51%) (p < 0.01), IL-6 (>63%) (p < 0.001), TNF-α (>40%) (p < 0.001), and NLRP3 (>29%) (p < 0.01) in colorectal tissues (p < 0.001)  ( tissues . HXZQ do tissues . HXZQ in< 0.001) . The res< 0.001) .IL10-/- mice spontaneously developed colitis, whereas upregulation of IL-10 alleviated CAC, which is consistent with our study  production and a strong positive correlation with the pro-inflammatory cytokine TNF-α (Acinetobacter activates the NLRP3 inflammasome and consequently mediates IL-1β production (Turicibacter and Desulfovibrio is increased under inflammatory conditions (Desulfovibrio has been reported to induce secretion of IL-6 and IL-8 from endothelial cells (Notably, changes in pro-inflammatory cytokine levels may also affect host’s intestinal microbiota. Accordingly, IL-33-deficient mice developed a dysregulated gut microbiota , and alt colitis . Disrupt colitis , which a colitis . The eff colitis . HXZQ inne TNF-α . Acinetooduction . The gronditions , and Desal cells .Intestinal microbiota directly influence host metabolism . HXZQ inNLRP3 transcription (Unsurprisingly, HXZQ activated Nrf2 signaling while increasing the levels of antioxidant enzymes such as CAT, HO-1, NQO-1, and SOD-1. Moreover, HXZQ inhibited the phosphorylation of IκBα, IKKα/β, and NF-κB as well as the expression of NLRP3. The accumulation of ROS during chronic inflammation leads to oxidative stress that aggravates CAC development . Nrf2 cocription .The present study has some limitations. The detailed relationship between HXZQ-mediated gut microbiota regulation and the anti-inflammatory and anti-oxidant effects that underlie its anti-CAC activity requires further investigation. Second, the main components involved in the therapeutic effect of HXZQ in the treatment of CAC need to be identified. Finally, the effects of HXZQ on other IBDs require further investigation.Altogether, HXZQ alleviates CAC by modulating the intestinal microbiota and metabolism, activating Nrf2-mediated antioxidant response, and inhibiting NF-κB-mediated NLRP3 in mice. Our data provide a reference for the use of HXZQ as a therapeutic agent or a combination agent for clinical CAC treatment.


Paenibacillus polymyxa DSM 365. The genome consists of a 5,788,318-bp chromosome, with a GC content of 45.48%. Annotation of the genome revealed a total of 5,246 genes . Gene function analysis indicated the ability to fix nitrogen (N2) and to produce value-added chemicals.We report the complete genome sequence of  Paenibacillus polymyxa DSM 365 is a Gram-positive plant growth-promoting rhizobacterium . To isolate DNA, cultures were grown overnight in tryptic soy broth at 30 °C and 200 rpm. Genomic DNA was extracted using the Wizard high-molecular-weight (HMW) DNA extraction kit . Library preparation and sequencing were conducted by Novogene Inc.  using the Illumina NovaSeq 6000 platform. To prepare the library for sequencing, genomic DNA was randomly sheared into short fragments. The fragments were end repaired, adenine tailed, and ligated with Illumina adapters. The quantified libraries (350-bp size) were pooled and sequenced to produce 6 Mb of paired-end 150-bp reads . In order to ensure accuracy and reliability, the reads were filtered using readfq software (v.10)  in 47 scaffolds, with a GC content of 45.48% and an average read coverage of 291×.acterium  with caphemicals –7. P. poe v.10) , 10, SPA , 10, SPe (v.10) , and ABye (v.10)  assemblye (v.10) . Assemble (v.10) . GapClose (v.10)  was usedGeneMarkS (v.4.10)  was used16–P. polymyxa DSM 365 was submitted to the National Center for Biotechnology Information (NCBI) database using the Prokaryotic Genome Annotation Pipeline (PGAP) (v.6.0) (e−5). Coding genes were predicted by Augustus (v.2.7) (nif operon), sporulation, acetoin utilization, biosynthesis of siderophores, polyketides, exopolysaccharides, and butanediol were detected.The whole-genome sequence of  (v.6.0) . Homolog (v.6.0)  was used (v.2.7)  with homP. polymyxa DSM 365 has been deposited in GenBank under the BioProject accession number PRJNA809744, the BioSample accession number SAMN26200526, and the Sequence Read Archive (SRA) accession number SRR18173204. The whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number JAKVDC010000000.The annotated genome sequence of


Data on willingness to participate in population-based long-COVID studies are sparse. We invited all citizens of Essen aged 18-74 years with a positive SARS-CoV-2 PCR test between Mar-Aug 2020 and assessed COVID-related symptoms in responders ∼1.5 years after infection.The invited population included 1282 infected citizens (48% women). At the time of testing 64% reported symptoms. We asked responders about past and current symptoms, hospitalization, smoking, sport, pre-existing conditions , subjective health status as compared to before infection, assessed BMI, and performed descriptive statistics.2), and more pre-existing conditions (23% vs 10%). Compared to before infection, 53% rated their current health worse, with a higher rate among inpatients (81%). After ∼1.5 years, 55% still reported symptoms: 25% fatigue, 20% concentration disorder, 18% breathing problems, 13% odor and 11% taste disorders. Persistent symptoms were more common in inpatients than in non-hospitalized (69% vs 53%).We investigated 255 participants  ∼20 month (median) after the PCR test. 95% reported symptoms at the time of testing: 67% fatigue, 58% taste disorders, 56% limb pain, 55% odor disorders, 54% headache, 50% cough, 43% fever; 10% needed hospitalization, 3% intensive care, 1.6% artificial ventilation. Compared to the non-hospitalized the formerly inpatients were more often male (62% vs 49%), older (56±13 vs 49±14 years), less often never smokers (42% vs 53%), had a higher BMI (31±7 vs 28±5 kg/mSymptomatic individuals are more likely to participate in a COVID19 follow-up study than asymptomatic ones. This may overestimate the number of individuals with long-term symptoms in population-based long-COVID study populations. However, persistent symptoms seem to be more likely in formerly inpatients compared to non-hospitalized individuals with former SARS-CoV-2 infection.• Symptomatic individuals are more likely to participate in a COVID19 follow-up study than asymptomatic ones.• Persistent symptoms seem to be more likely in formerly inpatients compared to non-hospitalized individuals with former SARS-CoV-2 infection.